Note: Descriptions are shown in the official language in which they were submitted.
CA 02384733 2002-03-12
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METHODS AND COMPOSITIONS UTILIZING RAD51
The present application is a continuation-in-part application U.S. Serial No.
09/455,300, filed
December 6, 1999, which is continuation-in-part application of 09/007,020,
filed January 14, 1998,
which claims priority to provisional applications 60/035,834, 60/045,668, and
60/119,578, filed
January 30, 1997, May 6, 1997, and February 10, 1999 respectively. The present
application is
also a continuation-in-part application of and claims priority to provisional
application 60/154,616,
filed September 17, 1999. Each of these applications is expressly incorporated
by reference herein
in its entirety.
FIELD OF THE INVENTION
The invention relates to methods of diagnosis, treatment and screening
utilizing Rad51 molecules.
BACKGROUND OF THE INVENTION
Homologous recombination is a fundamental process which is important for
creating genetic
diversity and for maintaining genome integrity. In Ecoli RecA protein plays a
central role in
homologous genetic recombination in vivo and promotes homologous pairing of
double-stranded
DNA with single-stranded DNA or partially single-stranded DNA molecules in
vitro. Radding, C. M.
(1988). Homologous pairing and strand exchange promoted by Escherichia coli
RecA protein.
Genetic Recombination. Washington, American Society for Microbiology. 193-230;
Radding, C. M.
(1991). J. Biol. Chem. 266: 5355-5358; Kowalczykowski, et al., (1994). Annu.
Rev. Biochem. 63:
991-1043. In the yeast Saccharomyces cerevisiae there are several genes with
homology to recA
gene; Rad51, Rad57 and Dmc1. Rad51 is a member of the Rad52 epistasis group,
which includes
Rad50, Rad51, Rad52, Rad54, Rad55 and Rad57. These genes were initially
identified as being
defective in the repair of damaged DNA caused by ionizing radiation and were
subsequently shown
to be deficient in both genetic recombination and the recombinational repair
of DNA lesions.
Game, J. C. (1983). Yeast Genetics: Fundamental and applied aspects. J.F.T.
Spencer, D.H.
Spencer and A.R.W. Smith, eds (New-Yorkapringer-verlag) : 109-137; Haynes, et
al., (1981 ). The
CA 02384733 2002-03-12
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molecular biology of the yeast Saccharomyces cerevisiae: Life cycle and
inheritance. J.N.
Strathern, E.W. Jones and J.M. Broach, eds (Cold Spring harbor, New York:Cold
Spring Harbor
laboratory press) : 371-414; Resnick, M. A. (1987). Meiosis, P.B. Moens, ed.
(New York: Academic
Press) : 157-210. During meiosis Rad51 mutants accumulate DNA double-strand
breaks at
recombination hot spots (Shinohara, et al., (1992). Cell 69: 457-470). Yeast
rad51 gene was cloned
and sequenced (Basile, et al., (1992). Mol. Cell. Biol. 12: 3235-3246;
Aboussekhara, et al., (1992)
Mol. Cell. Biol. 12: 3224-3234). Although yeast Rad51 gene shared homology
with E.coli recA
gene, the extent of homology was not very strong (27%). However, the extent of
structural
conservation between RecA protein and Rad51 protein became apparent when the
yeast Rad51
protein was isolated and was shown to form nucleoprotein filaments that were
almost identical to
the nucleoprotein filaments formed by RecA protein (Ogawa, et al., (1993). CSH
Symp. Quant.
Biol. 58: 567-576; Ogawa, T., et al., (1993). Science 259: 1896-1899; Story,
et al., (1993}. Science
259: 1892-1896). Recently genes homologous to E.coli recA and yeast rad51 were
isolated from
all groups of eukaryotes, including mammals (Morita, et al., (1993). Proc.
Natl. Acad. Sci. USA
90, 6577-6580; Shinohara, et al., (1993). Nature Genet. 4, 239-243; Heyer,
W.D. (1994).
Experientia 50, 223-233; Maeshima, et al., (1995). Gene 160: 195-200).
Phylogenetic analysis by
Ogawa and co workers suggested the existence of two sub-families within
eukaryotic RecA
homologs: the Rad51-like (Rad51 of human, mouse, chicken, S. cerevisiae, S.
pombe and Mei3 of
Neurospora crassa) and the Dmc1-like genes (S. cerevisiae Dmc1 and Lilium
longiflorum LIM15)
(Ogawa, supra). All these Rad51 genes share significant homology with residues
33-240 of the
E.coli RecA protein, which have been identified as a'homologous core' region.
Yeast and human Rad51 proteins have been purified and characterized
biochemically. Like E.coli
RecA protein, yeast and human Rad51 protein polymerizes on single-stranded DNA
to form a
right-handed helical nucleoprotein filament which extends DNA by 1.5 times
(Story, supra; Benson,
et al., (1994) EMBO J. 13, 5764-5771). Moreover like RecA protein Rad51
protein promotes
homologous pairing and strand exchange in an ATP dependent reaction (Sung, P.
(1994). Science
265, 1241-1243; Sung, P. and D. L. Robberson (1995). Cell 82: 453-461;
Baumann, et al., (1996)
Cell 87, 57-766; Gupta, et al., (1997) Proc. Natl. Acad. Sci. USA 94, 463-
468). Surprisingly,
polarity of strand exchange performed by Rad51 protein is opposite to that of
RecA protein (Sung
and Robberson supra) and the relevance of this observation remains to be seen.
Studies with mouse models show that targeted disruption of the Rad51 gene
leads to an embryonic
lethal phenotype (Tsuzuki, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 6236-
6240). Moreover
attempts to generate homozygous rad51-/-embryonic stem cells have not been
successful. These
results show that Rad51 plays an essential role in cell proliferation, a
surprise in view of the viability
of S.cerevisiae carrying rad51 deletions. It is also interesting to note that
Rad51 was found to be
associated with RNA polymerase II transcription complex (Maldonado, et al.,
(1996). Nature 381,
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86-89), the specificity and functional nature of these interactions remains to
be seen but these
observations point to a pleitropic role of hsRad51 in DNA metabolism.
While Rad51 transcripts and protein are present in all the cell types examined
thus far, the
highest transcript levels are found in tissues active in recombination,
including spleen,
thymus, ovary and testis (Morita, supra). Rad51 is specifically induced in
murine B cells
cultured with lipopolysaccharide, which stimulates switch recombination and
Rad51 localizes
to nuclei of switching B cells (Li, et al., (1996). Proc. Natl. Acad. Sci. USA
93: 10222-10227).
These findings are consistent with the view that Rad51 plays an important role
in lymphoid
specific recombination events such as V(D)J recombination and immunoglobulin
heavy
chain class switching. In spermatocytes undergoing meiosis, Rad51 is enriched
in the
synaptonemal complexes, which join paired homologous chromosomes (Haaf, et
al., (1995)
Proc. Natl. Acad. Sci. USA 92, 2298-2302; Ashley, et al., (1995) Chromosoma
104: 19-28;
Plug, et al., (1996). Proc. Natl. Acad. Sci. USA 93: 5920-5924). In cultured
human cells,
Rad51 protein is detected in multiple discrete foci in the nucleoplasm of a
few cells by
immunofluorescent antibodies. After DNA damage, the localization of Rad51
changes
dramatically when multiple foci form in the nucleus and stain vividly with
anti-Rad51
antibodies (Haaf, supra, 1995). After DNA damage the percentage of cells with
focally
concentrated Rad51 protein increases; the same cells show unscheduled DNA-
repair
synthesis.
Micronuclei (MN) originate from chromosomal material that is not incorporated
into daughter
nuclei during cell division. Different chemicals and treatment of cells induce
qualitatively
different types of micronuclei. MN caused by ionizing radiation or clastogens
(i.e.
5-azacytidine) mostly contain acentric chromosome fragments (Verhaegen, F.,
and Vral, A.
(1994). Radiation Res. 139, 208-213; Stopper, et al., (1995). Carcinogenesis
16, 1647-
1650). In contrast, MN induced by aneuploidogens (i.e. colcemid) result from
lagging whole
chromosomes and stain positively for the presence of kinetochores/ centromeres
(Marrazini
et al., 1994; Stopper, et al., (1994). Mutagenesis 9, 411-416). Determination
of MN
frequencies represents a good assay to measure genetic damage in cells, since
it is much
faster and simpler than karyotype analyses. In this light, the MN test has
been widely used
as a dosimeter of human exposure to radiation or clastogenic and aneugenic
chemicals, and
for the detection and risk assessment of environmental mutagens and
carcinogens (Heddle,
et al., (1991) Environmental Mol. Mutagenesis 18, 277-291; Norppa, et al.,
(1993).
Environmental Health Perspect. 101, Supp. 3, 139-143; Hahnfeldt, et al.,
(1994) Radiation
Res. 138, 239-245). However, although the MN assay is a convenient in situ
method to
monitor cytogenetic effects, the understanding of the connection between
initial DNA
damage and formation of MN is still poor.
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The tumor suppressor p53 prevents tumor formation after DNA damage by halting
cell cycle
progression to allow DNA repair or by inducing apoptotic cell death. Loss of
wild-type p53
function renders cells resistant to DNA damage induced cell cycle arrest and
ultimately leads
to genomic instabilities including gene amplifications, translocations and
aneuploidy. Some
of these chromosomal lesions are based on mechanisms that involve
recombinational
events (Lane, D. P. (1992). Nature 358: 15-16; Lane, D. P. (1993). Nature 362:
786-787;
Sturzbecher, et al., (1996). EMBO J. 15: 1992-2002) reported that wild-type
tumor
suppressor protein p53 interacts physically with human Rad51 protein and it
inhibits the
biochemical functions of Rad51 like ATPase and strand exchange. In vivo,
temperature
sensitive mutant p53 formed complexes with Rad51 only in wild type but not in
mutant
conformation. They suggested that gene amplifications and other types of
chromosome
rearrangements involved in tumour progression might occur not only as a result
of
inappropriate cell proliferation but as a direct consequence of a defect in
p53 mediated
control of homologous recombination processes due to mutations in the p53
gene. (Meyn, et
al., (1994). Int. J. Radiat. Biol. 66: S141-S149) showed that normal cells
transfected with a
dominant-negative p53 mutant acquired interference with the G1-S cell cycle
checkpoint and
showed up to an 80-fold elevation in Rad51 mediated homologous DNA
recombination rates
compared with the normal parental control cells. Thus, loss of normal p53
function may
cause a loss in control of normal DNA repair, recombination, and ultimately
replication,
resulting in inappropriate cell division and neoplastic growth. Breast tumour
cells have
mutated p53 genes and proteins and have various types of chromosomal
aberrations like
insertions, deletions, rearrangements, amplifications etc., indicative of
abnormally controlled
recombination.
Accordingly, the central role of Rad51 in cancer, DNA repair and recombination
is
recognized and characterized herein. Among other provisions, the invention
provides
methods of diagnosis and screening which focus on Rad51. Furthermore, the
invention
provides methods of using modulators of Rad51, preferably inhibitors, in
methods of
treatment.
SUMMARY OF THE INVENTION
In accordance with the objects outlined above, the present invention provides
methods of
diagnosing individuals at risk for a disease state which results in aberrant
Rad51 loci. The
methods comprise determining the distribution of Rad51 foci in a first tissue
type of a first
individual, and then comparing the distribution to the distribution of Rad51
foci from a second
normal tissue type from the first individual or a second unaffected
individual. A difference in
the distributions indicates that the first individual is at risk for a disease
state which results in
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aberrant Rad51 foci. Preferred disease states include cancer and disease
states associated
with apoptosis.
In an additional aspect, the present invention provides methods for
identifying apoptotic cells
and cells under stress associated with nucleic acid modification. The methods
comprise
determining the distribution of Rad51 foci in a first cell, and comparing the
distribution to the
distribution of Rad51 foci from a second non-apoptotic cell. A difference in
the distributions
indicates that the first cell is apoptotic or under stress.
In a further aspect, the present invention provides methods for identifying a
cell containing a
mutant Rad51 gene comprising determining the sequence of all or part of at
least one of the
endogenous Rad51 genes.
In an additional aspect, the invention provides methods of identifying the
Rad51 genotype of
an individual comprising determining all or part of the sequence of at least
one Rad51 gene
of the individual. The method may include comparing the sequence of the Rad51
gene to a
known Rad51 gene.
In a further aspect, the present invention provides methods for screening for
a bioactive
agent capable of binding to Rad51. The methods comprise adding a candidate
bioactive
agent to a sample of Rad51, and determining the binding of the candidate agent
to the
Rad51.
In an additional aspect, the invention provides methods for screening for a
bioactive agent
capable of modulating the activity of Rad51. The method comprises the steps of
adding a
candidate bioactive agent to a sample of Rad51, and determining an alteration
in the
biological activity of Rad51. The method may also comprise adding a candidate
bioactive
agent to a cell, and determining the effect on the formation or distribution
of Rad51 foci in the
cell.
In another aspect, the present invention provides methods for inhibiting cell
proliferation in
an individual comprising administering to the individual a composition
comprising a Rad51
inhibitor. Also provided herein is a method for inhibiting the growth of a
cell comprising
administering to said cell a composition comprising a Rad51 inhibitor. Such
methods can
further include the step of providing radiation or alkylating agents after
administration of said
Rad51 inhibitor. In preferred embodiments the methods are performed in vivo
and/or on
cancerous cells.
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In a further aspect, the invention provides methods of inducing apoptosis in a
cell comprising
increasing the activity of Rad51 in the cell. This can be done by
overexpressing an
endogenous Rad51 gene, or by administration of a gene encoding Rad51 or the
protein
itself.
In an additional aspect, the present invention provides composition comprising
a nucleic acid
encoding a Rad51 protein, and a nucleic acid encoding a tumor suppressor
protein. The
tumor suppressor protein may be p53 or a BRCA protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a digital image of photographs of cells that depict type I and
type II Rad51 foci,
respectively.
Figures 2A and 2B are digital images of photographs of two different breast
cancer cells from
a breast cancer cell line (BT20) that show Rad51 foci. The staining is
localized to the
nucleus, and does not occur in either the cytoplasm or the nucleolus.
Figures 3A, 3B, 3C and 3D show dynamic changes in the higher-order nuclear
organization
of Rad51 foci after DNA damage and cell-cycle arrest. (a-c) TGR-1 fibroblasts
were
irradiated with a lethal dose (900 rad) of'3'Cs and then allowed to recover
for various times.
Rad51 protein is stained (light), nuclei are counterstained with DAPI. Three
hours after
irradiation (a), Rad51 foci are distributed throughout the entire nuclear
volume. Many foci
have a double-dot appearance. After 16 hrs (b), clusters of Rad51 foci and
linear
higher-order structures are formed. Somatic pairing of linear strings of Rad51
foci is
observed. After 30 hrs (c), Rad51 clusters move towards the nuclear periphery
and are
eliminated into micronuclei. (d) Simultaneous staining of Rad51 protein
(light) and replicating
DNA (dark) in an exponentially growing, XPA fibroblast culture. BrdU was
incorporated into
DNA for 30 hrs and detected with red anti-BrdU antibody. Note that the Rad51-
positive cell is
devoid of BrdU label. Magnification 1000x.
Figure 4 depicts the exclusion of Rad51 -protein in micronuclei after DNA
damage. TGR-1
fibroblasts, two days after'3'Cs irradiation with a dose of 900 rad. Rad51
protein is stained
by (light), nuclei are counterstained with DAPI. Note the complete absence of
Rad51-protein
staining in nuclei. All Rad51 foci are excluded into micronuclei. Most
micronuclei exhibit
paired Rad51-protein structures. Magnification IOOOx.
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Figures 5A, 5B, 5C and 5D illustrates that apoptotic bodies (micronuclei)
contain Rad51
protein and fragmented DNA. (a and b) TGR-I nuclei, 3 hrs (right), 16 hrs
(middle), and 30
hrs (left) after'3'Cs irradiation. Rad51-protein foci show light staining. The
repair proteins
Rad52 (a) and Gadd45 (b) are detected by antibody probes (darker staining).
Nuclei are
counterstained with DAPI. Note that neither Rad52 nor Gadd45 foci co-localize
with Rad5l.
Only the Rad51 foci segregate into micronuclei. (c and d) Micronuclei induced
by treatment
of TGR-1 cultures with colcemid (c) and etoposide (d) contain Rad51 protein
(light staining,
left nucleus) and fragmented DNA (darker staining, right nucleus).
Magnification 1000x.
Figures 6A, 6B and 6C show the association of Rad51 protein with linear DNA
molecules. (a)
Mechanically stretched chromatin prepared from a'3'Cs-irradiated cell culture
and stained
with light anti-HsRad51 antibodies. The Rad51 signals appear as beads-on-a-
string on the
linearly extended chromatin fibers. (b and c) DNA fibers excluded from TGR-1
nuclei, one
day after'3'Cs irradiation. Preparations are not experimentally stretched.
Chromatin is
counterstained with DAPI. The DNA fibers are covered with Rad51 protein (c,
light staining),
whereas the remaining nuclei are devoid of detectable Rad51 foci. DNA-strand
breaks in
chromatin fibers are end labeled with fluorescent nucleotides (c, darker
staining co-localizing
with the Rad51 staining). Some fibers appear to form micronuclei.
Magnification 1000x.
Figures 7A, 7B, 7C, 7D, 7E and 7F show the linear higher-order structures of
Rad51 protein
in overexpressing nuclei and in colcemid-induced micronuclei. Rad51 protein is
stained with
anti-Rad51 antiserum, detected by green FITC fluorescence (light staining).
Preparations are
counterstained with DAPI, except the nucleus in b. (a and b) Human 710 kidney
cells
overexpressing Rad51 fused to a T1-tag epitope. Nuclei are filled with a
network of linear
Rad51 structures. Magnification IOOOx. (c) Subconfluent rat TGR 928.1-9 cells
overexpressing HsRad51. Nuclear staining is most prominent in cells during Go
and G,
phase of the cell cycle. Magnification IOOOx. (d) TGR 928.1-9 nucleus filled
with linear Rad51
structures. Magnification 1000x. (e and f) Linear Rad51 structures in
colcernid-induced
micronuclei. TGR-I fibroblasts were treated with colcemid for one day and then
allowed to
recover for two days. Note the absence of Rad51 staining in the nuclei.
Magnification 1000x.
Figure 8 is a schematic illustrating filter based assays to monitor strand
exchange by Rad51.
Single-stranded DNA used for making the nucleoprotein filament is unlabeled.
Rad51 is
shown as ovals. The duplex DNA is labeled with fluorophore (R, rhodamine) on
the
complementary strand that will be displaced after the completion of DNA strand
exchange.
The labeled displaced strand binds to the filter and is detected in screening
of the plate.
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Figure 9 shows a graph depicting DNA strand exchange reaction monitored by a
filter
binding assay. DNA joint molecules (intermediates in DNA strand exchange) are
formed
only when homologous DNA substrates are used. DNA joint molecules are not
formed when
the reaction is performed with either a heterologous DNA substrate or in the
absence of
RecA protein or with inactive RecA protein. Homologous DNA pairing and DNA
strand
exchange assays are highly dependent on the presence of DNA sequence homology,
recombinase protein, nucleotide and magnesium cofactors. Hence, by employing
appropriate controls, the specificity of the reaction and specific effects of
inhibitors are
established.
Figure 10 is a schematic illustrating homologous DNA pairing by a fluorescence
resonance
energy transfer (FRET) assay. Nucleoprotein filament is formed on a single-
stranded DNA
(black thick line) labeled with fluorescein (F). Recombinase is shown as
ovals. Duplex DNA
is labeled with rhodamine (R) on the complementary strand. Homologous pairing
and
subsequent DNA strand exchange results in quenching of emission from
fluorescein.
