Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
BRCA1 COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND
TREATMENT OF BREAST CAr~CE~
S 1. BACKGROUND OFTHE INVENTION
The present application is a continlling application of U. S. Provisional
Application Serial No. 60/015,863 filed July 8, 1996, the entire contents of which is
specifically incorporated herein by reference. The United States government has certain
rights in the present invention pursuant to grants DA57317, CA58318, P50-CA58183,
and 059-CA58183 from the National Cancer Institute.
1.1 FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology. More
particularly, certain embodiments concern methods and compositions comprising
BRCA1 compositions and methods for the diagnosis and treatment of breast cancer.Disclosed are methods and compositions useful in various pharmacological and
immunological applications.
1.2 DESCRIPTION OF THE RELATED ART
1.2.1 BREAST CANCER
Breast cancer is the most common fatal m~lign~ncy affecting females in
developed countries. The etiology of breast cancer involves a complex interplay of
genetic, hormonal, and dietary factors that are superimposed on the physiological status
of the host. Extensive genetic analysis of breast tumors has identified several alterations
in gene expression associated with the disease. At the molecular level, in addition to
frequently observed gene amplification (Escot et al.. 1986, Lidereau et al., 1988, Slamon
et al., 1987, van de Vijver et al.~ 1987, Varley et al., 1988), breast tumor development is
thought to be the consequence of l~nm~king one or more recessive genes by mutation.
Two genetic events serve to inactivate a recessive locus, and the resulting reduction to
homozygosity of the altered allele has been proposed to be an essential step in
tumorigenesis.
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Loss of heterozygosity (LOH) at six different regions of the human genome,
including chromosomes lq, 3p, llp, 13q, 17p and 17q, has been observed in a highpercentage of primary breast cancers (Ali et al., 1987, Chen et al., 1989, Devilee et al.,
1990, Devilee et al., 1989, Lundberg et al., 1987, Mackay et al., 1988a, Mackay et al.,
1988b, Futreal et al., 1992), and these allelic losses in tumor tissues suggest the locations
of potential tumor suppressor genes. Recently, the great excitement generated by the
cloning of the familial breast and ovarian cancer gene BRCAl on chromosome 17q (Miki
et al.~ 1994) has been somewhat tempered by failure to find mutations of the gene in
sporadic breast cancer (Futreal et al., 1994, Friedman et al., 1994, Shattucl~-Eiders et al.,
199~)- though BRCAI has been linked to greater than 45% of site-specific, inherited
breast cancers and 80% of families with breast and ovarian cancer (Easton et al., 1993),
no sporadic breast cancers and only about 10% of sporadic ovarian cancers have been
found to harbor BRCAI mutations (Futreal et al., 1994, Hosking et al., 1995, Merajver et
al., 1995).
Thus, the function of BRCAl in the pathogenesis of sporadic breast cancers,
which account for about 95% of all breast cancers (Claus et al., 199l)? has beenquestioned (Vogelstein et al., 1994). This contrasts with most other tumor suppressors,
such as RB and p 53, in which mutations are found in both familial and sporadic cancers
~Bookstein and Lee, 1991, Levine et al., 1991).
One explanation for these findings, which runs counter to established concepts
regarding tumor suppressor genes, suggests that BRCAI may be inactivated only infamilial breast cancers (Boyd, 1995; Castilla et al., 1994), and only a subset of these,
since other breast cancer genes, including BRCA2 on chromosome 13ql2-13 (Wooster et
al., 1994; 1995), have been mapped to different genetic loci. The pathogenesis of cancer
is a multistep process, and alternative pathways can eventually lead to the same or
similar consequences. Precedent exists in Wilms' tumor and hereditary nonpolyposis
colorectal cancer for different pathways to the same type of cancer (Vogelstein et al.,
1994).
Inactivation of both alleles of the WTI tumor suppressor gene, for example, has
been shown to be important for a substantial proportion of hereditary Wilms' tumors,
especially those occurring as part of the Denys-Drash syndrome, but not for sporadic
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Wilms' tumors (Pelletier et al., 1991; Coppes et al., 1993). Similar observations of
inactivation of the DNA mism~tc.h repair gene m sh2 in familial but not sporadic colon
cancers can be explained by the "mutator phenotype" generated by m sh2 mutation
(Cleaver, 1994; Vogelstein et al., 1994). Even without m sh2 inactivation, however, the
same steps in tumorigenesis can occur, albeit less frequently or rapidly.
1.2.2 ROLE OF BRCA1
The cloning of the familial breast and ovarian cancer gene BRCAI (Miki et al.,
1994) was a significant milestone in breast cancer research. Nonetheless, although
BRCAl has been linked to greater than 45% of site-specific, inherited breast cancers and
80% of families with breast and ovarian cancer (Easton et al., 1993), no sporadic breast
cancers and only about 10% of sporadic ovarian cancers have been found to harborBRCAI mutations (Miki et al., 1994; Futreat et al, 1994; Friedman et al., 1994;
Shattuck-Eiders et al., 1995; Hosking et al., 1995; Merajver et al., 1995). Thus the
general function of BRCAI in the pathogenesis of sporadic breast cancers, which
account for about 95% of such neoplasms (Claus et al, 1991), has been unproven to date
(Boyd, 1995; Castilla et al. 1994).
BRCA I complementary DNA encodes a 1863-amino acid protein whose
predicted structure includes two zinc finger domains near the NH,-terminus and an acidic
COOH-terminal domain, leading to speculation that the BRCAl protein is a transcription
factor (Miki et al, 1994; Vogelstein and Kinzler, 1994).
One and a half years after its isolation (Miki, et al., 1994), BRCAl, the gene on
human chromosome 17q21 responsible for almost 50% of inherited breast cancer,
remains an enigma. While mutations in BRCAl have been clearly linked to inherited
breast and ovarian cancer, no sporadic breast cancers, and only 10% of sporadic ovarian
cancers have been found to harbor BRCAl mutations (Futreal et al., 1994; Hosking et
al, 1995; Merajver et al., 1995). Rather. it has been suggested that BRCAI is
functionally inactivated by mislocation from its normal location within the nucleus to the
cytoplasm in spontaneous cancers (Chen et al., 1995). The defect responsible is
presumably in a protein required for the translocation of BRCAl to the nucleus, since
, . . . ~ , .,
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tagged, exogenous wild-type BRCAI is similarly mislocated in breast cancer cell lines
(Chen et al., 1996).
It has been speculated that BRCAI is a transcription factor, based on the presence
of a RING finger motif close to the N-terminus and a C-terminal segment rich in acidic
S residues (Miki et al., 1994). This would be consistent with the reported nuclear
localization of BRCAl. However, no direct evidence that BRCAl is a transcriptionfactor has yet been presented. In situ hybridization data have suggested that BRCAI
may play a critical role in cellular growth and differentiation, since BRCAl mRNA
appears to be generally expressed throughout developing mouse embryos, with
particularly high activity seen to correlate with tissues undergoing rapid proliferation and
differentiation (Lane et al., 1995; Marquis et al., 1995). Consistent with this,homozygous deletion of BRCA1 in mice is lethal in early embryogenesis (Gowen et al.
1996; Chia-Yang Liu et al. ). While these findings suggest potential roles for BRCAI, a
detailed characterization of BRCA I function at the molecular level has been hindered by
the lack of well characterized antibodies and sufficient purified protein with which to
assay function in vitro. Thus, there have been discrepancies in the literature concerning
the size and cellular location of BRCAl. While two groups have found BRCAI to be a
220 kDa nuclear protein (Chen et al., 1995; Scully et al, 1996), others using similar
antibodies have suggested BRCAI to be a 190 kDa secreted protein (Gudas et al., 1995;
Jensenetal., 1996).
1.2.3 FAMILIAL INHERITANCE OF BREAST CANCER
BRCAI, located on chromosome 17q21, is broadly believed to be responsible for
about 50% of familial breast and ovarian cancers. Based on the presence of a zinc finger
motif and an acidic activation domain, it has been speculated that BRCA I is a
transcription factor (Miki et al., 1994). However, to date, no gene activation or repressor
function has been documented. BRCA I may play a role in cellular growth and
differentiation since its mRNA is widely expressed in developing embryos, being
especially high in tissues where cells are rapidly proliferating and differentiating (Lane et
al., 1995; Marquis et al., 1995). In spite of a reported case of a woman with two mutated
alleles (Boyd et al., 1995), homozygous deletion of the BRCA I gene in mice is lethal in
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early embryogenesis (Gowan et al., 1996; Liu et al., 1996). The expression level and
phosphorylation by cdk2 kinase is regulated during the cell cycle (Chen et al., 1996). In
general, the data are consistent with a role for BRCA 1 in regulating cell proliferation and
differentiation.
s
1.2.4 T U M OR SUPPFUESSER FUNCTION OF B R C A1
As a tumor suppressor gene, it is paradoxical that mutations in BRCAI are clearly
linked to inherited breast and ovarian cancers, but are rarely found in sporadic tumors
(Miki et al., 1994; Futreal et al., 1994; Hosking e~ al., 1995). One suggestion is that~
although it is genetically intact, BRCAI may be functionally inactivated by mislocation
from the nuclear to cytoplasmic compartments in sporadic breast cancer cells (Chen et
al., 1995; 1996). However, the problem is either in nuclear transport, retention or
cytoplasmic confinement since epitope~tagged exogenous wild-type BRCAI protein is
also cytoplasmic in at least two lines of breast cancer cells (Chen et al., 1996).
Since BRCAI has a molecular mass of approximately 220 kDa, presumably it is
actively translocated from the cytoplasm to the nucleus by direct interactions with the
nuclear localization signal receptor or by indirect interactions with other NLS-containing
proteins (Hicks and Rikhel, Ig95; Dingwall and Laskey, 1991). The direct import of
karyophilic proteins through the nuclear pore complex requires energy (Newmeyer and
Forbes, 1988; Richardson et al., 1988) and a nuclear localization sequence (NLS) located
in the transport substrate (Dingwall et al., 1982; Kalderon et al., 1984) to which a
cytosolic receptor complex, importin-a and importin-~, binds (Gorlich et al., 1994;
1995). A GTP-binding protein, RAN, mediates the energy-dependent translocation of
the substrate-receptor complex through the nuclear pore complex (Moore and Blobel,
1993). After translocation, importin-~ dissociates from the complex in the vicinity of the
inner aspect of the nuclear envelope while importin-o~ accompanies the substrate to its
sites of function (Gorlich et al., 1995).
In contradiction to its nuclear localization reported by us (Chen et al., 1995;
1996) and others (Scully et al., 1996), there is a published report indicating that BRCAI
is a secreted protein (Jensen et al., 1996). Since the subcellular location of proteins is a
fundamental aspect of their function, it is important to solidify the data regarding the
. . .
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location of BRC~l in norrnal and cancer cells. More importantly, the subcellularcompartmentation of BRCAI is also a critical issue with regard to its role in breast
tumorigenesis.
1.3 DEFICIENCIES IN THE PRIOR ART
Therefore, what is lacking in the prior art are novel methods and compositions to
facilitate the diagnosis and treatment of breast cancer. Also what is lacking are methods
and compositions for deterrnining the subcellular localization of BRCA1 protein in
norrnal and suspected cancerous cells. Also needed are methods and compositions
comprising specific BRCAl-associated proteins which are responsible for the correct
translocation of the BRCAI gene product to the cell nucleus.
2. SUMMARY OF THE INVENTION
The present invention overcomes one or more of these and other drawbacks
inherent in the prior art by providing novel compositions and methods for their use in the
diagnosis and treatment of breast cancer.
In one aspect, the invention provides a method of localizing a BRCA1 protein or
peptide in a cell. The method generally involves contacting the cell with a labeled antibody
that specifically binds to a BRCAI or BRCAI-associated protein or peptide, underconditions effective to allow the formation of immune complexes; and determining the
location of the immune complexes in the cell. When such complexes are localized in the
cytoplasm of the cell, there is an indication of met~t~ci.~ or primary cancer of the cell.
Such information is useful in the early detecting and screening for cancers, and in
particular, breast and ovarian cancers, which the inventors have shown to be correlated with
cytoplasmic subcellular localization of BRCA1 protein. Preferably, the cells, are human,
and in particular, a breast or an ovarian cell.
A further object of the invention is a method of identifying a breast or ovariancancer cell in a sample. The method generally involves obtaining an ovarian or breast
tumor cell suspected of being cancerous and determining the subcellular location of a
BRCA1 protein or peptide in the tumor cell. As stated above, subcellular localization of
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the BRCAl protein or peptide to the cytoplasm of the cell has been demonstrated by the
inventors to be indicative of the presence of cancerl
Also provided, is a method of predicting susceptibility of an ovarian or breast cell
to cancer. The method generally involves identifying in the cell a cytoplasmically-
localized BRCAI or BRCAI-associated protein or peptide, wherein the presence of the
protein or peptide in the cytoplasm is indicative of susceptibility of the cell to cancer.
2.1 NUCLEIC ACID COMPOSITIONS
The invention provides nucleic acid sequences encoding a BRCAI-associated
protein (BAP). As used herein, a BAP gene means a nucleic acid sequence encoding a
BRCAl-associated protein. A preferred nucleic acid sequence encoding a BAP gene is a
nucleotide sequence which encodes the amino acid sequence of SEQ ID NO:I. It is
expected that the gene encoding BAP may vary in nucleic acid sequence from sample to
sample, but that the variation in nucleic acid sequence will not preclude hybridization
between sequences encoding BAP of each sample under strict hybridization conditions.
As used herein, a strain variant of BAP means any polypeptide encoded, in whole
or in part, by a nucleic acid sequence which hybridizes under strict hybridization
conditions to a nucleic acid sequence which encodes the amino acid sequence of SEQ ID
NO:l .
In the present invention, a BAP is also understood to mean a polypeptide that isimmunologically reactive with antibodies generated against the BAP protein of SEQ ID
NO:l .
Likewise, BRCAl is understood to mean a polypeptide that is capable of elicitingantibodies that are immunologically reactive with BRCA I and BRCA l-like gene
products, while BAP is understood to mean a polypeptide that is capable of eliciting
antibodies that are immunologically reactive with a BAP encoded by a nucleic acid
sequence which encodes the amino acid sequence of SEQ ID NO: 1.
As used herein, an active fragment of BAP includes BAPs which are modified by
conventional techniques, e.g., by addition~ deletion, or substitution, but which active
fragment exhibits substantially the same structure and function as BAP as described
herein. antigenicity according to conventional methods.
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Regarding BAP, the present invention concerns DNA segments, that can be
isolated from virtually any bacterial source, that are free from total genomic DNA and
that encode proteins having BAP-like activity. DNA segments encoding BAP-like
species may prove to encode proteins, polypeptides, subunits, functional domains, and
the like.
As used herein, the term "DNA segment" refers to a DNA molecule that has been
isolated free of total genomic DNA of a particular species. Therefore, a DNA segment
encoding BAP refers to a DNA segment that contains BAP coding sequences yet is
isolated away from, or purified free from, total genomic DNA of the species from which
the DNA segment is obtained. Included within the term "DNA segment", are DNA
segments and smaller fragments of such segments, and also recombinant vectors,
including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified BAP gene refers to
a DNA segment including BAP coding sequences and, in certain aspects, regulatorysequences, isolated substantially away from other naturally occurring genes or protein
encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a
functional protein, polypeptide or peptide encoding unit. As will be understood by those
in the art, this functional term includes both genomic sequences, extra-genomic and
plasmid-encoded sequences and smaller engineered gene segments that express, or may
be adapted to express, proteins, polypeptides or peptides. Such segments may be
naturally isolated, or modified synthetically by the hand of man.
"Isolated substantially away from other coding sequences" means that the gene ofinterest, in this case, a gene encoding BAP, forms the significant part of the coding
region of the DNA segment, and that the DNA segment does not contain large portions
of naturally-occurring coding DNA, such as large chromosomal fragments or other
functional genes or polypeptide coding regions. Of course, this refers to the DNA
segment as originally isolated, and does not exclude genes or coding regions later added
to the segment by the hand of man.
It will also be understood that amino acid and nucleic acid sequences may include
additional residues, such as additional N- or C-terminal amino acids or 5' or 3'sequences, and yet still be essentially as set forth in one of the sequences disclosed
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herein, so long as the sequence meets the criteria set forth above, including the
m~inten~nce of biological protein activity where protein expression is concerned. The
addition of terminal sequences particularly applies to nucleic acid sequences that may,
for example, include various non-coding sequences fl~nking either of the 5' or 3' portions
of the coding region or may include various upstream or downstream regulatory or- structural genes.
Naturally, the present invention also encompasses DNA segments that are
complementary, or essentially complementary, to the sequence set forth in SEQ IDNO:I. Nucleic acid sequences that are "complementary" are those that are capable of
base-pairing according to the standard Watson-Crick complementarity rules. As used
herein, the term "complementary sequences" means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotide comparison set
forth above, or as defined as being capable of hybridizing to a nucleic acid segment
which encodes the amino acid sequence of SEQ ID NO:I, under relatively stringentconditions such as those described herein.
The nucleic acid segments of the present invention, regardless of the length of the
coding sequence itself, may be combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other
coding segments, and the like, such that their overall length may vary considerably. It is
therefore contemplated that a nucleic acid fragment of almost any length may be
employed, with the total length preferably being limited by the ease of preparation and
use in the intended recombinant DNA protocol. For example, nucleic acid fragments
may be prepared that include a short contiguous stretch identical to or complementary to
a nucleic acid sequence which encodes the amino acid sequence of SEQ ID NO:l, such
as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in
length, with segments of about 3,000 being preferred in certain cases. DNA segments
with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about
50 base pairs in length (including all intermediate lengths) are also contemplated to be
useful.
It will be readily understood that "intermediate lengths", in these contexts, means
any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23.
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elc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153,
etc.; including all integers through the 200-500; 500-1,000; 1,000-2,000; 2,000-3,000;
3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 12,001,12,002, 13,001, 13,002 and the like.
It will also be understood that this invention is not limited to the particular
nucleic acid or amino acid sequences disclosed herein. Recombinant vectors and isolated
DNA segments may therefore variously include the BAP coding regions themselves,
coding regions bearing selected alterations or modifications in the basic coding region, or
they may encode larger polypeptides that nevertheless include BAP coding regions or
may encode biologically functional equivalent proteins or peptides that have variant
amino acids sequences.
If desired, one may also prepare fusion proteins and peptides, e.g., where the BAP
coding regions are aligned within the same expression unit with other proteins or
peptides having desired functions, such as for purification or immunodetection purposes
(e.g, proteins that may be purified by affinity chromatography and enzyme label coding
regions, respectively).
- Recombinant vectors form further aspects of the present invention. Particularly
useful vectors are contemplated to be those vectors in which the coding portion of the
DNA segment, whether encoding a full length protein or smaller peptide, is positioned
under the control of a promoter. The promoter may be in the form of the promoter that is
naturally associated with a BAP gene, as may be obtained by isolating the S' non-coding
sequences located upstream of the coding segment, for example, using recombinantcloning and/or PCRTM technology, in connection with the compositions disclosed herein.
In other embodiments, it is contemplated that certain advantages will be gained
by positioning the coding DNA segment under the control of a recombinant, or
heterologous, promoter. As used herein, a recombinant or heterologous promoter is
intended to refer to a promoter that is not normally associated with a BAP gene in its
natural environment. Such promoters may include BAP promoters normally associated
with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or
m~mm~ n cell. Naturally, it will be important to employ a promoter that effectively
directs the expression of the DNA segment in the cell type, organism, or even animal,
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chosen for expression. The use of promoter and cell type combinations for protein
expression is generally known to those of skill in the art of molecular biology, for
example, see Sambrook et al., 1989. The promoters employed may be constitutive, or
inducible, and can be used under the app~ul)l ;ate conditions to direct high level
expression of the introduced DNA segment, such as is advantageous in the large-scale
production of recombinant proteins or peptides.
Prokaryotic expression of nucleic acid segments of the present invention may be
performed using methods known to those of skill in the art, and will likely comprise
expression vectors and promoter sequences such as those provided by tac, trp, lac,
lacUVS or T7. When expression of the recombinant BAP, BAP-like, BRCA1, or
BRCAI-like proteins is desired in eukaryotic cells, a number of expression systems are
available and known to those of skill in the art. An exemplary eukaryotic promoter
system contemplated for use in high-level expression is the Pichia expression vector
system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant BAP,
BRCAI and/or related peptides, it is contemplated that longer DNA segments will most
often be used, with DNA segments encoding the entire BAP or BRCAI or functional
domains, epitopes, ligand binding domains, subunits, e~c. being most preferred.
However, it will be appreciated that the use of shorter DNA segments to direct the
expression of BAP or BRCAl peptides or epitopic core regions, such as may be used to
generate anti-BAP or anti-BRCA I antibodies, also falls within the scope of the
invention. DNA segments that encode peptide antigens from about 15 to about 100
amino acids in length, or more preferably, from about 15 to about 50 amino acids in
length are contemplated to be particularly useful.
The BAP gene and DNA segments may also be used in connection with somatic
expression in an animal or in the creation of a transgenic animal. Again, in such
embodiments, the use of a recombinant vector that directs the expression of the full
length or active BAP protein is particularly contemplated. Expression of a BAP
transgene in animals is particularly contemplated to be useful in the production of anti-
BAP antibodies for use in passive immunization methods and treatment of particular
breast cancers.
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2.2 RECOMBINANT EXPRESSION OF BAP AND BRCAI
As used herein, the term "engineered" or "recombinant" cell is intended to refer to
a cell into which a recombinant gene, such as a gene encoding a BAP or BRCAl hasbeen introduced. Therefore, engineered cells are distinguishable from naturally
occurring cells which do not contain a recombinantly introduced gene. Engineered cells
are thus cells having a gene or genes introduced through the hand of man.
Recombinantly introduced genes will either be in the form of a single structural gene, an
entire genomic clone comprising a structural gene and fl:~nking DNA, or an operon or
other functional nucleic acid segment which may also include genes positioned either
upstrearn and/or downstream of the promoter, regulatory elements, or structural gene
itself, or even genes not naturally associated with the particular structural gene of
interest.
Where the introduction of a recombinant version of one or more of the foregoing
genes is required, it will be important to introduce the gene such that it is under the
control of a promoter that effectively directs the expression of the gene in the cell type
chosen for engineering. In general, one will desire to employ a promoter that allows
constitutive (constant) expression of the gene of interest. Commonly used constitutive
eukaryotic promoters include viral promoters such as the cytomegalovirus (CMV)
promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40 early
gene promoter. The use of these constitutive promoters will ensure a high~ constant level
of expression of the introduced genes. The inventors have noticed that the level of
expression from the introduced genes of interest can vary in different clones, or genes
isolated from different strains or bacteria. Thus, the level of expression of a particular
recombinant gene can be chosen by evaluating different clones derived from each
transfection experiment; once that line is chosen? the constitutive promoter ensures that
the desired level of expression is permanently maintained. It may also be possible to use
promoters that are specific for cell type used for engineering, such as the insulin
promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in
anterior pituitary cell lines.
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The recombinant GST-BRCAl~Bglll gene fusion disclosed herein was deposited
with the American Type Culture Collection in E. coli DHSaTMF' under the terms of the
Budapest Treaty and was assigned the following accession number: ATCC 98100.
S 2.3 IMMUNODETECTION KITS
- In still further embodiments, the present invention concerns immunodetection
methods and associated kits. It is contemplated that the proteins or peptides of the
invention may be employed to detect antibodies having reactivity therewith, or,
alternatively, antibodies prepared in accordance with the present invention, may be
employed to detect BAP or BRCAI peptides. The kits may also be used in antigen or
antibody purification, as ~lo~l;ate.
In general, the preferred immunodetection methods will include first obtaining asample suspected of cont~ining a BAP or BRCAI-reactive antibody, such as a biological
sample from a patient, and contacting the sample with a first BAP or BRCA1 peptide
under conditions effective to allow the formation of an immunocomplex (primary
immune complex). One then detects the presence of any primary immunocomplexes that
are formed.
