Note: Descriptions are shown in the official language in which they were submitted.
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INHIBITOR OF BRN-3B AND ITS USE FOR THE TREATMENT OF~BREAST AND OVARIAN
CANCERS
Field of the invention
This invention relates to inhibitors of Brn-3b expression and/or activity and
screening
methods for the identification of such inhibitors. It further relates to the
use of said
inhibitors in the treatment of cancer.
Background to the invention
The POU (Pit-Oct-Unc) family of transcription factors was originally defined
on the
basis of a common 150-160 amino acid domain which was found in found in the
mammalian transcription factor Pit-1, Oct-l and Oct-2, and the nematode
regulatory
protein Uno-86 (for review see l, 2). The common POU domain constitutes the
DNA
binding domain of these proteins and consists of two portions, a POU-specific
domain
which is unique to the POU factors and a POU-homeodomain which is related to
the
homeobox found in a number of other transcription factors.
Following the identification of the original POU family transcription factors,
a number
of other members of this family have been defined (1-3) and shown to play
critical roles in
the regulation of gene expression. Moreover, mutations in the genes encoding
several of
these factors have been shown to be responsible for particular human diseases.
Thus, for
example, mutation in the gene encoding Pit-1 has been shown to result in a
failure of
pituitary gland development and consequent dwarfism in both mice and humans
(for
review see 4) whilst a mutation in the gene encoding Brn-4 has been shown to
result in X-
linked deafness (5). More recently, the gene encoding the POU factor Brn-3c
has been
shown to be mutated in a family with progressive late onset deafness (6).
Brn-3a, Brn-3b and Brn-3c are closely related members of the POU family which
are
encoded by different genes (7} and are expressed in distinct but overlapping
patterns in the
developing and adult nervous system (3, 8-11). in addition however, expression
of Brn-3a
and Brn-3b has also been detected in some non-neuronal cells such as cervical
epithelium
(9, 12).
Brn-3a has been shown to be over-expressed in aggressive neuroendocrine
tumours
(14), whilst both Brn-3a and Brn-3b are expressed at high levels in human
neuroblastomas
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(15, 16). Brn-3a and Bm-3b have previously been detected in the human breast
cancer cell
line MCF-7 (13). We have recently shown that mean Brn-3a levels are increased
over 300
fold in human samples exhibiting cervical intra-epithelial neoplasia grade 3
(CTN3)
compared to normal human cervical samples but Brn-3b levels are similar in the
two
groups of samples ( 12}.
Summary of the invention
The present invention is based on the finding that mammary tumour tissue which
has
reduced expression levels of the BRCA-1 gene shows elevated expression of the
Brn-3b
POU family transcription factor. The BRCA-1 gene was identified on the basis
of its
mutation in a number of cases of familial breast cancer indicating that its
inactivation can
cause this disease. Athough BRCA-1 does not appear to be mutated in cases of
sporadic
breast cancer, its expression has been shown to be reduced in a number of
cases.
The elevated expression of Brn-3b is not found in normal mammary cells, benign
tumours or malignant tumour samples which do not exhibit reduced levels of the
BRCA-1
IS gene. 1n contrast no correlation between the level of BRCA-1 expression and
the
expression of the related POU family transcription factor Brn-3a. Moreover,
Brn-3b but
not Brn-3a can strongly repress the BRCA-1 promoter approximately 20 fold in
transfections carried out in mammary tumour cells.
Thus, Brn-3b may play an important role in regulating expression of BRCA-1 in
mammary tumours with enhanced expression of Brn-3b resulting in reduced BRCA-1
expression and thereby being potentially involved in tumour development. The
repression
of Brn-36 expression by either pharmacological or by gene therapy procedures
represents a
potential method for treating breast cancers.
Thus, the invention provides an inhibitor of Brn-3b expression and/or activity
for use
in a method of treatment of the human of animal body. Such inhibitors are
useful, in
particular, in the treatment of breast cancer and/or ovarian cancer.
Screens may be carried out to identify inhibitors of Brn-3b expression and/or
activity.
Accordingly, the invention also provides:
a method for identifying an inhibitor of Brn-3b expression comprising:
(a) providing a test construct comprising a Brn-3b promoter operably linked to
a coding
seguence;
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(b) contacting a substance to be tested with the test construct under
conditions that would
permit the polypeptide encoded by the said coding sequence to be expressed in
the
absence of the said substance; and
(c) determining whether the said substance inhibits the expression of Brn-3b;
and
a method for identifying an inhibitor of Brn-3b activity comprising:
(a) providing a Brn-3b polypeptide or a homologue thereof, or a fragment
thereof;
(b) contacting a substance to be tested with the Brn-3b polypeptide under
conditions that
would permit activity of the polypeptide in the absence of said substance; and
(c) determining whether the said substance inhibits the activity of Brn-3b.
The invention further provides a pharmaceutical composition comprising an
inhibitor
of Brn-3b expression and/or activity and a therapeutically acceptable carrier
or diluent.
As noted above, the inhibitors of Brn-3b expression and/or activity may be
used in
treating breast or ovarian cancer and therefore the invention provides a
method of treatment
of treating a host suffering from breast cancer or ovarian cancer, which
method comprises
administering to the host a therapeutically effective amount of an inhibitor
of Brn-3a
expression and/or activity.
Brief description of the Figures
Figure 1 shows levels of Brn-3a mRNA as determined by RT/PCR assay in
normal/benign mammary material or in malignant mammary tumours from pre-
menopausal or post-menopausal women. Bar designates the mean with standard
deviation
(SD) and population size 'n' .
Figure 2 shows levels of Brn-3b mRNA as determined by RT/PCR assay in
normal/benign mammary material or in malignant mammary tumours from pre-
menopausal or post-menopausal women. Bar designates the mean with standard
deviation
(SD) and population size 'n' .
Figure 3 shows comparison of the mRNA levels of Brn-3b and BRCA-1 in malignant
mammary tumour material obtained from pre (panel a) or post (panel b)
menopausal
women or from benign mammary material (panel c). Brn-3b levels are shown as
solid
bars, BRCA-1 levels as open bars.