Figure 11 is a schematic illustrating strand exchange assay measured by FRET
assay. A
nucleoprotein filament is formed on unlabeled single-stranded DNA (thick black
line).
Recombinase is shown as ovals. Duplex DNA (thin line) is labeled. The
homologous DNA
strand is labeled with rhodamine (R) and its complementary strand is labeled
with fluorescein
(F). DNA strand exchange results in enhanced emission from fluorescein.
Figure 12 is a schematic illustrating a D-loop assay. Nucleoprotein filament
is formed on
unlabeled single-stranded DNA. Duplex DNA (either supercoiled or linear) is
end labeled
with 32P. The D-loop is formed after uptake of the single-stranded DNA into
the duplex DNA.
D-loops are trapped on nitrocellulose membranes because they contain single-
strand DNA
tails and/or single-strand regions and the unreacted duplex DNA is washed away
in the
filtrate.
Figure 13 depicts a photograph of a gel illustrating the down-regulation of
Rad51 protein in
MDA-MB-231 breast tumor cells by specific antisense oligodeoxynucleotides. The
lanes are
as follows: 1 and 8, untreated cells; 2, 200 mM AS3 (SEQ ID N0:1); 3, 200 nM
AS4 (SEQ
ID N0:2); 4, 200 nM AS3 (SEQ ID N0:1) 200 nM AS4 (SEQ ID N0:2); 5, 200 nM AS3
(SEQ
ID N0:1); 6, 200 nM AS6 (SEQ ID N0:4); 7, 200 nM AS3 (SEQ ID N0:1) 200 nM AS6
(SEQ
ID N0:4); 9, 200 nM AS4 (SEQ ID N0:2); 10, 200 nM AS6 (SEQ ID N0:4); 11, 200
nM AS4
(SEQ ID N0:2) 200 nM AS6 (SEQ ID N0:4).
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Figure 14 shows a graph which depicts the specific down-regulation of Rad51 by
antisense
oligonucleotides in MDA-MB-231 cells. Cells were treated for 48 hours with a
concentration
of 200 nM of a single antisense oligonucleotide, combinations of two different
antisense
oligonucleotides, or control treatments. Total protein extracts were analyzed
by SDS-PAGE
and Rad51 protein level was monitored by Western blotting using a polyclonal
Rad51
antibody. The levels of Rad51 protein were reduced, depending on the
oligonucleotide
utilized, 30% to 96% compared to control treatment cells. The amount of Rad51
present
after each treatment was quantitated. The average from two independent
experiments was
obtained and plotted in the bar graph. This graph shows that in addition to
single antisense
molecules, combinations of oligonucleotides work well, including the
combination of AS3 and
AS6 (SEQ ID NOS:1 and 4) which essentially completely inhibits the expression
of Rad51.
Figures 15A and 15B show the human Rad51 mRNA sequence wherein the regions
complementary to the antisense molecules SEQ ID NOS:1-9 are underlined.
Figures 16A-E show antisense oligonucleotides provided herein. Figure 16A
shows
antisense in the coding region. Figure 16B shows antisense in the 5'
untranslated region.
Figure 16C shows antisense in the 3' untranslated region. Figure 16D shows
sense
oligonucleotides. Figure 16E shows scrambled oligonucleotides.
Figure 17 shows a recombinasome of an embodiment of the present invention.
Figure 18 shows a schematic for double-stranded break repair in a eukaryotic
cell.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a series of discoveries relating to the
pivotal role that
Rad51 plays in a number of cellular functions, including those involved in
disease states.
Thus, it appears that the levels, function, and distribution of the Rad51
protein within cells
may be monitored as a diagnostic tool of cellular health or fate. In addition,
due to Rad51's
essential role in a number of cellular processes, Rad51 is an important target
molecule to
screen candidate drug agents which can modulate its biological activity.
Moreover, agents
which modulate its biological activity are provided herein for use in methods
of treatment.
Accordingly, in a preferred embodiment, the invention provides methods of
diagnosing
individuals at risk for a disease state. As will be appreciated by those in
the art, "at risk for a
disease state" means either that an individual has the disease, or is at risk
to develop the
disease in the future. By "disease state" herein is meant a disease that is
either caused by
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or results in aberrant Rad51 distribution or biological activity. For example,
as is more fully
described below, aberrant distribution of Rad51 foci in a cell can be
indicative of cancer,
apoptosis, cellular stress, etc., which can lead to the development of disease
states.
Similarly, disease states caused by or resulting in aberrant Rad51 biological
activity,
including alterations caused by mutation, changes in the cellular amount or
distribution of
Rad51, and changes in the biological function of Rad51, for example altered
nucleic acid
binding, filament formation, DNA pairing (i.e. D-loop formation), strand-
exchange, strand
annealing, recombination or DNA repair, are also included within the
definition of disease
states which are related to or associated with Rad51.
Thus, disease states.which may be evaluated using the methods of the present
invention
include, but are not limited to, cancer (including solid tumors such as skin,
breast, brain,
cervical carcinomas, testicular carcinomas, etc.), diseases associated with
premature or
incorrect apoptosis, including AIDS, cancers (e.g. melanoma, hepatoma, colon
cancer, etc.),
liver failure, Wilson disease, myelodysplastic syndromes, neurodegenerative
diseases,
multiple sclerosis, aplastic anemia, chronic neutropenia, Tupe I diabetes
mellitus, Hashimoto
thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma, leukemia,
solid tumors, and
autoimmune diseases), diseases associated with cellular stress which is
affiliated with
nucleic acid modification, including diseases associated with oxidative stress
such as
cardiovascular disease, immune system function decline, aging, brain
dysfunction and
cancer.
The present invention is directed to the use of Rad51 (and its analogs and
homologs) in a
variety of screening techniques. Thus, in one embodiment, Rad51 includes
homologues of
Rad51. In one aspect, Rad51 homologues can be defined by the Rad51 role in
recombinational repair. In another aspect, Rad51 genes encode proteins which
share
significant sequence identity with residues 33-240 of E.coli RecA protein,
which has been
identified as a homologous core region in the literature. Rad51 homologues
include RecA
and Rad51 homologues in yeast and in mammals. RecA and yeast Rad51 have been
cloned and are known in the art. Radding, Genetic Recom. 193-230 (1988);
Radding, J.
Biol. Chem. 266:5355-5358 (1991); Kowalczykoswski, et al., Annu. Rev.
Biochem., 63:991-
1043 (1994); Basile, et al., Mol. Cell. Biol., 12:3235-3246 (1992);
Aboussekhara, et al., Mol.
Cell. Biol., 12:3224-3234 (1992). Genes homologous to E. Coli recA and yeast
Rad51 have
been isolated from all groups of eukaryotes, including mammals. Morita, et
al., PNAS USA,
90:6577-6580 (1993); Shinohara, et al., Nature Genet., 4:239-243 (1993);
Heyer, Experentia,
50:223-233 (1994); Maeshima, et al., Gene, 160:195-200 (1995). Rad51 has been
identified in humans, mice, chicken, S. Cerevisiae, S. Pombe and Mei3 of
Neurospora
crassa. Human Rad51 homologues include Rad51A, Rad5lB, Rad51C, Rad51D, XRCC2
CA 02384733 2002-03-12
WO 01/19397 PCT/US00/25838
and XRCC3. Albala, et al., Genomics, 46:476-479 (1997); Dosanjh, et al.,
Nucleic Acids
Res, 26:1179(1998); Pittman, et al., Genomics, 49:103-11 (1998); Cartwright,
et al., Nucleic
acids Res, 26:3084-3089 (1998); Liu, et al., Mol Cell, 1:783-793 (1998).
In another embodiment, Rad51 is a dimer. The dimer may be a homodimer or a
heterodimer. In a preferred embodiment, the heterodimer is formed from two
different
homologues. In one embodiment, the homologues are selected from the group
consisting of
Rad51A, Rad51B, Rad51C, and Rad51D. In a preferred embodiment, the dimer
includes
Rad51 C or Rad51 B in any combination.
Also included with the definition of Rad51 are amino acid sequence variants.
These variants
fall into one or more of three classes: substitutional, insertional or
deletional variants. These
variants ordinarily are prepared by site specific mutagenesis of nucleotides
in the DNA
encoding the Rad51 protein, using cassette or PCR mutagenesis or other
techniques well
known in the art, to produce DNA encoding the variant, and thereafter
expressing the DNA in
recombinant cell culture as outlined above. However, variant Rad51 protein
fragments
having up to about 100-150 residues may be prepared by in vitro synthesis
using
established techniques. Amino acid sequence variants are characterized by the
predetermined nature of the variation, a feature that sets them apart from
naturally occurring
allelic or interspecies variation of the Rad51 protein amino acid sequence.
The variants
typically exhibit the same qualitative biological activity as the naturally
occurring analogue,
although variants can also be selected which have modified characteristics as
will be more
fully outlined below.
While the site or region for introducing an amino acid sequence variation is
predetermined,
the mutation per se need not be predetermined. For example, in order to
optimize the
performance of a mutation at a given site, random mutagenesis may be conducted
at the
target codon or region and the expressed Rad51 variants screened for the
optimal
combination of desired activity. Techniques for making substitution mutations
at
predetermined sites in DNA having a known sequence are well known, for
example, M13
primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using
assays
of Rad51 protein activities.
Amino acid substitutions are typically of single residues; insertions usually
will be on the
order of from about 1 to 20 amino acids, although considerably larger
insertions may be
tolerated. Deletions range from about 1 to about 20 residues, although in some
cases
deletions may be much larger.
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Substitutions, deletions, insertions or any combination thereof may be used to
arrive at a
final derivative. Generally these changes are done on a few amino acids to
minimize the
alteration of the molecule. However, larger changes may be tolerated in
certain
circumstances. When small alterations in the characteristics of the Rad51
protein are
desired, substitutions are generally made in accordance with the following
chart:
Chartl
Original Residue Exemplary Substitutions
Ala Ser
Arg Lys
Asn Gln, His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Pro
His Asn, Gln
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe Met, Leu, Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp, Phe
Val Ile, Leu
Substantial changes in function or immunological identity are made by
selecting substitutions
that are less conservative than those shown in Chart I. For example,
substitutions may be
made which more significantly affect: the structure of the polypeptide
backbone in the area of
the alteration, for example the alpha-helical or beta-sheet structure; the
charge or
hydrophobicity of the molecule at the target site; or the bulk of the side
chain. The
substitutions which in general are expected to produce the greatest changes in
the
polypeptide's properties are those in which (a) a hydrophilic residue, e.g.
seryl or threonyl is
substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue
having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an
electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a
bulky side chain,
e.g. phenylalanine, is substituted for (or by) one not having a side chain,
e.g. glycine.
The variants typically exhibit the same qualitative biological activity and
will elicit the same
immune response as the naturally-occurring analogue, although variants also
are selected to
modify the characteristics of the Rad51 proteins as needed. Alternatively, the
variant may
be designed such that the biological activity of the RAd51 protein is altered.
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In addition to Rad51, other proteins can be tested for their ability to effect
either Rad51
activity or other component activities. For example and referring to the
schematic in
Figure 17, it currently appears that in mammals, Rad51 is associated with a
variety of other
proteins, including, but not limited to, Rad51 B, Rad51 C, Rad51 D, Rad52,
Rad54,BRCA1,
BRCA2, p53, XRCC2, XRCC3, and RPA. Currently, it appears that a
"recombinosome",
comprising at least Rad51, Rad52, Rad54 and RPA, may function as the recA
equivalent in
mammals to exhibit double-stranded break repair. Accordingly, the
recombinosome may be
used in the assays outlined herein to either assay for alterations in Rad51
activity, alterations
in other components (e.g. Rad52, Rad54, etc.), or for alterations in
recombinosome activity.
Again, analogs and homologs of these other recombinosome proteins are included
as well.
In one embodiment, the method comprises first determining the distribution of
Rad51 foci in
a first tissue type of a first individual, i.e. the sample tissue for which a
diagnosis is required.
In some embodiments, the testing may be done on single cells. The first
individual, or
patient, is suspected of being at risk for the disease state, and is generally
a human subject,
although as will be appreciated by those in the art, the patient may be animal
as well, for
example in the development or evaluation of animal models of human disease.
Thus other
animals, including mammals such as rodents (including mice, rats, hamsters and
guinea
pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep,
pigs, etc., and
primates (including monkeys, chimpanzees, orangutans and gorillas) are
included within the
definition of patient.
As will be appreciated by those in the art, the tissue type tested will depend
on the disease
state under consideration. Thus for example, potentially cancerous tissue may
be tested,
including breast tissue, skin cells, solid tumors, brain tissue, etc.
Similarly, cells or tissues of
the immune system, including blood, and lymphocytes; cells or tissues of the
cardiovascular
system (for example, for testing oxidative stress).
In a preferred embodiment, the disease state under consideration is cancer and
the tissue
sample is a potentially cancerous tissue type. Of particular interest is
breast, skin, brain,
colon, prostate, and other solid tumor cancers. As outlined in the Examples,
cultured breast
cancer cells and primary invasive breast cancer cells all demonstrate an
increase in the
presence of Rad51 foci.
Similarly, several diseases caused by defective nucleotide excision repair
(NER) systems,
including Xeroderma pigmentosium, show increased Rad51 foci.
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In a preferred embodiment, primary cancerous tissue is used, and may show
differential
Rad51 staining. While the number of cells exhibiting Rad51 foci may be less
than for cell
lines, primary cancerous tissue shows an increase in Rad51 foci. Thus for
example, from
0.05 to 10% of primary cancerous cells exhibit differential Rad51 foci, with
from about 1 to
about 5% being common.
It should be noted that not all cancer cell lines exhibit aberrant Rad51
protein foci. For
example, the ovarian cancer cell line Hey does not show an increase in Rad51
foci.
Similarly, as outlined in the examples, transformed but non-malignant human
cells can show
an increased percentage of Rad51-positive cells (compared to non-transformed
cells) ,
although it is generally not as great as in tumor cells.
In a preferred embodiment, the disease state under consideration involves
apoptosis, and
includes, but is not limited to, including AIDS, cancers (e.g. melanoma,
hepatoma, colon
cancer, etc.), liver failure, Wilson disease, myelodysplastic syndromes,
neurodegenerative
diseases, multiple sclerosis, aplasitic anemia, chronic neutropenia, Type I
diabetes mellitus,
Hashimoto thyroiditis, ulcerative colitis, Canale-Smith syndrome, lymphoma,
leukemia, solid
tumors, and autoimmune diseases. This list includes disease states that
include too much
as well as too little apoptosis. See Peter et al., PNAS USA 94:12736 (1997),
hereby
incorporated by reference.
In a preferred embodiment, the disease state under consideration involves
cellular stress
associated with nucleic acid modification, including aging, cardiovascular
disease, declines
in the function of the immune system, brain dysfunction, and cancer.
The distribution of Rad51 foci is determined in the target cells or tissue. To
date, two main
types of Rad51 foci have been identified. As reported earlier (Haaf, 1995,
supra) in situ
immunostaining with Rad51 antibodies reveals three kinds of nuclei: 1 ) nuclei
that did not
show any staining at all ( no foci); 2) nuclei that showed weak to medium
staining and
showed only a few foci (Type I nuclei); and 3) nuclei that showed strong
staining and
showed many foci (Type II nuclei). In general, the staining is excluded from
the cytoplasm.
Type I and Type II patterns of nuclei staining are shown in Figure 1; many of
the foci have a
double-dot appearance, typical of paired DNA segments. In normal cells, type I
nuclei are
found in 7-10% of cells and type II nuclei in less than 0.4 to 1 % of cells,
with generally about
90% of the cells showing no foci. In contrast, some cells involved in disease
states show a
marked increase in Rad51 foci. As outlined herein and shown in the examples,
the numbers
of cells showing Rad51 foci in cells associated with disease states is
significantly increased.
Thus, in a preferred embodiment, the number of cells showing type 1 nuclei is
generally from
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about 5% to about 50% of the nuclei, with from about 10% to about 40%
generally being
seen. Thus, in a preferred embodiment, there is at least a 5% increase in the
type I foci, with
at least about 10 % being preferred, and at least about 30% being particularly
preferred.
Generally, to see this effect, at least about 100 cells should be evaluated,
with at least about
500 cells being preferred, and at least about 1000 being particularly
preferred.
Similarly, the number of cells showing type II nuclei also increases, with
from about 1 % to
about 10% of the nucleic exhibiting type II foci and from about 1 % to about
5% being
common. Thus, in a preferred embodiment, there is at least a 5% increase in
type II foci,
with at least about 10% being preferred, and at least about 30% being
particularly preferred.
In a preferred embodiment, both types of foci increase simultaneously. In
alternate
embodiments, only one type of foci increases. Similarly, an increase in both
types of foci
(i.e. an increase in any foci, irrespective of type) can also be evaluated
using the same
numbers.
The distribution of Rad51 foci can be determined in a variety of ways. In a
preferred
embodiment, a labeled binding agent that binds to Rad51 is used to visualize
the foci. By
"labeled" herein is meant that a compound has at least one element, isotope or
chemical
compound attached to enable the detection of the compound. In general, labels
fall into
three classes: a) isotopic labels, which may be radioactive or heavy isotopes;
b) immune
labels, which may be antibodies or antigens; and c) colored or fluorescent
dyes. The labels
may be incorporated into the compound at any position. Preferred labels are
fluorescent or
radioactive labels. The binding agent can either be labeled directly, or
indirectly, through the
use of a labeled secondary agent which will bind to the first binding agent.
The cells or
tissue sample is prepared~as is known for cellular or in situ staining, using
techniques well
known in the art, as outlined in the Examples.
In a preferred embodiment, the binding agent used to detect Rad51 protein is
an antibody.
The antibodies may be either polyclonal or monoclonal, with monoclonal
antibodies being
preferred. In general, it is preferred, but not required, that antibodies to
the particular Rad51
under evaluation be used; that is, antibodies directed against human Rad51 are
used in the
evaluation of human patients. However, as the homology between different
mammalian
Rad51 molecules is quite high (73% identity as between human and chicken, for
example), it
is possible to use antibodies against Rad51 from one type of animal to
evaluate a different
animal (mouse antibodies to evaluate human tissue, etc.). Thus, in a preferred
embodiment,
antibodies raised against eukaryotic Rad51 are used, with antibodies raised
against
mammalian Rad51 being especially preferred. Thus, antibodies raised against
yeast,
human, rodent, primate, and avian Rad51 proteins are particularly preferred.
In addition, as
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will be appreciated by those in the art, the protein used to generate the
antibodies need not
be the full-length protein; fragments and derivatives may be used, as long as
there is
sufficient immunoreactivity against the sample Rad51 to allow detection.
Alternatively, other
binding agents which will bind to Rad51 at sufficient affinity to allow
visualization can be
used.
Without being bound by theory, as outlined in the Examples, it does not appear
that the
quantitative amount of Rad51 protein is necessarily altered in cells
exhibiting the presence or
altered distribution of foci. However, in some circumstances the quantitative
amount of
Rad51 may be measured and correlated to the presence or absence of Rad51 foci.
In addition, the appearance of the foci may be used in the determination of
the presence of
aberrant Rad51 foci. As noted in the Examples, in some cases linear "strings"
of 5-10
Rad51 foci are formed, with somatic association of "homologous" strings of
similar length,
tightly paired at one of the ends. These structures are generally associated
with DNA fibers,
as is shown in the Figures. Thus, the formation of these types of structures
can be indicative
of aberrant Rad51 foci.