Contacting the chosen sample with the BAP or BRCAl peptide under conditions
effective to allow the forrnation of (primary) immune complexes is generally a matter of
simply adding the protein or peptide composition to the sample. One then incubates the
mixture for a period of time sufficient to allow the added antigens to form immune
complexes with, i.e., to bind to, any antibodies present within the sample. After this
time, the sample composition, such as a tissue section, ELISA plate, dot blot or western
blot, will generally be washed to remove any non-specifically bound antigen species,
allowing only those specifically bound species within the immune complexes to bedetected.
The detection of immunocomplex formation is well known in the art and may be
achieved through the application of numerous approaches known to the skilled artisan.
Detection of primary immune complexes is generally based upon the detection of a label
or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme
tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase
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being suitable. The particular antigen employed may itself be linked to a detectable
label, wherein one would then simply detect this label, thereby allowing the amount of
bound antigen present in the composition to be determined.
Alternatively, the primary immune complexes may be detected by means of a
second binding ligand that is linked to a detectable label and that has binding affinity for
the first protein or peptide. The second binding ligand is itself often an antibody, which
may thus be termed a "secondary" antibody. The primary immune complexes are
contacted with the labeled, secondary binding ligand, or antibody, under conditions
effective and for a period of time sufficient to allow the formation of secondary immune
complexes. The secondary immune complexes are then generally washed to remove any
non-specifically bound labeled secondary antibodies and the rem~ining bound label is
then detected.
For diagnostic purposes, it is proposed that virtually any sample suspected of
cont~ining the antibodies of interest may be employed. Exemplary samples includeclinical samples obtained from a patient such as blood or serum samples, cerebrospinal,
synovial, or bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even
perhaps urine samples may be employed. Furthermore, it is contemplated that suchembodiments may have application to non-clinical samples, such as in the titering of
antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical
samples may be from veterinary sources and may include such domestic animals as
cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be
used in accordance with the methods described herein.
In related embodiments, the present invention contemplates the preparation of
kits that may be employed to detect the presence of BAP- or BRCAI-specific antibodies
in a sample. Generally speaking, kits in accordance with the present invention will
include a suitable protein or peptide together with an immunodetection reagent, and a
means for containing the protein or peptide and reagent.
The immunodetection reagent will typically comprise a label associated with a
BAP or BRCAl peptide, or associated with a secondary binding ligand. Exemplary
ligands might include a secondary antibody directed against the first BAP or BRCA1
peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated
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label. Detectable labels linked to antibodies that have binding affinity for a human
antibody are also contemplated, e.g., for protocols where the first reagent is a BAP or
BRCAI peptide that is used to bind to a reactive antibody from a human sample. Of
course, as noted above, a number of exemplary labels are known in the art and all such
labels may be employed in connection with the present invention. The kits may contain
antigen or antibody-label conjugates either in fully conjugated form, in the form of
intermediates, or as separate moieties to be conjugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask,
bottle, syringe or other container means, into which the antigen may be placed, and
preferably suitably allocated. Where a second binding ligand is provided, the kit will
also generally contain a second vial or other container into which this ligand or antibody
may be placed. The kits of the present invention will also typically include a means for
cont~ining the vials in close confinement for commercial sale, such as, e.g., injection or
blow-molded plastic containers into which the desired vials are retained.
A hybridoma BRCA16B4 described herein producing mAbs against BRCAI
(aBRCAlBgl) was deposited with the American Type Culture Collection under the
provisions of the Budapest Treaty and was assigned the following accession number:
ATCC HB-12146. The isotype of this antibody is IgG1, K. Other antibodies specific for
BRCAI which are contemplated to be useful in the practice of the present invention
include aBRCAlN and aBRCA1, described in detail in Section 5.
2.11 VACCINE FORMULATION AND COMPOSITIONS
It is expected that to achieve an "immunologically effective formulation" it maybe desirable to a(1mini~ter BRCA1 or a BRCA1-associated protein to the human or
animal subject in a ph~rm~ceutically acceptable composition comprising an
immunologically effective amount of BRCAI or a BRCAI-associated protein mixed
with other excipients, carriers, or diluents which may improve or otherwise alter
stimulation of B cell and/or T cell responses, or immunologically inert salts, organic
acids and bases, carbohydrates, and the like, which promote stability of such mixtures.
Immunostimulatory excipients, often referred to as adjuvants, may include salts of
aluminum (often referred to as Alums), simple or complex fatty acids and sterol
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compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or
genetically modified protein toxins, and various particulate or emulsified combinations
thereof. BRCAl, BRCAI-derived peptides, or one or more BAPs may be formulated
within these mixtures, or each variant if more than one are present, would be expected to
comprise about 0.0001 to 1.0 milligrams, or more preferably about 0.001 to 0.1
milligrams, or even more preferably less than 0.1 milligrams per dose.
It is also contemplated that attenuated or~ni~mc may be engineered to express
recombinant BRCAl gene products or a BRCAI-associated protein and themselves be
delivery vehicles for the invention. Particularly preferred are attenuated bacterial species
such as Mycobacterium, and in particular M. bovis, M smegmatis, or BCG.
Alternatively, pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, or
Shigella, species may also be used in conjunction with the methods and compositions
disclosed herein.
The nal~ed DNA technology, often referred to as genetic immunization, has been
shown to be suitable for protection against infectious org~ni~m~. Such DNA segments
could be used in a variety of forms including naked DNA and plasmid DNA, and maya-lmini~tered to the subject in a variety of ways including parenteral, mucosal, and
so-called microprojectile-based "gene-gun" inoculations. The use of BRCAI or BAPgene nucleic acid compositions of the present invention in such immunization techniques
is thus proposed to be useful in the formulation of antibodies directed against such
proteins.
It is recognized by those skilled in the art that an optimal dosing schedule of a
vaccination regimen may include as many as five to six, but preferably three to five, or
even more preferably one to three :~(lmini.~trations of the immunizing entity given at
intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at
even longer intervals.
2.12 TRANSFORMED HOST CELLS AND RECOMBINANT VECTORS
Particular aspects of the invention concern the use of plasmid vectors for the
cloning and expression of recombinant peptides, and particular peptide epitopes
comprising either native, or site-specifically mutated BRCA1 or BRCAl-associated
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protein epitopes. The generation of recombinant vectors, transformation of host cells,
and expression of recombinant proteins is well-known to those of skill in the art.
Prokaryotic hosts are preferred for expression of the peptide compositions of the present
invention. An example of a preferred prokaryotic host is E. coli, and in particular, E coli
strains ATCC69791, BL21(DE3), JM101, XL1-Blue~, RR17 LE392, B, pl776 (ATCC No.
- 31537), and W3110 (F-, ~~, prototrophic, ATCC273325). Alternatively, other
Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, or
even other Gram-negative hosts including various Pseudomonas species may be used in
the recombinant expression of the genetic constructs disclosed herein.
In general, plasmid vectors cont~ining replicon and control sequences which are
derived from species compatible with the host cell are used in connection with these
hosts. The vector ordinarily carries a replication site, as well as marking sequences
which are capable of providing phenotypic selection in transformed cells. For example,
E. coli may be typically transformed using vectors such as pBR322, or any of itsderivatives (Bolivar et al., 1977). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also contain, or be
modified to contain, promoters which can be used by the microbial organism for
expression of endogenous proteins.
In addition, phage vectors cont~ining replicon and control sequences that are
compatible with the host microorganism can be used as transforming vectors in
connection with these hosts. For example, bacteriophage such as ~GEMT~ l 1 may be
utilized in making a recombinant vector which can be used to transform susceptible host
cells such as E. coli LE392.
Those promoters most commonly used in recombinant DNA construction include
the ~-lactamase (penicillinase) and lactose promoter systems (Chang et al.. 1978; Itakura
et al., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et
al., 1980). The use of recombinant and native microbial promoters is well-known to
those of skill in the art, and details concerning their nucleotide sequences and specific
methodologies are in the public domain, enabling a skilled worker to construct particular
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recombinant vectors and expression systems for the purpose of producing compositions
of the present invention.
In addition to the preferred embodiment expression in prokaryotes, eukaryotic
microbes, such as yeast cultures may also be used in conjunction with the methods
S disclosed herein. Saccttaromyces cerevisiae, or common bakers' yeast is the most
commonly used among eukaryotic microorg~ni~m~, although a number of other species
may also be employed for such eukaryotic expression systems. For expression in
Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb el al.,
1979; ~ing~m~n et al., 1979; Tschemper et al., 1980). This plasmid already contains the
trpL gene which provides a selection marker for a mutant strain of yeast lacking the
ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
The presence of the trpL lesion as a characteristic of the yeast host cell genome then
provides an effective environrnent for detecting transformation by growth in the absence
of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for 3-
phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al.,
1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase. In constructing suitable expression
plasmids, the termination sequences associated with these genes are also ligated into the
expression vector 3' of the sequence desired to be expressed to provide polyadenylation
of the mRNA and termination. Other promoters, which have the additional advantage of
transcription controlled by growth conditions are the promoter region for alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any
plasmid vector containing a yeast-compatible promoter, an origin of replication, and
terrnination sequences is suitable.
In addition to microorg~ni~m.~ cultures of cells derived from multicellular
organisms may also be used as hosts in the routine practice of the disclosed methods. In
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principle, any such cell culture is workable, whether from vertebrate or invertebrate
culture. However, interest has been greatest in vertebrate cells, and propagation of
vertebrate cells in culture (tissue culture) has become a routine procedure in recent years.
Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W13g, BHK, COS-7, 293 and MDCK cell lines. Expression
vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter
located in front of the gene to be expressed, along with any necessary ribosome binding
sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
For use in m~mm~ n cells, the control functions on the expression vectors are
often provided by viral material. For example, commonly used promoters are derived
from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because both are obtained easily
from the virus as a fragment which also contains the SV40 viral origin of replication
(Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there
is included the approximately 250 bp sequence extending from the HindIII site toward
the Bgn site located in the viral origin of replication. Further, it is also possible, and
often desirable, to utilize promoter or control sequences normally associated with the
desired gene sequence, provided such control sequences are compatible with the host cell
systems.
The origin of replication may be provided either by construction of the vector to
include an exogenous origin, such as may be derived from SV40 or other viral (e.g.,
Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal
replication mechz~ni~m. If the vector is integrated into the host cell chromosome, the
latter is often sufficient.
It will be further understood that certain of the polypeptides may be present inquantities below the detection limits of the Coomassie brilliant blue staining procedure
usually employed in the analysis of SDS/PAGE gels, or that their presence may bemasked by an inactive polypeptide of similar Mr. Although not necessary to the routine
practice of the present invention, it is contemplated that other detection techniques may
be employed advantageously in the visualization of particular polypeptides of interest.
Immunologically-based techniques such as Western blotting using enzymatically-,
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radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of
particular use in this regard. Alternatively, the peptides of the present invention may be
detected by using antibodies of the present invention in combination with secondary
antibodies having affinity for such primary antibodies. This secondary antibody may be
enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged.
Means for the labeling and detection of such two-step secondary antibody techniques are
well-known to those of skill in the art.
3. BRIEF DESCRIPTION OF THE DRAWINGS
The drawings form part of the present specification and are included to further
demonstrate certain aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in combination with thedetailed description of specific embodiments presented herein.
FIG. lA. A schematic showing the 3 overlapping cDNA clones used to
construct a full-length BRCAl cDNA. The 3 regions ofthe BRCAl cDNA used to rnakeGST fusion-proteins are also outlined.
FIG. lB. Identification of BRCAl as a 220-kDa protein in human cells.
S-methionine-labeled whole-cell extracts ~1 x 10 cells/lane) were immunoprecipitated
with either excess preimmune serum (Lane 1) or with anti-BRCAI polyclonal antibodies
(Lanes 2-7). Lanes 2, 4, and 6: single-step immunoprecipitates. Lanes 3, 5, and 7:
double immunoprecipitations. Grey arrowhead: potential co-precipitated protein.
FIG. 2A. Comparison of the mobility of in vitro translated BRCAl with that
from HBL 100 cells. Lanes 1, 2: BRCA1 from HBL100 cells (1 x 10 /lane) precipitated
with anti-BRCAl. In lane 2, the extract was treated with CIP prior to
immunoprecipitation. Lanes 3, 4, and 5: in vitro translated BRCA1 (1/20th total product)
immunoprecipitated with each of the three antisera.
FIG. 2B. Comparison of the mobility of recombinant, baculovirus-derived
BRCA1 with that from HBL100 cells. Lanes 1, 2: HBL100 cells (0.5 x 10 /lane). Lane
3: uninfected SF9 cells. Lanes 4-7: infected SF9 cells. Lanes 1 and 4:
immunoprecipitated with preimmune serum. Lanes 2, 3, and 5: immunoprecipitated with
anti-BRCAl. Lane 6: immunoprecipitated with anti-BRCAlBgl. Lane 7:
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immunoprecipitated with anti-BRCAlN. Immunoprecipitates were detected by westem
blotting and probing with anti-BRCAI monoclonal MAb 6B4.
FIG. 3A. BRCAl expression and phosphorylation is cell cycle dependent.
- Extracts from synchronized T24 cells were aliquoted, separated by SDS-PAGE, western
blotted, and probed as follows: Top Panel: I x 10 cells/lane probed for BRCAI using
anti-BRCAI antiserum. Middle Panel: I x 10 cells/lane probed for pl IORB using MAb
IID7. Lower Panel: 5 x 10 cells/lane probed for p84 with anti-N5-3. Lane I (U)
unsynchronized cells; Lane 2 (G1) I hr post-release; Lane 3 (Gl l) 11 hrs post-release;
Lane 4 (G18) 18 hrs post-release; Lane 5 (G24) 24 hrs post-release; Lane 6 (G33) 33 hrs
post-release; Lane 7 (M) cells treated with nocodazole (0.4 ~g/ml for 8 hrs).
FIG. 3B. BRCAl expression and phosphorylation is cell cycle dependent.
Phosphorylation of BRCAl . T~4 cells (2 x 10 /lane), synchronized as described above,
were pulsed with 300 ~Ci P ortho-phosphate for 4 hrs in phosphate-free medium and
then harvested in Iysis buffer and immunoprecipitated with anti-BRCAl. Lane 8:
immunoprecipitation with preimmune serum. Lanes 9-14 immunoprecipitation with anti-
BRCAl. The upper and lower panels in this figure are different exposures of the same
gel.
FIG.3C. BRCAl expression and phosphorylation is cell cycle dependent.
FACS analysis of the synchronized cells at the time points assayed showing the %distribution of cells in the various stages of the cell cycle.
FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H, FIG. 3I, FIG. 3J, FIG. 3K,
FIG. 3L, FIG. 3M, FIG. 3N, FIG. 30. BRCA 1 expression and phosphorylation is
cell cycle dependent. Immunofluorescence staining for BRCA I during cell cycle
progression. FIG. 3D, FIG. 3F. FIG. 3H~ FIG. 3J, FIG. 3L, FIG. 3N: DAPI staining for
DNA. FIG. 3E, FIG. 3G, FIG. 3I, FIG. 3K, FIG. 3M, FIG. 30: indirect-
immunofluorescence staining for BRCAl using anti-BRCAl antibody as the primary and
FITC-conjugated sheep- anti-mouse antibody as the secondary. FIG. 3D, FIG. 3E: 11 hrs
post release (Gl l); FIG. 3F, FIG. 3G: 24 hrs post-release (G24); FIG. 3H, FIG. 3I: 33 hrs
post-release (G33); FIG. 3J, FIG. 3K: metaphase; FIG. 3L, FIG. 3M: telophase; FIG. 3N,
FIG. 30: cells re-entering G I .
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FIG. 4. Phosphorylation of BRCA1 by various cyclin-dependent kinases.
Extracts from HBL100 celJs were precipitated with various anti-cyclin/cyclin-dependent
kinase antibodies. as shown. Precipitates were incubated in kinase buffer in the presence
of [~- P]ATP. and then washed and dissociated. The resultant supernatants were
reprecipitated using anti-BRCAI, separated by SDS-PAGE, and the gels dried and
autoradiographed.
FIG. 5A. Identification of BRCAl. Diploid human breast epithelial cells
(HBL100, about 1 x 107 cells per lane) were incubated with 35S-methionine (lanes 1 to 6)
[32P]phosphoric acid (lanes 7 and 8). Proteins from Iysates were then
immunoprecipitated by excess preimmune mouse serum (lanes 1, 4, and 8) or by mouse
polyclonal anti-BRCAl (lane 2). separated by SDS-polyacrylamide gel electrophoresis
and autoradiographed. Arrowheads indicate proteins coimmunoprecipitated by
anti-BRCAI serum. Immunoprecipitated proteins were dissociated from anti-BRCAl
and immunoprecipitated again with an excess of the same antibody to visualize only
BRCAI (lane 3). The same protein was immunoprecipitated by two different antibodies,
anti-BRCAI (lane 5) and C20 (lane 6). One protein species labeled with [32P]phosphate
was also immunoprecipitated by anti-BRCAI (lane 7) but not by preimmune serum (lane
8).
FIG. SB. Detection of full-length BRCAI in normal breast epithelial cells
and breast cancer cell lines. Established cell lines were obtained from American Type
Culture Tissue Collection. Malignant cells from pleural effusions, immediately after
being withdrawn from patients, were washed in 50:50 Ham's F-12-Dulbecco's modified
Eagle's medium (DMEM) and frozen in liquid nitrogen without passage, in the samemedium plus 50% fetal calf serum (FCS) and 10% dimethyl sulfoxide. Before fixation
for immunostaining the cells were washed, then plated for 12 hours in Ham's
F-12-DMEM plus 10% FCS. Viable cells were cytospun onto glass cover slips where
they were fixed as described for established cell lines. Human breast cell lines (5 x 1 o6
cells per lane) were labeled with [32P]phosphoric acid. Lane I, HBL 100 Iysate
immunoprecipitated by preimmune mouse serum. Cell Iysates in lanes 2 to 11
immunoprecipitated by anti-BRCAl: lane 2, T47D: lane 3. MCF7; lane 4, MB468; lane
5, MB175-7; lane 6~ MB-361; lane 7 MB-231; lane 8. MB-435S; lane 9, MB415; lane
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I ~, HS578T; and lane 11 , HBL 1 00. Sections 5-~m-~hick from randomly selected,forrnalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank
were immunostained by a modification of the avidin-biotin-horseradish peroxidasecomplex (ABC) method (Hsu et al., 1981). Anti-BRCAl was used at 1:100 dilution.
Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunostaining
- for BRCA1 did show positive immunostaining for the nuclear proliferation antigen
MiB 1 .
FIG. 5C. Full-length BRCA1 is expressed in tumor cell lines derived from
tissues other than breast. Human cell lines (~2 X lo6 per lane) were metabolically
labeled with 35S-methionine. One Iysate wàs immunoprecipitated by preimmune serum
(lane 1) and all others by anti-BRCA1 (lanes 2 to 12). Cell lines: lanes 1 and 2, T24
[transitional cell carcinoma (TCC) of the bladder]; lane 3, 5637 (TCC bladder); lane 4,
- DU145 (prostate carcinoma); lane 5, CAOV3 (ovarian carcinoma); lane 6, RD
(rhabdomyosarcoma); lane 7, HCTI 16 (colon carcinoma); lane, SW620 (colon
carcinoma); lane 9, C411 (cervical carcinoma); lane 10, MS751 (cervical carcinoma);
lane 11, SAOS-2 (osteosarcoma); and lane 12, U20S (osteosarcoma).
FIG. 6A. Localization of BRCA1 in normal and breast cancer cells.
Fractionation of HBL100 cells. Cells (1.5 x 107) were labeled with 35S-methionine; 5 x
Io6 cells were left unfractionated (total or T, lane 1) and the remainder were separated
into nuclear (N, lane 2), cytoplasmic (C, lane 3), and membrane (M, lane 4) fractions
(Chen e~ al., 1995; Abrams et al., 1982). For control of the fractionation procedure,
pl loRB served as a marker for nuclear distribution and GST for cytoplasmic distribution.
Small aliquots were incubated with GST beads, separated by SDS-PAGE, and stainedwith Coomassie Brilliant Blue to visualize the expected 26-kDa glutathione-S-transferase
(GST) band.
FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 6I.
Localization of BRCAl in normal and breast cancer cells. Detection of BRCAI
in the nuclei of intact HBL 100 cells by indirect immunofluorescence staining. (FIG. 6B.
FIG. 6D, FIG. 6F, FIG. 6H) OAPI staining to mark nuclei; (FIG. 6C, FIG. 6E, FIG. 6G.
FIG. 6I) immunofluorescence staining of the same cells. Indirect immunofluorescence
procedures have been described (Durfee et al., 1994). Briefly. cells grown on cover slips
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were fixed with 4% fonnaldehyde and 0.1% Triton X-100~ in phosphate-buffered saline
(PBS) and permeabilized with 0.05% Saponin in water. Fixed cells were then blocked
with 10% norrnal goat serum plus 0.5% NP-40~' in PBS, incubated with mouse
polyclonal anti-BRCAI primary antiserum (1:1000 dilution), washed, and incubated with
S fluorescein-tagged goat antibody to mouse immunoglobulin G. At the end for the
secondary antibody incubation, one drop of 4,6-diamidino-2-phenolindole propidium
iodide (DAPI) was added to the cells for 10 min to stain DNA. Cells were then viewed
and photographed under a fluorescence microscope. (FIG. 6B and FIG. 6C) Preimmune
serum as primary antibody; (FIG. 6D and FIG. 6E) anti-BRCAl as primary antibody;(FIG. 6F and FIG. 6G) anti-BRCAl preabsorbed with GST antigen; (FIG. 6H and FIG.61) anti-BRCAl preabsorbed with the GST-BRCAl fusion protein.
FIG.6J,FIG.6K,FIG.6L,FIG.6M,FIG.6N,FIG.60,FIG.6P,FIG.6Q.
Localization of BRCAl in normal and breast cancer cells. Detection of BRCAl
in the nuclei of cell lines derived from tissues other than breast. (FIG. 6J, FIG. 6L, FIG.
6N, FIG. 6P) DAPI staining; (FIG. 6K. FIG. 6M, FIG. 60, FIG. 6Q) BRCAI staining.(FIG. 6J and FIG. 6K) DU145 (prostate carcinoma) cells; (FIG. 6L and FIG. 6M) RAT2
fibroblasts; (FIG. 6N and FIG. 60) T24 (TCC bladder) cells; (FIG. 6P and FIG. 6Q) CVI
(monkey kidney epithelial) cells.
FIG.7A,FIG.7B,FIG.7C,FIG.7D,FIG.7E,FIG.7F,FIG.7G,FIG.7H,
FIG.7I,FIG.7J,FIG.7K,FIG.7L,FIG.7M,FIG.7N,FIG.70,FIG.7P.
Localization of BRCA 1 in normal and breast cancer cells. Cytoplasmic
localization of BRCAl in breast cancer cells. FIG. 7A through FIG. 7H breast cancer
line T47D; (FIG. 7K and FIG. 7L) breast cancer line MCF7; (FIG. 7M and FIG. 7N)
cells from primary malignant effusion #22550; (FIG. 70 and FIG. 7P) cells from primary
effusion #23159. (FIG. 7A, FIG. 7C, FIG. 7E, FIG. 7G~ FIG. 71, FIG. 7K, FIG. 7M,FIG. 70) DAPI staining; (FIG. 7B) preimmune serum as primary antibody; (FIG. 7D)polyclonal anti-BRCAI primary antiserum; (FIG. 7F) anti-BRCAI preabsorbed with
GST; (FIG. 7~) anti-BRCAI reabsorbed with GST-BRCAI fusion protein; (FIG. 71
through FIG. 7P) anti-BRCAl primary antibody, reabsorbed with
glutathione-S-transferase. Magnification is the same in FIG. 7B through FIG. 7D.
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FIG. 8A. Primary breast carlcer sections stained for BRCAI by the
immunoperoxidase method. Sections 5-~1m-thick from randomly selected,
formalin-fixed, paraffin-embedded~ breast cancer biopsies in the inventors' tumor bank
were immunostained by a modification of the avidin-biotin-horseradish peroxidasecomplex (ABC) method (Hsu et al., 1981). AntiBRCAl was used at 1:100 dilution.
Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunostaining
for BRCAI did show positive immunostaining for the nuclear proliferation antigenMiB 1. BRCA I localized to both cytoplasm and nuclei.