Figure 4 shows comparison of the mRNA levels of Brn-3a and BRCA-I in malignant
mammary tumour material obtained from pre (panel a) or post (panel b)
menopausal
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women or from benign mammary material (panel c). Brn-3a levels are shown as
solid
bars, BRCA-I levels as open bars.
Figure 5 shows comparison of BRCA-I levels in mammary tumours with low or high
levels of Bm-3b. Bar designates the mean with standard deviation (SD) and
population
size 'n'.
Figure 6 shows a luciferase reporter assay of MCF7 cells co-transfected with a
promoter/reporter construct containing the foil length BRCA-I promoter
together with
expression vector lacking any insert (V) or the same vector expressing either
Brn-3a or
Brn-3b. Values have been equalized relative to the activity obtained in the co-
transfection
with the reporter construct and empty expression vector (set at 100%) and are
the mean of
five determinations whose standard deviation is shown by the bars.
Detailed description of the invention
Any suitable inhibitor of Brn-3b expression or activity may be employed in the
present invention. For example, the expression of Brn-3b in a cell may be
reduced by
presence in that cell of a polynucleotide which can hybridize to the Brn-3b
mRNA.
Therefore a polynucleotide which is capable of hybridizing to Brn-3b mRNA can
constitute an appropriate inhibitor of Bm-3b expression. In this regard, two
approaches are
as follows:
(I) Antisense RNA
The delivery of a nucleic acid construct which allows the expression of an RNA
which
can hybridize to the Brn-36 mRNA. This results in the formation of an RNA-RNA
duplex
which may result in the direct inhibition of translation and/or the
destabilization of the
target message, for example, rendering susceptibility to nucleases. Therefore,
the nucleic
acid construct will typically lead to the expression of a polynucleotide which
hybridizes to
the ribosome binding region or the coding region of the Brn-3b mRNA.
(2) Antisense oligonucleotides
An oligonucleotide is delivered which hybridizes to the Brn-3b mRNA. Antisense
oligonucleotides are postulated to inhibit target gene expression by
interfering with one or
more aspects of RNA metabolism including processing, translation and metabolic
turnover.
Chemically modified oligonucleotides may be used and may enhance resistance to
nucleases and/or cell permeability.
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Antis ense
The coding sequence of Brn-3b is given in SEQ ff3 NO:1. An inhibitor of Brn-3b
comprises a polynucleotide which can hybridize to the Brn-3b mRNA. Typically
such a
polynucleotide will be an RNA molecule. Such a polynucleotide may hybridize to
all or
S part of the Brn-3b mRNA. Typically the polynucleotide will be complementary
to all of or
a region of the Brn-3b mRNA. For example, the polynucleotide rnay be the exact
complement of all or a part of Brn-3b mRNA. However, absolute complementarity
is not
required and preferred polynucleotides which have sufficient complementarity
to form a
duplex having a melting temperature of greater than 40°C under
physiological conditions
are particularly suitable for use in the present invention. The polynucleotide
may be a
polynucleotide which hybridises to the Brn-3b mRNA under conditions of medium
to high
stringency such as 0.03M sodium chloride and 0.03M sodium citrate at from
about 50 to
about 60 degrees centigrade.
It is preferred that the poiynucleotide hybridizes to the region of the mRNA
l5 corresponding to the coding sequence defined by nucleotides 213 to 392 of
SEQ lI7 NO:1.
The polynucieotide may hybridize to all or part of this region. However, a
polynucleotide
may be employed which hybridises to all or part of the 5'- or 3'-untranslated
region of the
mRNA. These regions correspond to nucleotides 1 to 212 and 1446 to 3110 of SEQ
B?
NO:1.
The polynucleotide will typically be at least 40, for example at least 60 or
at least 80,
nucleotides in length and up to 100, 200, 300, 400, 500, 600 or 700
nucleotides in length or
even up to a few nucleotides, such as five or ten nucleotides, shorter than
SEQ D7 NO: 1.
When the polynucleotide is an antisense RNA it may be expressed in a cell from
a
recombinant replicable vector. Such a replicable vector comprises a
polynucleotide which
when transcribed gives rise to antisense RNA. Preferably the poiynucleotide
giving rise to
the antisense RNA is operably linked to a control sequence which is capable of
providing
for the transcription of the polynucleotide giving rise to the antisense RNA.
The term
"operably linked" refers to a juxtaposition wherein the components described
are in a
relationship permitting them to function in their intended manner. A control
sequence
"operably linked" to a sequence giving rise to an antisense RNA is Iigated in
such a way
that transcription of the sequence is achieved under conditions compatible
with the control
sequences.
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The vectors may be for example, plasmid or virus vectors provided with an
origin of
replication, optionally a promoter for transcription to occur and optionally a
regulator of .
the promoter. The vectors may contain one or more selectable marker genes, for
example
an ampicillin resistance gene in the case of bacterial plasmid or a neomycin
resistance gene
for a mammalian vector. Vectors may be used in vitro, for example for the
production of
antisense RNA or used to transfect or transfom a host cell. The vector may
also be
adapted to be used in vivo, for example in a method of gene therapy.
Promoters/enhancers and other expression regulation signals may be selected to
be
compatible with the host cell for which the expression vector is designed. For
example,
mammalian promoters, such as b-actin promoters, rnay be used. Tissues-specific
promoters, in particular neuronal cell specific promoters (for example the
tyrosine
hydroxylase (TH), L7, or neuron specific enolase (NSE) promoters), are
especially
preferred. Viral promoters may also be used, for example the Moloney murine
leukaemia
virus long terminal repeat (MMLV LTR), the promoter roes sarcoma virus (RSV)
LTR
promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter,
herpes
simplex virus promoters or adenovirus promoters. All these promoters are
readily available in
the art. Preferred promoters are tissue specific promoters such as the casein
gene promoter.