Furthermore, in a preferred embodiment, particularly in disease states
involving apoptosis
and DNA damage, aberrant Rad51 foci includes the development of micronuclei
containing
Rad51. As shown in the Examples, evaluation of Rad51 foci over time, in
particular after
cellular stress, can lead to the concentration and exclusion of the Rad51 foci
(which are
associated with DNA) into micronuclei, which frequently is accompanied by
genome
fragmentation. This effect is seen in a wide variety of apoptotic cells, as is
shown in the
Examples, even in the absence of induced DNA damage, such as through the use
of
colcemid, a spindle poison, thus indicating the role of Rad51 in normal
apoptotic pathways.
In addition to the evaluation of the presence or absence of Rad51 foci, the
cells may be
evaluated for cell cycle arrest, as is outlined in the Examples.
Once the distribution of Rad51 foci has been determined for the target sample,
the
distribution of foci is compared to the distribution of Rad51 foci from a
second cell or tissue
type. As will be appreciated by those in the art, the second tissue sample can
be from a
normal cell or tissue from the original patient or a tissue from another,
unaffected individual,
which has been matched for correlation purposes. A difference in the
distribution of Rad51
foci as between the first tissue sample and the second matched sample
indicates that the
first individual is at risk for a disease state which results in aberrant
Rad51 foci.
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In a preferred embodiment, the difference in Rad51 foci distribution is an
increase in Rad51
foci, of either type 1 or type 2 foci, as outlined above. In an alternate
embodiment, the
difference in Rad51 foci distribution is a decrease in the number of Rad51
foci.
In some embodiments, there need not be a direct comparison. For example,
having once
shown that a particular normal tissue only contains a small percentage of
Rad51 foci, the
tissue or cells under evaluation may not need to be compared to a control
sample; the
presence of a higher percentage allows the diagnosis. Thus, for example, in
breast cancer,
the presence of at least 1 % of the cells containing Rad51 foci is indicative
that the patient is
at risk for breast cancer or in fact already has it.
In a preferred embodiment, a difference in the distribution of Rad51 foci, in
particular an
increase in Rad51 foci, indicates that the cell or tissue is cancerous.
In a preferred embodiment, a difference in the distribution of Rad51 foci, in
particular an
increase in Rad51 foci, indicates that the cell or tissue is apoptotic. These
differences can
include the association of Rad51 with DNA fibers, the association of Rad51
with damaged
DNA in micronuclei, or the presence of Rad51 in micronuclei.
In addition, in a preferred embodiment, the extent of aberrant distribution
indicates the
severity of the disease state. Thus, for example, high percentages of cells
containing Rad51
foci can be indicative of highly malignant cancer.
In addition to the evaluation of Rad51 foci, the presence or absence of
variant (mutant)
Rad51 genes may also be used in diagnosis of disease states. Mutant forms of
p53 have
been found in roughly 50% of known cancers, and it is known that Rad51 and p53
can
interact on a protein level. In addition, p53 and Rad51 have somewhat similar
biochemical
functions. Thus, the present discovery that Rad51 plays a pivotal role in some
cancers and
apoptosis thus suggests that variant Rad51, or incorrectly controlled Rad51
levels or
functions may be important in some disease states.
Accordingly, in a preferred embodiment, the present invention provides methods
for
identifying a cell containing a mutant Rad51 gene comprising determining the
sequence of all
or part of at least one of the endogenous Rad51 genes. By "variant Rad51 gene"
herein is
meant any number of mutations which could result in aberrant Rad51 function or
levels.
Thus, for example, mutations which alter the biochemical function of the Rad51
protein, alter
its half-life and thus its steady-state cellular level, or alter its
regulatory sequences to cause
an alteration in it's steady-state cellular level may all be detected. This is
generally done
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using techniques well known in the art, including, but not limited to,
standard sequencing
techniques including sequencing by PCR, sequencing-by-hybridization, etc.
Similarly, in a preferred embodiment, the present invention provides methods
of identifying
the Rad51 genotype of an individual or patient comprising determining all or
part of the
sequence of at least one Rad51 gene of the individual. This is generally done
in at least one
tissue of the individual, and may include the evaluation of a number of
tissues or different
samples of the same tissue. For example, putatively cancerous tissue of an
individual is the
preferred sample.
The sequence of all or part of the Rad51 gene can then be compared to the
sequence of a
known Rad51 gene to determine if any differences exist. This can be done using
any
number of known homology programs, such as Bestfit, etc.
In a preferred embodiment, the presence of a difference in the sequence
between the Rad51
gene of the patient and the known Rad51 gene is indicative of a disease state
or a
propensity for a disease state.
The present discovery relating to the role of Rad51 in cancer and apoptosis
thus provide
methods for inducing apoptosis in cells. In a preferred embodiment, the
methods comprise
increasing the activity of Rad51 in the cells. By "biological activity" of
Rad51 herein is meant
one of the biological activities of Rad51, including, but not limited to, the
known Rad51 DNA
dependent ATPase activity, the nucleic acid strand exchange activity, the
formation of foci,
single-stranded and double-stranded binding activities, filament formation
(similar to the recA
filament of yeast), pairing activity (D-loop formation), etc. See Gupta et
al., supra, and
Bauman et al., supra, both of which are expressly incorporated by reference
herein. As will
be appreciated by those in the art, this may be accomplished in any number of
ways. In a
preferred embodiment, the activity of Rad51 is increased by increasing the
amount of Rad51
in the cell, for example by overexpressing the endogenous Rad51 or by
administering a
gene encoding Rad51, using known gene-therapy techniques, for example. In a
preferred
embodiment, the gene therapy techniques include the incorporation of the
exogenous gene
using enhanced homologous recombination (EHR), for example as described in
PCT/US93/03868, hereby incorporated by reference in its entirety.
In a preferred embodiment, the cells which are to have apoptosis induced are
cancer cells,
including, but not limited to, breast, skin, brain, colon, prostate,
testicular, ovarian, etc.
cancer cells, and other solid tumor cells.
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In a preferred embodiment, the methods may also comprise subjecting the cells
to conditions
which induce nucleic acid damage, as this appears to provide a synergistic
effect, as
outlined above.
In a preferred embodiment, the methods further comprise increasing the
activity of p53 in the
cell, for example by increasing the amount of p53, as outlined above for
Rad51.
The present discoveries relating to the pivotal role of Rad51 in a number of
important cellular
processes and disease states also makes Rad51, and its associated proteins, an
important
target in drug screening. There are a wide variety of screens that can be
done, including
screening for alterations and modulations in Rad51 activity using Rad51
(including homologs
and analogs, and combinations of these), screening for alterations and
modulations in
Rad51 activity using recombinasomes, screening for alterations and modulations
in
recombinasome activity (namely, double stranded break repair) using the
recombinasome,
and screening for alterations and modulations in the activities of other
recombinasome
components using the recombinasome.
Thus, in a preferred embodiment, the present invention provides methods for
screening for a
bioactive agent which may bind to Rad51 and modulate its activity.
In a preferred embodiment, the methods are used to screen candidate bioactive
agents for
the ability to bind to Rad51. In this embodiment, the methods comprise adding
a candidate
bioactive agent to a sample of Rad51 and determining the binding of the
candidate agent to
the Rad51. By "candidate bioactive agent" or "candidate drugs" or grammatical
equivalents
herein is meant any.molecule, e.g. proteins (which herein includes proteins,
polypeptides,
and peptides), small organic or inorganic molecules, polysaccharides,
polynucleotides, etc.,
which are to be tested for the capacity to bind and/or modulate the activity
of Rad51.
Candidate agents encompass numerous chemical classes. In a preferred
embodiment, the
candidate agents are organic molecules, particularly small organic molecules,
comprising
functional groups necessary for structural interaction with proteins,
particularly hydrogen
bonding, and typically include at least an amine, carbonyl, hydroxyl or
carboxyl group,
preferably at least two of the functional chemical groups. The candidate
agents often
comprise cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic
structures substituted with one or more chemical functional groups.
Candidate agents are obtained from a wide variety of sources, as will be
appreciated by
those in the art, including libraries of synthetic or natural compounds. Any
number of
techniques are available for the random and directed synthesis of a wide
variety of organic
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compounds and biomolecules, including expression of randomized
oligonucleotides.
Alternatively, libraries of natural compounds in the form of bacterial,
fungal, plant and animal
extracts are available or readily produced. Additionally, natural or
synthetically produced
libraries and compounds are readily modified through conventional chemical,
physical and
biochemical means. Known pharmacological agents may be subjected to directed
or
random chemical modifications to produce structural analogs.
In a preferred embodiment, candidate bioactive agents include proteins,
nucleic acids, and
organic moieties.
In a preferred embodiment, the candidate bioactive agents are proteins. By
"protein" herein
is meant at least two covalently attached amino acids, which includes
proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally occurring
amino acids
and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid",
or "peptide
residue", as used herein means both naturally occurring and synthetic amino
acids. For
example, homo-phenylalanine, citrulline and noreleucine are considered amino
acids for the
purposes of the invention. "Amino acid" also includes imino acid residues such
as proline
and hydroxyproline. The side chains may be in either the (R) or the (Sj
configuration. In
the preferred embodiment, the amino acids are in the (S) or L-configuration.
If non-naturally
occurring side chains are used, non-amino acid substituents may be used, for
example to
prevent or retard in vivo degradations.
In a preferred embodiment, the candidate bioactive agents are naturally
occurring proteins or
fragments of naturally occuring proteins. Thus, for example, cellular extracts
containing
proteins, or random or directed digests of proteinaceous cellular extracts,
may be used. In
this way libraries of procaryotic and eukaryotic proteins may be made for
screening against
Rad51. Particularly preferred in this embodiment are libraries of bacterial,
fungal, viral, and
mammalian proteins, with the latter being preferred, and human proteins being
especially
preferred.
In a preferred embodiment, the candidate bioactive agents are peptides of from
about 5 to
about 30 amino acids, with from about 5 to about 20 amino acids being
preferred, and from
about 7 to about 15 being particularly preferred. The peptides may be digests
of naturally
occuring proteins as is outlined above, random peptides, or "biased" random
peptides. By
"randomized" or grammatical equivalents herein is meant that each nucleic acid
and peptide
consists of essentially random nucleotides and amino acids, respectively.
Since generally
these random peptides (or nucleic acids, discussed below) are chemically
synthesized, they
may incorporate any nucleotide or amino acid at any position. The synthetic
process can be
CA 02384733 2002-03-12
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designed to generate randomized proteins or nucleic acids, to allow the
formation of all or
most of the possible combinations over the length of the sequence, thus
forming a library of
randomized candidate bioactive proteinaceous agents.
In one embodiment, the library is fully randomized, with no sequence
preferences or
constants at any position. In a preferred embodiment, the library is biased.
That is, some
positions within the sequence are either held constant, or are selected from a
limited number
of possibilities. For example, in a preferred embodiment, the nucleotides or
amino acid
residues are randomized within a defined class, for example, of hydrophobic
amino acids,
hydrophilic residues, sterically biased (either small or large) residues,
towards the creation of
cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines,
tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the candidate bioactive agents are nucleic acids.
By "nucleic
acid" or "oligonucleotide" or grammatical equivalents herein means at least
two nucleotides
covalently linked together. A nucleic acid of the present invention will
generally contain
phosphodiester bonds, although in some cases, as outlined below, nucleic acid
analogs are
included that may have alternate backbones, comprising, for example,
phosphoramide
(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;
Letsinger, J. Org.
Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);
Letsinger et al., Nucl.
Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et
al., J. Am.
Chem. Soc. 110:4470 (1988}; and Pauwels et al., Chemica Scripta 26:141
91986)),
phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S.
Patent No.
5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321
(1989), O-
methylphophoroamidite linkages (see Eckstein, oligonucleotides and Analogues:
A Practical
Approach, Oxford University Press), and peptide nucleic acid backbones and
linkages (see
Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.
31:1008
(1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207
(1996), all of which
are incorporated by reference). Other analog nucleic acids include those with
positive
backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic
backbones
(U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et
al., Angew. Chem. Intl. Ed. English 30:423 (1991 ); Letsinger et al., J. Am.
Chem. Soc.
110:4470 (1988); Letsinger et al., Nucleoside 8~ Nucleotide 13:1597 (1994);
Chapters 2 and
3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed.
Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem.
Lett. 4:395
(1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett.
37:743 (1996)) and
non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033
and
5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate
Modifications
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in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids
containing one or
more carbocyclic sugars are also included within the definition of nucleic
acids (see Jenkins
et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are
described in
Rawls, C & E News June 2, 1997 page 35. All of these references are hereby
expressly
incorporated by reference. These modifications of the ribose-phosphate
backbone may be
done to facilitate the addition of additional moieties such as labels, or to
increase the stability
and half-life of such molecules in physiological environments. In addition,
mixtures of
naturally occurring nucleic acids and analogs can be made. Alternatively,
mixtures of
different nucleic acid analogs, and mixtures of naturally occuring nucleic
acids and analogs
may be made. The nucleic acids may be single stranded or double stranded, as
specified,
or contain portions of both double stranded or single stranded sequence. The
nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid
contains
any combination of deoxyribo- and ribo-nucleotides, and any combination of
bases, including
uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine,
isocytosine,
isoguanine, etc.
As described above generally for proteins, nucleic acid candidate bioactive
agents may be
naturally occuring nucleic acids, random nucleic acids, or "biased" random
nucleic acids.
For example, digests of procaryotic or eucaryotic genomes may be used as is
outlined
above for proteins.
In one aspect it is understood that when identifying agents which bind to
Rad51, the agent
either is exclusive of or in addition to the DNA on which Rad51 normally binds
to in the
process of recombinational activity.
In a preferred embodiment, the candidate bioactive agents are organic chemical
moieties, a
wide variety of which are available in the literature.
In another preferred embodiment, the candidate agent is a small molecule. The
small
molecule is preferably 4 kilodaltons (kd) or less. In another embodiment, the
compound is
less than 3 kd, 2kd or 1 kd. In another embodiment the compound is less than
800 daltons
(D), 500 D, 300 D or 200 D.
The candidate agents are added to a sample of Rad51 protein. As is outlined
above, all or
part of a full-length Rad51 protein can be used, or derivatives thereof.
Generally, the
addition is done under conditions which will allow the binding of candidate
agents to the
Rad51 protein, with physiological conditions being preferred.
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The binding of the candidate agent to the Rad51 sample is determined. As will
be
appreciated by those in the art, this may be done using any number of
techniques.
In one embodiment, the candidate bioactive agent is labelled, and binding
determined
directly.
Where the screening assay is a binding assay, one or more of the molecules may
be joined
to a label, where the label can directly or indirectly provide a detectable
signal. Various
labels include radioisotopes, fluorescent molecules, enzyme reporters,
colorimetric
reporters, chemiluminescers, specific binding molecules, particles, e.g.
magnetic or gold
particles, and the like. Specific binding molecules include pairs, such as
biotin and
streptavidin, digoxygenin and antidigoxygenin etc. For the specific binding
members, the
complementary member would normally be labeled with a molecule which provides
for
detection, in accordance with known procedures.
In some embodiments, only one of the components is labeled. For example, the
Rad51 may
be labeled at tyrosine positions using'ZSI. Alternatively, more than one
component may be
labeled with different labels; using'zsl for the Rad51, for example, and a
fluorophor for the
candidate agents.
In a preferred embodiment, the binding of the candidate bioactive agent is
determined
directly. For example, the Rad51 may be attached to a solid support such as a
microtiter
plate or other solid support surfaces, and labelled candidate agents added
under conditions
which favor binding of candidate agents to the Rad51 protein. Incubations may
be
performed at any temperature which facilitates optimal activity, typically
between 4 and 40°C.
Incubation periods are selected for optimum activity, but may also be
optimized to facilitate
rapid high through put screening. Typically between 0.1 and 1 hour will be
sufficient.
Excess reagents are washed off, the system is evaluated for the presence of
the label, which
is indicative of an agent which will bind to the Rad51. The agent which binds
can then be
characterized or identified as needed.
In a preferred embodiment, the binding of the candidate bioactive agent is
determined
through the use of competitive binding assays. In this embodiment, the
competitor is can be
any molecule known to bind to Rad51, for example an antibody to Rad5l, or one
of the
proteins known to interact with Rad51, including Rad52, Rad54, Rad55, DMC 1,
BRCA1,
BRCA2, p53, UBC9, RNA polymerase II, and Rad51 itself, any or all of which may
be used
in competitive assays. Either the candidate agents or the competitor may be
labeled, or both
may be labeled with different labels. In this embodiment, either the candidate
bioactive
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agent, or the competitor, is added first to the Rad51 sample for a time
sufficient to allow
binding, if present, as outlined above. Excess reagent is generally removed or
washed
away. The second component is then added, and the presence or absence of the
labeled
component is followed, to indicate binding.
In addition, filament formation assays may be done. In this embodiment,
nucleic acid
(generally single stranded) is assayed in the presence of candidate agents,
Rad51 and dyes
(particularly fluorescent dyes). In the absence of Rad51, the dye binds to the
nucleic acid.
However, upon addition of Rad51, the dye is displaced, and the signal changes.
In a preferred embodiment, methods for screening for a bioactive agent capable
of
modulating the activity of Rad51 comprise the steps of adding a candidate
bioactive agent to
a sample of Rad51, as above, and determining an alteration in the biological
activity of
Rad5l. "Modulating the activity of Rad51" includes an increase in activity, a
decrease in
activity, or a change in the type or kind of activity present. Thus, in this
embodiment, the
candidate agent should both bind to Rad51 (although this may not be
necessary), and alter
its biological or biochemical activity as defined above.
Thus, in this embodiment, the methods comprise combining a Rad51 sample and a
candidate bioactive agent, and testing the Rad51 biological activity as is
known in the art to
evaluate the effect of the agent on the activity of Rad51, including ATPase
activity, ATP
binding, strand exchange, etc.
In a preferred embodiment, methods for screening for a bioactive agent which
modulates the
strand exchange activity of Rad51 are done. In a preferred embodiment, FRET
assays that
exhibit changes in fluorescence, as outlined in the examples. The method
comprises
providing a preformed double stranded nucleic acid comprising a first nucleic
acid single
strand comprising a first fluor and a second nucleic acid single strand
comprising a second
fluor. The first and second nucleic acids are hybridized, quenching of one of
the fluors
occurs. A Rad51 nucleofilament is added comprising Rad51 and a third single
stranded
nucleic acid substantially complementary to one of the first or second
strands. The double
stranded nucleic acid is contacted with the nucleofilament in the presence of
a candidate
agent to form a mixture, and the mixture is assayed for strand exchange
activity.
In a preferred embodiment, the methods include both in vitro screening
methods, as are
generally outlined above, and in vivo screening of cells for alterations in
the presence,
distribution or activity of Rad51. Accordingly, in a preferred embodiment, the
methods
comprise the steps of adding a candidate bioactive agent to a cell, and
determining the effect
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on the formation or distribution of Rad51 foci in the cell. Generally, the
process provided
herein which determines the effect on foci, excludes candidate bioactive
agents already
known in the art such as methyl methanesulfonate.
The addition of the candidate agent to a cell will be done as is known in the
art, and may
include the use of nuclear localization signal (NLS). NLSs are generally
short, positively
charged (basic) domains that serve to direct the entire protein in which they
occur to the
cell's nucleus. Numerous NLS amino acid sequences have been reported including
single
basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys
Lys Lys Arg
Lys Val), Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acid
receptor-f3
nuclear localization signal (ARRRRP); NFKB p50 (EEVQRKRQKL; Ghosh et al., Cell
62:1019 (1990); NFKB p65 (EEKRKRTYE; Nolan et al., Cell 64:961 (1991); and
others (see
for example Boulikas, J. Cell. Biochem. 55(1):32-58 (1994), hereby
incorporated by
reference) and double basic NLS's exemplified by that of the Xenopus (African
clawed toad)
protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln
Ala Lys Lys Lys
Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al.,
J. Cell Biol.,
107:641-849; 1988). Numerous localization studies have demonstrated that NLSs
incorporated in synthetic peptides or grafted onto reporter proteins not
normally targeted to
the cell nucleus cause these peptides and reporter proteins to be concentrated
in the
nucleus. See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-
390, 1986;
Bonnerot, et al., Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987; Galileo, et
al., Proc. Natl.