FIG. 8B. Primary breast cancer sections stained for BRCAI by the
immunoperoxidase method. Sections 5-~m-thick from randomly selected,
formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank
were immunostained by a modification of the avidin-biotin-horseradish peroxidasecomplex (ABC) method (Hsu et al., 1981). Anti-BRCAl was used at 1:100 dilution.
Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunost:~ining
for BRCAI did show positive immunostaining for the nuclear proliferation antigenMiB 1. BRCA 1 localized only to cytoplasm.
FIG. 8C. Primary breast cancer sections stained for BRCAI by the
immunoperoxidase method. Sections 5-~1m-thick from randomly selected,
formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank
were immunostained by a modification of the avidin-biotin-horseradish peroxidasecomplex (ABC) method (Hsu et al., 1981). Anti-BRCAI was used at 1:100 dilution.
Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunostaining
for BRCA1 did show positive immunostaining for the nuclear proliferation antigenMiB1. BRCA1 staining absent. The small, round, dark signals in all sections are
Iymphocyte and stromal cell nuclei. Original magnification~ x400.
FIG. 9. Shown are NLS Deletion-Mutant Constructs. Schematic showing
- the positions and sequences of the three putative NLS motifs in BRCA1 together with the
respective changes made in each by PCR-based mutagenesis. As shown, these constructs
have been cloned, in-frame ~ith the FLAG epitope, into the pCEP4 vector, which directs
high-level expression of inserted cDNAs under the control of the CMV major-late
promoter.
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FIG. 10. Identification of a trans-activation domain in BRCA1 using a
yeast "one-hybrid" system. The various fragments of BRCAI shown were fused in-
frame to the GAL4 DNA-binding domain, expressed in the yeast vector pAS. These
constructs were then transfected into the yeast strain Y153, which harbors a GAL4
responsive b-galactosidase reporter gene b-galactosidase activity was determined either
qualitatively by streaking transformants onto plates and doing a colony lift assay, or
quantitatively by CPRG assay. These assays have been described by the inventors
previously (Durfee, et al., 1993).
FIG. 11. Schematic showing the regions of BRCAI used as bait in the yeast
two-hybrid screen. The putative Zn-finger, ~LS motifs! and trans-activation domain of
BRCAI are depicted on a schematic for the BRCAI cDNA. Below this are shown the
positions of the two regions used as bait in the yeast two-hybrid screen. These regions
were cloned in-frame with the Gal4 DNA-binding domain of the yeast expression vector
pAS. Numbers above the bars represent positions of amino acids within the sequence.
4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
4.1 THERAPEUTIC AND DIAGNOSTIC KITS FOR BAP OR BRCA1
Therapeutic kits comprising, in suitable container means, a BAP or BRCAI
composition of the present invention in a pharmaceutically acceptable formulation
represent another aspect of the invention. The BAP or BRCAl composition may be
native BAP or BRCAl, truncated BAP or BRCAl, site-specifically mutated BAP or
BRCAl, or BAP- or BRCAl-encoded peptide epitopes. or alternatively antibodies which
bind native BAP or BRCA I, truncated BAP or BRCA 1. site-specifically mutated BAP or
BRCAI, or BAP- or BRCAI-encoded peptide epitopes. In other embodiments, the BAP
or BRCAI composition may be nucleic acid segments encoding native BAP or BRCAl,
truncated BAP or BRCAI, site-speci~lcally mutated BAP or BRCA1, or BAP- or
BRCAI-encoded peptide epitopes. Such nucleic acid segments may be DNA or RNA,
and may be either native, recombinant, or mutagenized nucleic acid segments.
The kits may comprise a single container means that contains the BAP or BRCA I
composition. The container means may, if desired~ contain a pharmaceutically
acceptable sterile excipient. having associated with it. the BRCAl or BAP composition
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and, optionally, a detectable label or im~gin~ agent. The formulation may be in the form
of a gelatinous composition, e.g., a collagenous-BRCAI or BAP composition. or may
even be in a more fluid form that nonetheless forms a gel-like composition upon
~rlmini~tration to the body. In these cases, the container means may itself be a syringe,
pipette, or other such like apparatus, from which the BRCAI or BAP composition may
- be applied to a particular site. However, the single container means may contain a dry,
or Iyophilized, mixture of a BRCAI or BAP composition, which may or may not require
pre-wetting before use.
Alternatively, the kits of the invention may comprise distinct container means for
each component. In such cases, one container would contain the BAP or BRC~l
composition, either as a sterile DNA solution or in a Iyophilized form, and the other
container would include the matrix, which may or may not itself be pre-wetted with a
sterile solution, or be in a gelatinous, liquid or other syringeable form.
The kits may also comprise a second or third container means for cont~inin~ a
sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be
required to formulate the BAP or BRCAI component into a more suitable form for
application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral,
or intravenous forms. It should be noted, however, that all components of a kit could be
supplied in a dry form (Iyophilized), which would allow for "wetting" upon contact with
body fluids. Thus, the presence of any type of pharrnaceutically acceptable buffer or
solvent is not a requirement for the kits of the invention The kits may also comprise a
second or third container means for cont~inin~ a pharmaceutically acceptable detectable
im~ing agent or composition.
The container means will generally be a container such as a vial, test tube, flask,
bottle, syringe or other container means, into which the components of the kit may
placed. The matrix and gene components may also be aliquoted into smaller containers,
should this be desired. The kits of the present invention may also include a means for
containing the individual containers in close confinement tor commercial sale, such as,
e,~.~ injection or blow-molded plastic containers into which the desired vials or syringes
are retained.
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Irrespective of the number of containers, the kits cf the invention may also
comprise, or be packaged with, an instrument for assisting with the placement of the
ultimate matrix-gene composition within the body of an animal. Such an instrument may
be a syringe, pipette. forceps, or any such medically approved delivery vehicle.
s
4.2 AFFINITY CHROMATOGRAPHY
Affinity chromatography is generally based on the recognition of a protein by a
substance such as a ligand or an antibody. The column material may be synthesized by
covalently coupling a binding molecule, such as an activated dye, for example to an
insoluble matrix. The column material is then allowed to adsorb the desired substance
from solution. Next, the conditions are changed to those under which binding does not
occur and the substrate is eluted. The requirements for successful affinity
chromatography are:
I ) that the matrix must specifically-adsorb the molecules of interest;
2) that other cont~min~nts remain unadsorbed;
3) that the ligand must be coupled without altering its binding activity;
4) that the ligand must bind sufficiently tight to the matrix; and
5) that it must be possible to elute the molecules of interest without
destroying them.
A preferred embodiment of the present invention is an affinity chromatography
method for purification of antibodies from solution wherein the matrix contains BAP or
BRCA 1, or alternatively, peptide epitopes derived from either BAP or BRCA 1,
covalently-coupled to a suitable matrix such as e.g., Sepharose CL6B or CL4B. This
matrix binds the antibodies of the present invention directly and allows their separation
by elution with an appropriate gradient such as salt, GuHCI, pH, or urea. Another
preferred embodiment of the present invention is an affinity chromatography method for
the purification of BAP, BRCAI. or related peptide epitopes from solution. The matrix
binds the amino acid compositions of the present invention directly. and allows their
separation by elution with a suitable buffer as described above.
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4.3 METHODS OF NUCLEtC ACID DELIVERY AND DNA TRANSFECTION
In certain embodiments. it is contemplated that the nucleic acid segments
disclosed herein will be used to transfect appropriate host cells. Technology for
introduction of DNA into cells is well-known to those of skill in the art. Four general
methods for delivering a nucleic segment into cells have been described:
(I) chemical methods (Graham and Van der Eb, 1973);
(2) physical methods such as microinjection (Capecchi, 1980),
electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Yang
etal., 1990);
(3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and
(4) receptor-mediated mech~ni~m~ (Curiel et al., 1991; Wagner et al., 1992).
4.4 LIPOSOMES AND NANOCAPSULES
In certain embodiments, the inventors contemplate the use of liposomes and/or
nanocapsules for the introduction of particular peptides or nucleic acid segments into
host cells. Such formulations may be preferred for the introduction of pharmaceutically-
acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein.
The formation and use of liposomes is generally kno~vn to those of skill in the art (see for
exarnple, Couvreur et al., 1977 which describes the use of liposomes and nanocapsules in
the targeted antibiotic therapy of intracellular bacterial infections and diseases).
Recently, liposomes were developed with improved serum stability and circulation half-
times (Gabizon and Papahadjopoulos, 1988; Allen and Choun~ 1987).
Nanocapsules can generally entrap compounds in a stable and reproducible way
(Henry-Michelland etal., 1987). To avoid side effects due to intracellular polymeric
overloading, such ultrafine particles (sized around 0.1 llm) should be designed using
polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate
nanoparticles that meet these requirements are contemplated f'or use in the present
invention, and such particles may be are easily made, as described (Couvreur e~ al., 1977;
1988).
Liposomes are formed from phospholipids that are dispersed in an aqueous
medium and spontaneously t'orm multilamellar concentric bilayer vesicles (also termed
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multilamellar vesicles (MLVs). MLVs generally have diarneters of from 25 nrn to 4 ~m.
Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with
diameters in the range of 200 to 500 A, cont~ining an aqueous solution in the core.
In addition to the teachings of Couvreur et al. (1988), the following information
may be utilized in generating liposomal formulations. Phospholipids can forrn a variety
of structures other than liposomes when dispersed in water, depending on the molar ratio
of lipid to water. At low ratios the liposome is the preferred structure. The physical
characteristics of liposomes depend on pH, ionic strength and the presence of divalent
cations. Liposomes can show low perrneability to ionic and polar substances, but at
elevated temperatures undergo a phase transition which markedly alters their
permeability. The phase transition involves a change from a closely packed, ordered
structure, known as the gel state, to a loosely packed. Iess-ordered structure, known as
the fluid state. This occurs at a characteristic phase-transition temperature and results in
an increase in perrneability to ions, sugars and drugs.
Liposomes interact with cells via four different mech:lni~m.c: Endocytosis by
phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils;
adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components; fusion with the plasma
cell membrane by insertion of the lipid bilayer of the liposome into the plasma
membrane, with simultaneous release of liposomal contents into the cytoplasm; and by
transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without
any association of the liposome contents. It often is difficult to deterrnine which
mechanism is operative and more than one may operate at the same time.
4.5 METHODS FOR PREPARING BAP, BRCA1, AND ANTI-BAP OR ANTI_BRCA1
ABS
In another aspect, the present invention contemplates an antibody that is
immunoreactive with a polypeptide of the invention. As stated above~ one of the uses for
BRCA 1 and BRCA I -derived epitopic peptides or BAP and BAP-derived epitopic
peptides according to the present invention is to generate antibodies. Reference to
antibodies throughout the specification includes whole polyclonal and monoclonal
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antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties.
Antibody parts include Fab and F(ab)~ fragments and single chain antibodies. Theantibodies may be made in vivo in suitable laboratory :~nim~l.c or in vitro using
recombinant DNA techniques. In a preferred embodiment, an antibody is a polyclonal
S antibody.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an
immunogen comprising a polypeptide of the present invention and collecting antisera
from that immunized animal. A wide range of animal species can be used for the
production of antisera. Typically an animal used for production of anti-antisera is a
rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood
volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal. specific for BRCA 1 and
BRCAI-derived epitopes, or alternatively, BAP and BAP-derived epitopes, may be
prepared using conventional immunization techniques, as will be generally known to
those of skill in the art. A composition cont~ining antigenic epitopes of the particular
BRCAls and BAPs disclosed herein can be used to immunize one or more experimental
~nim~, such as a rabbit or mouse, which will then proceed to produce specific
antibodies against BAP or BRCAI peptides. Polyclonal antisera may be obtained, after
allowing time for antibody generation, simply by bleeding the animal and preparing
serum samples from the whole blood.
The amount of immunogen composition used in the production of polyclonal
antibodies varies upon the nature of the immunogen, as well as the animal used for
immunization. A variety of routes can be used to ;~mini~ter the immunogen
(subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). Theproduction of polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points following immunization. A second, booster
injection, also may be given. The process of boosting and titering is repeated until a
suitable titer is achieved. When a desired level of immunogenicity is obtained, the
immunized animal can be bled and the serum isolated and stored. and/or the animal can
be used to generate mAbs (below).
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One of the important features provided by the present invention is a polyclonal
sera that is relatively homogenous with respect to the specificity of the antibodies
therein. Typically, polyclonal antisera is derived from a variety of different "clones,"
i.e., B-cells of different lineage. mAbs, by contrast, are defined as coming from
S antibody-producing cells with a common ~-cell ancestor, hence their "mono" clonality.
When peptides are used as antigens to raise polyclonal sera, one would expect
considerably less variation in the clonal nature of the sera than if a whole antigen were
employed. Unfortunately7 if incomplete fragments of an epitope are presented, the
peptide may very well assume multiple (and probably non-native) conformations. As a
result, even short peptides can produce polyclonal antisera with relatively plural
specificities and unfortunately~ an antisera that does not react or reacts poorly with the
native molecule.
Polyclonal antisera according to present invention is produced against peptides
that are predicted to comprise whole, intact epitopes. It is believed that these epitopes
are, therefore, more stable in an immunologic sense and thus express a more consistent
immunologic target for the immune system. Under this model, the number of potential
B-cell clones that will respond to this peptide is considerably smaller and, hence, the
homogeneity of the resulting sera will be higher. In various embodirnents, the present
invention provides for polyclonal antisera where the clonality, i.e., the percentage of
clone reacting with the same molecular determinant, is at least 80%. Even higherclonality - 90%, 95% or greater - is contemplated.
To obtain mAbs, one would also initially immunize an experimental animal,
often preferably a mouse, with a BRCAI-cont~inin~ composition. One would then, after
a period of time sufficient to allow antibody generation, obtain a population of spleen or
Iymph cells from the animal. The spleen or Iymph cells can then be fused with cell lines,
such as human or mouse mveloma strains, to produce antibody-secreting hybridomas.
These hybridomas may be isolated to obtain individual clones which can then be
screened for production of antibody to the desired peptide.
Following immunization. spleen cells are removed and t'~lsed. using a standard
fusion protocol with plasmacvtoma cells to produce hybridomas secreting mAbs against
BAP or BRCA 1. Hybridomas which produce mAbs to the selected antigens are
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identified using standard techniques, such as ELISA and Western blot methods.
Hybridoma clones can then be cultured in liquid media and the culture supernatants
purified to provide the BAP- or BRCA I -specific mAbs.
It is proposed that the mAbs of the present invention will also find useful
S application in immunochemical procedures, such as EI,ISA and Western blot methods, as
well as other procedures such as immunoprecipitation, imrnunocytological methods, etc.
which may utilize antibodies specific to BAPs or BRCAl. In particular, BAP or BRCAI
antibodies may be used in immunoabsorbent protocols to purify native or recombinant
BAPs, BRCAl or BAP- or BRCAI-derived peptide species or synthetic or natural
variants thereof.
The antibodies disclosed herein may be employed in antibody cloning protocols
to obtain cDNAs or genes encoding BAPs or BRCAls from other species or org~ni~m~or to identify proteins having significant homology to BAP or BRCAI. They may also
be used in inhibition studies to analyze the effects of BAP or BRCAI in cells~ tissues, or
whole ~nim~lc. Anti-BRCAI or anti-BAP antibodies will also be useful in
immunolocalization studies to analyze the distribution of BRCAI or BAP protein under
different physiological conditions. A particularly useful application of such antibodies is
in purifying native or recombinant BAPs or BRCA1, for example, using an arltibody
affinity column. The operation of all such immunological techniques will be known to
those of skill in the art in light of the present disclosure.
4.6 RECOMBtNANT EXPRESSION OF BAP OR BRCA1
Recombinant clones expressing the BAP or BRCAl nucleic acid segments may be
used to prepare purified recombinant BRCAI (rBRCAl), purified rBRCA1-derived
peptide antigens or, alternatively~ purified recombinant BAP (rBAP~, purified rBAP-
derived peptide antigens, as well as mutant or variant recombinant protein species in
significant quantities. The selected antigens, and variants thereofi are proposed to have
significant utility in diagnosing and treating breast cancers. For example~ it is proposed
that rBAPs~ rBRCAls, peptide variants thereof, and/or antibodies against such rBAPs or
rBRCA l s may also be used in immunoassays to detect localization of BAP or BRCAl in
vivo or as vaccines or immunotherapeutics to treat breast cancers.
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Additionally, by application of techniques such as DNA mutagenesis, the present
invention allows the ready preparation of so-called "second generation" molecules
having modified or simplified protein structures. Second generation proteins will
typically share one or more properties in common with the full-length antigen~ such as a
particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be
provided on relatively short molecules prepared from knowledge of the peptide, or
encoding DNA sequence information. Such variant molecules may not only be derived
from selected immunogenic/ antigenic regions of the protein structure, but may
additionally, or alternatively, include one or more functionally equivalent amino acids
selected on the basis of similarities or even differences with respect to the natural
sequence.
4.7 ANTIBODY COMPOSITIONS A~D FORMULATIONS THEREOF
Means for preparing and characterizing antibodies are well kno~vvn in the art (See,
e.g, Harlow and Lane (1988); incorporated herein by reference). The methods for
generating mAbs generally begin along the same lines as those for preparing polyclonal
antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an
immunogenic composition in accordance with the present invention and collecting
antisera from that immunized animal. A wide range of animal species can be used for
the production of antisera. Typically the animal used for production of anti-antisera is a
rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large
blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal
antibodies.
As is well known in the art, a given composition may vary in its immunogenicity.It is often necessary therefore to boost the host immune system, as may be achieved by
coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other
albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be
used as carriers. Means for conjugating a polvpeptide to a carrier protein are well known
in the art and include glutaraldehyde. m-maleimidobenzoyl-N-hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
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mAbs may be readily prepared through use of well-known techniques, such as
those exemplified in U. S. Patent 4,196,265, incorporated herein by reference. Typically,
this technique involves immunizing a suitable animal with a selected immunogen
- composition, e.g, a purified or partially purified protein, polypeptide or peptide. The
S immunizing composition is :~ministered in a manner effective to stimulate antibody
- producing cells. Rodents such as mice and rats are preferred ;lnim~l~, however, the use
of rabbit, sheep or frog cells is also possible. The use of rats may provide certain
advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most
preferred as this is most routinely used and generally gives a higher percentage of stable
fusions.
Following immunization, somatic cells with the potential for producing
antibodies~ specifically B-lymphocytes (B-cells), are selected for use in the mAb
~- generating protocol. These cells may be obtained from biopsied spleens, tonsils or
Iymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of antibody-producing cells that
are in the dividing plasmablast stage, and the latter because peripheral blood is easily
accessible. Often, a panel of ~nim~l~ will have been immunized and the spleen of animal
with the highest antibody titer will be removed and the spleen Iymphocytes obtained by
homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately about 5 x 107 to about 2 x 1 o8 Iymphocytes.
The antibody-producing B Iymphocytes from the immunized animal are then
fused with cells of an immortal myeloma cell, generally one of the same species as the
animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing
fusion procedures preferably are non-antibody-producing, have high fusion efficiency,
and enzyme deficiencies that render them incapable of growing in certain selective media
which support the growth of only the desired fused cells (hybridomas).
- Any one of a number of myeloma cells may be used, as are known to those of
skill in the an (Goding, 1986; Campbell. 1984). For example, where the immunized- animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653. NSI/l.Ag 4 1,
Sp210-Agl4, FO, NSO/U, MPC-ll, MPCII-X45-GTG 1.7 and S194/SXX0 Bul; for
rats. one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,
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GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with
human cell fusions.
One preferred murine myeloma cell is the NS-I myeloma cell line (also termed
P3-NS-I-Ag4-l), which is readily available from the NIGMS Human Genetic Mutant
Cell Repository by requesting cell line repository number GM3573. Another mouse
myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or Iymph node cells
and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1
ratio, though the ratio may vary from about 20:1 to about l:l, respectively, in the
presence of an agent or agents (chemical or electrical) that promote the fusion of cell
membranes. Fusion methods using Sendai virus have been described (Kohler and
Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v)
PEG, by Gefter et al. (1977). The use of electrically in~ ced fusion methods is also
appropriate (Goding, 1986).
Fusion procedures usually produce viable hybrids at low fre~uencies, about
l x 10-6 to about 1 x 10-8. However, this does not pose a problem~ as the viable, fused
hybrids are differentiated from the parental, unfused cells (particularly the unfused
myeloma cells that would normally continue to divide indefinitely) by culturing in a
selective medium. The selective medium is generally one that contains an agent that
blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and
preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and
methotrexate block de novo synthesis of both purines and pyrimidines. whereas azaserine
blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT
medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating
nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are
defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl
transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but
they have a limited life span in culture and generally die within about two weeks.
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Therefore, the only cells that can survive in the selective media ~.re those hybrids formed
from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific
hybridomas are selected. Typically, selection of hybridomas is performed by culturing
the cells by single-clone dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the desired reactivity. The assay
should be sensitive, simple and rapid, such as radioimmunoassays, enzyme
imrnunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the
like.
The selected hybridomas would then be serially diluted and cloned into
individual antibody-producing cell lines, which clones can then be propagated
indefinitely to provide mAbs. The cell lines may be exploited for mAb production in
two basic ways. A sample of the hybridoma can be injected (often into the peritoneal
cavity) into a histocompatible animal of the type that was used to provide the somatic
and myeloma cells for the original fusion. The injected animal develops tumors
secreting the specific mAb produced by the fused cell hybrid. The body fluids of the
animal, such as serurn or ascites fluid~ can then be tapped to provide mAbs in high
concentration. The individual cell lines could also be cultured in vitro, where the mAbs
are naturally secreted into the culture medium from which they can be readily obtained in
high concentrations. mAbs produced by either means may be further purified. if desired,
using filtration, centrifugation and various chromatographic methods such as HPLC or
affinity chromatography.
4.8 IMMUNOASSAYS
As noted. it is proposed that native and synthetically-derived peptides and peptide
epitopes of the invention will find utilitv as immunogens, e.g., in connection with
vaccine development, or as antigens in immunoassays for the detection of reactive
antibodies. rurning first to immunoassays~ in their most simple and direct sense,
- preferred immunoassays of the invention include the various types of enzyme linked
immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it
will be readily appreciated that the utility of BRCAI-derived proteins and peptides is not
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limited to such assays, and that other useful embodiments include RIAs and other non-
enzyme linked antibody binding assays and procedures.
In preferred ELISA assays, proteins or peptides incorporating BAP, rBAP,
BRCA l, rBRCA 1, or BAP or BRCA I -derived protein antigen sequences are
immobilized onto a selected surface, preferably a surface exhibiting a protein affinity,
such as the wells of a polystyrene microtiter plate. After washing to remove
incompletely adsorbed material, one would then generally desire to bind or coat a
nonspecific protein that is known to be antigenically neutral with regard to the test
antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for
blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the
background caused by nonspecific binding of antisera onto the surface.
After binding of antigenic material to the well, coating with a non-reactive
material to reduce background, and washing to remove unbound material, the
immobilizing surface is contacted with the antisera or clinical or biological extract to be
tested in a manner conducive to immune complex (antigen/antibody) formation. Such
conditions preferably include diluting the antisera with diluents such as BSA, bovine
gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween~. These added
agents also tend to assist in the reduction of nonspecific background. The layered
antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably
on the order of about 25~ to about 27~C. Following incubation, the antisera-contacted
surface is washed so as to remove non-immunocomplexed material. A preferred washing
procedure includes washing with a solution such as PBS/Tween'~', or borate buffer.
Following formation of specific immunocomplexes between the test sample and
the bound antigen, and subsequent washing, the occurrence and the amount of
immunocomplex formation may be determined by subjecting the complex to a second
antibody having specificity for the first. Of course, in that the test sample will typically
be of human origin~ the second antibody will preferably be an antibody having specificity
for human antibodies. To provide a detecting means, the second antibody will preferably
have an associated detectable label~ such as an enzyme label, that will generate a signal,
such as color development upon incubating with an ~pplo~u~iate chromogenic substrate.