Vectors may further include sequences flanking the polynucleotide giving rise
to antisense
RNA which comprise sequences homologous to eukaryotic genomic sequences,
preferably
mammalian genomic sequences, or viral genomic sequences. This will allow the
introduction
of the polynucleotides of the invention into the genome of eukaryotic cells or
viruses by
homologous recombination. In particular, a plasmid vector comprising the
expression cassette
flanked by viral sequences, for example HSV1 or HSV2 sequences, can be used to
prepare a
viral vector, fpr example an HSV vector, suitable for delivering the
polynucleotides of the
invention to a mammalian cell. Other examples of suitable viral vectors
include retroviruses,
including ientiviruses, adenovinases and adeno-associated viruses. Gene
transfer techniques
using these viruses are will known to those skilled in the art. Retrovirus
vectors for example
maybe used to stably integrate the polynucleotide giving rise to the antisense
RNA into the
host genome. Replication-defective adenovirus vectors by contrast remain
episomal and
therefore allow transient expression.
Antisense oligonucleotides
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An antisense oligonucleotide will typically have a sequence such that it will
bind to the
Brn-3b mRNA. Therefore it will typically have a sequence which is the
complement of a
region of the sequence shown in SEQ )13 NO: 1. An antisense oligonucleotide
will generally be
from 6 to 40 nucleotides in length. Preferably it will be from I2 to 20
nucleotides in length.
Generally the oligonucleotide used will have a sequence that is absolutely
complementary
to the target sequence. However, absolute complementarity may not be required
and in general
any oligonucleotide having sufficient complementarity to form a stable duplex
(or triple helix
as the case may be) with the target nucleic acid is considered to be suitable.
The stability of a
duplex (or triplex) will depend inter alia on the sequence and length of the
hybridizing
oligonucleotide and the degree of complementarity between the antisense
oligonucleotide and
the target sequence. The system can tolerate less complementarity when longer
oligonucleotides are used. However oligonucleotides, especially
oligonucleotides of from 6 to
40 nucleotides in length, which have sufficient complementarity to from a
duplex having a
melting temperature of greater than 40°C under physiological conditions
are particularly
suitable for use in the present invention. The polynucleotide may be a
polynucleotide which
hybridises to under conditions of medium to high stringency such as 0.03M
sodium chloride
and 0.03M sodium citrate at from about 50 to about 60 degrees centigrade.
Antisense oligonucleotides may be chemically modified. For example,
phosphorothiaate
oligonucleotides may be used. Other deoxynucleotide analogs include
methylphosphonates,
phosphoramidates, phosphorodithioates, N3'PS'-phosphoramidates and
aligoribonucleotide
phosphorothioates and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide
methylphosphonates.
Alternatively mixed backbone oligonucleotides (MBOs) may be used. MBOs contain
segments of phosphothioate oligodeoxynucleotides and appropriately placed
segments of
modified oiigodeoxy- or oligoribonucleotides. MBOs have segments of
phosphorothioate
linkages and other segments of other modified oligonucleotides, such as
methyiphosphonate,
which is non-ionic, and very resistant to nucleases or 2'-O-
alkyloligoribonucleotides.
Administration
The vectors and antisense oligonucleotides of the invention, optionally with
an additional
therapeutic polypeptide or nucleic acid/vector encoding said therapeutic
polypeptide, may thus
be administered to a human or animal in need of treatment. Cancers which may
be treated
using the vectors, viral strains. antisense oligonucleotides and compositions
of the invention
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include breast or ovarian cancer and, in particular, breast or ovarian cancer
in which Btn-3b
expression is up-regulated such as non-familial breast cancer. The condition
of a patient
suffering from such a cancer can thus be improved.
The antisense oligonucleotides and compositions comprising antisense
oligonucleotides of
the invention together may be administered by direct injection into the site
to be treated, for
example mammary tissue. Preferably the antisense oligonucleotides are combined
with a
pharmaceutically acceptable carrier or diluent to produce a pharmaceutical
composition.
Suitable carriers and diluents include isotonic saline solutions, for example
phosphate-buffered
saline. The composition may be formulated for parenteral, intramuscular,
intravenous,
subcutaneous, intraocular or transdermal administration.
The dose at which an antisense oiigonucleotide is administered to a patient
will depend
upon a variety of factors such as the age, weight and general condition of the
patient, the cancer
that is beins treated and the stage which the cancer has reached, and the
particular antisense
oligonucieotide that is being administered. A suitable dose may however be
from 0.1 to 100
mg/kg body weight such as 1 to 40 mg/kg body weight.
The polynucleotides giving rise to antisense RNA of the invention may be
administered
directly as a naked nucleic acid construct. Uptake of naked nucleic acid
constructs by
mammalian cells is enhanced by several known transfection techniques for
example those
including the use of transfection agents. Example of these agents include
cationic agents (for
example calcium phosphate and DEAF-dextran} and lipofectants (for example
lipofectarn T''~
and transfectamT'~f )
Typically, nucleic acid constructs are mixed with the transfection agent to
produce a
composition.
Preferably the naked nucleic acid constn.ict, viral vector comprising the
polynucleotide or
composition is combined with a pharmaceutically acceptable Garner or diluent
to produce a
pharmaceutical composition. Suitable carriers and diluents include isotonic
saline solutions, for
example phosphate-buffered saline. The composition may be formulated for
parenteral,
intramuscular, intravenous, subcutaneous, intraocular or transdermal
administration.
The pharmaceutical composition is administered in such a way that the
polynucleotide of
the invention, viral vector for gene therapy, can be incorporated into cells
at an appropriate area.
When the polynucleotide of the invention is delivered to cells by a viral
vector, the amount of
virus administered is in the range of from 104 to 10'~ pfu, preferably from
105 to I07 pfu, more
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preferably about 106 pfu for herpes viral vectors and from 106 to 10~°
pfu, preferably from 10'
to 109 pfu, more preferably about 10$ pfu for adenoviral vectors. When
injected, typically 1-2
ml of virus in a pharmaceutically acceptable suitable Garner or diluent is
administered. When
the polynucleotide of the invention is administered as a naked nucleic acid,
the amount of
nucleic acid administered is typically in the range of from 1 ~g to 10 mg.