Acad. Sci. USA, 87:458-462, 1990. In general, the Rad51 foci will be evaluated
as is
generally discussed above.
In a preferred embodiment, the methods comprise adding a candidate bioactive
agent to a
cell, and determining the effect on double strand break repair, homologous
recombination,
sensitivity to ionizing radiation, and class switch recombination. Assays are
detailed in Park,
J. Biol. Chem. 270(26):15467 (1995) and Li et al., PNAS USA 93:10222 (1996),
Shinohara et
al., supra, 1992, all of which are hereby incorporated by reference.
In a preferred embodiment, the cells to which candidate agents are added are
subjected to
conditions which induce nucleic acid damage, including the addition of
radioisotopes (I'25, Tc,
etc., including ionizing radiation and uv), chemicals (Fe-EDTA, bis(1,10-
phenanthroline),
etc.), enzymes (nucleases, etc.).
A variety of other reagents may be included in the screening assays or kits,
below. These
include reagents like salts, neutral proteins, e.g. albumin, detergents, etc
which may be used
to facilitate optimal protein-protein binding and/or reduce non-specific or
background
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interactions. Also reagents that otherwise improve the efficiency of the
assay, such as
protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be
used. In general,
the mixture of components may be added in any order that provides for the
requisite binding.
In a preferred embodiment, the methods and compositions of the invention are
utilized in
high throughput screening (HTS) systems, generally comprising a robotic
system. The
systems outlined herein are generally directed to the use of 96 well
microtiter plates, but as
will be appreciated by those in the art, any number of different plates or
configurations may
be used. In addition, any or all of the steps outlined herein may be
automated; thus, for
example, the systems may be completely or partially automated.
As will be appreciated by those in the art, there are a wide variety of
components which can
be used, including, but not limited to, one or more robotic arms; plate
handlers for the
positioning of microplates; automated lid handlers to remove and replace lids
for wells on
non-cross contamination plates; tip assemblies for sample distribution with
disposable tips;
washable tip assemblies for sample distribution; 96 well loading blocks;
cooled reagent
racks; microtitler plate pipette positions (optionally cooled); stacking
towers for plates and
tips; and computer systems.
Fully robotic or microfluidic systems include automated liquid-, particle-,
cell- and organism-
handling including high throughput pipetting to perform all steps of the
screening process,
both in vitro and in vivo applications. This includes liquid, particle, cell,
and organism
manipulations such as aspiration, dispensing, mixing, diluting, washing,
accurate volumetric
transfers; retrieving, and discarding of pipet tips; and repetitive pipetting
of identical volumes
for multiple deliveries from a single sample aspiration. These manipulations
are cross-
contamination-free liquid, particle, cell, and organism transfers. This
instrument performs
automated replication of microplate samples to filters, membranes, and/or
daughter plates,
high-density transfers, full-plate serial dilutions, and high capacity
operation.
In a preferred embodiment, chemically derivatized particles, plates, tubes,
magnetic particle,
or other solid phase matrices can be used, particularly to bind one or more of
the
components of the assay. The binding surfaces of microplates, tubes or any
solid phase
matrices include non-polar surfaces, highly polar surfaces, modified dextran
coating to
promote covalent binding, antibody coating, affinity media to bind fusion
proteins or peptides,
surface-fixed proteins such as recombinant protein A or G, nucleotide resins
or coatings, and
other affinity matrix are useful in this invention to capture the required
components.
In a preferred embodiment, platforms for multi-well plates, multi-tubes,
minitubes, deep-well
plates, microfuge tubes, cryovials, square well plates, filters, chips, optic
fibers, beads, and
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other solid-phase matrices or plattorm with various volumes are accommodated
on an
upgradable modular plattorm for additional capacity. This modular platform
includes a
variable speed orbital shaker, electroporator, and multi-position work decks
for source
samples, sample and reagent dilution, assay plates, sample and reagent
reservoirs, pipette
tips, and an active wash station.
In a preferred embodiment, thermocycler and thermoregulating systems are used
for
stabilizing the temperature of the heat exchangers such as controlled blocks
or platforms to
provide accurate temperature control of incubating samples from 4~C to 100~C.
In a preferred embodiment, Interchangeable pipet heads (single or multi-
channel ) with
single or multiple magnetic probes, affinity probes, or pipetters robotically
manipulate the
liquid, particles, cells, and organisms. Multi-well or multi-tube magnetic
separators or
platforms manipulate liquid, particles, cells, and organisms in single or
multiple sample
formats.
In some preferred embodiments, the instrumentation will include a microscopes)
with
multiple channels of fluorescence; plate readers to provide fluorescent,
ultraviolet and visible
spectrophotometric detection with single and dual wavelength endpoint and
kinetics
capability, fluroescence resonance energy transfer (FRET), luminescence,
quenching, two-
photon excitation, and intensity redistribution; CCD cameras to capture and
transform data
and images into quantifiable formats; and a computer workstation. These will
enable the
monitoring of the size, growth and phenotypic expression of specific markers
on cells,
tissues, and organisms; target validation; lead optimization; data analysis,
mining,
organization, and integration of the high-throughput screens with the public
and proprietary
databases.
These instruments can fit in a sterile laminar flow or fume hood, or are
enclosed, self-
contained systems, for cell culture growth and transformation in multi-well
plates or tubes
and for hazardous operations. The living cells will be grown under controlled
growth
conditions, with controls for temperature, humidity, and gas for time series
of the live cell
assays. Automated transformation of cells and automated colony pickers will
facilitate rapid
screening of desired clones.
Flow cytometry or capillary electrophoresis formats can be used for individual
capture of
magnetic and other beads, particles, cells, and organisms.
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The flexible hardware and software allow instrument adaptability for multiple
applications.
The software program modules allow creation, modification, and running of
methods. The
system diagnostic modules allow instrument alignment, correct connections, and
motor
operations. The customized tools, labware, and liquid, particle, cell and
organism transfer
patterns allow different applications to be performed. The database allows
method and
parameter storage. Robotic and computer interfaces allow communication between
instruments.
In a preferred embodiment, the robotic workstation includes one or more
heating or cooling
components. Depending on the reactions and reagents, either cooling or heating
may be
required, which can be done using any number of known heating and cooling
systems,
including Pettier systems.
In a preferred embodiment, the robotic apparatus includes a central processing
unit which
communicates with a memory and a set of input/output devices (e.g., keyboard,
mouse,
monitor, printer, etc.) through a bus. The general interaction between a
central processing
unit, a memory, input/output devices, and a bus is known in the art. Thus, a
variety of
different procedures, depending on the experiments to be run, are stored in
the CPU
memory.
Once identified, the compounds having the desired pharmacological activity may
be
administered in a physiologically acceptable carrier to a host, as previously
described. The
inhibitory agents may be administered in a variety of ways, orally,
parenterally e.g.,
subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the
manner of
introduction, the compounds may be formulated in a variety of ways. The
concentration of
therapeutically active compound in the formulation may vary from about 0.1-100
wt.%.
The pharmaceutical compositions can be prepared in various forms, such as
granules,
aerosols, tablets, pills, suppositories, capsules, suspensions, salves,
lotions and the like.
Pharmaceutical grade organic or inorganic carriers and/or diluents suitable
for oral and
topical use can be used to make up compositions containing the therapeutically-
active
compounds. Diluents known to the art include aqueous media, vegetable and
animal oils
and fats. Stabilizing agents, wetting and emulsifying agents, salts for
varying the osmotic
pressure or buffers for securing an adequate pH value, and skin penetration
enhancers can
be used as auxiliary agents.
In a preferred embodiment, the modulators of Rad51 are inhibitors. A Rad51
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inhibitor as defined herein inhibits expression or translation of a Rad51
nucleic acid or the
biological activity of a Rad51 peptide by at least 30%, more preferably 40%,
more preferably
50%, more preferably 70%, more preferably 90%, and most preferably by at least
95%. In
one embodiment herein, a Rad51 inhibitor inhibits expression or translation of
a Rad51
nucleic acid or the activity of a Rad51 protein by 100%.
The biological activity of Rad51 is described above. As shown herein, in one
aspect, by
inhibiting the biological activity of Rad51, cell proliferation is inhibited.
In another aspect, a
Rad51 inhibitor is defined as a molecule that disrupts mammalian double
stranded break
repair. In a further aspect, a Rad51 inhibitor results in the cells containing
it to be more
sensitive to radiation and/or chemotherapeutic agents.
In one embodiment herein, inhibitors of Rad51 include those identified by the
methods
provided herein as well as known downregulators or inhibitors of Rad51 as
defined above.
In another aspect, Rad51 inhibitors can include known inhibitors of RecA
and/or known
inhibitors that sensitize cells to radiation and also affect aspects of
recombination in vivo.
Inhibitors of interest also include but are not limited to peptide inhibitors
of Rad51 (including
but not limited to amino acids 94-160 and 264-315 of p53 and Rad51 antibodies
(further
described below) including but not limited to single chain antibodies), small
molecules,
nucleotide analogues (including but not limited to ADP analogues), minor
groove DNA
binding drugs as inhibitors of Rad51 (including but not limited to distamycin
and derivatives
thereof), known radiation sensitizers (e.g., xanthine and xanthine derivatives
including
caffeine) on the biochemical activities of Rad51, antigenes against Rad5l,
particularly those
which inhibit transcription by locked hybrids, and antisense molecules. The
inhibitor can
inhibit Rad51 directly or indirectly, preferably directly by interacting with
at least a portion of
the Rad51 nucleic acid or protein. Additionally, the inhibitors herein can be
utilized
individually or in combination with each other.
Generally, the Rad51 antisense molecule is at least about 10 nucleotides in
length, more
preferably at least 12, and most preferably at least 15 nucleotides in length.
The skilled
artisan understands that the length can extend from 10 nucleotides or more to
any length
which still allows binding to the Rad51 nucleic acid. In a preferred
embodiment herein, the
length is about 100 nucleotides long, more preferably about 50 nucleotides,
more preferably
about 25 nucleotides, and most preferably about 12 to 25 nucleotides in
length.
The nucleic acids herein, including antisense nucleic acids, and further
described above, are
recombinant nucleic acids. A recombinant nucleic acid is distinguished from
naturally
occurring nucleic acid by at least one or more characteristics. For example,
the nucleic acid
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may be isolated or purified away from some or all of the nucleic acids and
compounds with
which it is normally associated in its wild type host, and thus may be
substantially pure. For
example, an isolated nucleic acid is unaccompanied by at least some of the
material with
which it is normally associated in its natural state, preferably constituting
at least about 0.5%,
more preferably at least about 5% by weight of the total nucleic acid in a
given sample. A
substantially pure nucleic acid comprises at least about 75% by weight of the
total nucleic
acid, with at least about 80% being preferred, and at least about 90% being
particularly
preferred. Alternatively, the recombinant molecule could be made
synthetically, i.e., by a
polymerase chain reaction, and does not need to have been expressed to be
formed. The
definition includes the production of a nucleic acid from one organism in a
different organism
or host cell.
The antisense molecules hybridize under normal intracellular conditions to the
target nucleic
acid to inhibit Rad51 expression or translation. The target nucleic acid is
either DNA or
RNA. In one embodiment, the antisense molecules bind to regulatory sequences
for Rad51.
In one embodiment, the antisense molecules bind to 5' or 3' untranslated
regions directly
adjacent to the coding region. Preferably, the antisense molecules bind to the
nucleic acid
within 1000 nucleotides of the coding region, either upstream from the start
or downstream
from the stop codon. In a preferred embodiment, the antisense molecules bind
within the
coding region of the Rad51 molecule. In a particularly preferred embodiment,
the antisense
molecule has a sequence selected from the group consisting of SEQ ID N0:1, SEQ
ID
N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, and SEQ ID N0:6. In one
embodiment,
the antisense molecules are not directed to the structural gene; this
embodiment is
particularly preferred when the antisense molecule is not combined with
another antisense
molecule.
In one embodiment combinations of antisense molecules are utilized. In one
embodiment, at
least antisense molecule is selected from the 3' untranslated region.
The term "antibody" is used in the broadest sense and specifically covers
single anti-Rad51
monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies) and anti-
Rad51 antibody compositions with polyepitopic specificity. The term
"monoclonal antibody"
as used herein refers to an antibody obtained from a population of
substantially
homogeneous antibodies, i.e., the individual antibodies comprising the
population are
identical except for possible naturally-occurring mutations that may be
present in minor
amounts. In a preferred embodiment, the antibodies are specific for a
particular homolog.
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The anti-Rad51 antibodies may comprise polyclonal antibodies. Methods of
preparing
polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies
can be raised in
a mammal, for example, by one or more injections of an immunizing agent and,
if desired, an
adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in
the mammal by
multiple subcutaneous or intraperitoneal injections. The immunizing agent may
include the
Rad51 polypeptide or a fusion protein thereof. It may be useful to conjugate
the immunizing
agent to a protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of
adjuvants which
may be employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol
may be selected by one skilled in the art without undue experimentation.
The anti-Rad51 antibodies may, alternatively, be monoclonal antibodies.
Monoclonal
antibodies may be prepared using hybridoma methods, such as those described by
Kohler
and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster,
or other
appropriate host animal, is typically immunized with an immunizing agent to
elicit
lymphocytes that produce or are capable of producing antibodies that will
specifically bind to
the immunizing agent. Alternatively, the lymphocytes may be immunized in
vitro.
The immunizing agent will typically include the Rad51 polypeptide or a fusion
protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are
desired, or spleen cells or lymph node cells are used if non-human mammalian
sources are
desired. The lymphocytes are then fused with an immortalized cell line using a
suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell [coding,
Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma cells of
rodent, bovine
and human origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma
cells may be cultured in a suitable culture medium that preferably contains
one or more
substances that inhibit the growth or survival of the unfused, immortalized
cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas typically
will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances
prevent the
growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level
expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are murine
myeloma
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lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center,
San Diego, California and the American Type Culture Collection, Rockville,
Maryland.
Human myeloma and mouse-human heteromyeloma cell lines also have been
described for
the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001
(1984);
Brodeur et al., Monoclonal Antibody Production Technigues and Applications,
Marcel
Dekker, Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the
presence of monoclonal antibodies directed against Rad51. Preferably, the
binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are
known in
the art. The binding affinity of the monoclonal antibody can, for example, be
determined by
the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting
dilution procedures and grown by standard methods [coding, su ra . Suitable
culture media
for this purpose include, for example, Dulbecco's Modified Eagle's Medium and
RPMI-1640
medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in
a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the
culture medium or ascites fluid by conventional immunoglobulin purification
procedures such
as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis,
dialysis, or affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those
described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies
of the
invention can be readily isolated and sequenced using conventional procedures
(e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as
a preferred source of such DNA. Once isolated, the DNA may be placed into
expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese
hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin
protein, to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The
DNA also may be modified, for example, by substituting the coding sequence for
human
heavy and light chain constant domains in place of the homologous murine
sequences [U.S.
Patent No. 4,816,567; Morrison et al., su ra or by covalently joining to the
immunoglobulin
coding sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide.
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Such a non-immunoglobulin polypeptide can be substituted for the constant
domains of an
antibody of the invention, or can be substituted for the variable domains of
one antigen-
combining site of an antibody of the invention to create a chimeric bivalent
antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well known in the art. For example, one method involves
recombinant
expression of immunoglobulin light chain and modified heavy chain. The heavy
chain is
truncated generally at any point in the Fc region so as to prevent heavy chain
crosslinking.
Alternatively, the relevant cysteine residues are substituted with another
amino acid residue
or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of
antibodies to produce fragments thereof, particularly, Fab fragments, can be
accomplished
using routine techniques known in the art.
The anti-Rad51 antibodies of the invention may further comprise humanized
antibodies or
human antibodies. Humanized forms of non-human (e.g., murine) antibodies are
chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')Z or other antigen-binding subsequences of antibodies) which contain
minimal
sequence derived from non-human immunoglobulin. Humanized antibodies include
human
immunoglobulins (recipient antibody) in which residues from a complementary
determining
region (CDR) of the recipient are replaced by residues from a CDR of a non-
human species
(donor antibody) such as mouse, rat or rabbit having the desired specificity,
affinity and
capacity. In some instances, Fv framework residues of the human immunoglobulin
are
replaced by corresponding non-human residues. Humanized antibodies may also
comprise
residues which are found neither in the recipient antibody nor in the imported
CDR or
framework sequences. In general, the humanized antibody will comprise
substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of
the FR regions are those of a human immunoglobulin consensus sequence. The
humanized
antibody optimally also will comprise at least a portion of an immunoglobulin
constant region
(Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-
525 (1986);
Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596
(1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a
humanized antibody has one or more amino acid residues introduced into it from
a source
which is non-human. These non-human amino acid residues are often referred to
as
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WO 01/19397 PCT/US00/25838
"import" residues, which are typically taken from an "import" variable domain:
Humanization
can be essentially performed following the method of Winter and co-workers
[Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et
al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the
corresponding sequences of a human antibody. Accordingly, such "humanized"
antibodies
are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially
less than an intact
human variable domain has been substituted by the corresponding sequence from
a non-
human species. In practice, humanized antibodies are typically human
antibodies in which
some CDR residues and possibly some FR residues are substituted by residues
from
analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including
phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991
); Marks et al.,
J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et
al. are also
available for the preparation of human monoclonal antibodies (Cole et al.,
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al.,
J. Immunol.,
147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing
of human
immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous
immunoglobulin genes have been partially or completely inactivated. Upon
challenge,
human antibody production is observed, which closely resembles that seen in
humans in all
respects, including gene rearrangement, assembly, and antibody repertoire.
This approach
is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications: Marks et
al.,
Bio/Technolo4v 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994);
Morrison,
Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996);
Neuberger, Nature Biotechnolo4y 14, 826 (1996); Lonberg and Huszar, Intern.
Rev.
Immunol. 13 65-93 (1995).
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have
binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for the Rad51, the other one is for any other
antigen, and preferably
for a cell-surface protein or receptor or receptor subunit. In a preferred
embodiment, one of
the binding specificities is for the Rad51, the other one is for a tumor
suppressor antigen
subunit or a tumor antigen subunit. In one embodiment, one of the binding
specificities is for
the Rad51, the other one is for c-abl or ATM (ataxia telangiectasia mutated).
In another embodiment, the antibodies bind to Rad51 only when it is complexed
with another
protein. The antibody may bind to both Rad51 and the other protein, or it may
only bind to
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Rad51, wherein the epitope is not exposed on Rad51 in its uncomplexed form. As
discussed below, Rad51 complexes include Rad51 complexed with oncogene
products such
as c-abl, ATM, tumor suppressor gene products, Rad51 homologs, other Rad
proteins such
as Rad54, 55 and 57 and all the proteins forming a Rad51 recombisome.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant
production of bispecific antibodies is based on the co-expression of two
immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have different
specificities
[Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random
assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a
potential
mixture of ten different antibody molecules, of which only one has the correct
bispecific
structure. The purification of the correct molecule is usually accomplished by
affinity
chromatography steps. Similar procedures are disclosed in WO 93/08829,
published 13
May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining
sites) can be fused to immunoglobulin constant domain sequences. The fusion
preferably is
with an immunoglobulin heavy-chain constant domain, comprising at least part
of the hinge,
CH2, and CH3 regions. It is preferred to have the first heavy-chain constant
region (CH1)
containing the site necessary for light-chain binding present in at least one
of the fusions.
DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the
immunoglobulin
light chain, are inserted into separate expression vectors, and are co-
transfected into a
suitable host organism. For further details of generating bispecific
antibodies see, for
example, Suresh et al., Methods in Enzymolo4y, 121:210 (1986).
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted
cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO
92/200373; EP 03089]. It is contemplated that the antibodies may be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents.
For example, immunotoxins may be constructed using a disulfide exchange
reaction or by
forming a thioether bond. Examples of suitable reagents for this purpose
include
iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for
example, in U.S.
Patent No. 4,676,980. Phage display methods can be used to identify epitopes.
In an embodiment provided herein, the invention provides methods of treating
disease states
requiring inhibition of cellular proliferation. In a preferred embodiment, the
disease state
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requires inhibition of at (east one of Rad51 expression, translation or the
biological activity of
Rad51 as described herein. As will be appreciated by those in the art, a
disease state
means either that an individual has the disease, or is at risk to develop the
disease.
Disease states which can be treated by the methods and compositions provided
herein
include, but are not limited to hyperproliferative disorders. More particular,
the methods can
be used to treat, but are not limited to treating, cancer (further discussed
below),
autoimmune disease, arthritis, graft rejection, inflammatory bowel disease,
proliferation
induced after medical procedures, including, but not limited to, surgery,
angioplasty, and the
like. Thus, in one embodiment, the invention herein includes application to
cells or
individuals afflicted or impending affliction with any one of these disorders.
The compositions and methods provided herein are particularly deemed useful
for the
treatment of cancer including solid tumors such as skin, breast, brain,
cervical carcinomas,
testicular carcinomas, pancreas, prostate, colon, etc.. More particularly,
cancers that may
be treated by the compositions and methods of the invention include, but are
not limited to:
Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),
myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic
carcinoma
(squamous cell, undifferentiated small cell, undifferentiated large cell,
adenocarcinoma),
alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,
chondromatous
hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,
leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma,
gastrinoma,
carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid
tumors,
Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),
large bowel
(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);
Genitourinar)i tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma],
lymphoma,
leukemia), bladder and urethra (squamous cell carcinoma, transitional cell
carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma,
teratoma,
embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial
cell
carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma
(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom, angiosarcoma,
hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma),
fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma,
malignant
lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell
tumor chordoma,
osteochronfroma (osteocartilaginous exostoses), benign chondroma,
chondroblastoma,
chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous s~rstem:
skull
(osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges
(meningioma,
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meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma,
ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,
schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma,
glioma, sarcoma); Gynecolo4ical: uterus (endometrial carcinoma), cervix
(cervical
carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous
cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma],
granulosa-
thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant
teratoma), vulva
(squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma,
fibrosarcoma,
melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid
sarcoma
[embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematolo4ic: blood
(myeloid
leukemia [acute and chronic], acute lymphoblastic leukemia, chronic
lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome),
Hodgkin's
disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant
melanoma, basal
cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic
nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:
neuroblastoma. Thus,
the term "cancerous cell" as provided herein, includes a cell afflicted by any
one of the above
identified conditions.
The individual, or patient, is generally a human subject, although as will be
appreciated by
those in the art, the patient may be animal as well. Thus other animals,
including mammals
such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs,
rabbits, farm
animals including cows, horses, goats, sheep, pigs, etc., and primates
(including monkeys,
chimpanzees, orangutans and gorillas) are included within the definition of
patient. In a
preferred embodiment, the individual requires inhibition of cell
proliferation. More preferably,
the individual has cancer or a hyperproliferative cell condition.
The compositions provided herein may be administered in a physiologically
acceptable
carrier to a host, as previously described. Preferred methods of
administration include
systemic or direct administration to a tumor cavity or cerebrospinal fluid
(CSF).
In a preferred embodiment, these compositions can be administered to a cell or
patient, as is
outlined above and generally known in the art for gene therapy applications.
In gene therapy
applications, the antisense molecules are introduced into cells in order to
achieve inhibition
of Rad51. "Gene therapy" includes both conventional gene therapy where a
lasting effect is
achieved by a single treatment, and the administration of gene therapeutic
agents, which
involves the one time or repeated administration of a therapeutically
effective DNA or RNA.
It has already been shown that short antisense oligonucleotides can be
imported into cells
where they act as inhibitors, despite their low intracellular concentrations
caused by their
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WO 01/19397 PCT/US00/25838
restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad.
Sci. USA 83,
4143-4146 [1986]). The oligonucleotides can be modified to enhance their
uptake, e.g. by
substituting their negatively charged phosphodiester groups by uncharged
groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques suitable for
the transfer of
nucleic acid into mammalian cells in vitro include the use of liposomes,
electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation
method, etc.
The currently preferred in vivo gene transfer techniques include transfection
with viral
(typically retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et
al., Trends in Biotechnology 11, 205-210 [1993]).
In some situations it is desirable to provide the nucleic acid source with an
agent that targets
the target cells, such as an antibody specific for a cell surface membrane
protein or the
target cell, a ligand for a receptor on the target cell, etc. Where liposomes
are employed,
proteins which bind to a cell surface membrane protein associated with
endocytosis may be
used for targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling,
proteins that target intracellular localization and enhance intracellular half-
life. The technique
of receptor-mediated endocytosis is described, for example, by Wu et al., J.
Biol. Chem. 262,
4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For
review of gene marking and gene therapy protocols see Anderson et al., Science
256, 808-
813 (1992).
The antisense molecules can be combined in admixture with a pharmaceutically
acceptable
carrier vehicle. Therapeutic formulations are prepared for storage by mixing
the active
ingredient having the desired degree of purity with optional physiologically
acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol,
A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
Acceptable
carriers, excipients or stabilizers are nontoxic to recipients at the dosages
and
concentrations employed, and include buffers such as phosphate, citrate and
other organic
acids; antioxidants including ascorbic acid; low molecular weight (less than
about 10
residues) polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as
glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and other
carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols
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such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or
nonionic
surfactants such as Tween, Pluronics or PEG.
Dosages and desired drug concentrations of pharmaceutical compositions of the
present
invention may vary depending on the particular use envisioned. The
determination of the
appropriate dosage or route of administration is well within the skill of an
ordinary physician.
Animal experiments provide reliable guidance for the determination of
effective doses for
human therapy. Interspecies scaling of effective doses can be performed
following the
principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies
scaling in
toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al.,
Eds., Pergamon
Press, New York 1989, pp. 42-96.
In one aspect, the Rad51 inhibitors herein induce sensitivity to alkylating
agents and
radiation. Induced sensitivity (also called sensitization or hypersensitivity)
can be measured
by the cells tolerance to radiation or alkylating agents. For example,
sensitivity, which can
be measured, i.e., by toxicity, occurs if it is increased by at least 20%,
more preferably at
least 40%, more preferably at least 60%, more preferably at least 80%, and
most preferably
by 100% to 200% or more.
In an embodiment herein, the methods comprising administering the Rad51
inhibitors
provided herein further comprise administering an alkylating agent or
radiation. For the
purposes of the present application the term ionizing radiation shall mean all
forms of
radiation, including but not limited to alpha, beta and gamma radiation and
ultra violet light,
which are capable of directly or indirectly damaging the genetic material of a
cell or virus.
The term irradiation shall mean the exposure of a sample of interest to
ionizing radiation, and
term radiosensitive shall refer to cells or individuals which display unusual
adverse
consequences after receiving moderate, or medically acceptable (i.e.,
nonlethal diagnostic or
therapeutic doses), exposure to ionizing irradiation. Alkylating agents
include BCNU and
CENU. Particularly preferred are DNA damaging agents. A preferred agent is
cisplatin.
In one embodiment herein, the Rad51 inhibitors provided herein are
administered to prolong
the survival time of an individual suffering from a disease state requiring
the inhibition of the
proliferation of cells. In a preferred embodiment, the individual is further
administered a DNA
damaging agent, such as radiation or an alkylating agent.
In a preferred embodiment, kits are provided. The kits can be utilized in a
variety of
applications, including determining the distribution of Rad51 foci, diagnosing
an individual at
risk for a disease state, including cancer, diseases associated with
apoptosis, and diseases
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WO 01/19397 PCT/US00/25838
associated with stress (including oxidative stress, hypoxic stress, osmotic
stress or shock,
heat or cold stress or shock). The kits include a Rad51 binding agent, that
will bind to the
Rad51 with sufficient affinity for assay. Antibodies are preferred binding
agents. The kits
further include a detectable label such as is outlined above. In one
embodiment, the Rad51
binding agent is labeled; in an additional embodiment, a secondary binding
agent or label is
used. Thus for example, the binding agent may include biotin, and the
secondary agent can
include streptavidin and a fluorescent label. Additional reagents such as
outlined above can
also be included. Furthermore, the kit may include packaging and instructions,
as required.
The identification of the crucial role of Rad51 in a number of cellular
processes and disease
states also identifies a number of methods and compositions relating to
combinations of
Rad51 and other tumor suppressor genes. Thus, Rad51 may function interactively
with a
number of tumor suppressor genes and thus compositions comprising combinations
of these
genes may be useful in methods of gene therapy treatment and diagnosis.
Accordingly, in a preferred embodiment, compositions comprising a nucleic acid
encoding a
Rad51 protein and at least one nucleic acid encoding a tumor suppressor gene
are provided.
Suitable tumor suppressor genes include, but are not limited to, p53, and the
BRCA genes,
including BRCA1 and BRCA2 genes. Thus, preferred embodiments include
compositions of
nucleic acids encoding a) a Rad51 gene and a p53 gene; b) a Rad51 gene and a
BRCA1
gene; c) a Rad51 gene and a BRCA2 gene; d) a Rad51 gene, a p53 gene, and a
BRCA
gene; and e), a Rad51 gene, a p53 gene, a BRCA1 gene and a BRCA2 gene.
Other compositions provided herein are either the nucleic acids encoding a
complex
comprising Rad51 or a Rad51 complex. A Rad51 complex as defined herein is a
composition which Rad51 associates with in vivo. Examples include but are not
limited to
Rad51 in a complex with Rad54, Rad55, Rad57, any of the tumor suppressor genes
described herein or oncogene gene products. In one embodiment the composition
comprises the epitope to which an antibody binds to the Rad51 complex.
In an additional embodiment, the compositions comprise recombinant proteins.
By
"recombinant" herein is meant a protein made using recombinant techniques,
i.e. through the
expression of a recombinant nucleic acid as depicted above. a recombinant
protein is
distinguished from naturally occurring protein by at least one or more
characteristics. For
example, the protein may be isolated or purified away from some or all of the
proteins and
compounds with which it is normally associated in its wild type host, and thus
may be
substantially pure. For example, an isolated protein is unaccompanied by at
least some of
the material with which it is normally associated in its natural state,
preferably constituting at
CA 02384733 2002-03-12
WO 01/19397 PCT/US00/25838
least about 0.5%, more preferably at least about 5% by weight of the total
protein in a given
sample. a substantially pure protein comprises at least about 75% by weight of
the total
protein, with at least about 80% being preferred, and at least about 90% being
particularly
preferred. The definition includes the production of a protein from one
organism in a different
organism or host cell. Alternatively, the protein may be made at a
significantly higher
concentration than is normally seen, through the use of a inducible promoter
or high
expression promoter, such that the protein is made at increased concentration
levels.
Alternatively, the protein may be in a form not normally found in nature, as
in the addition of
an epitope tag or amino acid substitutions, insertions and deletions, as
discussed below.
In a preferred embodiment, these compositions can be administered to a cell or
patient, as is
outlined above and generally known in the art for gene therapy applications.
The following examples serve to more fully describe the manner of using the
above-
described invention, as well as to set forth the best modes contemplated for
carrying out
various aspects of the invention. It is understood that these examples in no
way serve to limit
the true scope of this invention, but rather are presented for illustrative
purposes. All
references cited herein are specifically incorporated by reference in their
entirety.
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EXAMPLES
Example 1
Immunofluorescent Staining of Human Breast Cancer Cells
Breast tumour cells have mutated p53 and have various types of chromosomal
aberrations
like insertions, deletions, rearrangements, amplifications etc. Recombination
proteins such
as Rad51 could evidently participate in such processes. In order to better
understand the
role of uncontrolled recombination and its role in tumour formation and
progression, the
status of Rad51 protein in breast tumour cells by staining them with anti
Rad51 antibodies
was done.
Detailed methods of cloning and expression of HsRad51 gene in E.coli,
purification of
recombinant HsRad51 protein with six histidine residues at it's aminoterminal
end and
preparation of ployclonal antibodies against HsRad51 protein were described
previously by
Haaf, Golub et al. 1995, supra, which is expressly incorporated herein by
reference.
Immunofluorescent staining with anti-Rad51 protein antibodies. Monolayer
cultures of
different cell substrates (see table 1 ) were grown in Dulbecco's MEM medium
supplemented
with 10% fetal bovine serum and antibiotics. The cells were detached from
culture flasks by
gentle trypsinization, pelleted and resuspended in phosphate buffered saline
(PBS; 136 mM
NaCI, 2 mM KCI, 10.6 mM Na2HP04, 1.5 mM KHZP04 [pH 7.3]) prewarmed at
37°C. For
immunofluorescence staining standard protocols were used (Haaf 1995, supra).
Cultured
cells were washed and resuspended in PBS. The density of somatic cells was
adjusted to
about 105 cells per ml in PBS. Aliquots (0.5 ml) of the cell suspension were
centrifuged onto
clean glass slides at 800 rpm for 4 min, in a Cytospin (Shandon, Pittsburg).
Immediately after
cytocentrifugation, the slides were fixed in -20°C methanol for 30 min
and then immersed in
ice-cold acetone for a few seconds to permealize the cells for antibody
staining. Following
three washes with PBS, the preparations were incubated at 37°C with
rabbit anti-HsRad51
antiserum, diluted 1:50 with PBS containing 0.5% bovine serum albumin, in a
humidified
incubator for 30 min. The slides were washed three times for 10 min each and
then
incubated for 30 min with fluorescein-isothiocyanate (FITC)- conjugated anti-
rabbit IgG
diluted 1:20 with PBS. After three washes with PBS, the preparations were
counterstained
with 4',6-diamidino-2- phenylindole (DAPI; 0.1 ug/ml for 1 min) and mounted in
antifade {90%
(vol/vol) glycerol/0.1 m tris-HCI pH 8.0)/2.3% 1,4-diazabicyclo[2.2.2]octane
(DABCO)}.
Digital Imaging Microscopy. Images were taken with a Zeiss epifluorescence
microscope
with a thermo-electronically cooled charge coupled device (CCD) camera (model
PM512;
Photometrics, Tucson, AZ) which was controlled by an Apple Macintosh computer.
Grey
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scale source images were captured separately with filter sets for fluorescein
and DAPI.
Gray scale source images were pseudocolored and merged using ONCOR Image and
ADOBE Photoshop software. It is worth emphasizing that although a CCD imaging
system
was used, all antibody signals were clearly visible by eye through the
microscope.
To study the possible involvement of Rad51 in tumorigenesis we compared the
the in situ
localization of of Rad51 protein homologs in different cell substrates i.e.
mortal fibroblast
strains, virus-transformed non-malignant cell lines and tumor cell lines (see
table). a specific
rabbit antiserum raised against human Rad51 protein was used in these studies.
These
antibodies reacted mainly with Rad51 protein in mammalian cell extracts as
judged by
Western blotting (see fig No 2 in (Haaf, Golub et al. 1995). Immunostaining of
different cells
showed that HsRad51 is concentrated in small and discrete sites (foci) through
out
nucleoplasm and is largely excluded from nucleoli and cytoplasm. At least 250
nuclei of
exponentially growing cultures were analyzed for each experiment. As reported
earlier (Haaf,
Golub et al. 1995) immunostaining revealed three kinds of nuclei: 1 ) nuclei
that did not show
any staining at all ( no foci), 2) nuclei that showed weak to medium staining
and showed
only a few foci (Type I nuclei) 3) nuclei that showed strong staining and
showed many foci
(Type II nuclei). In normal fibroblast control cells, we found type I nuclei
in about 10% of cells
and type II nuclei in less than 0.4 to 1 % of cells and about 90%of the cells
showed no foci.
Use of preimmune serum, as well as omission of either the primary or secondary
antibody,
resulted in the absence of focally concentrated nuclear immunofluorescence.
As reported earlier (Haaf, Golub et al. 1995) in normal (mortal) fibroblast
control cells (Hs68)
we found type I nuclei in 7% -10% of cells and type II nuclei in less than
0.4% of cells, where
as 90% or more of the cells showed no foci (Table 1). In contrast all breast
tumor cell lines
tested (BT20, SrBr3, McF7) exhibited 1-5% of type II nuclei and 10-38% of type
I nuclei
(Table 1 ). Transformed but non-malignant human cells, i.e. SV 40 transformed
fibroblasts
(LNLB, 63L7), EBV-transformed lymphoblasts (GM 01194), and adenovirus-
transformed
kidney cells (293) also showed an increased percentage of Rad51-positive cells
(compared
to normal fibroblasts), however the numbers observed were lower than in tumor
cells.
Interestingly, some tumor substrates i.e. the ovarian cancer line Hey; did not
show a
significant increase of Rad51-positive cells.
As demonstrated earlier (Haaf, Golub et al. 1995), when the normal fibroblast
cells were
exposed to DNA damaging agents like 137Cs, there was a significant increase of
cells
containing type I and type II nuclei (Table 2). It is worth emphasizing that
non-irradiated
breast tumor cells show approximately the same percentage of Rad51-positive
nuclei as
Hs68 fibroblasts exposed to 900 rad Cs137 which kills 99% of cells (Table 2).
The
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immunofluorescent patterns of (non-irradiated) breast cancer cells (Figure 1 )
and fibroblasts
that were exposed to DNA damaging agents are identical.
When the breast cancer cells were exposed to Cs137, the increase in the number
of cells
with type I and type II nuclei was even more dramatic than in normal (Hs68) or
transformed
(LNLB) fibroblasts (Table 2). Up to 40% of irradiated breast cancer cells
showed type I nuclei
and 11 %-18% showed type I I nuclei.
In order to rule out any artifacts that would arise due to the examination of
cultured breast
cancer cells, we then examined the breast tissue obtained directly from the
patient for
Rad51 positive staining. Immunohistochemical evaluation revealed definite
nuclear staining
of invasive breast carcinoma cells. Specifically, nuclear reactivity could be
demonstrated in
sections obtained from three paraffin-embedded samples. The nuclear staining
appeared
granular in some areas, and in others, occupied the entire nucleus. The actual
number of
invasive carcinoma cells that fluoresced was quite small, and estimated to be
less than 5%
of the nuclei seen in three samples with definite reactivity Figure 2).
Nuclear staining was not
identical in normal breast epithelium or lactating breast tissue. Bright
nuclear reactivity was
seen in positive control testicular tissue, specifically, in the cells lining
the seminiferous
tubules. Background staining did not appear to be problematic.
Increase in immunofluorescence of HsRad51 in breast cancer cells can result
from either
increase in the amount of Hsrad51 in these cells or it could be seen as a
result of
re-organization of Hsrad51 in these nuclei in response to damage related
activities. We think
that the latter is true because there was no apparent increase in the amount
HsRad51 in
breast cancer cells as shown by the Western blots (data not shown).