Thus~ for e~ample. one will desire to contact and incubate the antisera-bound surface
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with a urease or peroxidase-conjugated anti-human IgG for a period of time and under
conditions that favor the development of immunocomplex formation (e.g, incubation for
2 hours at room temperature in a PBS-containing solution such as PBS-Tween~).
After incubation with the second enzyme-tagged antibody, and subsequent to
washing to remove unbound material. the amount of label is quantified by incubation
with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-
ethyl-ben7thi~7nline)-6-sulfonic acid (ABTS) and H2O2, in the case of peroxidase as the
enzyme label. Quantitation is then achieved by measuring the degree of color
generation, e.g., using a visible spectrum spectrophotometer.
ELISAs may be used in conjunction with the invention. In one such ELISA
assay, proteins or peptides incorporating antigenic sequences of the present invention are
immobilized onto a selected surface, preferably a surface exhibiting a protein affinity
such as the wells of a polystyrene microtiter plate. After washing to remove
incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a
nonspecific protein that is known to be antigenically neutral with regard to the test
antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk.
This allows for blocking of nonspecific adsorption sites on the immobilizing surface and
thus reduces the background caused by nonspecific binding of antisera onto the surface.
4.9 IMMUNOPRECIPITATION
The anti-BRCA 1 and anti-BAP antibodies of the present invention are
particularly useful for the isolation of BRCAI and BAP antigens by
immunoprecipitation. Immunoprecipitation involves the separation of the target antigen
component from a complex mixture, and is used to discriminate or isolate minute
amounts of protein.
In an alternative embodiment the antibodies of the present invention are useful
for the close juxtaposition of two antigens. T his is particularly useful for increasing the
localized concentration of antigens, e.g ~ enzyme-substrate pairs.
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4.10 WESTERN BLOTS
The compositions of the present invention will find great use in immunoblot or
western blot analysis. The anti-BRCAI and anti-BAP antibodies may be used as high-
affinity primary reagents for the identification of proteins immobilized onto a solid
support matrix~ such as nitrocellulose, nylon or combinations thereof. In conjunction
with immunoprecipitation, followed by gel electrophoresis, these may be used as a single
step reagent for use in detecting antigens against which secondary reagents used in the
detection of the antigen cause an adverse background. This is especially useful when the
antigens studied are immunoglobulins (precluding the use of immunoglobulins binding
bacterial cell wall components), the antigens studied cross-react with the detecting agent,
or they migrate at the sarne relative molecular weight as a cross-reacting signal.
Immunologically-based detection methods in conjunction with Western blotting
(including en~ymatically-, radiolabel-, or fluorescently-tagged secondary antibodies
against the toxin moiety) are considered to be of particular use in this regard.
4.1 1 VACCINES
The present invention contemplates vaccines for use in both active and passive
immunization embodiments. Immunogenic compositions proposed to be suitable for use
as a vaccine may be prepared most readily directly from the novel immunogenic proteins
and/or peptide epitopes described herein. Preferably the antigenic material is extensively
dialyzed to remove undesired small molecular weight molecules and/or Iyophilized for
more ready formulation into a desired vehicle.
The preparation of vaccines that contain peptide sequences as active ingredientsis generally weli understood in the art, as exemplified by U. S. Patents 4,608,251;
4,601,903; 4.599~231; 4,599,230; 4,596~792; and 4,578,770, all incorporated herein by
reference. Typically, such vaccines are prepared as injectables, either as liquid solutions
or suspensions~ solid forms suitable for solution in~ or suspension in~ liquid prior to
injection may also be prepared. The preparation may also be emulsified. The active
immunogenic ingredient is often mixed with excipients that are pharrnaceuticallyacceptable and compatible with the active ingredient. Suitable excipients are, for
example. water. saline. dextrose, glycerol. ethanol, or the like and combinations thereof.
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In addition, if desired, the vaccine may contain minor ~nounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the
effectiveness of the vaccines.
A composition comprising BAP, BRCAI or BRCAI-derived proteins and/or
native or modified epitopic peptides therefrom could also be the basis for humanvaccines. The preparation of such compositions that are essentially free from endotoxin
can be achieved by following the published methodology, for example, U. S. Patent
4,271,147 (incorporated herein by reference) discloses methods for the preparation of
~eisseria meningitidis membrane proteins for use in vaccines.
BAP, BRCAI, BRCAI-derived and BAP-derived epitope-based vaccines may be
conventionally a~lminictered parenterally, by injection, for example, either
subcutaneously or intramuscularly. Additional formulations that are suitable for other
modes of administration include suppositories and, in some cases, oral formulations. For
suppositories, traditional binders and carriers may include, for example, polyalkalene
glycols or triglycerides: such suppositories may be forrned from mixtures containing the
active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations
include such normally employed excipients as, for exarnple, pharmaceutical grades of
marmitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. These compositions take the form of solutions. suspensions,
tablets, pills~ capsules, sustained release formulations or powders and contain 10-95% of
active ingredient, preferably 25-70%.
The proteins may be formulated into the vaccine as neutral or salt forms.
Pharmaceutically acceptable salts, include the acid addition salts (formed with the free
arnino groups of the peptide) and those that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric. mandelic, and the like. Salts formed with the free carboxyl groups may also be
derived t'rom inorganic bases such as~ t'or example, sodium, potassium. ammonium,
calcium, or terric hydroxides. and such organic bases as isopropylamine~ trimethylamine,
2-ethylamino ethanol. histidine, procaine, and the like.
The vaccines may be :~imini.~tered in a manner compatible with the dosage
formulation. and in such amount as will be therapeutically effective and immunogenic.
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The quantity ;o be ~tlmini~tered depends on the subject to be treated, including, e.g, the
capacity of the individual's immune system to synthesize antibodies, and the degree of
protection desired. Precise amounts of active ingredient required to be Atlmini~tered will
be readily determinable by the skilled practitioner. However, suitable dosage ranges are
of the order of several hundred micrograms active ingredient per vaccination. Suitable
regimes for initial ~(lminictration and booster shots are also variable, but are typified by
an initial ~tlmini~tration followed by subsequent inoculations or other ~imini.strations.
The manner of application may be varied widely. Any of the conventional
methods for a(lmini~tration of a vaccine are applicable. These are believed to include
oral application on a solid physiologically acceptable base or in a physiologically
acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine
will depend on the route of ~mini~tration and will vary according to the size of the host.
Various methods of achieving adjuvant effect for the vaccine includes use of
agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1
percent solution in phosphate buffered saline, admixture with synthetic polymers of
sugars (Carbopol~) used as 0.25% solution, aggregation of the protein in the vaccine by
heat treatment with temperatures ranging between about 70~ and about 101~C for 30
second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated
F(ab) antibodies to albumin, mixture with bacterial cells such as C. parvum or
endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in
physiologically acceptable oil vehicles such as mannide monooleate (Aracel-ATM) or
emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DATM) used as a block
substitute may also be employed.
In many instances, it will be desirable to have multiple a-lmini~trations of thevaccine, usually not exceeding six vaccinations, more usually not exceeding fourvaccinations and preferably one or more. usually at least about three vaccinations. The
vaccinations will normally be at from two to twelve week intervals, more usually from
three to five week intervals. Periodic boosters at intervals of 1-5 years. usually three
years, will be desirable to maintain protective levels of the antibodies. The course of the
immunization may be followed by assays for antibodies for the supernatant antigens.
The assays may be performed by labeling with conventional labels. such as
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radionuclides, enzymes, r.uorescers, and the like. These techni~ues are well known and
may be found in a wide variety of patents, such as U. S. Patent Nos 3,791,932;
4,174,384 and 3,949,064, as illustrative of these types of assays.
Of course, in light of the new technology on DNA vaccination, it will be
understood that virtually all such vaccination regimens will be appropriate for use with
DNA vectors and constructs, as described by Ulmer et al. (1993), Tang et al. (1992), Cox
etal. ( 1993), Fynan etal. ( 1993), Wang etai. ( 1993a; 1993b) and ~hitton etal. (1993),
each incorporated herein by reference. In addition to par~llLe~dl routes of DNA
inoculation, including intramuscular and intravenous injections, mucosal vaccination is
also contemplated. as may be achieved by ~(lmini~tering drops of DNA compositions to
the nares or trachea. It is particularly contemplated that a gene-gun could be used to
deliver an effectively immunizing amount of DNA to the epidermis (Fynan et al., 1993).
4.12 PHARMACEUTICAL COMPOSITIONS
The pharmaceutical compositions disclosed herein may be orally ~lminictered,
for example, with an inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or
they may be incorporated directly with the food of the diet. For oral therapeutic
~minictration, the active compounds may be incorporated with excipients and used in
the form of ingestible tablets, buccal tables, troches, capsules. elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations should contain at least
0.1 % of active compound. The percentage of the compositions and preparations may, of
course, be varied and may conveniently be between about 2 to about 60% of the weight
of the unit. The amount of active compounds in such therapeutically useful compositions
is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the following: a
~inder, as gum tragacanth, acacia, cornstarch. or gelatin; excipients. such as dicalcium
phosphate; a disintegrating agent, such as corn starch. potato starch, alginic acid and the
like; a lubricant. such as magnesium stearate; and a sweetening agent~ such as sucrose,
lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of
wintergreen. or cherry flavoring. When the dosage unit forrn is a capsule. it may contain,
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in addition to materials of the above type, a liquid carrier. Various other materials may
be present as coatings or to otherwise modify the physical form of the dosage unit. For
instance, tablets~ pills, or capsules may be coated with shellac, sugar or both. A syrup of
elixir may contain the active compounds sucrose as a sweetening agent methyl andpropylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of
course, any material used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In addition, the active
compounds may be incorporated into sustained-release ~,epa-dtion and formulations.
The active compounds may also be ~mini~tered parenterally or intraperitoneally.
Solutions of the active compounds as free base or pharmacologically acceptable salts can
be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol~ liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorg~nicm~.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous p,~ala1ion of sterile
injectable solutions or dispersions. In all cases the form must be sterile and must be fluid
to the extent that easy syringability exists. It must be stable under-the conditions of
manufacture and storage and must be preserved against the cont~min~ting action of
microorg~ni.sm~, such as bacteria and fungi. The carrier can be a solvent or dispersion
medium cont~3ining, for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof,
and vegetable oils. The proper fluidity can be maintained, for example, by the use of a
coating, such as lecithin, by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. The prevention of the action of microorganisms
can be brought about by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid~ thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents~ f'or example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by the use in
the compositions of agents delaying absorption, for example, aluminum monostearate
and gelatin.
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Sterile injectable solutions are prepared by incorporating the active compounds in
the required amount in the ;~p~lupllate solvent with various of the other ingredients
enumerated above. as required, followed by filtered sterilization. Generally, dispersions
- are prepared by incorporating the various sterilized active ingredients into a sterile
vehicle which contains the basic dispersion medium and the required other ingredients
from those enumerated above. In the case of sterile powders for the ~lep~dlion of sterile
injectable solutions, the preferred methods of ple~rdlion are vacuum-drying and freeze-
drying techniques which yield a powder of the active ingredient plus any additional
desired ingredient from a previously sterile-filtered solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media. coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
For oral prophylaxis the polypeptide may be incorporated with excipients and
used in the forrn of non-ingestible mouthwashes and dentifrices. A mouthwash may be
l)~c~a~,d incorporating the active ingredient in the required amount in an appropriate
solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active
ingredient may be incorporated into an antiseptic wash cont~ining sodium borate,glycerin and potassium bicarbonate. The active ingredient may also be dispersed in
dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be
added in a therapeutically effective amount to a paste dentifrice that may include water,
binders, abrasives. flavoring agents, foaming agents. and humectants.
The phrase "pharmaceutically-acceptable" refers to molecular entities and
- compositions that do not produce an allergic or similar untoward reaction when
~lministered to a human. The preparation of an aqueous composition that contains a
protein as an active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid solutions or suspensions; solid
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forms suitable for solution in, or suspension in, liquid prior to ;njection can also be
prepared. The preparation can also be emulsified.
The composition can be formulated in a neutral or salt forrn.
Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free
arnino groups of the protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be
derived from inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylarnine,
histidine, procaine and the like. Upon formulation, solutions will be ~ mini~tered in a
manner compatible with the dosage forrnulation and in such amount as is therapeutically
effective. The formulations are easily ~lministered in a variety of dosage forrns such as
injectable solutions, drug release capsules and the like.
For parenteral ~mini~tration in an aqueous solution, for example, the solution
should be suitably buffered if necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. These particular aqueous solutions are especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal ~(lmini.~tration. In this
connection, sterile aqueous media which can be employed will be known to those of skill
in the art in light of the present disclosure. For example, one dosage could be dissolved
in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypoderrnoclysis fluid
or injected at the proposed site of infusion, (see for example, "Remington's
Pharrnaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the condition of the subject being
treated. The person responsible for ~dmini.~tration will, in any event, deterrnine the
appropriate dose for the individual subject. Moreover, for human ~lministration~pl~aldlions should meet sterility, pyrogenicity, general safety and purity standards as
required by FDA Oi'fice of Biologics standards.
4.13 EPITOPIC CORE SEQUENCES
The present invention is also directed tO protein or peptide compositions. free
from total cells and other peptides, which comprise a purified protein or peptide which
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incorporates an epitope .hat is immunologically cross-reactive with one or mole of the
antibodies of the present invention.
As used herein, the term "incorporating an epitope(s) that is immunologically
cross-reactive with one or more anti-BRCAI antibodies" is intended to refer to a peptide
or protein antigen which includes a primary, secondary or tertiary structure similar to an
epitope located within a BAP or BRCAI polypeptide. The level of similarity will
generally be to such a degree that monoclonal or polyclonal antibodies directed against
the BAP or BRCAI polypeptide wilJ also bind to. react with, or otherwise recognize, the
cross-reactive peptide or protein antigen. Various immunoassay methods may be
employed in conjunction with such antibodies, such as, for example, Western blotting,
ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identification of BAP or BRCA l epitopes such as those derived from BAP or
BRCAI or BRCA l-like gene products and/or their functional equivalents, suitable for use
in vaccines is a relatively straightforward matter. For example, one may employ the
methods of Hopp, as taught in U.S. Patent 4,554,101, incorporated herein by reference,
which teaches the identification and preparation of epitopes from amino acid sequences
on the basis of hydrophilicity. The methods described in several other papers, and
software programs based thereon, can also be used to identify epitopic core sequences
(see, for example, Jameson and Wolf, 19~8; Wolf et al., 1988; U.S. Patent Number4,554,101). The amino acid sequence of these "epitopic core sequences" may then be
readily incorporated into peptides~ either through the application of peptide synthesis or
recombinant technology.
Preferred peptides for use in accordance with the present invention will generally
be on the order of about 5 to about 25 amino acids in length, and more preferably about 8
to about 20 amino acids in length. It is proposed that shorter antigenic peptide sequences
will provide advantages in certain circumstances. for example, in the preparation of
vaccines or in immunologic detection assays. Exemplary advantages include the ease of
preparation and purification, the relatively low cost and improved reproducibility of
~ production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized
through the preparation of synthetic peptides whicll include modified and/or extended
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epitopic/immunogenic core sequences which result in a "universal" epitopic peptide
directed to BAP, BAP-related, BRCAl or BRCA1-related sequences. It is proposed that
these regions represent those which are most likely to promote T-cell or B-cell
stimulation in an animal, and, hence, elicit specific antibody production in such an
animal.
An epitopic core sequence, as used herein, is a relatively short stretch of arnino
acids that is "complementary" to, and therefore will bind, antigen binding sites on BAP
or BRCAl epitope-specific antibodies. Additionally or alternatively, an epitopic core
sequence is one that will elicit antibodies that are cross-reactive with antibodies directed
against the peptide compositions of the present invention. It will be understood that in
the context of the present disclosure, the term "complementary" refers to amino acids or
peptides that exhibit an attractive force towards each other. Thus, certain epitope core
sequences of the present invention may be operationally defined in terrns of their ability
to compete with or perhaps displace the binding of the desired protein antigen with the
corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly
crucial, so long as it is at least large enough to carry the identified core sequence or
sequences. The smallest useful core sequence expected by the present disclosure would
generally be on the order of about 5 amino acids in length, with sequences on the order of
8 or 25 being more preferred. Thus, this size will generally correspond to the smallest
peptide antigens prepared in accordance with the invention. However, the size of the
antigen may be larger where desired, so long as it contains a basic epitopic core
sequence.
The identification of epitopic core sequences is known to those of slcill in the art,
for example, as described in U.S. Patent 4 554,101~ incorporated herein by reference.
which teaches the identification and preparation of epitopes from arnino acid sequences
on the basis of hydrophilicity. Moreover, numerous computer programs are available for
use in predicting antigenic portions of proteins (see e.g.. Jameson and Wolf, 1988: Wolf
et al., 1988). Comp-lterized peptide sequence analysis programs (e.g., DNA Star(~
software, DNAStar, Inc. Madison, WI) may also be useful in designing synthetic
BRCAI peptides and peptide analogs in accordance with the present disclosure. The
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peptides provided by this invention are idea' targets for use as vaccines or
immunoreagents for the detection of BAP, BRCAI and BAP- or BRCAl-encoding
genes, or alternatively the detection of either BAP, BRCA 1 or BRCA l-like gene
product(s). In this regard, particular advantages may be realized through the preparation
of synthetic peptides that include epitopic/immunogenic core sequences. These epitopic
core sequences may be identified as hydrophilic and/or mobile regions of the
polypeptides or those that include a T cell motif. It is known in the art that such regions
represent those that are most likely to promote B cell or T cell stimulation, and, hence,
elicit specific antibody production.
To confirm that a protein or peptide is immunologically cross-reactive with, or a
biological functional equivalent of, one or more epitopes of the disclosed peptides is also
a straightforward matter. This can be readily determined using specific assays, e.g., of a
single proposed epitopic sequence, or using more general screens, e.g., of a pool of
randomly generated synthetic peptides or protein fragments. The screening assays may
be employed to identify either equivalent antigens or cross-reactive antibodies. In any
event, the principle is the same, i.e., based upon competition for binding sites between
antibodies and antigens.
Suitable competition assays that may be employed include protocols based upon
immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like. In
any of the competitive assays, one of the binding components, generally the known
element, such as the BRCAI-derived peptide, or a known antibody, will be labeled with
a detectable label and the test components, that generally remain unlabeled, will be tested
for their ability to reduce the arnount of label that is bound to the corresponding reactive
antibody or antigen.
As an exemplary embodiment, to conduct a competition study between a BAP or
a BRCAl and any test antigen, one would first label BAP or BRCAI with a detectable
- labeh such as, e.g, biotin or an enzymatic, radioactive or fluorogenic label, to enable
subsequent identification. One would then incubate the labeled antigen with the other.
test, antigen to be examined at various ratios (e.g., 1:1, 1:10 and 1:100) and, after mixing,
one would then add the mi~cture to an antibody of the present invention. Preferably. the
kno~vn antibody would be immobilized, e.g, by attaching to an ELISA plate. The ability
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of the mix~ure to bind to the antibody would be determined by de~ecting the presence of
the specifically bound label. This value would then be compared to a control value in
which no potentially competing (test) antigen was included in the incubation.
The assay may be any one of a range of imrnunological assays based upon
hybridization, and the reactive antigens would be detected by means of detecting their
label, e.g, using streptavidin in the case of biotinylated antigens or by using a
chromogenic substrate in connection with an enzymatic label or by simply detecting a
radioactive or fluorescent label. An antigen that binds to the same antibody as BRCAl,
for example, will be able to effectively compete for binding to and thus will significantly
reduce BRCA1 binding, as evidenced by a reduction in the amount of label detected.
The reactivity of the labeled antigen, e.g, a BAP or BRCAl composition, in the
absence of any test antigen would be the control high value. The control low value
would be obtained by incubating the labeled antigen with an excess of unlabeled BAP or
BRCAl antigen, when competition would occur and reduce binding. A significant
reduction in labeled antigen reactivity in the presence of a test antigen is indicative of a
test antigen that is "cross-reactive", i.e., that has binding affinity for the sarne antibody.
"A significant reduction", in terms of the present application, may be defined as a
reproducible (i.e., consistently observed) reduction in binding.
In addition to the peptidyl compounds described herein, the inventors also
contemplate that other sterically similar compounds may be formulated to mimic the key
portions of the peptide structure. Such compounds, which may be termed
peptidomimetics, may be used in the same manner as the peptides of the invention and
hence are also functional equivalents. The generation of a structural functionalequivalent may be achieved by the techniques of modeling and chemical design known
to those of skill in the art. It will be understood that all such sterically similar constructs
fall within the scope of the present invention.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope
within their sequence. are readily achieved using conventional synthetic techniques such
as the solid phase method (~.g.. through the use of a commercially-available peptide
synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide
antigens synthesized in this manner may then be aliquoted in predetermined amounts and
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stored in conventional manners, such as in aqueous solutions or, even more pre.~erably, in
a powder or Iyophilized state pending use.
In general, due to the relative stability of peptides. they may be readily stored in
aqueous solutions for fairly long periods of time if desired, e.g., up to six months or
more~ in virtually any aqueous solution without appreciable degradation or loss of
antigenic activity. However, where extended aqueous storage is contemplated it will
generally be desirable to include agents including buffers such as Tris or phosphate
buffers to m~int~in a pH of about 7.0 to about 7.5. Moreover, it may be desirable to
include agents which will inhibit microbial growth, such as sodium azide or Merthiolate.
For extended storage in an aqueous state it will be desirable to store the solutions at 4~C,
or more preferably, frozen. Of course~ where the peptides are stored in a Iyophilized or
powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that
may be rehydrated with a predetermined amount of water (preferably distilled) or buffer
prior to use.
4.14 SITE SPECI~IC MUTAGENESIS
Site-specific mutagenesis is a technique useful in the plel)a d~ion of individual
peptides, or biologically functional equivalent proteins or peptides, through specific
mutagenesis of the underlying DNA. The technique, well-known to those of skill in the
art, further provides a ready ability to prepare and test sequence variants, for example,
incorporating one or more of the foregoing considerations, by introducing one or more
nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide sequences which
encode the DNA sequence of the desired mutation. as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient size and sequence
complexity to form a stable duplex on both sides of the deletion junction being traversed.
Typically, a primer of about 14 to about 25 nucleotides in length is preferred, with about
5 to about 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art, as
exemplified by various publications. As will be appreciated, the technique typically
employs a phage vector which exists in both a single stranded and double stranded form.
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Typical vectors useful in site-directed mutagenesis include vectors such as the M13
phage. These phage are readily commercially-available and their use is generallywell-known to those skilled in the art. Double-stranded plasmids are also routinely
employed in site directed mutagenesis which elimin~tes the step of transferring the gene
of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first
obtaining a single-stranded vector or melting apart of two strands of a double-stranded
vector which includes within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared,
generally synthetically. This primer is then annealed with the single-stranded vector, and
subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment,
in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex
is formed wherein one strand encodes the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex vector is then used to transform
ap,o~ ,;ate cells, such as E. coli cells, and clones are selected which include recombinant
vectors bearing the mutated sequence arrangement.
The ~ ~dtion of sequence variants of the selected peptide-encoding DNA
segments using site-directed mutagenesis is provided as a means of producing potentially
useful species and is not meant to be limiting as there are other ways in which sequence
variants of peptides and the DNA sequences encoding them may be obtained. For
example, recombinant vectors encoding the desired peptide sequence may be treated with
mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details
regarding these methods and protocols are found in the teachings of Maloy et al., 1994;
Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis etal., 1982, eachincorporated herein by reference~ for that purpose.
The PCRTM-based strand overlap extension (SOE) (Ho etal., 1989) for site-
directed mutagenesis is particularly preferred for site-directed mutagenesis of the nucleic
acid compositions of the present invention. The techniques of PCRTM are well-known to
those of skill in the art, as described hereinabove. The SOE procedure involves a two-
step PCRTM protocol, in which a complementary pair of internal primers (B and C) are
used to introduce the appropriate nucleotide changes into the wild-type sequence. In two
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separate reactions, fl~3nking PCRTM primer A (res;riction site incorporated into the oligo)
and primer D (restriction site incorporated into the oligo) are used in conjunction with
primers B and C, respectively to generate PCRTM products AB and CD. The PCRTM
products are purified by agarose gel electrophoresis and the two overlapping PCRTM
fragments AB and CD are combined with flanking primers A and D and used in a second
PCRTM reaction. The amplified PCRTM product is agarose gel purified, digested with the
appropriate enzymes, ligated into an expression vector, and transformed into E. coli
JMIOI, XLI-BlueTM (Stratagene, La Jolla, CA), JMI05, or TGI (Carter et al., 1985)
cells. Clones are isolated and the mutations are confirmed by sequencing of the isolated
plasmids. Beginning with the native BRCAI gene sequence~ suitable clones and
subclones may be made from which site-specific mutagenesis may be perforrned.