Where the polynucleotide giving rise to the antisense RNA is under the control
of an
inducible promoter, it may only be necessary to induce gene expression for the
duration of the
treatment. Once the condition has been treated, the inducer is removed and
expression of the
polypeptide of the invention ceases. This will clearly have clinical
advantages. Such a system
may, for example, involve administering the antibiotic tetracycline, to
activate gene expression
via its effect on the tet repressor/VP16 fusion protein.
The use of tissue-specific promoters will be of assistance in the treatment of
disease using
the polypeptides, polynucleotide and vectors of the invention. For example,
several
neurological disorders are due to aberrant expression of particular gene
products in only a small
subset of cells. It will be advantageous to be able express therapeutic genes
in only the relevant
affected cell types, especially where such genes are toxic when expressed in
other cell types.
The routes of administration and dosages described above are intended only as
a guide
since a skilled physician will be able to determine readily the optimum route
of administration
and dosage for any particular patient and condition.
Screening for inhibitors of Brn-3b expression and/or activity
The invention provides a method for identifying an inhibitor of Brn-3b
expression
comprising:
{i) providing a test construct comprising a Brn-3b promoter operably. linked
to a coding
sequence;
(ii) contacting a substance to be tested with the test construct under
conditions that would
permit expression of the polypeptide encoded by the said coding sequence to be
expressed
in the absence of the said substance; and
(iii) determining whether the said substance inhibits the expression of Brn-
3b.
Any suitable assay format may be used for identifying an inhibitor of Brn-3b
expression.
Typically, however, the assay is catxied out using a cell harbouring a
promoter:reporter
polypeptide construct. A typical assay is as follows:
- a defined number of cells are inoculated, in for example 100p,1 of growth
medium, into the
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wells of a plastics micro-titre plate in the presence of a substance to be
tested.
- optical density (OD) at 590nm may be measured as may expression of the
reporter
potypeptide according to any method appropriate for the reporter polypeptide
being used.
- the micro-titre plates are covered and incubated at 37 °C in the
dark.
- the OD is read again and expression of the reporter polypeptide assayed at
convenient time
intervals. The change in OD is a measure of cell proliferation. Control
experiments can be
carned out, in which the substance to be tested is omitted.
Also the substance may be tested with any other known promoter to exclude the
possibility that the test substance is a general inhibitor of gene expression.
Any reporter polypeptide may be used, for example GUS or GFP are used. GUS is
assayed by measuring the hydrolysis of a suitable substrate, for example S-
bromo-4-chloro-3-
indolyl-~i-D-glucoronic acid (X-gluc)or 4-methyiumbelliferyt-~3-glucuronide
(MUG). The
hydrolysis of MUG yields a product which can be measured fluorometrically. GFP
is
quantified by measuring fluorescence at 590nm after excitation at 494nm. These
methods are
1 S well known to those skilled in the azt.
Alternatively the coding sequence may be the Brn-3b coding sequence itself In
such an
experiment a mammary cancer cell line which exhibits Brn-3b overexpression
could be used.
The expression of Bm-3b may be followed by for example, Northern/RNA blotting,
Westem/antibody blotting, RNA in situ hybridization or immunotocalisation.
The invention further provides a method for identifying an inhibitor of Brn-3b
activity
comprising:
(i) providing a Brn-3b polypeptide or a homologue thereof, or a fragnnent
thereof;
(ii) contacting a substance to be tested with the Brn-3b polypeptide under
conditions that
would permit activity of the potypeptide in the absence of said substance; and
{ii) determining whether the said substance inhibits the activity of Brn-3b.
Suitable Brn-3b for the assay can be obtained, for example, recombinantly by
any method
known to those skilled in the art. Any suitable format may be used for.
identifying an inhibitor
of Brn-3b.
Atso the substance may be tested with any other known transcription factor to
exclude the
possibility that the test substance is a general inhibitor of transcription
factors activity.
In addition to the Brn-3b polypeptide, the reaction mixture can contain a
suitable buffer.
A suitable buffer includes any suitable biological buffer that can provide
buffering capability at
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a pH conducive to the reaction requirements of the Brn-3b polypeptide.
Test substances
A substance which inhibits the expression of Brn-3b may do so by binding
directly to the
promoter, thus preventing the initiation of transcription. Alternatively a
substance could bind to
a protein which is associated with the promoter and is required for
transcription. This may
result in reduced levels of transcription.
The Brn-3b promoter:reporter polypeptide constructs of the invention may
include the
untranslated region of the Brn-3b gene. Therefore a substance may reduce Brn-
3b expression
by binding to the untranslated region of the Brn-3b gene. This could prevent
the initiation of
translation. Alternatively a substance could bind to a protein associated with
the untranslated
region and prevent the protein associating with the untranslated region.
A substance which inhibits the activity of Brn-3b may do so by binding to one
or both of
the enzymes. Such enzyme inhibition may be reversible or irreversible. An
irreversible
inhibitor dissociates very slowly from its target enzyme because it becomes
very tightly bound
1 S to the enzyme, either covalently or non-covalently. Reversible inhibition,
in contrast with
irreversible inhibition, is characterised by a rapid dissociation of the
enzyme-inhibitor complex.
The test substance may be a competitive inhibitor. In competitive inhibition,
the enzyme
can bind substrate (forming an enzyme-substrate complex) or inhibitor (enzyme-
inhibitor
complex) but not both. Many competitive inhibitors resemble the substrate and
bind the active
site of the enzyme. The substrate is therefore prevented from binding to the
same active site. A
competitive inhibitor diminishes the rate of catalysis by reducing the
proportion of enzyme
molecules bound to a substrate.
The inhibitor may also be a non-competitive inhibitor. In non-competitive
inhibition,
which is also reversible, the inhibitor and substrate can bind simultaneously
to an enzyme
molecule. This means that their binding sites do not overlap. A non-
competitive inhibitor acts
by decreasing the turnover number of an enzyme rather than by diminishing the
proportion of
enzyme molecules that are bound to substrate.
The inhibitor can also be a mixed inhibitor. Mixed inhibition occurs when an
inhibitor
both effects the binding of substrate and alters the turnover number of the
enzyme.