The molecular basis and the consequence of the increase in HsRad51 in breast
cancer
cells is not clear. Since Rad51 protein interacts with other proteins of the
Rad52 epistasis
group and these multiprotein complexes are involved in the recombinational
repair of
double-strand breaks (Hays, et al., (1995). Proc. Natl. Acad. Sci. USA 92:
6925-6929;
Johnson, R. D. and L. S. Symington (1995). Mol. Cell. Biol. 15: 4843-4850), it
is tempting to
speculate that these foci are the sites where repair/recombination events are
taking place.
Since p53 is known to interact with Rad51 it Twill be interesting to see the
colocalization of
p53 and Rad51 protein in these complexes. It is quite possible that these foci
contain either
wild type or mutant p53 and other breast cancer related proteins like BRCA1,
BRCA2 or the
newly discovered STG1 protein. We propose that the increase in the
immunofluorescence
of Rad51 in the breast cells can be used as an important cytological marker
for cell
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proliferation and malignant cell growth. Further experimentation will be done
to validate this
proposal and to understand the role of increase in Rad51 foci and
carcinogenesis.
Table 1: Percentage of nuclei containing discrete foci enriched with HsRad51
protein.
Cell Substrate No foci Type I Type II
_______________________________________________________________________________
______________________
Hs68 Normal fibroblasts 90% 10% 0%
93% 7% 0%
LNL8 (N1 00847) Transformed fibroblasts9% 1%
90%
(SV 40) 90% 8% 2%
63L7 Transformed fibroblasts 94% 6% 0%
(SV40) 94% 3% 3%
GM01194 Transformed lymphoblasts 91 % 7% 2%
(EBV) 90% 9% 1%
92% 8% 0%
80% 18% 2%
80% 19% 1
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293 Cells Transformed kidney 75% 23% 2%
cells
(Adenovirus) 83% 15% 2%
82% 17% 1
BT20 Breast cancer line 86% 10% 4%
82% 13% 5%
78% 17% 5%
SrBr3 Breast cancer line 74% 25% 1
McF7 Breast cancer line 57% 38% 5%
88% 10% 2%
Tera2 Testicular teratoma 76% 23% 1
77% 22% 1
Hey Ovarian cancer line 94% 5% 1
98% 2% 0%
HeLa Cervix (?) tumor cells 67% 31 % 2%
46 .
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Table 2: Percentage of nuclei containing discrete foci enriched with HsRad51
protein.
Cell substrate Treatment No foci Type I Type II
Hs68 None 90% 10% 0%
(normal None 93% 7% 0%
fibroblasts) 6 hrs after 10 rad 96% 4% 0%
Cs137
6 hrs after 50 rad Cs137 96% 4% 0%
6 hrs after 150 rad Cs137 92% 7% 1
6 hrs after 450 rad Cs137 88% 8% 4%
6 hrs after 900 rad Cs 137 91 % 4% 5%
LNLB(N100847) None 90% 9% 1%
(SV40-transformedNone 90% 8% 2%
fibroblasts) 6 hrs after 150 88% 11 % 1
rad Cs137
6 hrs after 300 76% 19% 5%
rad Cs137
6 hrs after 900 78% 17% 5%
rad Cs137
BT20 None 86% 10% 4%
(breast cancer None 82% 13% 5%
cells) None 78% 17% 5%
6 hrs after 300 rad Cs137 44% 41 % 11
6 hrs after 900 rad Cs137 52% 30% 18%
Example 2
Nuclear foci of human recombination protein Rad51
in nucleotide excision repair defective cells
Eurkaryotic cells have several different mechanisms for repairing damaged DNA
(for review
see R. Wood, 1996). One of the major pathway is nucleotide excision repair
(NER), which
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excises damage within oligomers that are 25-32 nucleotides long. Patients with
recessive
heredity disorder XP have defects in one of several enzymes, which participate
in ER.
There are seven XP groups (XP-A to XP-G), which have defects in the initial
steps of the
DNA excision repair.
DNA damage is removed several-fold faster from transcribed genes than from non-
transcribed, mainly due to preferential NER of the transcribed strand (for
review see
Hanawalt, 1994). This mechanism does not function in Cockayne's syndrome (CS)
patients.
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NER defective cells, evidently, sustain increased amount of DNA damage. Thus
we
evaluated NER defective cells from XP and CS cells for an increased amount of
Rad51
protein foci.
To study possible effect of NER on localization of HsRad51 in somatic tissue
culture cells,
we compare in situ localization of the protein in normal fibroblasts,
different XP cells and CS-
B cells. A policlonal rabbit antiserum raised against human Rad51 protein was
used in this
study. These antibody reacted in mammalian cell extract mainly with Rad51
protein as
judged by Western Blotting (see FIG. 2 in Haaf et al., 1995). Immunostaining
of different cell
lines showed that HsRad51 is concentrated in small and discrete sites (foci)
throughout
nucleoplasm and is largely excluded from nucleoli and cytoplasm. As discussed
above,
immunostaining revealed three kinds of nuclei, types I, II and III. The
results are shown in
Table 3.
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Table 3: Percentage of nuclei containing discrete foci
enriched with HsRad51 protein
Cell substrate No Type Type
foci I* II*
Hs68 Normal fibroblasts90% 10% 0%
Normal fibroblasts
63L7 94% 6% 0%
63L7 (confluent) FA fibroblasts 94% 3% 3%
6935 FA fibroblasts 92% 6% 2%
6914 Normal 72% 21 % 7%
6914 lymphoblasts 72% 25% 3%
6914 67% 24% 9%
GM01194 Normal 91 7% 2%
%
GM01194 lymphoblasts 90% 9% 1
GM01194 92% 8% 0%
FA lymphoblasts
GM07063 90% 8% 2%
GM07063 FA lymphoblasts96% 4% 0%
GM13020 FA lymphoblasts92% 7% 1%
GM13022 86% 13% 1%
GM13022 78% 20% 2%
GM13023 94% 5% 1%
GM13071 81% 15% 4%
GM13071 74% 23% 3%
*Type I nuclei show only few (<15) foci and/or weak to medium HsRad51
immunofluorescence, whereas Type II cells show many and/or strongly
fluorescing foci.
250 nuclei were analyzed for each experiment.
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In normal (mortal) fibroblast control cells, LNL8 and NF, we found type I
nuclei in 5-9% cells
and type II nuclei in 1-7% cells, where as 88-90% of the cells showed no foci
(Table 3). Use
of preimmune serum, as well as omission of either the primary or secondary
antibody,
resulted in the absence of focally concentrated nuclear immunofluorescence.
XP-V cells are normal in NER, but have defect in postreplication repair
process (Boyer et al.,
1990; Griffiths et al., 1991; Wang et al., 1991, 1993). As we expected, these
cells showed
the same distribution pattern of nuclear HsRad51 as control cell lines (Table
3).
Distribution of HsRad51 foci in CS-B cells also was similar to the cells with
normal NER
(Table 3). This result was also anticipated. CS-B cells are defective in NER
which is
coupled with transcription (Venema et al., 1990). Transcribed genes,
evidently, comprise
only a small part of the whole genomic DNA and damage in transcribed genes,
therefore,
should be accounted for only a very small fraction of the damage in genomic
DNA.
XP-A, XP-B, XP-F and XP-G cells are all defective in NER. XP-A cells have
defect in XPA
protein, which carries out a crucial rate-limiting step in NER-recognition of
DNA lesion (Jones
and Wood, 1993). The protein makes a ternary complex with ERCC1 protein and
XPF
protein, which is defective in XP-F cells (Park and Sankar, 1994). XP-B and XP-
G cells are
defective in different steps of NER which follow damage recognition (Reviewed
in Ma et al.,
1995).
XP-A and XP-F cell lines have increased amount of cells with HsRad51 protein
foci (Table
3). In contrast, XP-B and XP-G cells have about the same level of HsRad51
protein foci, as
cells with normal NER (Table 3). This result could be easily understood if we
assume, that
1 ) formation of HsRad51 foci is caused by DNA damage, b) DNA lesion is
excluded from the
pool of damage DNA which cause Rad51 foci formation as soon as XPA/XPF/ERCC1
complex binds to the lesion. DNA damage in XP-Band XP-G cells is recognized by
NER
system, but the damage cannot be proceeded and removed by the system. Such
unremoved damage, evidently, is not considered as a substrate for Rad51
protein involved
repair as soon as the damage is recognized by NER complex XPA/XPF/ERCC1 as a
substrate for NER, even if defect in subsequent steps of NER makes its
removing
impossible.
Induction of principal DNA repair system (SOS respond) in E. coli is, assumed
to be
triggered by formation of single-stranded DNA (ssDNA) which results from DNA
damage
(reviewed in Little and Mount, 1982). DNA damage in XP-A cells is not
recognized by NER
and, therefore, at least a considerable part of DNA damage is not proceeded to
formation of
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ssDNA regions. Nevertheless, Rad51 foci are effectively formed in XP-A cells
and their
amount could be further increased by UV or -irradiation (Tables 4 and 5).
Evidently, ssDNA
is not a primary signal for HsRad51 protein foci formation.
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Table 4: Percentage of nuclei containing discrete foci enriched with HsRad51
protein
Cell substrateTreatment No foci Type I* Type II*
LNL8 (control)No treatment90% 9% 1%
90% 8% 2%
NF (control)No treatment
" 88% 5% 7%
XPA No treatment89% 5% 6%
" 51 % 39% 10%
No treatment72% 20% 8%
XPB " 55% 34% 7%
No treatment86% 11 % 3%
a
XPD 86% 11 % 3%
No treatment
" 87% 8% 5%
XPF No treatment63% 28% 9%
48% 41% 11%
No treatment
XPG " 64% 25% 8%
None 88% 7% 5%
XPV 85% 9% 6%
94% 5% 1
CBS 89% 11 % 0%
87% 8% 5%
*Type I nuclei show only a few (<15) foci and/or weak to medium HsRad51
immunofluorescence, whereas type II cells show many and/or strongly
fluorescing foci.
250 nuclei were analyzed for each experiment.
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Table 5: Percentage of nuclei containing discrete foci enriched with HsRad51
protein
Cell substrateTreatment No fociType Type II*
I*
LNL8 (control)No treatment 90% 9% 1%
No treatment 90% 8% 2%
6 hrs after 150 rad 88% 11% 1%
Cs137
6 hrs after 300 rad
Cs137
76% 19% 5%
6 hrs after 900 rad
Cs137
None 78% 17% 5%
XPA** 3 hrs after 300 rad'3'Cs
51 % 39% 10%
None
61 % 24% 15%
6 hrs after 900 rad'3'Cs
None 72% 20% 8%
5 hrs after 5 J/m2 59% 25% 16%
UV
5 hrs after 15 J/m2
UV
59% 34% 7%
None
53% 31 16%
%
5 hrs after 800 rad'3'Cs
27 hrs after 800 rad'3'Cs55% 26% 19%
CBS**
87% 8% 5%
60% 21 19%
%
77% 6% 17%
*Type I nuclei show only a few (<15) foci and/or weak to medium HsRad51
immunofluorescence, whereas Type II cells show many and/or strongly
fluorescing foci.
150 nuclei were analyzed for each experiment.
**Induction of HsRad51 foci in Xeroderma pigmentosum (Type A) implies that
single
stranded DNA molecules are not the primary signal.
***Induction of HsRad51 foci in cells from patients with Cockayne's syndrome
implies
that the induction is not dependent on transcription.
In conclusion, human recombination protein HsRad51 is concentrated in multiple
discrete
foci in nucleoplasm of cultured human cells. After treatment of cells with DNA
damaging
agents, the percentage of cells with HsRad51 protein immunofluorescence
increases.
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Xeroderma pigmentosum (XP) cells XP-A with inactive protein XPA, responsible
for lesion
recognition by nucleotide excision repair (NER) system have increased
percentage of cells
with HsRad51 protein foci. XP-F cells, defective in XPF protein, which forms
complex with
XPA protein, also have increased level of the HsRad51 protein foci. In
contrast, XP-B and
XP-G cells with defects in different steps ER, which follow the damage
recognition, as well
as XP-V cells (normal level of NER) and Cockayne's syndrome (CS) cells (defect
in NER,
responsible for preferential repair of the transcribed DNA strand) have normal
level of
HsRad51 protein foci. Evidently, formation of HsRad51 protein foci is caused
by DNA
damages. DNA damages, however, do not participate in causing formation of
HsRad51
protein foci, as soon as they are recognized by NER system, even if the system
is blocked
on one of the step, leading to DNA repair.
Example 3
Higher order nuclear structures of Rad51 and its exclusion into
micronuclei after cell damage
Previous studies have revealed a time- and dose-dependent increase of nuclear
HsRad51
protein foci after DNA damage introduced into the genome by various agents
(Haaf et al.,
1995, supra). Here we show that when the damaged cells are allowed to recover,
these
Rad51 foci form specific higher-order nuclear structures. Finally, all the
focally concentrated
Rad51 protein is eliminated into micronuclei that undergo apoptotic genome
fragmentation.
Treatment of cells with clastogens and aneuploidogens implements a mechanism
that
affects the nuclear distribution of Rad51 protein and targets Rad51 foci, most
likely along
with irreversibly damaged DNA into micronuclei. To examine the role of Rad51
protein in
DNA repair and cell proliferation, we have analyzed the intranuclear
distribution of
overexpressed Rad51 protein during the cell cycle and in cell populations
proceeding
through apoptosis.
Experimental Procedures
Cell Culture. The sources of the cell lines were as follows. Rat TGR-1 cells,
J. Sedivy,
Brown University; mouse 3T3-Swiss cells, ATCC; human 293 kidney cells, ATCC;
human
teratoma cells, B. King, Yale University; human LNL8 fibroblasts, S. Meyn,
Yale; human XPA
and XPF fibroblasts, P Glazer, Yale.
Monolayer cultures were grown in D-MEM medium supplemented with 10% fetal
bovine
serum and antibiotics. The cells were detached from culture flasks by gentle
trypsination,
pelleted and resuspended in phosphate-buffered saline (PBS; 136 mM NaC1, 2 mM
KCI,
10.6 mM NazHPO,, 1.5 mM KHzP04, pH 7.3).prewarmed at 37°C.
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To induce DSBs in DNA and recombinational repair, cell cultures were exposed
to a "'Cs
irradiator at doses of 900 rad and then allowed to recover for various time
spans. In another
experiment, cells were treated with 10 uM 5-aza-dC for 24 hrs. This
hypomethylating base
analog is a potent DNA-strand breaker (Snyder, et al., (1989). Mutation Res.
226, 185-190;
Haaf, 1995). Incubation of cells with the spindle poison colcemid (1 Ng/ml for
24 hrs) resulted
in the formation of multinuclei and micronuclei containing entire chromosomes.
Under the
experimental conditions chosen, colcemid does not cause chromsome breakage.
Treatment
with etoposide (Sedivy), a drug that inhibits DNA topoisomerase II, is a
classic system for
inducing apoptosis in cells (Mizumoto, et al., (1994). Mol. Pharmac. 46, 890-
895).
Antibody Probes. HsRad51 protein, expressed in E. coli, was isolated and used
for
preparation of rabbit polyclonal antibodies. Western blotting experiments
revealed that rabbit
antiserum does not react significantly with any other proteins in mammalian
cells except
Rad51 (Haaf et al., 1995). Similarly, polydonal antibodies against HsRadS2, a
structural
homolog of yeast Rad52, were raised in the rat, as is known in the art. Mouse
monoclonal
antibody 30T14 recognizes Gadd45, a ubiquitously expressed mammalian protein
that is
induced by DNA damage (Smith, et al., (1994). Science 266, 1376-1380).
Monoclonal
antibodies H4 and H14 bind specifically to the large subunit of RNAPII
(Bregman et al.,
(1995) J. Cell Biol. 129, 287-298). Monoclonal antibody Pab246 against amino
acids 88-93
of mouse p53 was purchased from Santa Cruz Biotechnology, Inc.
Immunofluorescent Staining. Harvested cells were washed and resuspended in
PBS.
Cell density was adjusted to 105 cells/ml. 0.5 ml aliquots of this cell
suspension were
centrifuged onto clean glass slides at 800 rpm for 4 min, using a Shandon
Cytospin.
Immediately after cytocentrifugation, the preparations were fixed in absolute
methanol for 30
min at -20°C and then rinsed in ice-cold acetone for a few seconds.
Following three washes
with PBS, the preparations were incubated at 37°C with rabbit anti-
HsRad51 antiserum,
diluted 1:100 with PBS, in a humidified incubator for 30 min. For some
experiments, the
slides were simultaneously labeled with rat anti-HsRad52 antiserum or mouse
monoclonal
antibody. The slides were then washed in PBS another three times for 10 min
each and
incubated for 30 min with fluorescein-isothiocyanate (FITC)-conjugated anti-
rabbit IaG,
appropriately diluted with PBS. Rad52, Gadd45, p53, and RNAPII were detected
with
rhodamine, conjugated anti-rat IgG or anti-mouse IgG+IgM. After three further
washes with
PBS, the preparations were counterstained with 1 ug/ml 4,6-diamidino-2-
phenylindole (DAPI)
in 2xSSC for 5 min. The slides were mounted in 90% glycerol, 0.1 M Tris-HCI,
pH 8.0, and
2.3% 1,4-diazobicyclo-2,2,2-octane (DABCO).
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For preparation of chromatin fibers, cells were centrifuged onto a glass slide
and covered
with 50 NI of 50 mM Tris-HCI, pH 8.0, 1 mM EDTA, and 0.1 % SDS. The protein-
extracted
chromatin was mechanically sheared on the slide with the aid of another slide
(Heiskanan, et
al., (1994) BioTechniques 17, 928-933) and then fixed in methanol/acetone.
Fluorescence In Situ End Labeling (FISEL). FISEL detects cell death
(apoptosis) in situ
by quantitating DNA strand breaks in individual nuclei. It uses terminal
transferase (TdT) to
label the 3'-ends in fragmented genomic DNA with biotinylated nucleotide. 100
u1 of reaction
mix contain I NI (25 Units) TdT (Boehringer Mannheim), 20 u1 5xTdT buffer
(supplied with the
enyzme), 1 u1 0.5 mM biotin-II-dUTP, 3 u1 0.5 mM dTTP, and 75 NI ddHzO.
Cytological
preprations are incubated at 37°C for 1 hr with this reaction mix.
Washing the slides for 3x5
min in PBS is sufficient to terminate the reaction. The incorporated biotin-
dUTP is detected
with rhodarnine-conjugated avidin.
In Situ Labeling of DNA-Replication Synthesis. The base analog BrdU is
incorporated in
place of thymidine into the DNA of replicating cells. In order to mark cycling
cells, 10 ug/ml
BrdU were added to the culture medium 30 hrs before cell harvesting. Depending
on the cell
substrate, this corresponds to one or two population doublings. At the end of
the labeling
period, slides were prepared as described above. After Rad51-protein staining,
the
preparations were again fixed in a 3:1 mixture of methanol and acetic acid for
several hours
at -20°C. Since the anti-BrdU antibody only recognizes BrdU
incorporated into chromosomal
DNA if the DNA is in the single-stranded form, the slides were denatured in
70% formamide,
2xSSC for 1 min at 80°C and then dehydrated in an alcohol series. BrdU
incorporation was
visualized by indirect anti-BrdU antibody staining. First, the preparations
were incubated
with mouse monoclonal anti-BrdU antibody (Boehringer Mannheim), diluted 1:50
with PBS,
for 30 min. The slides were washed with PBS and then incubated with
rhodamine-conjugated anti-mouse IgG, diluted 1:20 with PBS, for another 30
min. Only cells
with intense BrdU labeling of the entire nucleus were considered BrdU-positive
and scored
as cycling cells.