4.15 BIOLOGICAL FUNCTIONAL EQUIVALENTS
Modification and changes may be made in the structure of the peptides of the
present invention and DNA segments which encode them and still obtain a functional
molecule that encodes a protein or peptide with desirable characteristics. The following
is a discussion based upon chzlnging the amino acids of a protein to create an equivalent,
or even an improved, second-generation molecule. The amino acid changes may be
achieved by ch~n~ing the codons of the DNA sequence, according to Table 1.
For example, certain amino acids may be substituted for other amino acids in a
protein structure without appreciable loss of interactive binding capacity with structures
such as~ for example, antigen-binding regions of antibodies or binding sites on substrate
molecules. Since it is the interactive capacity and nature of a protein that defines that
protein's biological functionai activity, certain amino acid sequence substitutions can be
made in a protein sequence, and~ of course, its underlying DNA coding sequence, and
nevertheless obtain a protein with like properties. It is thus contemplated by the
inventors that various changes may be made in the peptide sequences of the disclosed
compositions~ or corresponding DNA sequences which encode said peptides without
appreciable loss of their biological utility or activity.
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TABLE 1
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Asparticacid Asp D GAC GAU
Glutamic acid Glu E GAA GAG
Phenylalanine Phe F UUC UW
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
In m~king such changes, the hydropathic index of amino acids may be
considered. The importance of the hydropathic amino acid index in conferring
interactive biologic function on a protein is generally understood in the art (Kyte and
Doolittle, 1982, incorporated herein by reference). It is accepted that the relative
hydropathic character of the amino acid contributes to the secondary structure of the
resultant protein~ which in turn defines the interaction of the protein with other
molecules. for example, enzymes~ substrates, receptors, DNA, antibodies. antigens, and
the like. Each amino acid has been assi~ned a hydropathic index on the basis of their
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hydrophobicity and ~;harge characteristics (Kyte and Doolittle, 1982), .hese are:
isoleucine (+4.5); valine (~4.2); leucine (+3.8); phenyl~l~nine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); gh3t~m~te (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); Iysine (-3.9); and arginine (~.5).
It is known in the art that certain amino acids may be substituted by other amino
acids having a similar hydropathic index or score and still result in a protein with similar
biological activity, i.e., still obtain a biological functionally equivalent protein. In
m~king such changes, the substitution of amino acids whose hydropathic indices are
within +2 is preferred, those which are within +1 are particularly pler~l-ed, and those
within "0.5 are even more particularly preferred. It is also understood in the art that the
substitution of like amino acids can be made effectively on the basis of hydrophilicity.
U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local
average hydrophilicity of a protein, as governed by the hydrophilicity of its ~dj~Pnt
arnino acids, correlates with a biological property of the protein.
As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have
been assigned to amino acid residues: arginine (+3.0); Iysine (+3.0); aspartate (+3.0 + 1);
glllt~m~t~- (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
threonine (-0.4), proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4) It is understood that an amino acid can be
substituted for another having a similar hydrophilicity value and still obtain abiologically equivalent, and in particular, an immunologically equivalent protein. In
such changes, the substitution of amino acids whose hydrophilicity values are within +2
is preferred, those which are within +1 are particularly preferred, and those within +0.5
are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on therelative similarity of the amino acid side-chain substituents, for example, their
hydrophobicity, hydrophilicity. charge~ size. and the like Exemplary substitutions which
take various of the foregoing characteristics into consideration are well known to those of
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skill in the art and include: argiiline and Iysine; glllt~mz~te and aspartate7 serine and
threonine; glutamine and asparagine; and valine, leucine and isoleucine.
4.16 NUCLEAR TRANSPORT
Nuclear transport is a multi-step process. Following synthesis in the cytoplasm,proteins that contain an active nuclear localization sequence (NLS) are transported to the
nucleus~ entering through pore complexes in the nuclear envelope (Silver, 1991). There
are several critical steps in this process, involving multiple proteins. First, the NLS of
the proteins to be imported must be recognized. Second, the proteins are brought to the
nuclear pore complex. Third, the pore complex mediates selective entry of these
proteins. In addition, there are several examples of proteins that are tethered in the
cytoplasm by other proteins that release them for nuclear transport in response to specific
signals. The NF-kB/IkB and the Hsp90/glucocorticoid receptor interactions are
paradigms of such regulation (Sanchez e~ al., 1985; Ghosh and Baltimore, 1990). The
phosphorylation status of a protein may also affect its localization. For example, the
yeast transcription factor PHO4 is prevented from translocating to the nucleus when it is
phosphorylated by the PHO850-PHO85-cyclin-CDK complex (O'Neill et al., 1996). The
mislocation of BRCAl in advanced breast tumor cells may be due to failure in any one
of these steps. However, it is unlikely that a major nuclear transport system is defective
in these tumor cells, since they are viable. Most likely subtle regulators, such as proteins
required for modification of BRCAI in order to expose its NLS, or a protein thatspecifically recognizes the NLS of BRCAI. may fail to function properly.
4.17 ABBREVIATIONS
cdk = cyclin dependentkinase
CIP = calf intestinal alkaline phosphatase
GST = glutathione-S-transferase
IP = immunoprecipitation
FACS = fluorescence-activated cel l sorting
BSA = bovine serum albumin
p.c. = post coitum
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EGF = epidermal grov~th factor
SDS = sodium dodecyl sulfate
PAGE = polyacrylamide gel electrophoresis
S 5. EXAMPLES
The following examples are included to demonstrate pl~re.led embodiment~ of
the invention. It should be appreciated by those of skill in the art that the techniques
disclosed in the exarnples which follow represent techniques discovered by the inventors
to function well in the practice of the invention, and thus can be considered to constitute
preferred modes for its practice. However, those of skill in the art should, in light of the
present disclosure, appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
5.1 EXAMPLE 1 -- CHARACTERIZATION OF THE BRCA1 NUCLEAR
PHOSPHOPROTEIN
This exarnple describes the isolation and characterization of polyclonal antisera
specific for different regions of the BRCAl protein. These were used to confirm that
BRCAI is a 220 kDa phosphoprotein in human cells, and that it is expressed and
phosphorylated in a cell cycle dependent manner and is localized to the nuclei of normal
cells.
5.1.1 MATERIALS AND METHODS
5.1.1.1 GENERATION OF ANTIBODlES SPECIFIC FOR HUMAN BRCAI
Mouse polyclonal antisera were generated as previously described using purified
GST-BRCAI fusion-proteins expressed in bacteria as immunogens (Durf'ee et al., 1994).
Two of the antisera (anti-BRCAlBgl and anti-BRCAl, raised against amino acids 341-
748 and 762-1315, respectively) are described herein. A third antiserum (anti-BRCAlN)
- was generated using a GST fusion-protein encoding the first 302 amino acids of BRCAl.
The monoclonal anti-BRCA I antibody (MAb 6B4) was developed against GST-
BRCA I Bgl bv standard methods (Harlow and Lane, 1988).
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5.1.1.2 GENERATION OF A FULL-LENGTH BRCA1 CDNA
A full-length BRCAl cDNA was obtained by using a PCR-generated fragment of
exon 11 to probe a human cDNA library (Zhu, et al., 1995). Three overlapping clones
were identified that together encoded the entire cDNA. Convenient restriction sites
within these clones were then used to assemble a full-length clone (see FIG. lA) in
pBSK (Stratagene, La Jolla, CA).
5.1.1.3 IN VITRO TRANSCRIPTION AND TR~NSLATION
In vitro translated BRCA 1 was generated from the cDNA using the TNT
reticulocyte-lysate transcription and translation kit (Promega, Madison, WI) according to
the manufacturer's instructions. Immunoprecipitations were done using 1/20th of the
total reaction volume.
I S 5.1.1.4 IMMUNOPRECIPITATION, IP/WESTERN AND WESTERN ANALYSES
Immunoprecipitation with the three polyclonal antisera against BRCAI was done
according to standard protocols (Chen et al., 1989), using the various antisera at a
dilution of 1: 1000. After immunoprecipitation, proteins were separated on 6.5%
polyacrylamide gels. Proteins labeled with S-methionine (300 ~Ci for 90 min. in
methionine-free medium) were detected by autoradiography. For IP/western studies, the
immunoprecipitates were transferred to ImmobilonT:~s membranes (Millipore, Bedford,
MA) and probed with MAb 6B4 at a dilution of 1: 1000, according to standard procedures
(Durfee et al., 1994). Double immunoprecipitations were done as described earlier. For
the detection of pllORB and p84 by western analysis, MAb IID7 and anti-N5-3,
respectively, were used as primary antibodies as described (Durfee e~ al., 1994).
Treatment of extracts with calf intestinal alkaline phosphatase prior to
immunoprecipitation was perforrned as described (Zhu~ et al., 1995).
5.1.1.5 INDIRECT IMMUNOFLUORESCENCE
Indirect immunofluorescence was performed as described (C~len. el al., 1995;
Durfee, et al.. 1994).
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5.1.1.6 PRODUCTION OF BRCA1 IN INSECT CELLS
The full-length BRCAl cDNA, with an engineered NotI site immediately 5' of
the initial methionine codon, was subcloned into pAcHLT-B (Pharmingen, San Diego,
CA) using the Notl and Smal sites, so as to generate a poly-histidine tagged BRCAl,
expressed from the baculovirus polyhedrin promoter. This construct was co-transfected
into SF9 cells (obtained from ATTC, Rockville, MD) together with BaculoGold~) viral
DNA (Pharmingen), according to the manufacturer's protocols. After 5 days, the culture
medium was collected, and a plaque assay done, as per the m~nllf~.turer's protocols.
After I week, recombinant plaques were identified and picked. Several plaque-purified
viruses were then screened for BRCA 1 production by using nickel-affinity
chromatography to purify expressed protein from infected SF9 cells. Purified BRCAI
was detected either by Coomassie Brilliant Blue staining or by western blotting with
MAb 6B4.
5.1.1.7 FACS ANALYSIS
Cells (5 x 10 ) were trypsinized, fixed in 70% cold ethanol, washed in PBS, and
resuspended in I ml PBS containing 200 llg/ ml RNase A and 20 ~g/ml propidium
iodide for 30 min at 37~C. Flow cytometric analysis was done using a FACSCalibur~
flow cytometer (Becton-Dickinson, San Jose. CA).
5.1.1.8 KINASE ASSAY
Cells were Iysed in Lysis 250 buffer as described (Chen, e~ al., 1989), and the
extracts immunoprecipitated with antibodies against various cyclins and cyclin
dependent kinases (CDC2, CDK2, CDC4. cyclin D, cyclin E, and cyclin A: all purchased
from Santa Cruz Biotech. Santa Cruz, CA). The precipitates were washed and then
resuspended in 50 ILll CDC2 kinase buffer (75 mM HEPES, pH 7.5, 100 mM MgC12; 25
mM EGTA, 3 mM DTT, I ~g/ml BSA) and incubated in the presence of 10 ,L1Cj [Y- P]ATP for 30 min at 30~C. The reactions were tllen washed once in cold kinase bLlffer, and
the complexes dissociated and re-immunoprecipitated with anti-BRCAI. as previously
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described (Chen, et al., 199'). The resultant precipitates were separated by SDS-PAGE,
and the gel dried and exposed to X-ray film.
5.1.2 RESULTS
5.1.2.1 ANTIBODY COMPOSITIONS AGAINST DISTINCT BRCA1 REGIONS
Mouse polyclonal antisera have been generated against human BRCAl using
GST fusion-proteins encoding three different regions of the BRCA 1 protein as
irnmunogens (depicted schematically in FIG. IA). As shown in FIG. IB (lanes 2, 4, and
6), each of the three independent antisera immunoprecipitated a protein, running as a
doublet band, with a molecular mass of 220 kDa from whole-cell Iysates of S-
methionine-labeled HBLI00 (human breast epithelial) cells. This protein was not seen
using preimmune serum (FIG. 1 B, lane 1). Since several other proteins are co-
immunoprecipitated with BRCA1, the specificity of each antiserum was further
confirmed using a double immunoprecipitation protocol (Chen, et al., 1995), in which
the first round precipitates were denatured, so as to disrupt any protein complexes, and
then re-immunoprecipitated with the same antibody. This more stringent protocol
resulted in the detection of only the 220 kDa doublet by all three antisera (FIG. I B, lanes
3, 5, and 7), strongly suggesting that only the doublet is specifically recognized by the
three independent antisera and that, most likely. it is BRCAI. Many of the other bands
detected in the single-step immunoprecipitation are also seen with preimmune serum and
are thus likely to be non-specific. However, some bands in addition to the 220 kDa
doublet are not detected with preimmune serum. These may be proteins that form
complexes with BRCAI. The most compelling of these being co-immunoprecipitated
with all three antisera, e.g., bands at 110 kDa (FIG. IB).
To further confirm that the 220 kDa protein is BRCAl, a full-length cDNA for
BRCAI was generated. This was used to drive in VitYo translation of the gene product,
which was then immunoprecipitated with the three antisera. As shown in FIG. 2A all
three antisera recognize a 220 kDa protein from the in vitro translation mixt~lre that co-
migrates with the lower band of the do~lblet detected in whole-cell Iysates (FIG. 2A,
lanes 1. 3~ 4~ and 5). The upper band was shown to incorporate radioactive phosphate,
suggesting that it might be a phosphorvlated form of BRCAI (Chen. ~ l. 1995).
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Con.~ tent with this, treatment of cell l~sates with calf intestinal ~IkAline phosphatase
(CIP) prior to immunoprecipitation generated a single band with the sarne mobility as the
faster migrating band of the original doublet (FIG. 2A~ lane 2).
5.1.2.2 BRCA11SA220 KDA PROTEININ BACULOVIRUSSYSTEMS
Since the in vitro translated product is produced in rather small ~uantities,
presumably due to the size of the transcript, a baculovirus-based expression system was
prepared for BRCAl to generate greater quantities of full-length BRCAI that could be
readily purified using nickel-affinity chromatography. Several plaque-purified
recombinant viruses were analyzed and found to produce a 220 kDa protein that could be
isolated by nickel-affinity chromatography from Iysates of infected SF9 cells. This
protein was not detected in Iysates of uninfected SF9 cells. When extracts from infected
SF9 cells were immunoprecipitated using each of the three antisera, and the
immunoprecipitated protein detected by probing a western blot with the anti-BRCAI
monoclonal antibody MAb 6B4, a 220 kDa protein was detected that co-migrated with
endogenous BRCAI from HBLI00 cells (FIG. 2B). This protein could not be detectedin uninfected cells or by irnmunoprecipitating with preimmune serum. That the 6B4
monoclonal antibody against BRCA I recognizes a 220 kDa protein in all the
immunoprecipitates provides further strong support for the conclusion that the three
antisera all recognize the same 220 kDa protein which is BRCAI.
5.1.2.3 EXPRESSION OFBRCA1 FOLLOWSTHE CELL CYCLE
High level BRCAI mRNA expression in mice has been shown to correlate with
tissues undergoing rapid proliferation combined with differentiation (Lane, et al., 1995;
Marquis, e~ al.. 1995). Thus, it was reasoned that BRC~I expression might vary with
cell cycle stage. To investigate this possibility~ the e:~pression of BRCAI was analyzed
in synchronized T24 bladder carcinoma cells. These cells are conveniently arrested in
G0 by contact inhibition and display very high synchrony upon replating at low density
in fresh medium. FIG. 3A depicts an IP/western t'or BRCAI using extracts made either
at various times following release of T24 cells f'rom density arrest, or from T24 cells
arrested in M-phase using nocodazole (0.4 ~lg/ml t'or 8 hrs). The cell cycle distribution
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profile at each time-point, deterrnined by FACS analysis, is presented in FIG. 3B. The
phosphorylation status of Rb in the same extracts was used as an additional indicator of
cell cycle progression (FIG. 3B, middle panel), and staining for p84 (FIG. 3A, bottom
panel) served to quantify loading. While BRCAI is readily detected in unsynchronized
cells ~lane l), it is expressed at very low levels in early Gl, such that it is undetectable by
western analysis until 18 hrs post-release (lane 4). This corresponds to late Gl, since the
cells still have a 2N DNA content by FACS analysis, but Rb has already become
phosphorylated (FIG. 3B, lane 4). As the cells enter S-phase, BRCAI expression rapidly
increases to a maximum. (FIG. 3A, lanes 5, 6). Although the expression level decreases
somewhat in M-phase~ it remains high overall (lane 7).
5.1.2.4 IMMUNOSTAINING OF BRCA1 DURING THE CELL CYCLE
Given the alterations in expression level of BRCAI in parallel with cell cycle
progression, it was of interest to determine whether differences in the immunostaining
pattern could also be detected. To do this, T24 cells were again arrested in G0 by density
arrest and then stimulated to enter the cell cycle synchronously by replating on coverslips
at low density. At various times, cells were fixed and stained, as previously described
(Chen et al., l 995; Durfee et al., I 994), for BRCA 1 expression by indirect
immunofluorescence, and for DNA using DAPI. The results are presented in FIG. 3Dthrough FIG. 30. l I hours post release, BKCAl is just detectable as homogenous
nuclear staining (FIG. 3D and FIG. 3E). As cells progress into S-phase (24 and 33 hrs
post release) staining intensifies (consistent with the IP/western data) and becomes
punctate (FIG. 3F, FIG. 3G, FIG. 3~, FIG. 31). During mitosis, BRCAI staining appears
to surround the chromosomes as they align on the metaphase plate (FIG. 3J and FIG. 3K)
and then move apart (FIG. 3L and FIG. 3M). As the cells re-enter G l ~ weak,
homogenous nuclear staining returns (FIG. 3N and FIG. 30), confirming that BRCAI is
expressed throughout the cell cycle, even though it is undetectable by western analysis in
early Gl (FIG. 3A).
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5.1.2.5 PHOSPHORYLATION OF'BRCA1 IS CELL_CYCLE DEPEND~NT
As shown in FIG. 2A, BRCA I is phosphorylated in vivo, resulting in the
detection of both hypo- and hyper-phosphorylated species as a 220-kDa doublet byimmunoprecipitation. The dependence of this phosphorylation on cell cycle progression
was investigated by pulse-labeling synchronized T24 cells with 32p ortho phosphate at
various times following release from G0 arrest, or following nocodazole arrest. Cell
extracts were then immunoprecipitated with anti-BRCA I . This analysis revealed
BRCAl to be phosphorylated in a cell cycle dependent manner that paralleled its
expression: becoming evident in mid/late Gl, rising to a maximum in S-phase, and then
rçm~inin~ elevated through M-phase (FIG. 3A and FIG. 3D through FIG. 30).
5.1.2.6 BRCA1 PIIOSPHORYLATION BY SPECIFIC CYCLIN_DEPENDENT PROTEIN
KINASES
Since BRCA I phosphorylation is cell cycle-dependent, it was determined
whether any known cell cycle-dependent protein kinases could phosphorylate BRCAl.
To do this, cell Iysates were irnrnunoprecipitated with antibodies directed against various
CDKs and cyclins. The precipitates were then incubated in kinase buffer in the presence
of L~-32P]ATP. Finally, the precipitates were washed, dissociated, and re-
immunoprecipitated with anti-BRCAI antibodies. The resulting precipitates were then
separated by SDS-PAGE, and the gels dried and autoradiographed. The results of atypical study, presented in FIG. 4, show BRCAI to be phosphorylated by cyclins D and
A, both complexed to cdk2. This is consistent with the initiation of BRCA I
phosphorylation in mid Gl and with its continued phosphorylation during S-phase.
5.3 DISCUSSION
Antisera specific for BRCAI has been characterized and used to analyze BRCAI
expression in normal cells. The results con~lrmed and extended previous observation
that BRCAI is a 220 kDa nuclear phosphoprotein in normal cells. It was shown that the
polyclonal antisera. raised against three different regions of the BRCAl protein, all
specifically reco~nized a 220 kDa protein in whole-cell Iysates. Immunostaining
recon~lrmed previoLIs observations that BRCAl is a nuclear protein in normal cells
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(Chen et al., 1995). Confirrning that the 220 kDa protein is bona fide rull-length
BRCAI, both in vitro translated BRCA1 and recombinant BRCA1 expressed using the
baculovirus system were shown to co-migrate with BRCAI from HBL100 cells. In
human cells, BRCAl migrates as a doublet, the upper band of the doublet being a
phosphorylated forrn of BRCA 1. The 220 kDa size is fully consistent with the predicted
molecular weight for full-length BRCAI, and is in agreement with previous data (Chen
et al., 1995; Scully et al., 1996). Others have reported detecting a 190-kDa protein using
antibodies raised against the same immunogen as Scully et al. (Scully et al., 1996; Gudas
et al., 1995; Jensen et al., 1996) BRCAl is reported to undergo alternative splicing (Miki
et al., 1994) and it is possible that the 190 kDa species is an alternatively spliced variant
of BRCAl expressed in some cell types. However. the sarne size protein was also
detected in cells transfected with a retrovirus expressing a full-length BRCAl cDNA
(Holt et al., 1996). The same group of authors also described a baculovirus-derived,
recombinant BRCAI with a molecular weight of 180 kDa (Jensen et al., 1996). In the
present invention, baculovirus-derived BRCAl was expressed as a 220 kDa protein that
co-migrated with BRCAl from HBL100 cells. The fact that this 220 kDa protein could
be detected by three separate criteria: nickel-affinity chromatography, imrnuno-precipitation with three independent BRCAl-specific antisera, and western blotting with
a BRCAl-specific monoclonal antibody, make it very unlikely that this protein is not
correctly synthesized.
There are three possible explanations for these differences. First, production of
proteins using baculovirus requires introduction of the cDNA of interest into the viral
genome by homologous recombination. It is possible that inaccurate recombinationwould generate an incomplete protein; multiple plaque-purified viruses were exarnined,
and all were found to produce a 220-kDa protein. Second, it was noted that BRCAI is
susceptible to proteolytic degradation and have occasionally seen lower molecular weight
degradation products in addition to the full-length 220-kDa protein. This secondpossibility may also explain the detection of a 190-kDa protein in human cell extracts.
Finally, it is possible that the peptide antisera used in the other studies are not specific for
BRCAl, but for some other protein such as the EGF receptor.
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Several lines of evidence suggest that BRCAI plays a critical role in the
regulation of cell growth and determination, at least in mice. BRCAI nullizygous mice
die at the egg cylinder stage of development (5-6 days p.c.), suggesting a role for
BRCA I in early cell fate determination (Chia-Yang Liu, et al., 1996)
Whether BRCAl has a similar role in humans is unclear. A developmentally
norrnal woman has been described carrying gerrnline nonsense mutations in both alleles
of BRCA1 (Boyd et al., 1995). It is possible that in humans there is functional
redlln~l~ncy between BRCAl and another protein, or that different mutations havevarying effects. In this regard it is worth noting that homozygous-null individuals are yet
to be reported among the extensively studied Ashkenazi Jewish population that has a
high incidence of breast and ovarian cancer due to a founder BRCAl mutation (Friedman
et al., 1995). In situ hybridization studies show ubiquitous BRCAl expression in early
mouse embrvos, and the timing of this expression, as well as that seen in breastepithelium during puberty, is consistent with BRCA I expression being highest in tissues
that are undergoing rapid growth and differentiation (Gowen et al., 1996; Scully et al.,
1996). Consistent with these observations, BRCAl was found to be expressed in a cell-
cycle-dependent manner. Initial expression is detected in mid-Gl at, or just prior to, the
restriction point. Expression builds to a maximum in S-phase and then remains high
through out M-phase, falling to low levels again in Gl. In parallel with the increase in
expression seen as cells transit Gl and enter S-phase. BRCAl becomes phosphorylated,
apparently by cyclins D and A, both complexed to cdk2. That BRCAI is phosphorylated
in parallel with its synthesis suggests that the phosphorylated species may be the active
forrn of the protein and that its activity is regulated by cyclin-dependent kinases.