A substance which inhibits the activity of Brn-3b may also do so by binding to
the
substrate. The substance may itself catalyze a reaction of the substrate, so
that the substrate is
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not available to the enzyme. Alternatively the inhibitor may simply prevent
the substrate
binding to the enzyme. .
Suitable candidate substances include antibody products (for example,
monoclonal and
polyclonal antibodies, single chain antibodies, chimaeric antibodies and CDR-
grafted
antibodies) which are specific for Brn-3b Furthermore, combinatorial
libraries, defined
chemical entities, peptide and peptide mimetics, oligonucleotides and natural
product libraries
may be screened for activity as inhibitors of Brn-3b in assays such as those
described below.
The candidate substances may be chemical compounds. The candidate substances
may be used
in an initial screen of, for example, ten substances per reaction, and the
substance of these
batches which show inhibition tested individually.
Inhibitors of Brn-3b expression and/or activity
An inhibitor of Brn-36 expression and/or activity is one which produces a
measurable
reduction in Brn-3b expression and/or activity in the assays described above.
Preferred
substances are those which inhibit Brn-3b expression and/or activity by at
least 10%, at least
I 5 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%,
at least 80%, at least
90%, at Ieast 95% or at least 99% at a concentration of the inhibitor of 1 pg
mi '', l Opg mf',
100~.g ml'', 500pg ml'', lmg mf'° lOmg mf', I OOmg ml''. The percentage
inhibition represents
the percentage decrease in expressionlactivity in a comparison of assays in
the presence and
absence of the test substance. Any combination of the above mentioned degrees
of percentage
inhibition and concentration of inhibitor may be used to define an inhibitor
of the invention,
with greater inhibition at lower concentrations being preferred.
Candidate substances which show activity in assays such as those described
below can
then be tested on mammary cancer cell lines for example. Candidate inhibitors
could be tested
far their ability to attenuate Brn-3b expression in mammary cancer cell lines
in which Brn-3b is
up-regulated and also for the effect on BRCA-1 expression in mammary cancer
cell lines in
which BRCA-1 is down-regulated.
Human use
Inhibitors of Brn-3b expression and/or activity identified by the screening
procedures
described above may be used to treat breast or ovarian cancer and, in
particular, breast or
ovarian cancer in which Brn-3b expression is up-regulated such as non-familial
breast cancer.
The condition of a patient suffering from a cancer can therefore be improved
by administration
of such an inhibitor. A therapeutically effective amount of such an inhibitor
may be given to a
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human patient in need thereof.
The formulation of an inhibitor for use in preventing or treating breast Or
ovarian cancer
will depend upon factors such as the nature of the substance identified,
whether a
pharmaceutical or veterinary use is intended, etc. Typically an inhibitor is
formulated for use
with a pharmaceutically acceptable carrier or diluent. For example it may be
fornmlated for
topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular,
transdermal or oral
administration. A physician will be able to determine the required route of
administration for
each particular patient. The pharmaceutical carrier or diluent may be, for
example, an isotonic
solution.
The dose of inhibitor may be determined according to various parameters,
especially
according to the substance used; the age, weight and condition of the patient
to be treated; the
route of administration; and the required regimen. Again, a physician will be
able to determine
the required route of administration and dosage for any particular patient.
The following Example illustrates the invention.
EXAMPLE
Materials and Methods
Unless indicated otherwise, the methods used are standard biochemistry and
molecular biology
techniques. Examples of suitable general methodology textbooks include
Sambrook et al.,
Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current
Protocols in
Molecular Biology (1995), John Wiley and Sons, Inc.
Normal and breast cancer samples
Total RNA from normal mammary gland and from malignant breast tumours (from
pre
and post menopausal women) was obtained from the Candis Tissue Bank Research
Centre
Liverpool University
Reverse transcri~tase/polymerase chain reaction assay
About 0.I pg of RNA from each sample was used as a template for cDNA
synthesis. Th
synthesized cDNA was used in RT-PCR assays as previously described (17) using
the
following oligonucleotide primers: Brn-3a: 5' GTCGACATGGACTCGGACACG-3', 3'-
ACGGTGAATGACTCCCCCGA-5 ; Brn-3b: S'GGAGAAGAAGCGCAAGC-3',
3'CTGAGAACGGGAGAGGTCT-5'. Amplification of the invariantly expressed human
cyclophilin mRNA used as a control was carried out in parallel using the
following primers: 5'-
TTGGGCCGCGGTACTCCTTTCA-3', and 3'-TTTCGTATGGCCCAGGACCG-5'.
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in all cases, 20~e1 of each PCR product was fractionated on a 2% agarose gel
and blotted
onto Hybond-N+ nylon membrane (Amersham International, Little Chalfont, United
Kingdom) and hybridized with homologous complimentary 3'P-lablled probes.
Membranes
were exposed to films, (Eastman Kodak Co., Rochester, NY) and the subsequent
autoradio5naphs were then analyzed using a densitometer (Bio-Rad Laboratories,
Hercules,
CA). We have previously shown that this blotting procedure, in conjunction
with the RT-PCR
conditions used, allows accurate quantification of the Brn-3a and Brn-3b mRNAs
relative to the
constitutively expressed cyclophilin mRNA (9, 12, 17).
Plasmid constructs
The Brn-3a and Brn-3b expression vectors contain full length cDNA clones under
the
control of the moloney murine leukaemia virus promoter and have previously
been described
(7. 17). The BRCA-1 promoterlreporter constnzcts contain 4 kilo-bases or 400
bases of
upstream sequence containing the BRCA-1 a and (3 promoters cloned into the
pGL2 luciferase
vector.