Overexpression of HsRad51 Protein in Mammalian Cells. Human kidney cells (line
293,
ATCC CRL1573) were stably transformed by plasmid pEG9 15. This plasmid carries
the
whole coding sequence of the HsRad51 gene inserted in frame with the 5'-end
terminal
sequence of vector pEBVHisB (Invitrogen). The resulting cell lines 710 and 717
constitutively express Rad51 protein fused to a T7-tag epitope (Haaf et al.,
1995).
Digital Imaging Microscopy. Images were taken with a Zeiss epitluorescence
microscope
equipped with a thermoelectronically cooled charge coupled device (CCD) camera
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(Photometries CH250), which was controlled by an Apple Macintosh computer.
Gray scale
source images were captured separately with filter sets for FITC, rhodamine,
and DAPI.
Gray scale images were pseudocolored and merged using ONCOR Image and ADOPE
Photoshop software. It is worth emphasizing that although a CCD imaging system
was
used, the immunofluorescent signals described here were clearly visible by eye
through the
microscope.
Dynamic Nuclear Distribution of Rad51 Protein after DNA Damage Nuclear foci of
mammalian Rad51-recombination protein can be induced significantly after
irradiation of cell
cultures with Cesium ('3'Cs). Since Western blots have not shown a dramatic
net increase
in Rad51 protein in irradiated cells, we conclude that DNA damage mainly
affects its nuclear
distribution (Haaf et al., 1995). To gain insight into the radiation-induced
perturbations in
nuclear organization and the possible role of Rad51 protein in repair
processes, we have
analyzed the topological rearrangements of Rad51-protein foci in rat TGR-I
fibroblasts that
have sustained DNA damage. TGR-I is an immortal rat cell line with a stably
diploid
karyotype. After'3'Cs irradiation with a dose of 900 red which kills 99% of
cells, rat Rad51
protein was visualized in situ using polyclonal antibodies raised against
HsRad51. The
percentage of cells with cytologically detectable Rad: 1-protein foci started
to increase in the
first three hours (Table 6). Rad51-positive nuclei contained up to several
dozen discrete foci
throughout their nucleoplasm. Immunofluorescence staining was largely excluded
from the
cytoplasm. Many of these nuclear Rad51 foci had a double-dot appearance,
typical of
paired DNA segments (Figure 3a).
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Table 6. Induction of Rad51 Foci after'3'Cs Irradiation of TGR-1 cells and
Their
Elimination into Micronuclei
Percentage Percentage of CellsPercentage of Cells
of with with
Cells withoutType la Foci Type 118 Foci
reatment Foci in in in in
Nuclei Micronuceli Nuclei Micronuclei
None 93% 6% 0% 1 % 0%
3 hrs after
900 rad 8% 0.4% 11 % 0.6%
137CS 80%
16 hrs 9% 8% 1 % 9%
after
900 rad
'3'Cs 73%
1% 13% 1% 13%
30 hrs
after
900 rad
,3'Cs 72% 0% 4% 0% 6%
4 days
after
900 rad
'3'Cs 90%
aType I nuclei and micronuclei show weak to medium HsRad51 immuno-
fluorescence,
whereas type II cells show strongly fluorescing foci. 1000 cells were anlayzed
for each
experiment.
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When irradiated cells were then cultured for various times to allow repair of
induced DNA
damage and apoptosis to occur, significant changes in the distribution of
Rad51-protein foci
were detected. Nuclear foci coalesced into larger clusters with extremely high
immunofluorescence intensity after 6-20 furs. Only a few discrete foci
remained singly in the
nucleoplasm. In a percentage of nuclei linear strings of 5-10 Rad51-protein
foci were formed
(Figure 3b). Immediately striking was the somatic association of "homologous"
strings of
similar length. These strings were always tightly paired at one of their ends.
The dynamics
of the Rad51-protein foci after induction of DNA damage are clear evidence for
a
higher-order organization of nuclear structure that accompanies DNA repair
and/or
programmed cell death.
One to two days after'3'Cs irradiation with a lethal dose the coalesced Rad51
clusters
showed a highly non-random localization towards the nuclear periphery (Figure
3c). Finally,
the Rad51 structures were excluded into micronuclei. The nucleoplasm was
virtually cleared
of Rad51 protein and only aggregated Rad51 foci in MN were remaining (Figure
4;Table 6).
Similar to the situation seen earlier in interphase nuclei, many MN displayed
paired Rad51
foci and higher-order structures. The highest number of MN (approximately
three per cell)
as well as the highest number of Rad51-positive MN(approximately 30%) were
observed 16
hrs after irradiation (Table 7). However, at each time point analyzed the
majority of
radiation-induced MN did not show detectable Rad51-protein foci.
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Table 7. Rad51 Foci in Micronuclei of Different Cell Substrates
Number of Percentage Percentage of
Cell substrateMicronuclei of Rad51-Negative
Treatment in 1000 Rad51-PositiveMicronuclei
Cells Micronuclei
TGR01
None 93 14% 86%
3 hrs after
900
rad'3'Cs 279 22% 78%
16 hrs after
900
rad'3'Cs 2719 28% 72%
4 days after
900
rad'3'Cs 1040 20% 80%
LNL8
None n.d. 23% 77%
None n.d. 26% 74%
XPA
None n.d. 18% 82%
Teratoma
None n.d. 10% 90%
3T3-Swiss
None 472 125% 88%
1000 cells were analyzed for each experiment
a
Segregation of Rad51-Protein Foci into Micronuclei An increased rate of MN is
also
observed in 5-azadeoxycytidine (5-aza-dC)-treated cell cultures (Guttenbach,
et al., (1994)
Exp. Cell Res. 211, 127-132; Stopper et al., 1995, supra). This
hypomethylating base
analog induces inhibition of chromatin condensation, leading to instability of
the affected
chromosome regions (Haaf, 1995). Its cytotoxic effects are at least partially
due to the
induction of single- and double-strand breaks in DNA. Like'3'Cs irradiation, 5-
aza-dC can
induce the formation of Rad51-protein foci in nuclei and its elimination into
MN. Rat TGR-1
and human LNL8 fibroblast cultures treated with non-lethal doses of 5-aza-dC
displayed MN
with focally concentrated Rad51 protein in 5-10% of their cells (Table 8).
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Table 8. Induction of Rad51 Foci by 5-Azadeoxycytidine
Percentage Percentage of CellsPercentage of Cells
of with with Type
Cells withoutType la Foci Ile Foci
Foci
Cell type in in in in
Treatment Nuclei Micronuclei Nuceli Micronuclei
TGR-1
None 93% 6% 0% 1 % 0%
5-aza-dCb86% 5% 4% 1 % 4%
LNL8
None 92% 6% 1 % 1 % 0%
5-aza-dCb89% 3% 1 % 2% 5%
a Type I nuclei and micronuclei show weak to medium HsRad51 immuno-
fluorescence,
whereas type II cells show strongly fluorescing foci. 500 cells were analyzed
for each
experiment.
° 10-5 M 5-aza-dC were added to the culture medium 24 h rs before cell
harvest.
Rapidly dividing cell cultures always exhibit a baseline MN frequency even
without exposure
to clastogens or aneuploidogens. In five different substrates studied, LNLB,
XPA, teratoma,
3T3-Swiss, and TGR-1 cells, 10-30% of these spontaneously occuring, non-
induced MN
exhibited Rad51-protein foci (Table 7). This further links Rad51-protein foci
and MN
formation.
Rad52 and Other Repair Proteins Are Not Excluded into Micronuclei Studies in
yeast
(Shinohara et al., 1992, supra; Milne, G., and Weaver, D. (1993). Genes Dev.
7, 1755-1765)
and humans (Shen, et al., (1996). J. Biol. Chem. 271, 148-152) have shown
physical
interaction between Rad51 and Rad52 proteins both in vitro and in vivo. Double
immunofluorescence with rabbit anti-Rad51 and rat anti-Rad52 antibodies
on'3'Cs irradiated
TGR-1 cells showed that both proteins are enriched in nuclear foci but they do
not
co-localize. Rad52-protein foci remained in the nucleus throughout the entire
time course,
while Rad51-protein foci were segregated into MN (data not shown). The same
holds true
for Gadd45 (data not shown) an inducible DNA-repair protein that is stimulated
by p53
(Smith et al., 1994, supra). Biochemical evidence further suggests specific
protein-protein
association between HsRad51 and p53 (Sturzbecher et al., 1996, supra).
However, after
anti-p53 antibody staining the RadS1 foci were not particularly enriched with
p53 protein
(data not shown). In addition, HsRad51 was reported to be associated with a
RNA
polymerase II (RNAPII) holoenyme (Maldonado et al., 1996, supra). Afthough
RNAPII was
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irnmunolocalized in discrete discrete nuclear foci, as reported previously
(Bregman et al.,
1995, supra), transcription complexes did not coincide with Rad51 foci (data
not shown).
Association of Rad51 Protein with DNA Fibers In a few (<1%) cells of
irradiated and
drug-treated cultures, we observed very elongated Rad51 structures, up to
several hundred
micrometer io length, that were eliminated from the nuclei. Since these fiber-
like structures
stained DAPI-positively, they are thought to contain single DNA molecules of
several
megabases covered with Rad51 (data not shown). Fluorescence in situ end
labeling
(FISEL) demonstrated that these DNA fibers contain fragmented DNA typical of
apoptosis
(data not shown). Sometimes the DNA fibers appeared to leak out of the nucleus
through
holes in the nuclear membrane and condense into micronuclei. In all cell
substrates studied,
a high percentage of MN displayed genome fragmentation (data not shown).
The association of Rad51 protein with DNA was also visible on experimentally
extended
chromatin fibers from irradiated cells. SDS lysis and mechanical stretching of
nuclear
chromatin across the surface of a glass slide can cause complete deattachment
of DNA
loops from the nuclear matrix, producing highly elongated, linear chromatin
fibers (Haaf, T.,
and Ward, D.C. (1994). Hum. Mol. Genet. 3, 629-633.; Heiskanen et al., 1994,
supra).
Immunofluorescence staining revealed linear strings of Rad51 label on these
stretched DNA
fibers (data not shown). By comparison with YAC hybridlzat-on signals on
similar
preparations (Haaf and Ward, 1994, supra), the length ofthe Rad51 fibers was
estimated 1-2
Mb.
Rad51-Protein Foci and Apoptosis To determine whether Rad51-positive MN
specifically
detect exposure to clastogens, analyses were performed in rat TGR-I cells with
the
aneuploidogen colcemid. This mitotic spindle poison causes lagging of whole
chromosomes
that are excluded into MN. Surprisingly, when colcemid-treated cells were
allowed to
recover for 24 hrs in drug-free medium, over 30% of the induced MN contained
very brightly
fluorescing Rad51 foci (Table 9). Some MN contained rod-like linear structures
(data not
shown) similar to those observed in Rad51-overexpressing cells. Most of these
Rad51-positive MN, 24 hrs after colcemid, did not contain fragmented DNA, as
determined by
simultaneous FISEL (Table 9). When cells were grown for one or two more days
in the
absence of the drug, the percentage of Rad51-containing MN decreased
dramatically. In
addition, the Rad51 protein was no longer concentrated in discrete foci but
appeared to
disperse throughout the entire MN volume. At the same time most MN became
apoptotic
and by FISEL their degraded DNA showed incorporation of fluorescent
nucleotides. Thus,
we conclude that mitotic arrest after colcemid triggers a cascade that induces
the elimination
of Rad51 protein into MN and drives apoptotic events. Our results seem to be
consistent
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with the hypothesis that apoptosis is a special form of aberrant mitosis
(Ucker, D.S. (1991 ).
New Biologist 3, 103-1009; Shietal., 1994, supra).
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Table 9. Rad51 Foci and Apoptosis in Colcemid-Induced Micronuclei of TGR-1
Cells
Treatment Number Percentage
of of Cells
micro- Showings
nuclei Rad51-/Rad51+/ Rad 51+/ Rad51-/
in
1000 cellsFISEL- FISEL- FISEL+FISEL+
None 93 75% 12% 2% 11
Colcemidb n.d. 85% 6% 0% 9%
1 day of recovery1293 54% 31 % 1 % 14%
2 days of recovery1061 45% 45% 6% 40%
3 days of recovery769 43% 7% 4% 46%
aApoptotic cells show fluorescence in situ end labeling (FISEL+), while cells
without genome
fragmentation show absence of labeling (FISEL-). "Rad51+" cells with Rad51
foci, "Rad51-+
cells without foci.
bTGR-1 cells were grown for 24 hrs in medium contaiing 0.1 Ng/ml colcemid to
induce
micronucleus formation (without inducing DNA damage). 185 of the colcemid-
treated cells
were arrested at metaphase, 17% showed multinuclei (>10 micronuclei), and 65%
had no
micronuclei. The cells were then allowed to recover for various times in the
absence of the
drug. 500 micronuclei were analyzed for each experiment.
Another more classical way for inducing apoptosis in vitro is the exposure of
TGR-1 cells to
the topoisomerase II inhibitor etoposide. After adding etoposide to the
culture medium, the
percentage of apoptotic cells steadily increased (Table 10). After 36 hrs half
of the cells
showed genome fragmentation and stained FISEL-positively. The nuclear events
of
apoptosis were accompanied by the appearance of Rad51 protein in nuclei and
MN. These
results indicate that different stimuli (e.g., irradiation and DNA-damaging
drugs,
topisomerase inhibitors, and aneuploidogens) that condem cells to apoptosis
can induce
focal concentration of Rad51 protein and its exclusion into MN.
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Table 10. Induction of Rad51 Foci and Apoptosis by Etoposide Treatment of TGR-
1 Cells
TreatmentPercentagePercentagePercentage of Percentage of Cells
. Cells with with
of of Cells Type 1e Foci Type Ila Foci
Apoptoticwithout
Cellsb Foci
in in in in
Nuclei MicronucleiNuclei Micronuclei
None 6% 93% 6% 0% 1 % 0%
2 hrs
after n.d. 92% 4% 1 % 1 % 2%
etoposide'
5 hrs n.d 92% 3% 2% 1% 2%
after
etoposide
17% 87% 8% 2% 1 % 2%
12 hrs
after
etoposide24% 79% 3% 8% 1 % 9%
18 hrs
after 33% 82% 2% 2% 6% 8%
etoposide
24 hrs 47% 83% 2% 5% 1 % 9%
after
etoposide
36 hrs
after
etoposide
aType I nuclei and micronuclei show weak to medium HsRad51 immunofluorescence,
whereas type II cells show strongly fluorescing foci. 500 cells were analyzed
for each
experiment.
bDetected by fluorescence in situ end labeling (FISEL+).
'Cells were grown in medium containing etoposide for the indicated times.
Higher-Order Nuclear Organization of Overexpressed Rad51 Protein Human 293
cells
were transfected with the HsRad51 gene. The resulting cell lines 710 and 717
constitutively
expressed a HsRad51-fusion protein. This overexpressed protein formed brightly
fluorescing linear structures inside the nucleus (Figure 7a). Some nuclei were
completely
filled with a network of rod-like structures (Figure 7b). Identical Rad51
structures were
observed in transformed rat TGR 928.1-9 cells, stably expressing the HsRad51
protein
without a tag epitope (data not shown). This suggests that Rad51 protein is
able to
assemble into higher-order structures within the highly ordered interphase
nucleus. The
linear nature of RadS 1 structures in overexpressing cells is reminscent of
the strings of
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Rad51-protein foci after DNA damage and colcemid treatment and in meiotic
cells (Haaf et
al., 1995).
However, in contrast to the situation after DNA damage, the overexpressed
HsRad51 protein
is not eliminated into MN. The numbers of Rad51-positive MN were not radically
different in
Rad51-overexpressing human 717 cells versus in 293 control cells and in rat
928.1-9
overexpressers versus in parental TGR-1 cells. This means that Rad51
overexpression
alone does not cause apoptosis. In exponentionally growing unsynchronized
cultures, 14%
of both 717 and 293 cells (500 cells were analyzed for each experiment) and 8%
of both
928.1-9 and TGR-1 cells showed cleavage of the cell's DNA by FISEL. We
conclude that
the segregation of Rad51 into MN is a specific behavior of apoptotic cells and
precedes
genome fragmentation.
Cell-Cycle Arrest of Cells with Focally Concentrated RadS1 Protein
Simultaneous
Rad51-protein immunofluorescence and antibromodeoxyuridine (BrdU) antibody
staining
demonstrated that nuclei with focally concentrated Rad51 protein do not
undergo
DNA-replication synthesis (data not shown). BrdU was incorporated into
replicating DNA of
unsynchronized cell cultures for 30 hrs. Rapidly growing transformed cell
lines (293, LNLB,
XPA, and XPF) which showed detectable Rad51 immunolabling in a percentage of
nuclei
even without induction of DNA damage as well as Rad51 overproducers (928.1-9
and 717)
were analyzed. For each experiment, 100 nuclei with prominent Rad51 foci and
100 nuclei
without detectable Rad51 foci were stained with fluorescent anti-BrdU
antibody. In the
widely different substrates tested, 80%-100% of the cells with focally
concentrated Rad51
protein were found to be BrdU-staining negative (Table 11). In contrast, 30%-
90% of the
cells without Rad51 foci from the same cultures showed BrdU incorporation,
indicative of
cycling cells. The BrdU-substituted DNA was located in discrete replication
sites throughout
the nucleus as reported previously (Nakayasu, H., and Berezney, R. (1989). J.
Cell Biol.
108, 1-11; Fox, et al., (1991) J. Cell Sci. 99, 247-253). This suggests that
even without
induction of DNA damage the cells with Rad51 foci are arrested during the cell
cycle or enter
S phase delayed of the Rad51-foci negative cells.
Table 11 Induction of Rad51 Foci after'3'Cs Irradiation of TGR-1 cells and
Their
Elimination into Micronuclei
Percentage Percentage of CellsPercentage of Cells
of with with
Cells withoutType la Foci Type Ila Foci
Foci
in in in in
Treatment Nuclei MicronuceliNuclei Micronuclei
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None 93% 6% 0% 1 % 0%
3 hrs after
900 rad 8% 0.4% 11 % 0.6%
137CS 80%
16 hrs 9% 8% 1 % 9%
after
900 rad
'3'Cs 73%
1 % 13% 1 % 13%
30 hrs
after
900 rad
,3'Cs 72% 0% 4% 0l0 6%
4 days
after
900 rad
'3'Cs 90%
aType I nuclei and micronuclei show weak to medium HsRad51 immuno-
fluorescence,
whereas type II cells show strongly fluorescing foci. 1000 cells were anlayzed
for each
experiment.
Rat TGR-1 cells are capable of normal physiological withdrawal into the
quiescent (Go)
phase of the cell cycle as well as resumption of growth following the
appropriate stimuli
(Prouty, et al., (1993). Oncogene 8, 899-907). In TGR 928.1-9 cells
o~erexpressing a
HsRad51 transgene(s), Go arrest upon serum starvation dramatically induced
HsRad:
1-protein foci (Table 12). Synchronous re-entry into the cell cycle after
feeding reduced the
percentage of HsRad51-foci positive cells to very low levels. However, new Go
arrest upon
contact inhibition following three population doublings increased the number
of cells with
nuclear Rad51 foci again. We therefore conclude that cells with prominent
nuclear Rad51
foci are most likely in Go or G1 phase of the cell cycle.