Consistent with this, there are two consensus cdk phosphorylation sites within BRCA I .
5.2 EXAMPLE 2 -- PREPARATION OF BRCAI ANTIBODIES
To characterize BRCA1, the inventors generated polyclonal antibodies to BRCA1
(anti-BRCA I ) by creating a glutathione-S-transferase (GST)-BRCA I fusion protein
cont~ining amino acids encoded by a 3' portion of BRC~A I exon 11.
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5.2.1 FUSION PROTEINS
In the creation of glutathione-S-transferase (GST)-BRCAl fusion constructs, the
PCRTM was used to amplify two exon BRCAl fragments from WI 38 cell (human diploid
lung) genomic DNA. A fragment of~l.9 kb was amplified with two 27-nucleotide
primers synthesized according to the published BRCA 1 sequence: BRCA9
[5'-TTGCAAACTGAAAGATCTGTAGAGAGT-3'~ (SEQ ID NO:2), upstream of a
BglII site, and BRCA7 [5'-TTCCAAGCCCGTTCCTCTTTCTTCCAT-3'], (SEQ ID
NO:3) downstream of a BamHI site. The amplified genomic DNA was then digested
with BglII and BamHI to create a 1.8-kb fragment from codons 762 to 1315. This
fragment was purified and subcloned into the GST expression vector pGEX-2T to create
pGST-BRCA 1. For creation of a second plasmid, GST-BRCA l -Bgl, another
27-nucleotide primer, BRCA8 [5'-GATTTGAACACCACTGAGAAGCGTGCA-3']
(SEQ ID NO:4), be~innin~ at codon 245, and primer BRCA9 were used to amplify a
3.2-kb fragment comprising almost all of exon 11. This fragment was then digested with
BgnI to create a 1.2-kb fragment from codons 341 to 748, which was subcloned into a
modified pGEX-2T.
Each of the two fusion proteins was expressed in Escherichia coli and purified
with glutathione-Sepharose beads for use as an antigen in mice. Serum from immunized
mice was then preabsorbed on GST affinity columns. The serum raised against the first
GST-BRCAl protein was used in all studies illustrated in the figures. Preimmune serum
was obtained from the same mice and used at the same dilution. Anti-BRCAI serum
specifically immunoprecipitated a protein with a molecular mass of 220-kDa in HBL 100
human diploid breast epithelial cells metabolically labeled with 35S-methionine (FIG.
SA). The protein migrated at approximately the size predicted from the 1863-amino acid
sequence (Miki et al, 1994). Because anti-BRCAI serum coprecipitated at least five
proteins other than BRCAI, a double immunoprecipitation involving denaturation was
performed and detected only the 220-kDa protein (FIG. SA, lane 3).
Immunoprecipitation was performed by labeling HBL 100 cells with 35S-methionine~Iysing them in Iysis-250 buffer, and immunoprecipitating with anti-BRCA 1 ~ as
previously described for retinoblastoma protein (Chen ~t ~ 1989).
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Immunoprecipitated proteins were boiled in a denaturin~ buffer [20 mM Tris-HCI
(pH 7.4), 50 mM NaCI, 1% SDS, and S mM dithiothreitol] for 5 min, diluted 10-fold
with a Iysis-50 buffer containing different detergents [20 mM TrisHCI (pH 7.4), 50 mM
- NaCI, 1% NP-40, and 1% deoxycholate], and reimmunoprecipitated by anti-BRCAI in
the same buffer. This doubly immunoprecipitated protein was then washed with
- Iysis-250 buffer before separation by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
Two additional polyclonal antibodies were used in similar studies. C20, directedagainst an epitope near the COOH-terminus, and BRCAI-Bgl, raised against a fusion
protein with sequences encoded by the more 5' portion of exon 11, identified the same
protein as the first antibody (FIG. SA, lane 6). Rabbit polyclonal antibody C20, raised
against a synthetic peptide corresponding to amino acids 1843 to 1862 of BRCAI, was
purchased from Santa Cruz Biotechnology, Inc. In the creation of
glutathione-S-transferase (GST)-BRCA I fusion constructs, the PCRTM was used to
amplify two exon BRCAI fragments from WI 38 cell (human diploid lung) genomic
DNA. A fragment of ~1.9 kb was amplified with two 27-nucleotide primers synthesized
according to the published BRCA I sequence: BRCA9
[5'-TTGCAAACTG~AAGATCTGTAGAGAGT-3'] (SEQ ID NO:2), upstream of a
BgllI site, and BRCA7 [5'-TTCCAAGCCCGTTCCTCTTTCTTCCAT-3'] (SEQ ID
NO:3), downstream of a BamHI site. The amplified genomic DNA was then digested
with BglII and BamHI to create a 1.8-kb fragment from codons 762 to 1315. This
fragment was purified and subcloned into the GST expression vector pGEX-2T to create
pGST-BRCA 1.
For creation of a second plasmid, GST-BRC'A l -Bgl, another 27-nucleotide primer~
BRCA8 [5'-GATTTGAACACCACTGAGAAGCGTGCA-3'] (SEQ ID NO:4)~
beginning at codon 245~ and primer BRCA9 were used to amplify a 3.2-kb fragment
comprising almost all of exon 11. This fragment was then digested with Bglll to create a
1.2-kb fragment from codons 341 to 748, which was subcloned into a modified
pGEX-2T. Each of the two fusion proteins was expressed in Escherichia coli and
purified with gl~ltathione-Sepharose beads for use as an antigen in mice. Serum from
immunized mice was then preabsorbed on GST af'finity columns. The serum raised
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against the first CiST-BTACl protein was used in all studies illustrated in the figures.
Preirnmune serum was obtained from the same mice and used at the same dilution. The
same results were obtained when each step in the double immunoprecipitation was
performed with a different polyclonal antibody.
These immunological results demonstrated that the 220-kDa protein is the
BRCAI gene product. The immunoprecipitate of Iysate from HBL100 cells labeled with
[32P]phosphoric acid contained only a single, more slowly migrating species (lane 7) and
thus showed that BRCA 1 is a phosphoprotein.
BRCAl is present not only in normal breast epithelial cells like the l~BL100 line,
but in all breast cancer lines tested (FIG. 5B). It appears to be expressed largely intact in
these cells, because the proteins identified by 32P labeling and immunoprecipitation with
anti-BRCAI all migrated in the gel at ~220 kDa. Thus BRCAI is not mutated by
truncation in most breast cancer cell lines. In tumor lines derived from tissues other than
breast, BRCAl appears to be more abundant than in breast cancer lines; it can beIS detected more easily in bladder, cervical, colon, and other cancers by labeling with
35S-methionine (FIG. 5C).
5.2.2 SUBCELLULAR LOCALIZATION OF BRCA1
To determine the subcellular localization of BRCAl, the inventors fractionated
HBL100 cells into nuclear. cytoplasmic. and membrane components (Chen et al., 1994;
Abrams et al., 1982). BRCA I was detected in norrnal cells mainly in nuclei (FIG. 6A).
Furthermore, indirect immunostaining of intact cells, including HBL100, several other
normal cell lines, and tumor cells derived from tissues other than breast or ovary also
localized BRCAl to nuclei (FIG. 6B through FIG. 6Q; Table 2). In contrast~ BRCAIwas detected mainly in the cytoplasm of almost all breast cancer cell lines tested (FIG. 6J
through FIG. 6Q; Table ~). In 14 of 17 cell lines established from breast cancers,
BRCAl staining was principally cytoplasmic For two other breast cancer lines, both
nuclear and cytoplasmic staining was observed in the same cells. One line.
MDA-MB361, which was originally derived from a brain metastasis (Cailleau ~ c~l.1978), contained two distinct populations of cells: A less abundant fraction of lar~er.
more heterogeneous cells in which BRCAl localized to the nuclei, and a more abundant
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fraction of smaller, more homogeneous cells in which BRCA 1 localized to the
cytoplasm. Similar results were obtained by cell fractionation in several of the same cell
lines. These results suggest that BRCA1 is located aberrantly in the cytoplasm of most
breast and ovarian cancer cell lines.
Next primary cells from malignant pleural effusions and biopsy sections from
- patients with breast cancer were examined. In all of the primary malignant effusion cells,
obtained from 17 different patients, BRCAI was also located primarily in the cytopl~m
(FIG. 7N and FIG. 7P; Table 2). Other tumor cells grown in suspension (such as
leukPmi~ lines CEM, HL60, and Molt4) or metastatic to pleura (K562 and U937) stained
mainly in nuclei (Table 2).
Breast tumor cells in culture and from malignant pleural effusions were all
derived from advanced. metastatic cancers. To determine whether BRCAl also localized
aberrantly in primary tumors, the inventors used the same polyclonal anti-BRCAl serum
to stain cells in tissue sections. Complete or partial localization of BRCAl was shown in
the cytoplasm of most breast cancer cells (FIG. 8A~ FIG. 8B and FIG. 8C). In 50
biopsies, BRCAl staining was mainly cytoplasmic in 6 (12%), cytoplasmic and nuclear
to a variable extent in 34 (68%), primarily nuclear in 10 (20%), and absent in 2(4%)
(Table 2). These results demonstrate abnormal subcellular localization of BRCAl in
primary breast tumors as well as those that are distantly metastatic. Complete
mislocation of BRCAI appears to be more common in end-stage breast cancer. but
nonetheless occurs to a variable extent in the great majority of tumors in a random
survey. The 4% of tumors that lack BRCAl altogether may represent f~mili~l cases;
such a percentage corresponds well with the similar, small incidence of BRCA I
mutations in breast cancers of all kinds (Claus et al., 1991). Note that in the stromal cells
and Iymphocytes from the tumor in FIG. 8C, staining for BRCAI is nuclear, whereas
breast tumor cells in the same sections fail to stain at all with the same procedure.
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TABLE 2
SUBCELLULAR LOCATION OF BRCA1 IN CELL LINES, PRIMARY TUMOR CELLS
FROM MALIGNANT PLEURAL EFFUSIONS, AND TISSUE BIOPSY SECTIONS
Tissue ortumor Cases (n) BRCAI Location BRCAl
of origin Nucleus Cytoplasm Both Absent
Established lines
Normal fibroblast 2 2 0 0 0
Renal epithelium 1 1 0 0 0
Bladder carcinoma 3 3 0 0 0
Cervical carcinoma 2 2 0 0 0
Leukemiaorlymphoma 4 4 0 0 0
Osteosarcoma 2 2 0 0 0
Prostate carcinoma 1 l 0 0 0
Rhabdomyosarcoma 2 2 0 0 0
Breast epithelium 1 1 0 0 0
Breast adenocarcinoma 18 1 15 2 0
Ovarian carcinoma 3 l 2 0 0
Malignant e~usions
Breast adenocarcinoma 17 0 17 0 0
Ovariancarcinoma 8 0 8 0 0
Leukemiaorlymphoma 2 2 0 0 0
Fixed tissue sections
Infiltrating Iymphocytes 50 50 0 0 0
Breastcarcinoma 50 8 6 34 2
The BRCA I amino acid sequence does not have typical bipartite nuclear
localization signals (NLSs) (Miki et al., 1994), but does contain at least two other
putative NLSs (Boulikas. 1994). These signals, NKLKRKRRP (SEQ ID NO:S), amino
acids 419 to 427; and NRLRRKS (SEQ ID NO:6), amino acids 609 to 615) are similar to
sequences found in estrogen. progesterone, and other steroid hormone receptor molecules
(Boulikas, 1994; Arriza e~ al . 1987; Danielsen el al. . 1986; Green et al, 1986; Kastner el
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al., 1990; Laudet et al., 1991). To be activated and to redistribute from a primarily
cytoplasmic location to the nucleus, steroid hormone receptors require binding to their
ligands. conforrnationai changes, and perhaps dimerization (Forrnan and Samuels, 1990;
- Jensen, 1991). BRCA1 may norrnally localize to the nucleus in a similar manner, by
S dissociation from proteins that anchor it in the cytoplasm, as a passenger with other
nuclear proteins, or aRer modification to expose its own potential NLS. Similar transport
mech~ni~m~ have been demonstrated for other transcription factors including SV40 large
T antigen and c-Fos (Schneider et al., 1988; Roux et al., 1990; Moll et al., 1991). The
mutations of molecules involved in the pathway of BRCA1 transport from its site of
synthesis to sites of action in the nucleus may be alternative ways to inactivate the same
crucial protein in many sporadic breast cancers.
5.3 EXAMPLE 3 __SEQUENCES OF BRCA1 INTERACT WITH lMPORTIN-a SUBUNIT
OF THE NUCLEAR TRANSPORT SIGNAL RECEPTOR
The BRCAl gene product is a nuclear phosphoprotein that is aberrantly localized
in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential
mech~ni.cm for the nuclear transport of BRCA1 protein, three regions of highly charged,
basic residues 503KRKRRPs08, (SEQ ID NO:7) 606PKKNRLRRKS651,(SEQ ID NO:8)
and 651KKKKYN656 (SEQ ID NO:9) were identified as potential nuclear localizationsignals (NLSs). These three regions were subse~uently mutated to 503KLP3 jo8,
607KLS6~5, and 6'1KLA6j6. respectively. Wild-type and mutated proteins were tagged
with the flag epitope, expressed in human DU145 cells~ and detected with the M2
monoclonal antibody. In DU14~ cells the KLP mutant completely fails to localize in
nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear
2S localization. The KLN protein is always located in nuclei.
Consistently, hSRPla (importin-a), a component of the NLS receptor complex.
was identified in a yeast two-hybrid screen using BRCA I as the bait. The specificity of'
the interaction between BRCAI and importin-a was further demonstrated by showingthat the '03KRKRRP'~'~ (SEQ ID NO:7) and 606PKKNRLRRKS6'l (SEQ ID NO:8)
regions, but not 6'1KKKKYN6'6 (SEQ ID NO:9), are critical for this interaction. To
determine if the cytoplasmic mislocation of endogenous BRCA I in breast cancer cells is
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due to a deFlciency of the cells, wild-type BRCAI protein tagged with the flag epitope
was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that,
in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of
the construct in four non-breast cancer cell lines resulted in nuclear localization. These
data support the possibility that the mislocation of the BRCAl protein in breast cancer
cells may be due to defect in the cellular m~rhinery involved in the NLS
receptor-mediated pathway of nuclear import.
5.3.1 EXPERIMENTAL PROCEDURES
5.3.1.1 CELL CULTURE AND DNA TRANSFECTIONS
Human cell lines DUl45 (prostate cancer), T24 (bladder cancer), T47D, ZR75,
MB231, MB468, MDA330~ MCF7 (breast cancer), HBLI00 (normal breast epithelial
cells immortalized with SV40), and CVI (monkey kidney cell line) were grown at 37 ~C
in a humidified 10% C02-cont~inin~ atmosphere in Dulbecce's modified Eagle's medium
(DMEM; Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf
serum (Hyclone Laboratories, Inc.) on plastic surfaces. Each 10-cm dish of cells grown
to 60% confluency was transfected with 10 ~lg of plasmid DNA using the calcium
phosphate method (Kingston, 1994). The calcium phosphate precipitate was left in the
culture medium for 6-8 h. At that time the medium was drained, and the cells were refed
with fresh medium.
5.3.1.2 NLS MUTACENESIS
To introduce mutations into the three putative nuclear localization sequences ofBRCAl, a PCRTM strategy was used. Briefly, the following external primers and internal
primers with HindIlI restriction sites (underlined, below) were used to create in-frame
deletions of each NLS sequence and the addition of a leucine residue. The external
primers used for all of the NLS mutations were 5'-
GATTTGAACACCACTGAGAAGCGTGCA-3' [745 to 771 of BRCAI cDNA] (SEQ
ID NO:4) and 5'-CTTTAAGGACCCAGGTGGGCAGAGAA-3' [2791 to 2765] (SEQ
ID NO:10). For the KLP mutation the following internal primers were used. IA: 5'-
CCTTTTAAGCTTTAATTTATTTGTGAAGGGGACGCTC-3' [1521 to 1495] (SEQ
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ID NO: 11) and 1 B 5'-~CTTAAAGCTTCCTACATCAGGCCTTCATCCTGA-3' (SEQ
ID NO:~2). For the KLS mutation the following internal primers were used. 2A
5 '-CTCCCAAGCTTAGGTGCTTTTGAATTGTGGATATTT-3 ' ~ 1830- 1806~ (SEQ ID
NO:13) and 2B 5'-CCTCCCAAGCTTTCTTCTACCAGGCATATTCATGCGC-3'
[1854 to 1879] (SEQ ID NO:14). The KLN mutation was generated with the followinginternal primers:
3A 5'-CCTCCCAAGCTTTATCTCTTCACTGCTAGAACAACT-3' [1962 to 1939]
(SEQ ID NO: 15); and
3B 5 '-CCTCCCAAGCTTAACCAAATGCCAGTCAGGCACAGC-3 ' [1978 to 2101]
(SEQ ID NO: 16).
Plasmid BSK-BRCAla that contains a full-length BRCAl cDNA (Chen e~ al.~
1996) was used as the template for PCRTM amplifications using each pair of internal and
external primers. The resulting DNA fragments were gel purified and cut with Afl~I and
HindIII for the N-terminal cDNA fragments, and with KpnI and HindIII for the
C-terminal cDNA fragments. The N- and C-terminal fragments were then used to
replaced the Af~lI/KpnI fragment in pBSK-BRCAla. Ligation ofthe HindIII site at each
of the NLS sites generated in-frame deletions and additions of CTT codons for leucine
residues. The AJlII/KpnI fragments from pBSK-BRCAI-KLP, KPS, and KLN] were
then used to replace a similar fragment in the expression vector pCEP-FlagBRCAI
(Chen e~ al., 1996) to generate pCEP-FlagBRCAI ;.LP' pCEP-FlagBRCAI KLS and
PCEP FlagBRCA I KLN
5.3.1.3 TRANSIENT EXPRESSION Ar~lD IMMUNOSTAINING
Cells were transfected with pCEP-FlagBRCAI~ LP~ pCEP-FlagBRCAl~,;LS or
pCEP-FlagBRCAl~LN for expression of the epitope- (Flag, Kodak, IBI) tagged NLS
mutated proteins and pCEP-FlagBRCA1 for Flag-tagged wild-type BRCAI. After
replating and growth on coverslips for 30 hours, the cells were fixed and indirectl~
immunostained with the M' Flag mAb (Kodak. IBI) using previously described
procedures (Mancini ~ l . 1994). The microscopic images were acquired using a
H~mm~mat.~u Color Chilled 3CCD camera attached to a Zeiss AxiophotTM fluorescence
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microscope. The image files were digitally processed for presentation using Adobe
Photoshop~).
5.3.1.4 IMMUNOPRECIPITATION AND WESTERN BLOTS
The BRCAl proteins were immunoprecipitated as described (Chen et al., 1995)
using mouse anti-BRCAl-Bgl antibodies (Chen et al., 1995). After SDS/PAGE,
epitope-tagged BRCAI protein was detected in Western blots using the anti-Flag M2
mAB (Kodak, IBI) and endogenous BRCAI was detected with the BRC,41-Bgl antibodies
(Chen et al.~ 1995).
5.3.1.5 IDENTIFICATIO~ OF BRCA1 ACTIVATION DOMAIN
The identification of an activation domain in BRCAI was done by a yeast
one-hybrid assay in Saccharomyces cerevisiae strain Yl 53, which contains a lacZreporter under the control of a promoter with GAL4-binding sites in the ~ LI~:alll
activating sequence of GALI (UASG) (Durfee etal., 1993). The BRCAl deletion
constructs in FIG. 2A and FIG. 2B were obtained by translationally fusing the
DNA-binding domain of GAL4 (Keegan et cll., 1986; Ma and PCashne, 1987) in pAS
(Durfee etal.. 1993) to cDNA fragments obtained from pBSK-BRCAla (Chen etal.,
1996a) using convenient restriction sites. ~-Galactosidase activity was deterrnined by
colony color and quantitated using chlorophenyl red ~-D-galactopyranoside in assays as
described previously (Durfee et ai., 1993).
5.3.1.6 YEAST TWO_HYBRID SCREEN
A cDNA library prepared from human B Iymphocytes was screened as described
previously (Durfee et al., 1993). The protein from pAS-BRCA3.5 (see FIG. 2A and FIG.
2B) sewed as the "bait," which consisted of amino acids 1-1142 of BRCAl fused to the
GAL4 DNA-binding domain (Keegan et cll., 1986; Ma and PCashne. 1987) in plasmid
pAS (Durfee et cll., 1993).
Interactions between the NLS of BRCAI and Importin-oc-Yeast strain Y153 was
co-transfected with pAS-BRCA3.5~ pAS-KLP, pAS-KLS, or pAS-KLN and
pACT-importin ~20-259 (see FIG. 3A) and assayed for ~-galactosidase activity as
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described (Durfee etal., 1993). For importin-a expression, a cD~A encoding aminoacids 220-529 was fused to the activation domain of GAL4 (Keegan et al., 1986; Ma and
PCashne, 1987) in pACT (Durfee etal., 1993). pAS-KLP, pAS-KLS, mad pAS-KLN
were constructed by fusing BRCAl 1-1142 cDNAs from pBSK-BRCAl-KLP, KLS, and
S KLN to the DNA-binding domain of GAL4 in pAS (Durfee et al., 1993).
~-galactosidase activity was assayed as described above.
5.3.2 RESULTS
5.3.2.1 NUCL~AR LOCALIZATION SEQUENCE IN BRCAI
To initiate the study of BRCAl nuc}ear transport, NLS motif~s] were identified.
By analysis of the amino acid sequence, three possible nuclear localization sequences
were found in BRCA 1. The three regions of highly charged, basic residues are
503-KRKRRP-508 (SEQ ID NO:7), 606-PKKNRLRRKS-615 (SEQ ID NO:8) and
651-KKKKYN-656 (SEQ ID NO:9) (FIG. 9). To deterrnine if these sequences are
functional in nuclear localization, PCRTM mutagenesis that generated in frame deletions
and addition of a leucine residue at each of the sites was perforrned. Each of the mutated
proteins was expressed in DU 145 cells as fusions containing an N-terminal Flag epitope,
(Kodak, IBI) in plasmids pCEP-FlagBRCAlKLp, pCEP-FlagBRCAlKLs and
pCEP-FlagBRCAlKLN. To demonstrate the expression of full-length tagged protein in
these studies, IP Westerns were done using anti-BRCAl and anti-Flag M2 antibodies for
precipitations and detection. A Flag-tagged BRCAI protein with the same mobility as
full-length endogenous BRCA I was expressed.
The subcellular localization of each of the mutated as well as wild-type proteins
were determined by immunostaining with the anti-Flag M2 mAB (Kodak, IBI).
Consistent with previous studies, wild-type Flag-BRC,~I 1 protein is located in the
nucleus. The 651 -KLN-656 mutation is also nuclear indicating that the residues.651 -KKKKYN-656 (SEQ ID NO:9), are not important for nuclear transport of
Flag-BRCAlKLN. In contrast. the 503-KLP-508 and 607-KI.S-615 mutations both result
in cytoplasmic localization of Flag-BRCAl~Lp and Flag-BRCAl~Ls indicating that both
of these stretches of basic residues are critical for nuclear import. During the course of
these studies, it was noted that Flag-BRCAl~Ls, when overexpressed, can, in some
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instances, localize in the nucleus. The occasional nuclear staining when this mutated
protein is overexpressed could be the result of a slightly higher affinity for the cytosolic
nuclear receptor than that observed with the KLP mutation. It is emphasized thatFlag-tagged BRCAl,jLp was never observed in the nucleus.
5.3.2.2 IDENTIFICATION OF BRCA1~ TER~CTING PROTEINS
Nuclear transport of BRCAI clearly requires interactions with other cellular
proteins. The inventors elected to use the yeast two-hybrid method to identify and clone
genes encoding BRCAI-interacting proteins. Since BRCAl has been proposed to be atranscription factor (Miki etal.~ 1994), it may therefore have transactivation activity.