Transient transfection
MCF7 cells were routinely grown in Dulbecco's modified Eagle's medium
containing L-
gIutamate and phenol red which was supplemented with 10% foetal calf serum and
l Ong of
insulin per ml. Before experiments were carried out subconfluent cells were
maintained in
phenol red-free Dulbecco's modified Eagle's medium containing 10% dextran
coated charcoal-
striped foetal calf serum prepared according to the method described by
Migliaccio et crl. , (18}
and long of insulin per ml for 72 hours. The medium was replaced by ~ ml of
fresh medium
12 hours prior to transfection. Transfection of piasmid DNA was carried out
according to the
method of Gorman (19). Routinely Sug of reporter DNA and SlZg of each
expression vector
were transfected into 5x10' cells and the cells harvested after 72 hours. The
amount of DNA
taken up by the cells in each case was measured by slot blotting of lSpl of
the extract and
hybridization with a probe derived from the ampicillin resistance gene in the
plasmid vector
(20). Differences in the intensity of the bands were measured by densitometry
and used to
equalize the volumes of extracts used for subsequent assay. All transfections
also included an
internal reporter gene encoding the renilla luciferase with a dual luciferase
assay being carried
out to control for transfection efficiency. Luciferase assays were done as
described by the
manufacturers (Promega) protocol with results measured on a Turner 20-E
iuminometer.
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Results
To quantitate the level of Brn-3a and Brn-3b mRNAs we used a reverse
transcriptase/polymerase chain reaction (RT/PCR) assay which we have
previously used to
reliably quantitate these mRNAs in small amounts of clinical and other
material {I2, 15). This
assay was used to compare the levels of Brn-3a and Brn-3b mRNAs in normal
breast tissue or
benign breast tumour material with that obtained from malignant breast tumours
from pre- or
post-menopausal women. The mean levels of Brn-3a {figure I ) and Brn-3b
(figure 2) both
showed some increase in the material prepared from the pre-menopausal
malignant tumours
compared to that obtained from normal/benign tumour material and a further
increase was
observed in both cases in the post-menopausal tumour material but this was not
statistically
significant (p>0.05 in all comparisons of normal versus tumour levels).
Interestingly, however, clear differences were noticed in the distribution
pattern of Brn-3a
and Brn-3b expression levels in the different tumour samples. Thus, Brn-3a
levels appeared to
show a continuous distribution between the different samples. In contrast, Brn-
3b levels
I 5 exhibited a bipartite distribution in the different tumour samples with
approximately 40% of the
samples showing low levels of Brn-3b within the range observed for the normal
samples whilst
the remaining samples showed much higher levels outside the normal range. This
bipartite
distribution was observed for tumour samples obtained from both pre-and post-
menopausal
women.
To determine whether this bipartite distribution had any functional
significance, we
searched for differences between the tumour samples with respectively low or
high levels of
Brn-3b expression. In particular, we examined the level in the different
samples of the mRNA
encoding BRCA-1 protein which is mutated in many cases of familial breast
cancer (21, 22)
and which has also been reported to exhibit reduced expression in non-
hereditary (sporadic)
breast cancer (23). Most interestingly, the levels of BRCA-1 in the different
samples showed
an inverse expression pattern to that observed with Brn-3b. Thus, the samples
with low levels
of Brn-3b exhibited high levels of BRCA-1 which was observed in the samples
derived from
both pre-menopausal (figure 3a) and post-menopausal (figure 3b) women,
although the effect
was particularly marked in the samples from pre-menopausal women. In contrast
no significant
variation in BRCA-1 mRNA levels was noted in different samples of normal
mammary
material or material from benign tumours which also had similar levels of Brn-
3b (figure 3c).
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In contrast to the results with Brn-3b, the level of BRCA-I mRNA in malignant
material
from pce-menopausal (figure 4a) or post-menopausal (figure 4b) women showed no
correlation
with the level of Brn-3a mRNA in the different samples. Moreover, similar
levels of BRCA-1
mRNA were observed in the normal or benign material despite the different Brn-
3a levels in
the different samples (figure 4c). The specific association of high Brn-3b
levels with low
BRCA-I expression was confirmed by dividing the samples into those with high
Brn-3b and
low Brn-3b. As illustrated in figure 5, BRCA-I levels were highly elevated in
the sample
group with low Brn-3b compared to the level in those with high Brn-3b. This
effect was
statistically significant (p<0.001 comparing BRCA-I levels in the high and low
Brn-3b
expressing groups).
These findings therefore raise the possibility that the Brn-3b transcription
factor may have
an inhibitory effect on expression of BRCA-I, with the high levels of Brn-3b
observed in some
malignant mammary tumour samples being associated with a low level of BRCA-I.
To
investigate this possibility further, we co-transfected MCF7 cells with
expression vectors
encoding Brn-3a or Brn-3b and a construct in which a four kilo-base fragment
of the BRCA-1
promoter drives expression of a luciferase reporter gene.
In this experiment (figure 6} the activity of the promoter was reduced by
approximately
50% in the sample co-transfected with Brn-3a compared to the level observed in
the sample co-
transfected with the corresponding expression vector lacking any insert.
However, a much
more dramatic inhibitory effect was observed in the sample co-transfected with
the Brn-3b
expression vector where promoter activity was reduced to approximately 5% of
that observed
in the sample transfected with empty expression vector. Similar results were
also obtained with
a reporter construct containing only 400 base pairs of the BRCA-1 promoter
indicating that this
effect was dependent on this short promoter region (data not shown). This
effect was specific
to the BRCA-I promoter, since transfection of the same Bm-3b expression vector
with
reporters containing an oestrogen response element resulted in a strong
stimulatory effect on the
promoter in accordance with our previous results (13) (data not shown). Hence
Brn-3b can
indeed directly inhibit the BRCA-I promoter in co-transfections into breast
cancer cells.
CA 02343354 2001-03-13
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References
I . Vernjzer, C.P. and van der Vliet, P.C. POU domian transcription factors.
Biochimica et.
Biophysica Acta 1173:1-21, 1993.
2. Ryan, A.K. and Rosenfeld, M.G. POU domain family values:- flexibility,
partnerships
and developmental codes. Genes artd Developme~et 11:1207-1225, 1997.
3. He, X., Treacy, M.N., Simmons, D.M., Ingraham, H.A., Swanson, L.S. and
Rosenfeld,
M.G. Expression of a large family of POU-domain regulatory genes in mammalian
brain
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4. Anderson, B. and Rosenfeld, M.G. Pit-1 determines cell types during
development of
the anterior pituitary gland. Jour>7al of Biological C'hemist~y 269:29335-
29338, 1994.