Table 12. Rad51 Foci in Micronuclei of Different Cell Substrates
Number of Percentage Percentage
of of
Cell substrate Micronuclei Rad51-Positive Rad51-Negative
in 1000
Treatment Cells Micronuclei Micronuclei
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TGR01
None 93 14% 86%
3 hrs after
900
rad'3'Cs 279 22% 78%
16 hrs after
900
rad'3'Cs 2719 28% 72%
4 days after
900
rad'3'Cs 1040 20% 80%
LNL8
None n.d. 23% 77%
None n.d. 26% 74%
XPA
None n.d. 18% 82%
Teratoma
None n.d. 10% 90%
3T3-Swiss
None 472 125% 88%
1000 cells were analyzed for each experiment
(On ward)Example 4
Rad51 Biochemical Assays Compatible for High Throughput Screening
As discussed above, homologous pairing and DNA strand exchange are unique
properties of
recombination proteins like Rad51 and RecA protein. Several assays are
available to detect
the homologous pairing and strand exchange activity of Rad5l. Strand exchange
reactions
catalyzed by human Rad51 are monitored with oligonucleotide substrates. These
substrates
are very convenient and easy to use because of machine synthesis and labeling
of
oligonucleotides either with fluorophores or with biotin. Rad51 (or RecA)
protein carries out
strand exchange in three distinct phases: I) presynapsis, during which RecA
protein binds
cooperatively and stoichiometrically to single-stranded DNA and forms a right
handed helical
nucleoprotein filament; II) synapsis, in which duplex DNA is taken up into the
nucleoprotein
filament and homologously aligns; and III) DNA strand displacement, which
produces a
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recombinant (heteroduplex) double-stranded DNA molecule and a displaced single-
stranded
DNA molecule (Figure 8).
Intermediates of the reaction known as joint molecules (also referred to as D-
loops) and the
final products can be monitored either by filter assays or by gel
electrophoresis.
A given test inhibitor can inactivate Rad51 by interfering with any one of
these three steps.
Hence, test Rad51 inhibitors can be added at the stage of presynapsis,
synapsis or strand
displacement stage of DNA strand exchange. These methods can be used to
determine
whether the inhibitors are acting by a) interfering with the cooperative
polymerization of
Rad51 on single-stranded DNA, b) affecting the pairing of the filament to the
homologous
DNA target or, c) affecting the process of strand exchange by inhibiting
hydrolysis of ATP.
Filter binding DNA strand exchange assays (solid phase-based) of Rad51
activity
which are compatible with high throughput screening. Filter binding assays are
based
on single-stranded DNA binding to nitrocellulose membranes under the
appropriate salt
conditions. The linear duplex DNA is labeled with bases linked to
fluorophores, 32P, or biotin
and the single-stranded DNA substrates are unlabeled. After uptake of the
double-stranded
DNA into the nucleoprotein filament, DNA base pair switching displaces the
complementary
strand of the parental duplex DNA. As a result of DNA strand displacement,
hybrid DNA
intermediates of the DNA strand exchange reaction contain single-stranded DNA
tails, and
one of the products of strand exchange is single-stranded DNA; both of which
can be
trapped on nitrocellulose filters. The unreacted linear double-stranded DNA
cannot bind to
the membrane and is washed away in the filtrate. Since the initial single-
stranded DNA used
to make the nucleoprotein filament is not labeled, it is not detected.
The filter binding assays are easy to use and extremely reliable. Typical
results of DNA
strand exchange by RecA protein as monitored by the filter assays is shown in
Figure 9.
These assays can be performed in high throughput mode, for example, using a 96
well
format on manifolds fitted with nitrocellulose membranes.
Fluorescence spectroscopy-based assays for monitoring the DNA strand exchange
activity of human Rad51 are highly specific and compatible with high
throughput
screening. Assays based on fluorescence to measure DNA pairing and DNA strand
exchange by human Rad51 protein have been developed. This approach enables one
to
distinguish homologous DNA pairing from subsequent DNA strand exchange.
Homologous
pairing of a single-stranded oligonucleotide with a duplex oligonucleotide is
measured by
fluorescence resonance energy transfer (FRET). Energy transfer between two
fluorescent
CA 02384733 2002-03-12
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dyes indicates their proximity. In the case of DNA, the proximity of two
complementary
strands labeled with dyes can be determined by FRET. For example, when a 5'-
Watson
strand labeled with fluorescein comes into proximity with a complementary 3'-
Crick strand
labeled with rhodamine, the overlap between its emission spectrum and the
excitation
spectrum of rhodamine allows the nonradiative transfer of energy to rhodamine
by
fluorescence resonance energy transfer (Bazemore et al., J. Biological Chem.,
272(23):14672-14682 (1997) and Bazemore, et al., PNAS USA, 94:11863-11868
(1997)).
Homologous DNA pairing assay by FRET. A test oligonucleotide labeled at its 3'
end with
fluorescein is used to form the nucleoprotein filament with Rad51. Rhodamine
is attached to
the 5' end of the complementary strand in duplex DNA. Homologous pairing
between the
two DNA molecules juxtaposes the two fluorescent molecules, resulting in
nonradiative
energy transfer from fluorescein to rhodamine when fluorescein is excited at
493 nm, near its
excitation peak. As a result of the energy transfer, the fluorescence emission
from
fluorescein is quenched and that from rhodamine is enhanced (Figure 10).
To measure homologous pairing of a test single-stranded oligonucleotide with
its
homologous duplex oligonucleotide, an 83-mer oligonucleotide (minus strand)
labeled at its
3' end with fluroescein is preincubated with 1.2 uM Rad51 protein in a
reaction mixture
containing 1 mM MgCl2, 25 mM HEPES (pH 7.4), 1 mM DTT, 2 mM ATP and 100 Ng of
BSA
per ml for 4 minutes at 37°C. The concentration of MgCl2 is increased
to 30 mM, and finally
3 NM duplex DNA (labeled with rhodamine at the 5' end of the plus strand) is
added.
DNA strand exchange assay by FRET. When the fluorescein and rhodamine are
juxtaposed by 20 A on opposite complementary strands, the emission from the
fluorescein is
quenched and that from rhodamine is enhanced as a result of energy transfer.
To measure
the strand exchange activity, both fluorophores are present in the duplex
where they are
juxtaposed. When strand exchange is completed, the two labeled strands are
separated
from each other as monitored by the enhanced emission from fluorescein (Figure
11 ).
To measure DNA strand exchange by FRET, Rad51 protein is added to unlabeled
single-
strand oligonucleotide for 4 min. at 37°C followed by the addition of
the filament to a reaction
mixture containing 30 mM MgCl2 and 3 uM duplex oligonucleotide (labeled on the
3' end of
the minus strand with fluorescein and on the 5' end of the plus strand with
rhodamine). The
final concentrations of ssDNA and protein are 3 NM and 1.2 NM, respectively.
Fluorescence emission spectra are recorded from 502 to 620 nm upon excitation
at 493 nm
on an SLM 8000C (SLM Aminco, Urbana, IL) or similar spectrofluorimeter.
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Example 5
Determination of Lead Compound Specificity
As discussed above, modulators for Rad51 biological activity can be assayed in
a number of
ways. The following assays can be used to assay for a change in biological
activity to
initially identify inhibitors, or to determine the specificity of identified
inhibitors: D-loop assay,
DNA dependent ATPase assay, nucleoprotein filament assay, and complementary
single-
strand hybridization assay. These assays are unique features of the Rad51
protein and
determine the specificity of, for example, small molecules that inhibit Rad51
protein activity.
D-loop assay. The non-enzymatic uptake of a homologous single-stranded DNA by
a
negatively supercoiled DNA leads to the formation of a DNA displacement loop
(D-loop,
Figure 12) (Holloman et al., PNAS, USA, 76:1638-1642 (1975)). However,
nonenzymatic
formation of D-loops is seen only at elevated temperatures (65°C).
RecA and Rad51 enzymes both catalyze D-loop formation under physiological
conditions.
Negative superhelicity is not required in these reactions catalyzed by RecA or
Rad51. Only
members of RecA and Rad51 protein families can catalyze the formation of D-
loops under
physiological conditions.
Several standard assays monitor D-loop formation by DNA recombination enzymes.
Most
commonly used assays use duplex DNA (either supercoiled DNA or a linear duplex
DNA)
and a linear single-stranded DNA (either an oligonucleotide or a relatively
longer linear
single-stranded DNA) as substrates. The D-loop products are analyzed either by
filter
assays or by gel electrophoresis. Filter assays are simple, fast and samples
can be
analyzed in a plate format. In these experiments, the target duplex DNA is
labeled and the
single-stranded DNA substrate is unlabeled. After uptake of the single-
stranded DNA into
duplex DNA, the tails of the unincorporated single-stranded DNA of the hybrid
molecules are
trapped on the filter and only the D-loops can be detected. Unreacted double-
stranded DNA
(superhelical DNA or linear duplex DNA) do not bind to the membrane. If
necessary, the D-
loop products can also be monitored by following the separation of hybrids by
gel
electrophoresis.
DNA dependent ATPase assay. The ATP binding domain is highly conserved
throughout
evolution in the homologues of RecA protein (Heyer, Experentia, 50:223-233
(1994)). The
unique feature of the ATPase activity of RecA and Rad51 is that this activity
is DNA
dependent. Rad51 hydrolyzes ATP only in the presence of single-stranded DNA
and has no
ATPase activity in the absence of DNA.
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To monitor ATP hydrolysis, labeled ATP is incubated with single-stranded DNA
and Rad51.
The reaction mixture is incubated at 37°C for 30 min. and an aliquot of
the reaction is applied
directly onto CEL 300 PEI/UV2~ thin layer chromatography plates to separate
the product of
hydrolysis (ADP) from the substrates. Non-DNA dependent ATPases are used as
controls
for these reactions. Small molecule compounds that inhibit the ATPase activity
of Rad51
would not be expected to affect the activity of other ATPase enzymes.
Assay of nucleoprotein filament formation. Rad51 protein binds cooperatively
to DNA to
form a right-handed helix. The resulting protein-DNA complex is an active
nucleoprotein
filament which catalyzes DNA pairing and DNA strand exchange reactions. The
DNA helix
inside the filament is extended 1.5 times the size of B-form duplex length.
This structure of
the nucleoprotein filament is a hallmark feature of RecA and Rad51 proteins
and is DNA
sequence independent. The DNA inside the filament is completely protected from
phosphodiesterases, as RecA and Rad51 proteins bind to and protect the
phosphate
backbone from cleavage. Formation of nucleoprotein filaments is easily
monitored by
protein-based filter binding assays. Another DNA binding protein is used as a
control.
Complementary single-strand DNA renaturation assay. Rad51 protein promotes the
hybridization of complementary strands of DNA under specific conditions in
which the
spontaneous renaturation of complementary strands does not occur.
Hybridization activity is
easy to monitor in a DNA micro array format or by filter binding assays. Other
single-strand
annealing proteins, such as SSB, will be used as controls.
Example 6
Down-regulation of Human Rad51 Protein by Antisense Oligodeoxynucleotides in
Human
Breast, Brain and Prostate Cells
An essentially complete reduction in the expression of Rad51 protein by using
specific
human Rad51 antisense oligodeoxynucleotides in a variety of human tumor cell
lines has
been achieved herein. The human Rad51 mRNA sequence is shown in Figure 15A and
15B
wherein the regions complementary to the antisense molecules SEQ ID NOS:1-9
are
underlined. Figure 16 shows the antisense oligonucleotides.
Specific antisense oligonucleotides targeted against the 5' untranslated
region (AS4 (SEQ ID
N0:2), AS5 (SEQ ID N0:3)), the 3' untranslated region (AS6, 7, 8, and 9 (SEQ
ID N0:4, 5, 9
and 6, respectively) and the coding region (AS3 (SEQ ID N0:1) of Rad51 mRNA
were used
herein. Also independently tested herein were AS1 (SEQ ID N0:7) and AS2 (SEO
ID N0:8)
as indicated in Ohnishi, et al., Biochemical and Biophysical Res. Comm.,
245:319-324
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(1998), incorporated by reference herein in its entirety. AS1 (SEQ ID N0:7),
AS2 (SEQ ID
N0:8) and AS3 (SEQ ID N0:1 ) target the coding sequence of both mouse and
human
Rad51 mRNA.
These antisense oligonucleotides were tested against human cell lines derived
from breast,
brain and prostate tumors. Combinations of two antisense oligonucleotides
reduced the
levels of Rad51 protein 30% to 96% compared to sense, scrambled and untreated
controls.
Figure 13 shows the levels of Rad51 protein after treatment of MDA-MB-231
cells with some
of the most effective antisense oligonucleotides used. Quantitation is shown
in Figure 14.
Combination of AS3 and AS6 induces a near complete reduction of Rad51.
Similar results were obtained in other human breast (MCF-7), brain (U87 and
U251 ), and
prostate tumor cell lines (LnCAP and DU145) tumor cell lines.
The degree of reduction of Rad51 protein with antisense oligonucleotides in
MDA-MB-231
breast cancer cells are as follows: AS3 (SEQ ID N0:1 ), 70%; AS4 (SEQ ID
N0:2), 70%;
AS5 (SEQ ID N0:3), 40%; AS6 (SEQ ID N0:4), 80%; AS7 (SEQ ID N0:5), 90%; AS9
(SEQ
ID N0:6), 50%. AS8 (SEQ ID N0:9) was lethal, Rad51 protein levels were
undetectable.
SEQ ID NOS:7, 8, 10, 11 and 12 had no determinable reduction. Similar results
ocurred with
MCF-7 breast cancer cells and U87 glioblastoma cells.
Further experiments will include the following cell lines, WT p53 and p3,
BT20, BT549,
MCF7, MDA-MB-231, MDA-MB-468, and HMEC-4144-2.
Example 7
Cytotoxicity and Cell Growth Assays and Sensitization of Human Tumor Cells to
Radiation
and DNA-damaging Chemicals
A sulforhodamine B-based optical density assay of protein in cultured cells
will be used
(Skenen, et al., J. Natl. Cancer Inst., 82:1107-12 (1990); Skehan, et al.,
Cell Biol. Toxicol.
2:357-368 (1986) as a cell-based high throughput drug screen for inhibitors of
Rad51
activity. The phenotypes screened for are cytotoxicity and growth inhibition
in target tumor
(breast, brain and prostate) and control (non tumorigenic) cell lines. Cells
are placed in 96-
well microtiter plates. Next, these drugs are introduced after one day of
culture, and treat for
an additional 96 hours. Assays begin at the beginning of drug treatment, at 48
hours and at
96 hours. Qualitative changes are monitored by comparing the amount of
cellular protein
present at the beginning of the drug incubation period with the amount of
protein present in
control and test cultures at day 3 and day 5 of growth. Other time points will
be added if
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necessary. Quantitative drug-induced changes in culture growth will be
evaluated using the
doubling time and fractional growth rate (Skehan, Assays of cell growth and
cytotoxicity, Cell
Growth and apoptosis: A practical approach, G. Studzinski, ed., 2nd Ed., pp
169-191 ).
Also determined is Rad51 activity in these samples using methods outlined
above to
determine whether the biological effects measured are specific to alterations
in Rad51
activity. This can be done by comparing the results to antisense
oligodeoxynucleotides that
specifically down-regulate Rad51 protein.
Sensitization of human tumor cells to radiation and DNA-damaging chemicals.
Following DNA damage, cells may survive by undergoing transient cell arrest
and regrowth,
or may remain in permanent cell cycle arrest; or the cells may simply die. The
extent to
which Rad51 down-regulation sensitizes cells to DNA damage can be determined
by
assaying how Rad51 down-regulation shifts the dose response curves for DNA
damaging
agents (radiation, BCNU, cyclophosphamide, cis-platin) in systems that measure
growth,
survival and death. The effects on cell growth are screened using the
sulforhodamine B
assay described above. Assayed is the effect on cell survival using a
clonigenic assay to
determine the surviving fraction of clonigenic cells. Apoptosis is assayed
using either a flow
cytometric assay for subdiploid fractions or by using the TUNEL method, which
utilizes
terminal deoxynucleotide transferase to incorporate fluorescein-conjugated
deoxyuridine
triphosphate into DNA nicks formed in apoptotic cells. As noted above,
parallel experiments
with antisense oligodeoxynucleotides will assure the specificity.
Example 8
Human Tumor Xenografts Implanted in Nude Mice
Human tumors in a human host and human tumors transplanted into athymic mice
respond
similarly to antineoplastic drugs, and therefore xenografts grown in athymic
mice provide an
invaluable preclinical model to validate candidate agents identified in cell-
based assays.
There are two aims to these experiments. One is to determine whether Rad51
down-
regulation has a growth inhibitory effect. The second evaluates Rad51 down-
regulation as a
sensitizer to DNA damage. The experimental blueprint for these studies in
human tumor
xenografts is: a) define growth curves for the untreated xenograft and for
xenografts treated
with inhibitors of Rad51. This aim will also determine how growth is related
to Rad51 down
regulation; b) choose appropriate doses of DNA damaging agents that will allow
us to
compare the responses of the control and Rad51 down-regulated cell lines; c)
quantitate the
amount of sensitization to DNA damage.
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Implantation and growth of human tumor xenografts in nude mice. The target
tumor
cell lines chosen herein can all form tumors in nude mice. 1 x 105, 1 x 106,
and 1 x 10' cells
taken from cell culture in the log phase of growth will be injected into the
flanks of nude mice,
and the tumor will be measured daily for 50 days, until the tumor reaches
2000mm3, or until
the animal becomes debilitated. Each group will have 5 mice. Generally tumors
are treated
when they reach a volume of 50-100mm3.
Determination of lethal dose. This is a simple determination of lethality of
the lead
compound found by escalating single doses in mice which are indexed to
intracellular
concentrations that down-regulate Rad51 in culture. Toxicity will be
investigated by physical
exam and organ histopathology.
Treatment of tumors with lead compounds. Once tumors reach a treatable size,
animals
will be injected with escalating doses of the lead compound. Tested will be
both systemic
and local routes of administration. The tumors will be examined for Rad51
activity and for
volume. Toxicity of the compound in these animals will be assayed by weight
gain, serum
chemistries and organ histopathology (including liver, lung, kidneys, heart,
gastrointestinal
tract and brain).
Dose finding for DNA damage. The growth characteristics of the cell lines are
established
and then various doses of DNA damaging agents (for example, 0, 5, 10 and 15 Gy
for
radiation) will be used to define a series of growth curves that describe
response. This
information will allow selection of doses to compare control cell lines to the
Rad51 down-
regulated cell lines. Each group will have at least 5 animals and
appropriately matched
controls.
Sensitization of radiation and chemotherapy treatments by lead compounds.
Control
and Rad51 down-regulated cell lines will be grown and treated with DNA
damaging agents at
times and doses determined above. If the growth rates of control and down-
regulated lines
are similar, direct comparisons can be made between growth delays caused by
radiation in
sets of modified and unmodified cell lines. However, if their growth rates
differ, a dose will
be chosen that will allow us to measure the average size of tumors at a
specific point in time.
If the difference between the average sizes of control (i.e., not down-
regulated) tumors
treated and not treated with DNA damaging agents is DU and the difference
between the
average sizes of down-regulated tumors treated and not treated with DNA
damaging agents
is 0M, our hypothesis would predict that DU - OM > 0. Studies include 4 groups
of 15
animals for each lead compound. Assuming a normal distribution, this would
provide 90%
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power to detect a difference DU - DM of 1.5 times the standard deviation,
assuming a one-
tailed hypothesis test is a = 0.05.
Other references specifically incorporated by reference are Haaf, T. (1995)
Pharmac. Ther.
65, 19-46; Haaf, T., and Schmid,M. (1991) Exp. Cell Res. 192, 325-332; and
Owaga, et al,
(1993) Science 259, 1896-1899.
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