The presence of such activity would confound a two-hybrid assay. To functionallyidentify potential transactivation domains in BRCAI, various domains of BRCAI
protein were fused in-frame with the DNA-binding domain of GAL4 (FIG. 2A and FIG.
2B) in plasmid pAS (Durfee et al., 1993). If these fusion proteins contain an activation
domain, they will activate the GAL4 UASG-responsive ,B-galactosidase reporter (Durfee
et al., 1993) after transfection into the Y153 strain. Through this analysis the inventors
defined a strong activation domain located between amino acids 1142 and 1646 (FIG. 2A
and FIG. 2B). This activation domain was deleted in BRCA3.5 (FIG. 2A and FIG. 2B),
which only contains amino acids 1-1142 of BRCAI. BRCA3.5 was then fused to the
GAL4 DNA-binding-domain of pAS vector as the bait for screening BRCAl-interacting
proteins as described previously (Durfee et al., 1993). Four different clones were
isolated and sequenced. Then compared with GenBankTM, the inventors found that one
is novel, one has homology to an uncharacterized zinc finger domain-cont~ining protein,
and two bear sequence homology to previously cloned cDNAs (Table 3). Interestingly,
the sequence of hBRAP21 is identical to that of the nuclear localization signal receptor
hSRPla (Weis et al., 1995)~ also known as importin-a (Gorlich el al., 1994) or
karyopherin-a (Radu et al., 1995).
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TABLE3
SUMMARY OF CLONES ENCODING BRCA1-tNTERACTING PROTEINS
Clone Insert sizea ~-Galactosidase Similarity with known sequences
activity
hBRAP2 0.8 ++++ With sequence conserved in S. cerevisiae
and Caenor~abditis elegans
hBRAP12 1.2 ~++ Part of an uncharacterized zinc finger
domain-cont~ining protein
hBRAP14 1.1 ++ Novel sequence
hBRAP21 0.9 ++ Identical to the nuclear localization signal
receptor(hSRPl a/importin-(x)
alnsert size is given in kilobase pairs.
5.3.2.3 INTER~CTION OF IMPORTIN a WITH BRCAl NLS
To investigate the potential interaction of the NLS of BRCAI with
hSRPla/importin-a and to obtain additional evidence concerning the functional NLS of
BRCAI, a yeast two-hybrid assay were used. This approach takes advantage of the
ability of hSRPla/importin-a to interact with BRCAII 1142 in the yeast-two hybrid
system and activate a GAL4 UASG-responsive ~3-galactosidase gene. In this assay,pAS-BRCA3.5 with wild-type BRCAI amino acid sequence 1-1142 or the same region
containing the mutated NLS sequences, KLP, KLS and KLN, cloned into the pAS
expression vector (pAS-KLP~ pASKLS, and pASKLN) were used.
A region of importin-a from amino acid 2~0 to 529, which is known to interact
with BRCAI I 1142~ was translationally fused to the activation domain of GAL~ in pACT
(Durfee et al., 1993). ~n two-hybrid assays, expression of pAS-BRCA3.5 encoding
wild-type BRCAII II,,,, produced blue colonies and had a 100 fold increase in
~-galactosidase activity over that of the negative control. untransfected Y153 cells.
Consistent with the ability of Flag-BRCAl~;LN to translocate into the nucleus. the assay of
pAS-KLN also resulted in blue colonies and a 150 fold increase in ~-galactosidase
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activity. In agreement .~vith the inability of Flag-BRCA1KLp to be imported into the
nucleus, this mutation in BRCAl1 l~42 resulted in white colonies and no increase of
~-galactosidase activity over background. Interestingly, expression of pAS-KLS
demonstrated a ten-fold increase in ,~-galactosidase activity over background. As noted
.
earller~ thlS lncrease ln actlvlty IS conslstent wlth the lmmunostalmng data for thls
mutation that showed occasional nuclear localization when Flag-BRC~1KLs is
overexpressed.
5.3.2.4 BRCA1 CYTOPLASMIC LOCALIZATION IN BREAST CANCER CELLS
Previously, the inventors transfected an expression plasmid Cont:~ining
flag-tagged BRCAI into two breast cancer cell lines, T47D and MB468, and one
immortalized non-breast ,epithelial cell line, HBLI00. The flag-tagged BRCAI protein
was found in the cytoplasm of the T47D and MB468 cells and the nucleus of HBLI00cells by iimmunostaining with anti-flag M2 monoclonal antibody. To confirm this
observation and to verify the e~cpression of full-length flag-tagged BRCA1 protein, the
inventors repeated this study using four non-breast cancer and six breast cancer cell lines
listed in Table 4. Nuclear localiz~tion of flag-BRCAl is observed in normal monkey
kidney cells CV 1 and in DU 145, T24, and HBL 100 cells (Table 4). In contrast,
cytoplasmic localization of this protein is seen in ZR75 and MB231 and in MB468~MDA330, and MCF7 breast tumor cells (Table 4). These data suggest an altered
transport or retention system for the BRCAl protein in breast cancer cells.
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TABLE 4
SUMMARY OF FLAG BRCA1 STAINING RESULTS
Cell line Localization
Anti-BRCAIU M2
CV I Nb N
DUI45 N N
T24 N N
HBLI00 N N
T47D CC C
MB468 C C
MDA330 N, C C
MB23 1 C C
ZR75 C C
MCF7 N,C C
aChen et al. (1995); included for comparison with M2 staining.
bN, nucleus
CC, cytoplasm.
5.3.3 DISCUSSION
The identification of two regions of charged, basic amino acids between 503 and
508 and between 606 and 615 that are both crucial for efficient nuclear transport of the
BRCAl protein further supports this notion. The distance between these two motifs is
much greater than the 10 amino acids separating the bipartite sites of nucleoplasmin
(Robbins et al., 1991). The structure and function of the NLSs in BRCAI is similar to
other nuclear proteins in which two NLSs are more widely spaced such as those in the
polyoma large T antigen (l~ichardson et al., 1986)~ influenza A virus NSI protein
(Greenspan et Ql., 1988)~ and adenovirus DNA-bindhl~ protein (Morin et al., 1989).
While the inventors cannot rule out the possibility that other sequences are also required
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for translocation of BRCAI from the cytoplasrr, to the nucleus, the NLS at 503-508 iS
essential for this process.
This observation was supported by results showing that mutation of the NLS at
503-508 in BRCA1 completely abolished its interactions with importin-a. The NLS at
606-615 in BRCAl is less critical, because mutation of this NLS did not completely
~imini~h the nuclear import of BRCAl. The inventors' results indicating that BRCAI is
a nuclear protein with a functional NLS are at odds with the report indicating that the
protein is membrane-bound, and secreted (Jensen etal., 1996). Such a discrepancy is
puzling but may be explained by cross-reactivity of the peptide antisera to the epiderrnal
growth factor receptor (Wilson et al., 1996).
Using mouse polyclonal antibodies specific for the BRCAI protein~ the inventors
have consistently found BRCAI to be a 220-kDa nuclear protein that is aberrantlylocated in the cytoplasm of ad~anced breast cancer cells (Chen et al., 1995; Chen et al.,
1996b). However, Scully et al. (Scully et al., 1996) reported that the 220-k~a BRCAI
protein remains in the nucleus of some breast cancer cell lines. Although the precise
reason for this discrepancy is unclear, one cannot exclude the possibility of less specific
antibodies, potential immunostaining artifacts, or both. By ectopically expressing
epitope-tagged BRCAI protein and using the specific anti-flag M2 monoclonal antibody,
the inventors have circumvented the difficulties in obtaining highly specific antibodies
against BRCAI. Through this completely .different approach, wild-type flag-tagged
BRCA1 expressed in breast cancer cells remains in the cytoplasm. This result further
suggests that its mislocation in breast cancer cells is not due to mutations of BRCAl
itself. Rather, the aberrant localization seems to be the result of alterations in the cells,
perhaps at the level of nuclear transport Of BRCAl .
In this regard, the demonstration here that BRCAI interacts with the importin-a
subunit of the nuclear transport receptor complex could be an important clue. However,
if there is a problem with the importin-a subunit or the importin-substrate complex, why
is it manifested in breast epithelial cells? Does this indicate an unsuspected specificity of
importin-a for B~CAI? And. does the defect in the function of BRCAI reside in the
cytoplasm or the nucleus? Once translocation across the nuclear pore complex occurs,
importin-a is reported to accompany the transport s-lbstrate to its areas of nuclear
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function (Gorlich etal., 1995b). If there is a problem with BRCAI dissociating from
importin-a in the nucleus, perhaps BRCAl protein is immediately exported from the
nucleus, resulting in the appearance of cytoplasmic localization. Co-localization studies
similar to those in Gorlich et al. (Gorlich et al., 1995b) using normal and breast cancer
cells might address this possibility.
An alternative possibility is that, in breast cancer cells, there is a problem in the
regulation of the nuclear transport of BRCAI. The known mech~ni~m~ for regulating
nuclear trans-location (reviewed in Refs. 12 and 13) are as follows: (a)
phosphorylation/dephosphorylation, e.g. c-rel and vjun and cell cycle-regulated proteins
such as cyclin B-Cdk complex and pencllllin; (b) cytoplasmic retention by rrl~kin~ of the
NLSs as seen in dorsal, NF KB, the glucocorticoid receptor, and the periodicity protein;
or (c) more general regulation at the level of the nuclear pore complex. Perturbations in
the gene products in any of these regulatory systems could potentially result incytoplasmic localization of BRCAI in breast epithelial cells. The possibility that some
of the other BRCAl-interacting proteins identified in the two-hybrid screen could have
this kind of role in breast cancer cells is being investigated.
Interestingly, there are other reports of mislocation to the cytoplasm of a nuclear
tumor suppressor protein in breast and other types of cancer cells. Of 27 breast cancer
cases examined, 37% demonstrated cytoplasmic staining for pS3, which by sequencing
was revealed to be wild type (Moll et al. 1992). In another study, wild-type p53 was
found located in the cytoplasm of human cervical carcinoma cell lines with integrated
human papillomavirus-18 or - 16 (Liang et al., 1993). Both of these studies suggest that
the tumor suppressor function of normal p53 can, in some cases, be inactivated by
cytoplasmic mislocation (Moll et al. 1992; Liang et al., 1993). These data are similar to
the inventors' observations for BRCA I and seem to suggest a global alteration of
subcellular compartmentation in breast cancer cells. If this is the case~ then BRCAI and
p53 along with, perhaps. other nuclear regulatory proteins may be retained in the
cytoplasm of these cells~ the composite effect of which may contribute to their
-- tumorigenesis.
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5.4 EXAMPLE 4 -- IDENTIFICATION OF SPECIFIC PROTEINS REQUIRED FOR
CORRECT BRCA1 LOCALIZATION
Fragments of BRCA I that exclude any potential intrinsic trans-activation activity
are fused, in-frame, with the Gal4 DNA-binding domain. These are used to screen a
cDNA library, fused to the Gal4 activation domain~ using a yeast strain cont~inin~ a Gal4
responsive b-galactosidase gene. The clones are analyzed based on the following criteria.
First, whether they map to chromosomal regions known to undergo frequent LOH in
breast cancer. Second, whether large scale deletions in the genes are detected by
Southern analysis of DNA from tumor lines in which BRCA1 is mislocated. And third,
whether more subtle mutations in these genes can be detected in the tumor lines by RT-
PCRTM-based single-strand conformational polymorphism (RT-PCR~M/SSCP) assay.
Genes in which mutations are found are strong candidates for the gene responsible for
translocation of BRCAI to the nucleus.
First, the pattems of proteins that co-immunoprecipitate with either wild-type
BRCAI or mutant proteins lacking the NL~ motif are compared. Second, proteins that
co-immunoprecipitate with wild-type BRCAI in extracts from norrnal cells, but not from
extracts of breast tumor lines, are compared. The genes encoding these candidatemediators of BRCAl nuclear transport are then be cloned by standard procedures.
To confirm the specificity of interaction and further define the region of BRCAIto which the identified proteins bind, in vitro binding and in vivo co-immunoprecipitation
assays are performed using wild-type BRCAl, NLS-deficient mutants, and deletion
mutants encompassing other regions of BRCAI .
5.5 EXAMPLE 5 -- EXPRESSION OF BAPS RESCUE TRANSPORT OF ENDOGENOUS OR
ECTOPICALLY E,YPRESSED BRCA1 INTO THE NUCLEUS OF BREAST TUMOR CELLS
To determine if the genes identified in aim 2 can complement the defect in
BRCAl localization in breast cancer cells, it is necessary to clone full-length cDNAs. In
addition, antibodies are raised against the protein products to permit their detection. If
mutations in the identified ~~ene(s) are responsible for tl~e mislocalization of BRCAI in
the tumor lines, then these antibodies are also used in screening tumor samples.
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The cDNAs identified are expressed in breast cancer cell lines under the cont;olof a tetracycline-inducible promoter, so that expression is regulated by the presence of
tetracycline in the culture medium. This avoids potentially toxic effects due to the
expression of these proteins. The effect of their expression on the localization of
endogenous and/or exogenous wild-type BRCAI is monitored by immunofluorescence
and cell fractionation assays.
5.6 EXAMPLE 6 -- DEFECTIVE TR~NSPORTATION IS A COMMON CAUSE OF
BRCA1 MISLOCALI~ATION IN ADVANCED BREAST TUMOR CELLS
Wild-type BRCAl may be expressed in norrnal cells and breast tumor lines.
When the protein fails to move into the nucleus in breast tumor cells, this suggests that
the advanced breast tumor cells have a defective transportation process for BRCAI rather
than mutations in BRCAI itself.
This exarnple is designed to confirm that a defective nuclear transport system is a
common cause of the mislocation of BRCAI protein in advanced breast cancer cells.
This is done by analyzing BRCAI localization in 15 additional cell lines in which
BRCAI is mislocated. Initially, transiently transfected cells expressing these constructs
are analyzed for protein localization by indirect immunofluorescence staining with the
anti-FLAG antibody. Subsequently, the localization can be confirrned by cellularfractionation coupled with western blotting analysis to detect the exogenous protein by
SDS-PAGE in stably transfected lines~ as previously described (Chen, et al., 1995). As
described above, the nuclear-matrix associated protein, p84(N5) is used as a control for
nuclear transport function. Immortalized human breast epithelial cells (HBL100), and
CV I cells can be used as controls for normal BRCA1 localization.
Tagged BRCAI proteins with deletions in each of the three NLS motifs may be
constructed and expressed in normal cells, so as to identify the motif required for
transportation of BRCAI into the nucleus. If the three putative NLS motifs are not
responsible for the transportation~ a series of systematic deletion mutants can be
generated and similarly tested to define the functional NLS motif. Once defined, the
NLS mutants can be used as tools to screen the BRCAI-associated proteins identified in
aim 2 to deterrnine if any interact with the NLS.
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To determine which NLS of BRCAl is important for nuclear import, the
inventors have constructed 3 BRCAI deletion mutants, each specifically removing one
of the putative NLS motifs (amino acids: 500-508, 606-614, and 650-655). These have
been cloned, in-frame with the FLAG epitope, into the pCEP4 vector (FIG. 9).
The mutant proteins can be transiently expressed in normal cells, and their
localization determined by indirect-immunofluorescence using the anti-FLAG antibody
M2 (Kodak). It is possible that any one of the NLS motifs can promote BRCAI
translocation to the nucleus. Thus, double and triple deletion mutants are also
constructed to test this possibility. From previous experience in deterrnining the NLS of
the Rb-associated protein, mitosin (Zhu, et al., 1995), it is clear that the rules for
determining functional NLS motifs are not absolute, and it may be necessary to broaden
the spectrum of potential NLS candidate sequences. Should this be the case, a series of
systematic BRCAI deletion mutants can be generated and tested for their ability to
translocate to the nucleus. These studies are useful in identifying residues crucial for
nuclear transport of BRCA 1.
5.7 EXAMPLE 7 -- SPECIFIC BAPS ARE REQUIRED FOR
CORRECrBRCA1 LOCALIZATION
The yeast two-hybrid system has been very successful in isolating a total of 25
Rb-associated proteins (Durfee ~t al., 1993). An advantage of this method is that it is
able to detect fairly weak interactions and is more sensitive than co-immunoprecipitation
assays that depend on efficiency of metabolic labeling, affinity of the
immunoprecipitating antibody, and the half-life of the complex assayed.
Two screens for BRCA1-associated proteins have been initiated. As mentioned
above, it has been suggested that BRCAI is a transcription factor. This is based on the
presence of a putative DNA-binding, Zn-finger domain in the N-terminal region of the
protein, three putative NLS motifs, and a potential acidic trans-activation domain
towards the C-terminus. The nature of the yeast two-hybrid assay requires that the bait
protein does not contain intrinsic trans-activation activity. Thus. the inventors first
screened a series of BRCAI deletion mutants to identify regions that contain such
activity. As shown in ~IG. 10. a region of about 600 amino acids towards the C-
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terminus contains strong trans-activation activity. Therefore, the inventors have used
fragments of BRCAI not encoding this region as baits to identify associated proteins.
Two BRCAl expression plasmids have been constructed: pAS-BRCA2.5 (amino
- acids 303-1142) and pAS-BRCA3.5 (amino acids 1-1142) (FIG. Il). These create
hybrid molecules between sequences for the DNA-binding domain of the yeast
transcription factor Gal4 (amino acids 1-147; (Keegan et al., 1986)) and portions of the
BRCAI protein. These constructs have been used to screen a cDNA library from human
B-lymphocytes, which was cloned into a second expression plasmid Cont~3ining
sequences for the Gal4 activation domain II ~amino acids 768-881; (Ma and Ptashne,
1987)). An additional screen using a mouse embryonic cDNA library is currently in
progress. As first demonstrated with Gal4-Gal80 interactions (Ma and Ptashne, 1988)
and later generalized (Fields and Song, 1989), if the two proteins expressed in yeast are
able to interact, the resulting complex can activate transcription from promoters
cont~ininE Gal4-binding sites derived from the upstream activating sequence of the Gall
gene. Previously, the inventors successfully identified several important proteins that
interact with the retinoblastoma protein T antigen-binding domain through this method
(Durfee et al., 1993). This method was then quickly spread to the entire community as a
powerful tool for identifying novel protein-protein interactions.
In the first screen, a total of four clones showing high ~3-galactosidase activity
were selected. These 4 clones have been further characterized (Table 5). One of these
clones (hBRAP21) is identical, over the 400 bp region sequenced~ to the human gene
hS~Pla, which was recently identified as a human homologue of a Xenopus protein,importin (Gorlich et al., 1994; Weis et a/., 1995). Importin/hSRPla functions in NLS
recognition and cooperates with other nuclear import proteins, such as Ran/TC4, to
promote the binding of NLS containing peptides to the nuclear pore complex, where they
are then translocated across the nuclear envelope (Weis et al., 1995). Interestingly, there
- appear to be at least 5 forrns of importin - all closely related~ differin~ by only a few
amino acids (Gorlich et a/., 1994). The sequence is identical to the major form, at least
over the 400-bp region sequenced. ~lowever, subtle differences may be apparent in the
region not yet sequenced. The presence of several closely related importin molecules
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raises the possibility that each may be involved in the nuclear transport of specific
subsets of proteins (Gorlich et al., 1994).
TABLE 5
5CLONES ISOLATED USING THE YEAST TWO HYBRID SYSTEM
Clone CPRG cDNA Repeat- Similarity with Know Sequences
Activity size (kb) Assay
Activity
hBRAP2 3000-6000 0.8 ++++ Identical to a sequence conserved
hBRAP12 790-850 1.2 +++ in S. cerevisiae and C. elegans
Part of an uncharacterized zinc-finger
hBRAP14 220-240 1.1 ++ Novel sequence
hBRAP21 150-160 0.9 ++ Similar to the Nuclear Localization
Signal Receptor (hSRP l a/importin)
The analysis of BRCAI-associated proteins by co-immunoprecipitation provides
a useful complement to the yeast two-hybrid screen described above. This method has
two significant advantages over the yeast two-hybrid system. First, it is not restricted by
the presence of a potential trans-activation domain in BRCAI . Thus, protein interactions
with full-length BRCAl can be addressed. This is important, since although defects in
proteins that interact with the NLS of BRCAI are prime suspects for the mislocation of
BRCAI in advanced breast cancers, it is equally likely that proteins interacting with
other regions of BRCA I are important in this process, perhaps by causing
conformational changes in BRCAI to reveal an otherwise hidden NLS motif.
Thus, proteins that interact with other regions of BRCAI, not present in the yeast
two-hybrid screen, could be critical for the nuclear transport of BRCAI. For example, a
protein that tethers BRCAI in the cytoplasm through phosphorylation, similar to the
action of PHO80-PHO85-CDK on PHO4 (O'Neill et al., 1996), or by directly m~.~king
the NLS, similar to the action of IkB on NF-kB (Ghosh and Baltimore, 1990), need not
necessarily bind to the NLS of BRCAl. The second advantage of this method is that it
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provides a more directed search for proteins involved in BRCAl transport than does the
yeast two-hybrid screen.
At least five proteins with molecular weights of: 40, 41, 49, 95, and 140 kDa are
specifically co-immunoprecipitated with BRCAl in HBI,I00 cells. Further analysisincludes the following: First, proteins that co-immunoprecipitate with wild-type BRCAI
in normal cells and breast tumor cells may be compared. This should identify interacting
proteins that are potentially involved in BRCAI import and which may be defective in
cancer cells. Second, differences in the co-immunoprecipitated proteins between the
NLS mutants and wild-type BRCAI in normal cells are determined. This is done to
identify proteins that specifically interact with the NLS of BRCAI. Taken together,
these results permit the identification of proteins that are critical for BRCA1 nuclear-
import. To ensure efficient co-immunoprecipitation~ therefore, the inventors over-
express the FLAG-tagged BRCAI protein in the cells by transfection. To avoid anypotentially toxic effect that this expression may have, the tetracycline-inducible system
can be used. The FLAG-tag provides a convenient handle to mark exogenous protein,
since it is located at the N-terminus of the protein and is thus less likely to induce steric
hindrance between BRCAI and any interacting proteins. In addition, the high affinity
monoclonal antibody against FLAG (M2, Kodak) is highly specific, thus reducing
problems of cont~min~tion of the co-precipitate with unrelated, cross-reacting proteins.
The genes encoding the identified proteins may be cloned according to
procedures previously established in the laboratory for cloning Rb-associated proteins
(Qian e~ al., 1993). Briefly, sufficient protein are purified by co-immunoprecipitation to
permit peptide sequencing to be done. A comparison of the obtained peptide sequences
with the GenBank database reveals if the genes are novel and may provide additional
clues as to their function. Novel genes are cloned by screening a cDNA library with
degenerate oligonucleotides based on the peptide sequences obtained. The identity of the
cloned genes and the co-immunoprecipitated proteins are confirmed in several ways.
First, the SDS-PAGE mobility of the co-immunoprecipitated protein(s) and the in vitro
transcribed/tr~n~l~tccl product of the cloned cDNA(s) are compared. Second, antibodies
against the cloned gene(s) are developed and tested for their ability to recognize the
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BRCA1-associated proteins in immunoprecipitation/western studies. Third, the in vitro
and in vivo interaction of the cloned genes with BRCAl are performed.
5.8 EXAMPLE 8 -- GENERATION OF TRANSGENIC MICE CARRYING THE
~OXP-FLANKED BRCAI GENE
This strain was constructed using conventional gene targeting techniques (Lee etal., 1992; Liu et al., 1996). A targeting plasmid was constructed using an 8.0 kb
HindIII-BamHI DNA fragment containing Brcal [exons 9, 10, and part of 11]. l~irst, a
single loxP site derived from pGEM-30 (Gu et a/.? 1993) was inserted into a unique SacI
site within intron 9 using EcoRI-linkers. A second set of-loxP sites in conjunction with a
neomycin resistance cassette derived from P12-neor [obtained from H. Gu, NH-I/NIAID~
was then inserted into a unique X7~oI site within intron 10. The resulting plasmid was
designated Brcal-loxP.