5. de Kok, Y.J.M., van der Maarel, S.M., Bitner-Glindzicz, M., Huber, L,
Monaco, A.P.,
Malcolm, S., Pembrey, M.E., Ropers, H-H. and Cremers, F.P.M. Association
between
X-linked deafness and mutations in the POU domain gene POU3F4. Science 267:685-
688,
1995.
6. Vahaua, O., Morell, R., Lynch, E.D." Weiss, S., Kagen, M.F., Ahitau, N.,
Morrow, 3.E.,
Lee, M.K." Skvorak, A.B., Morton, C.C., Blumenfeld, A., Frydman, M., Friedman,
T.B., King,
M-C. and Avraham, K.B. Mutation in transcription factor POU4F3 associated with
progressive
hearing loss in humans. Scie~tce 279:1950-1954, 1998.
7. Theil, T., McLean-Hunter, S., Zornig, M. and Moroy, T. Mouse Brn-3 family
of POU
transcription factors: a new amino terminal domain is crucial for the
oncogenic activity of
Brn-3A. Nucleic Acids Research 21:5921-5929, 1993.
8. Gerrero, M.R., McEvilly, R.J., Tuner, E., Lin, C.R., O'Connell, S., Jenne,
K.J., Hobbs,
M.V. and Rosenfeld, M.G. Brn 3.0: A POU domain protein expressed in the
sensory immune
and endocrine systems that functions on elements distinct from known octamer
motifs.
Proc.Natl.AcadSci. USA 90:10841-10845, 1993.
9. Lillycrop, K.A., Budhram-Mahadeo, V.S., Lakin, N.D., Terrenghi, G., Wood,
J.N.,
Polak, J.M. and Latchman, D.S. A novel POU family transcription factor is
closely related to
Brn-3 but has a distinct expression pattern in neuronal cells. Narcliec Acids
Research
20:5093-5096, 1992.
10. Turner, E.E., Jenne, K.J. and Rosenfeld, M.G. Bm-3.2: A Bm-3-related
transcription
factor with distinctive central nervous system expression and regulation by
retinoic acid.
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Neuron 12:205-218, 1994.
11. Ninkina, N.N., Stevens, G.E.M., Wood, J.N. and Richardson, W.D. A novel
Brn-3 like
POU transcription factor expressed in subsets of rat sensory and spinal cord
neurons. Nucleic
Acids Research 21:3175-3 I 82, 1993.
12. Ndisdang, D., Morris, P.J., Chapman, C., Ho, L., Singer, A. and Latchman,
D.S The
HPV activating transcription factor Brn-3a is over expressed in CIN3 lesions.
.louYnal of
Clinicallnvestigation IOl:I687-1692, 1998.
13. Budhram-Mahadeo, V., Parker, M. and Latchman, D. S.
The POU domain factors Brn-3a and Brn-3b interact with the estrogen receptor
and
differentially regulate transcriptional activity via an ERE. Molecular and
Cellular° Biology
18:1029-1041, 1997.
14. Leblond-Francillard, M., Picon, A., Bertagna, X. and Keyzer, Y. High
expression of
the POU factor Brn-3a in aggressive neuroendocrine tumors. .lou>"nal of
Clinical
~rTdc~crinvlogry and Mclahvlisni 82:89-94, 1997.
15. Smith, M.D. and Latchman, D.S. The functionally antagonistic POU family
transcription factors Brn-3a and Brn-3b show opposite changes in expression
during the growth
arrest and dii~erentiation of human neuroblastoma cells. Irrtematiotral .lour-
nal of Cancer
67:653-660, 1996.
16. Deans, Z.C., Dawson, S.J., Kiliman, M.W., Wallace, D., Wilson, M.C. and
Latchman,
D.S. Differential regulation of genes encoding synaptic proteins by the Oct-2
transcription
factor. Mol Brain Re,r. 51:1-7, 1997.
17. Morris, P.J., Theil, T., Ring, C.J.A., Lillycrop, K.A., Mdroy, T. and
Latchman, D.S.
The opposite and antagonistic effects of the closely related POU family
transcription factors on
the activity of a target promoter are dependent upon differences in the POU
domain. Molecular
and Cellulaf~ Biolo~ry 14:6907-6914, 1994.
18. Migliaccio, A., Pagano, M. and Auricchio, F. Immediate and transient
stimulation of
protein tyrosine phosphorylation by estradiol in MCF-7 cells. Oncogene 8:2183-
2191, 1993.
19. Gorman, C.M. High ef~icency gene transfer into mammalian cells. In: DNA
cloning, a
pnatical appr°oach, edited by Glover, D.M. IRI, Press, 1985, p. 143-
190.
20. Abken, H. and Reifenrath, B. A procedure to standardize CAT reporter gene
assay.
Nucleic Acids Resecn~ch 20:3527, 1992.