A pMC I -tk cassette was placed at the 5'end of the construct to generate the final
targeting vector: Brcal -loxP ko. The construct was linearized by BamHI and
individually transfected into ES cells [an early passage of E14-1 (Handyside et al.,
1989)], as described previously (Lee et al., 1992; Liu et al., 1996). Colonies doubly
resistant to G418 and FIAU were isolated from which the DNA was analyzed by
Southern blotting to identify clones cont~ining a site specific integration of the
Brcal-floxP gene resulting from homologous recombination.
The presence of the Neo cassette in intron 10 could disrupt Brcal expression by
interfering with RNA processing. To elimin~te this possibility, targeted ES cells are
transfected with pIC-Cre (Sternberg et al., 1981) to initiate the transient expression of
Cre recombinase. Under these conditions, it is expected that Cre-mediated excision will
only occur once in some cells (Gu et al., 1994). Since there are three possible
recombination events, a screen of 100 clones subsequent to transfection is used to
generate the desired recombinant that has deleted the Neo cassette but not exon 10.
ES cells with the desired placement of loxP sites are independently injected into
C57BL/6 blastocysts, which are implanted into the uteri of pseudo-pregnant F~ [CBA X
C57BL/6] female foster mice to generate chimeras. Germline transmission are tested by
back crossing chimeric male mice with C57BL/6 females. Because the loxP sites are
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inserted into intron sequences, they are not expected to affect endogenous Brcal gene
function (Gu et al., 1994). Germline chimeras are used to establish Brcal-loxP
heterozygous and homozygous transgenic lines.
5.9 EXAMPLE 9 -- ANALYSIS OF EFFECTS OF ~RCAI DELETION ON THE
DEVELOPMENT AND FUNCTION OF THE MAMMARY GLAND
The ability to conditionally and temporally initiate the excision of the Brcal gene
in the breast epithelial cells of mice will present a unique opportunity to investigate the
role of this gene. In virgin females, transgenes driven by the WAP promoter can be
activated cyclically during estrus in about 30~/O of the secretory alveolar structures
(Robinson et al., 1995). With the onset of pregnancy, the number of alveoli recruited for
differentiation and activation of the WAP transgenes begins to increase around day 15
reaching maximum during lactation (Robinson e~ al., 1995). As discussed above, by
regulating the expression the rtTA with the WAP promoter, it should be possible to
initiate the homozygous excision of the Brcal gene by treatment of the females with
doxycycline during estrus or anytime after mid-pregnancy. Subse4uent to doxycycline
treatment, the inventors will conduct a detailed and comprehensive histological
ex~rnin~tion of m~mm~ry glands of control and treated animals. In addition, the
inventors plan to include an ex~min~tion of differentiation markers such as endogenous
milk protein gene expression.
One concern about this approach is asynchrony in the development of alveolar
secretory units as monitored by WAP expression (Robinson et al., 1995). For example,
if the Brcal gene is deleted in most of the epithelial cells of only 30~~O of the secretory
units during estrus [assuming high efficiency of Cre expression and excision], how
would this effect the development of the total gland? Obviously, it is very difficult to
predict the outcome which will have to be ascertained experimentally.
There is also a concern about the fate of the Brcal~/~ cells in subsequent diestrus
and proestrus although there is evidence for their persistence as non-expressing, but
differentiated cells [(Robinson et al., 1995) and references therein]. As the recruitment
of secretory subunits increases during pregnancy, this becomes less of a concernalthough it is completely unknown what the fate of Brcal~l~ cells will be during
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involution after weaning, should the glands progress to that stage, or for that matter, what
the effect of no functional Brcal protein will be on this process.
5.10 EXAMPLE 10 -- GENERATION OF DOUBLE TRANSGENIC MICE CARRYING
BRCA1-LOXP AND THE TETR~CYCLINE-INDUCIBLE CRE RECOMBINASE
Homozygous transgenic lines with high expression of Cre recombinase, as
described above, are mated with homozygous Brcal-loxP mice. This initially generates
obligate heterozygotes for both WPA-rtTA;Cre and Brcal-loxP. These F~ heterozygotes
are then be bred to generate an F2 population in which I out of 16 mice is expected to be
homozygous for both alleles. Breeding these animals generates obligate
WPA-rtTA;Cre/Brcal-loxP homozygotes in the F3 generation that may then be used for
the targeted-excision studies.
Doxycycline is given to female mice in their drinking water [or. alternatively,
through sub cutaneous slow release pellets, Innovative Research of America] to induce
expression of the Cre recombinase. Initially, four time points are used for the
~mini~tration of doxycycline; estrus, late pregnancy [days 15-18 ], and day 3 oflactation. Previous studies have shown that the WAP promoter is capable of activating
reporter gene expression at these time points. Thus, the inventors expect rtTA expression
to be induced at these times (Robinson et al., 1995). With doxycycline treatment, rtTA
should activate expression of the Cre recombinase, which in turn will catalyze the
targeted excision of Brcal exon 10. Negative controls are female littermates that have
not been treated with doxycycline. To confirm that Brcal exon 10 is deleted following
induction of Cre, histological sections are made from the m~rnm~ry glands of mice from
each of the treatment time points. These are then examined for Brcal by standardimmunohistochemical methods using BRCAI antibodies that specifically recognize the
C-terminal region of exon 11 (Chen e~ al., 1 996b).
Alternatively, microdissection followed by PCRT~ analysis as described
previously (Liu et al., 1996) may be used to screen directly for genomic deletion of exon
10. Having confirmed that the excision event can be induced, groups of mice are
followed for the development of m~mms~ry glands and the genesis of tumors. as outlined
below.
.. . .
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Subsequent to treatment of WPArtTA;Cre/Brcal-loxP females with doxycycline~
breast is prepared for e~min~tion as follows. In 4 month virgins either untreated or
treated with doxycycline during estrus, the tissues [eight breasts per animal] are collected
for analysis according to the schedule shown. In pregnant Anim~ doxycycline
S treatment is ~lministf~red beginning on day 14 and tissues collected according to a
prescribed schedule.
Using standard histological procedures (Liu ef al., 1996), serial sections of four
breasts per animal are analyzed for abnormal gland development [e.g., in the ductal tree
or alveolar structures], and for an overall increase or decrease in the number of secretory
units compared to controls. To confirm the deletion of the Brcal gene, microdissection
PCRTM is done on individual alveolar secretory units (Liu et al, 1996). Two breasts per
animal serve for this analysis. If deletion of Brcal perturbs mammary gland
development, then the inventors might expect that abnormalities of m~mm~ry epithelial
cells will be revealed by histocytochemistry studies. Markers of differentiationcommonly used are milk protein genes, specifically for the inventors' purposes [~-casein
and WDNMl [early pregnancy], and whey acidic protein and a-lactalbumin [later near
the end of gestation] (Robinson et al., 1995). Two breasts per animal serve as a source of
tissue for either immunohistochemistry studies [antibodies can be obtained from L.
Hennighausen, NIH-NIDDK], or in situ hybridization.
5.11 EXAMPLE 11 -- ANALYSIS OF TUMOR DEVELOPMENT
Although the inventors predict that females treated with doxycycline will develop
breast tumors, the time course of tumor formation is not predictable. Once the
experimental animals are treated with doxycycline, both these animals and the untreated
controls will be routinely examined for breast tumors. If tumors develop~ the tissues may
be examined histologically. Microdissection PCRTM (Liu et al.~ 1996) and
immunohistochemistry with BRCAl antibodies are used (Chen et al., 1996b) to verify
that the tumors are derived from cells in which Brcal was deleted. The penetrance of
tumor formation can be established by determining the percentage of treated animals that
develop tumors compared to untreated controls. To be a useful model the penetrance
needs to be high, and, ideally, tumor formation should occur at a predictable time
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subsequent to Brcal disruption. It is also important to compare the nurnber of tumor foci
to the frequency of induced homozygous deletion.
Once the occurrence of tumors has been characterized, the dynamics of tumor
development may be determined by sacrificing mice for histological e~min~tion ofbreasts over several time-points, from the initial deletion event to the time at which overt
tumors are ~etect~ble. In this way, it should be possible to document tumor formation
from its earliest stages until the last stages of m~lign:~ncy, including metastasis. This
approach was recently used successfully in documenting the dynamics of melanotrophic
tumor formation in Rb+/- mice (Nikitin and Lee, 1996).
5.12 EXAMPLE 12 -- INTERACTIONS OF BRCA1 WITH HBRApl2
In yeast two-hybrid screens using with BRCA1 (minus its activation domain) as
the bait, the inventors have identified four cDNAs that encode putative BRCAl-
interacting proteins (Table 6).
TABLE 6
SUMMARY OF CLONES ENCODING BRCAI INTERACTING PROTEINS
Clone Insert Size (kb) ,B-Galactosidase Similarity with Known
Activity Sequences
hBRAP2 0.8 ++++ Sequence conserved in S.
cerevisiae and C. elegans
hBRAP12 1.2 +++ Part of a zinc-fmger domain-
containing protein
hBRAP14 1.1 ++ None known
hBRAP2 1 0.9 ++ Nuclear Localization Signal
Receptor (hSRP 1 a/importin
~)
The primary amino acid sequence of the hBRAP12 protein is shown in SEQ ID
NO:I. Notably, it contains eight zinc fingers of the C2H2 class that are highly
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homologous to each other but are not homologous to any other proteins, including known
zinc finger domains in the GenBank.
To determine the subcellular localization of hBRAP12, a full-length cDNA was
translationally fused to the green fluorescent protein. The subcellular localization of the
fusion protein is nuclear in CV1 and HBLI00 cells.
The fact that hBRAP12 is a nuclear protein is consistent with an interaction with
BRCAl. In vitro interactions may be be confirmed by GST pull-down assays as
previously applied for E2F-retinoblastoma protein interactions (Shan et al., 1992). In
vivo interactions between BRCA 1 and hBRAP 12 is ascertained using reciprocal
immunoprecipitation combined with Western blotting (Shan et al., 1992).
The interaction of hBRAP12 with BRCA1 may be assessed at two levels. First,
their capacity to associate in vitro is determined by assaying the ability of GST-
hBRAP12 bound to glutathione-agarose beads, to bind 35S-methionine labeled, in vitro
translated BRCAl or baculovirus-made BRCAl (Chen et al., 1996b). A negative control
is GST alone. The converse study using different fragments of GST-BRCAl fusion
proteins bound to glutathione beads (Chen et al., 1996b) and in vitro translatedhBRAP12 is also done as previously described (Shan et al., 1992). Second~ the in vivo
interaction of hBRAP12 and BRCAl is tested by co-immunoprecipitation assays of
whole cell extracts (Shan et al., 1992) using antibodies generated against hBRAP12 and
those available for BRCAl (Chen et al., 1995). Highly specific monoclonal antibodies
for BRCAl raised against two different epitopes in exon 11 of BRCAl may also be used
in in vivo and in vitro binding assays. Mouse antisera against hBR~P12 has been
generated using GST-hBRAP 12 fusion proteins expressed in E. coli as previously
described (Chen et al., 1995). Before use, the hBRAP12 antiserum is preabsorbed using
GST-glutathionine beads (Chen et al., 1995). The co-immunoprecipitation assays are
performed either using cells that co-express sufficient quantities of endogenous proteins~
or using cells co-transfected with expression vectors for either tagged (flag) or untagged
BRCA I and hBRAP 12.
To accomplish this objective, a series of hBRAP12 deletion mutants including theN- and C-terminus and zinc-finger domains may be generated and tested for binding to
full-length BRCAl, as described above. The inventors' initial hypothesis is that~ in
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addition to DNA-binding, the zinc finger domain will also be important for the BRCAI
interaction. In any event, these studies should be effective in determining the region of
hBRAP12 required for binding to BRCAl.
Using a strategy similar to that described in the previous paragraph, deletion
mutants of BRCAI may be assayed for their ability to interact with GST-hBRAP12 in an
in vitro binding assay, as described above. The RING-finger domain of BRCA1 appears
to be important for this interaction, and GST-fusions have been prepared for either the
wild-type N-terminal region of BRCAI or the same region with a single point mutation
(T to G substitution) which results in a Cys61 to Gly found in a familial breast cancer
case (Johannsson, et al, 1996). This mutation disrupts the RING-finger domain ofBRCAI and, if its involved in protein-protein interactions, should be negative for
interactions with hBRAP 12.
If hBRAP12 is a DNA-binding transcription factor, it should recognize a specificDNA sequence. To identify this sequence, one may use the method of random sequence
selection and PCRTM (Perkins et al., 1991; Blackwell and Weintraub, 1990) to define the
consensus DNA binding site for BRCA1. Importantly, the identification of the cognate
binding site for hBRAP12 permits functional testing of hBRAP12 activator function as
described. The identification of the cognate binding site is also important for the
identification of the downstream target genes for hBRAP 12.
Complementary DNAs encoding hBRAP12, hBRAP12-Z8 (the Zn finger domain
alone) ~Zn-hBRAP12 (minus the Zn finger), were translationally fused to GST using the
pGEX-3X vector. The cDNAs for these constructions were generated using standard
PCRTM and cloning methodologies. After expression in E. coli, the bacterial Iysates were
incubated with glutathione agarose beads and washed extensively to remove non-specific
binding proteins. After quantification of GST-hBRAP12, GST-hBRAPl2-Z8 and ~Zn-
hBRAP 12 protein-binding, the respective beads were used to screen the random
sequence DNA library.
For the selection and amplification of hBRAP12 binding sites, 10 llg of random
oligonucleotides is incubated with beads bound with ten llg of GST-~Zn-hBRAP12
protein in a binding buffer containing ZnS04 and 100 ~lg/ml of tRNA as a non-specific
competitor. The purpose of this first incubation is to remove DNA that binds non-
... . .. . . . .
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specifically. Next, either GST-hBRAP or GST-hBRAPI2Z8 is incubated with the pre-bound random DNA library. After extensive washing with low salt buffer~ the DNA is
eluted with high salt buffer (1.0 M NaCI) and precipitated with ethanol in the presence of
tRNA carrier. The recovered DNA will then be subjected to PCRTM amplification and
the binding process repeated four times. After the fifth round of selection and PCRTM,
the DNA is cleaved with BamHI and HindIII, purified on 15% acrylamide gels and
ligated into pBSK vector (Stratagene). Random colonies are picked, and
minipreparations of plasmid DNA sequenced according to standard procedures. Fromthis sequence analysis, consensus binding sequences may be identified. The progress of
the selection will be monitored using gel shift analysis (Shan et al., 1992). To do this,
the PCRTM reaction is performed with a primer end-labeled with 32p-~ATP using T4polynucleotide kinase. Specific binding by the consensus binding site is determined by
gel shift analyses using specific and non-specific competitor oligonucleotides (Shan et
a/., 1992), and/or DNAaseI footprinting (Cao et al., 1988) with wild-type hBRAP12 or
with ~Zn-hBRAP 12 proteins. An alternative to affinity purification. is to use
electrophoretic mobility shifts to isolate the DNA fragments (Kinzler and Vogelstein,
1989; Caubin et al., 1994) which is amplified by PCRTM and then selected and amplified
four additional times. The resulting fr~Ements are digested and ligated into pBSK, as
described above.
. .
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TABLE 7
SUMMARY OF BRCAl LOCALIZATION RESULTS BY DIFFERENT METHODS
Localization
Cell line anti-BRCA1 flag-BRCA1 GFP-BRCA1 mAb 17F8
Non breast cancer cell lines
CVI N N N N
DU 145 N N N/D N
T24 N N N N
Saos-2 N N/D N N
5637 N N/D N/D N
HBLI00 N N N N
Breast cancer cell lines
T47D C C C C
MB468 C C C C
MDA330 N,C* C N/D N/D
MB231 C C C C
ZR75 C C N/D C
MCF7 N,C* C C C
MB435S C N/D C C
MB415 C N/D C C
SKBR-3 C N/D N/D C
MB 175-7 C N/D C C
Hs578T C N/D N/D C
MB361 C N/D C C
BT483 C N/D C C
BT20 C N/D N/D C
N: nucleus C: cytoplasm N/D: not done
*: small portion of cells shows nuclear localization
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5.13 EXAMPLE 13 -- AMINO ACID SEQUENCE OF THE BRCA1-ASSOCIATED PROTEIN
DERIVED FROM HBR~P12
A full length cDNA for hBRAP12 was isolated from a human fibroblast library
using the panial cDNA from a yeast two-hybrid screen as the hybridization probe. The 8
zinc fingers begin at residue 208 and end at 431. The Cys and His residues in each finger
are in bold.
hBRAP Primary Sequence (SEQ ID NO:1)
MIQAQESITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASK
PDALFKLEQGEQPWTIEDGIHSGACSDIWKVDHVLERLQSESLVNRRKPCHEH
0 DAFENIVHCSKSQFLLGQNHDIFDLRGKSLKSNLTLVNQSKGYEIKNSVEFTG
NGDSFLHANHERLHTAIKFPASQKLISTKSQFISPKHQKTRKLEKHHVCSECG
KAFIKKSWLTDHQVMHTGEKPHRCSLCEKAFSRKFMLTEHQRTHTGEKPYECP
ECGKAFLKKSRLNIHQKTHTGEKPYICSECGKGFIQKGNLIVHQRIHTGEKPY
ICNECGKGFIQKTCLIAHQRFHTGKTPFVCSECGKSCSQKSGLIKHQRIHTGE
KPFECSECGKAFSTKQKLIVHQRTHTGERPYGCNECGKAFAYMSCLVKHKRIH
TREKQEAAKVENPPAERHSSLHTSDVMQEKNSANGATTQVPSVAPQTSLNISG
LLANRNWLVGQPWRCAASGDNSGFAQDRNLVNAVNVVVPSVINYVLFYVTE
NP
. ~ ,
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All of the compositions and methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present disclosure. While the
compositions and methods of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that variations may be applied
to the composition, methods and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and scope of the invention.
More specifically, it will be apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described herein while the same
or similar results would be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the spirit, scope and concept
of the invention as defined by the appended claims. Accordingly, the exclusive rights
sought to be patented are as described in the claims below.
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7. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Board of Regents, The University of Texas
System
(B) STREET: 201 W. 7th
(C) CITY: Austin
(D) STATE: Texas
(E) COUNTRY: U.S.
(F) POSTAL CODE (ZIP): 78701
(G) TELEPHONE: (512) 418-3000
(H) TELEFAX: (512) 474-7577
(ii) TITLE OF INVENTION: BRCAl COMPOSITIONS AND METHODS FOR THE
DIAGNOSIS AND TREATMENT OF BREAST CANCER
(iii) NUMBER OF SEQUENCES: 16
(iv) COMPVTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 532 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Ile Gln Ala Gln Glu Ser Ile Thr Leu Glu Asp Val Ala Val Asp
1 5 10 15
Phe Thr Trp Glu Glu Trp Gln Leu Leu Gly Ala Ala Gln Lys Asp Leu
Tyr Arg Asp Val Met Leu Glu Asn Tyr Ser Asn Leu Val Ala Val Gly
Tyr Gln Ala Ser Lys Pro Asp Ala Leu Phe Lys Leu Glu Gln Gly Glu
Gln Pro Trp Thr Ile Glu Asp Gly Ile His Ser Gly Ala Cys Ser Asp
Ile Trp Lys Val Asp His Val Leu Glu Arg Leu Gln Ser Glu Ser Leu
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Val Asn Arg Arg Lys Pro Cys His Glu His Asp Ala Phe Glu Asn Ile
100 105 llO
Val His Cys Ser Lys Ser Gln Phe Leu Leu Gly Gln Asn His Asp Ile
115 120 125
Phe Asp Leu Arg Gly Lys Ser Leu Lys Ser Asn Leu Thr Leu Val Asn
130 135 140
Gln Ser Lys Gly Tyr Glu Ile Lys Asn Ser Val Glu Phe Thr Gly Asn
145 150 155 160
Gly Asp Ser Phe Leu His Ala Asn His Glu Arg Leu His Thr Ala Ile
165 170 175
Lys Phe Pro Ala Ser Gln Lys Leu Ile Ser Thr Lys Ser Gln Phe Ile
180 185 190
Ser Pro Lys His Gln Lys Thr Arg Lys Leu Glu Lys His His Val Cys
195 200 205
Ser Glu Cys Gly Lys Ala Phe Ile Lys Lys Ser Trp Leu Thr Asp His
210 215 220
Gln Val Met His Thr Gly Glu Lys Pro His Arg Cys Ser Leu Cys Glu
225 230 235 240
Lys Ala Phe Ser Arg Lys Phe Met Leu Thr Glu His Gln Arg Thr His
245 250 255
Thr Gly Glu Lys Pro Tyr Glu Cys Pro Glu Cys Gly Lys Ala Phe Leu
260 265 270
Lys Lys Ser Arg Leu Asn Ile His Gln Lys Thr His Thr Gly Glu Lys
275 280 285
Pro Tyr Ile Cys Ser Glu Cys Gly Lys Gly Phe Ile Gln Lys Gly Asn
290 295 300
Leu Ile Val His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Ile Cys
305 310 315 320
Asn Glu Cys Gly Lys Gly Phe Ile Gln Lys Thr Cys Leu Ile Ala His
325 330 335
Gln Arg Phe His Thr Gly Lys Thr Pro Phe Val Cys Ser Glu Cys Gly
340 345 350
Lys Ser Cys Ser Gln Lys Ser Gly Leu Ile Lys His Gln Arg Ile His
355 360 365
Thr Gly Glu Lys Pro Phe Glu Cys Ser Glu Cys Gly Lys Ala Phe Ser
370 375 380
Thr Lys Gln Lys Leu Ile Val His Gln Arg Thr His Thr Gly Glu Arg
385 390 395 400
., .. ., . . ,.. , ~ . .. .
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Pro Tyr Gly Cys Asn Glu Cys Gly Lys Ala Phe Ala Tyr Met Ser Cys
405 410 415
Leu Val Lys His Lys Arg Ile His Thr Arg Glu Lys Gln Glu Ala Ala
420 425 430
Lys Val Glu Asn Pro Pro Ala Glu Arg His Ser Ser Leu His Thr Ser
435 440 445
Asp Val Met Gln Glu Lys Asn Ser Ala Asn Gly Ala Thr Thr Gln Val
450 455 460
Pro Ser Val Ala Pro Gln Thr Ser Leu Asn Ile Ser Gly Leu Leu Ala
465 470 475 480
Asn Arg Asn Val Val Leu Val Gly Gln Pro Val Val Arg Cys Ala Ala
485 490 495
Ser Gly Asp Asn Ser Gly Phe Ala Gln Asp Arg Asn Leu Val Asn Ala
500 505 510
Val Asn Val Val Val Pro Ser Val Ile Asn Tyr Val Leu Phe Tyr Va
515 520 525
Thr Glu Asn Pro
530
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
TTGCAAACTG AAAGATCTGT AGAGAGT 27
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TTCCAAGCCC GTTCCTCTTT CTTCCAT 27
(2) INFORMATION FOR SEQ ID NO: 4:
... ..
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- I 19 -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
GATTTGAACA CCACTGAGAA GCGTGCA 27
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C~ STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Asn Lys Leu Lys Arg Lys Arg Arg Pro
1 5
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Asn Arg Leu Arg Arg Lys Ser
1 5
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Lys Arg Lys Arg Arg Pro
(2) INFORMATION FOR SEQ ID NO: 8:
CA 022~91~4 1999-01-0~
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- 120-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
~D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Pro Lys Lys Asn Arg Leu Arg Arg Lys Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Lys Lys Lys Lys Tyr Asn
t2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CTTTAAGGAC CCAGGTGGGC AGAGAA 26
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CCTTTTAAGC TTTAATTTAT TTGTGAAGGG GACGCTC 37
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
CA 022~91~4 l999-01-0~
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- 121 -
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CCTTAAAGCT TCCTACATCA GGCCTTCATC CTGA 34
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CTCCCAAGCT TAGGTGCTTT TGAATTGTGG ATATTT 36
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPT}ON: SEQ ID NO: 14:
CCTCCCAAGC TTTCTTCTAC CAGGCATATT CATGCGC 37
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CCTCCCAAGC TTTATCTCTT CACTGCTAGA ACAACT 36
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
, . . , . . .. , . , . .. , ~ ~ . ~ .. . ... .
CA 02259154 1999-01-05
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-122-
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CCTCCCAAGC TTAACCAAAT GCCAGTCAGG CACAGC 3 6