CA 02343354 2001-03-13
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1/4
SEQUENCE LISTING
(1)
GENERAL
INFORMATION:
(i) APPLICANT:
(A) NAME: UNIVERSITY COLLEGE LONDON
(B) STREET: Gower Street
(C) CITY: London
(E) COUNTRY: United Kingdom
(F) POSTAL CODE (ZIP): WC1E 6BT
(ii) TITLE OF INVENTION: TREATMENT OF CANCER
(iii) NUMBER OF SEQUENCES: 1
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOSIMS-DOS
(D) SOFTWARE: PatentIn Release #1Ø
Version #1.30 (EPO)
(2)
INFORMATION
FOR
SEQ
ID
N0:
1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3110 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:213..1445
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
AGACCTCGGC ACCCGTTCAG ACTGACAGCA GAGGCGGCGA.AGGAGCGCGT AGCCGAGATC 60
AGGCGTACAG AGTCCGGAGG CGGCGGCGGG TGAGCTCAAC TTCGCACAGC CCTTCCCAGC 120
3 0 TCCAGCCCCG GCTGGCCCGG CACTTCTCGG AGGGTCCCGG CAGCCGGGAC CAGTGAGTGC I80
CTCTACGGAC CAGCGCCCCG GCGGGCGGGA AG ATG ATG ATG ATG TCC CTG AAC 233
Met Met Met Met Ser Leu Asn
1 5
AGC AAG CAG GCG TTT AGC ATG CCG CAC GGC GGC AGC CTG CAC GTG GAG 281
3 5 Ser Lys Gln Ala Phe Ser Met Pro His G1y Gly Ser Leu His Val Glu
10 15 20
CCC AAG TAC TCG GCA CTG CAC AGC ACC TCG CCG GGC TCC TCG GCT CCC 329
Pro Lys Tyr Ser Ala Leu His Ser Thr Ser Pro Gly Ser Ser Ala Pro
25 30 35
40 ATC GCG CCC TCG GCC AGC TCC CCC AGC AGC TCG AGC AAC GCT GGT GGT 377
SUBSTITUTE SHEET (RULE 26)
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2l4
Ile Ala Pro Ser Ala Ser Ser Pro Ser Ser Ser Ser Asn Ala Gly Gly
40 45 50 . 55
GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GGC GGA GGC CGA AGC 425
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Arg Ser
60 65 70
AGC AGC TCC AGC AGC AGT GGC AGC AGC GGC GGC GGG GGC TCG GAG GCT 473
Ser Ser Ser Ser Ser Ser Gly Ser Ser Gly G1y G1y G1y Ser Glu Ala
75 80 85
ATG CGG AGA GCC TGT CTT CCA ACC CCA CCG AGC AAT ATA TTC GGC GGG 521
Met Arg Arg Ala Cys Leu Pro Thr Pro Pro Ser Asn ile Phe Gly Gly
90 95 100
CTG GAT GAG AGT CTG CTG GCC CGC GCC GAG GCT CTG GCA GCC GTG GAC 569
Leu Asp Glu Ser Leu Leu Ala Arg Ala Glu Ala Leu Ala Ala Val Asp
105 110 115
ATC GTC TCC CAG AGC AAG AGC CAC CAC CAC CAT CCA CCC CAC CAC AGC 617
ile Val Ser Gln.Ser Lys Ser His His His His Pro Pro His His Ser
120 125 130 135
CCC TTC AAA CCG GAC GCC ACC TAC CAC ACT ATG AAT ACC ATC CCG TGC 665
Pro Phe Lys Pro Asp Ala Thr Tyr His Thr Met Asn Thr Ile Pro Cys
140 145 150
ACG TCG GCC GCC TCT TCT TCA TCG GTG CCC ATC TCG CAC CCT TGC GCG 713
Thr Ser Ala Ala Ser Ser Ser Ser Val Pro Ile Ser His Pro Cys Ala
155 160 165
TTG GCG GGC ACG CAC CAC CAC CAC CAC CAT CAC CAC CAC CAC CAC CAC 761
Leu Ala Gly Thr His His His His His His His His His His His His
170 175 180
CAA CCG CAC CAG GCG CTG GAG GGC GAG CTG CTG GAG CAC CTG AGT CCC 809
Gln Pro His Gln Ala Leu Glu Gly Glu Leu Leu Glu His Leu Ser Pro
185 190 ~ 195
3 0 GGG CTG GCC CTG GGC GCT ATG GCG GGC CCC GAC GGC GCT GTG GTG TCC 857
Gly Leu Ala Leu Gly Ala Met Ala Gly Pro Asp Gly Ala Val Val Ser
200 205 210 215
ACG CCG GCT CAC GCG CCG CAC ATG GCC ACC ATG AAC CCC ATG CAC CAA 905
Thr Pro Ala His Ala Pro His Met Ala Thr Met Asn Pro Met His Gln
3 5 220 225 230
GCA GCG CTC AGC ATG GCC CAC GCG CAC GGG CTG CCG TCG CAC ATG GGC 953
Ala Ala Leu Ser Met Ala His Ala His Gly Leu Pro Ser His Met Gly
235 240 245
TGC ATG AGC GAC GTG GAC GCC GAC CCG CGG GAC CTG GAG GCA TTC GCC 1001
40 Cys Met Ser Asp Val Asp Ala Asp Pro Arg Asp Leu Glu Ala Phe Ala
250 255 260
SUBSTITUTE SHEET (RULE 26)
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GAG CGC TTC AAG CAG CGA CGC ATC AAG CTG GGG GTG ACC CAG GCA GAT 1049
Glu Arg Phe Lys Gln Arg Arg Ile Lys Leu Gly Val Thr Gin Ala Asp
265 270 275
GTG GGC TCC GCG CTG GCC AAC CTC AAG ATC CCC GGC GTG GGC TCG CTT 1097
val Gly Ser Ala Leu Ala Asn Leu Lys Ile Pro Gly Val Gly Ser Leu
280 285 290 295
AGC CAG AGC ACC ATC TGC AGG TTC GAG TCC CTC ACA CTG TCC GAC AAT 1145
Ser Gln Ser Thr Ile Cys Arg Phe Glu Ser Leu Thr Leu Ser His Asn
300 305 310
AAT ATG ATC GCG CTC AAA CCC ATC CTG CAG GCA TGG CTC GAG GAG GCC 1193
Asn Met Ile Ala Leu Lys Pro Ile Leu Gln Ala Trp Leu G1u Glu Ala
315 320 325
GAG AAG TCC CAC CGC GAG AAG CTC ACC AAG CCT GAA CTC TTC AAT GGC 1241
Glu Lys Ser His Arg Glu Lys Leu Thr Lys Pro G1u Leu Phe Asn Gly
330 335 340
GCG GAG AAG AAG CGC AAG CGC ACG TCC ATC GCT GCG CCA GAG AAG CGC 1289
A1a Glu Lys Lys Arg Lys Arg Thr Ser Ile Ala Ala Pro Glu Lys Arg
345 350 355
TCG CTC GAA GCC TAC TTT GCC ATT CAG CCT CGG CCC TCC TCT GAA AAG 1337
Ser Leu Glu Ala Tyr Phe Ala Ile Gln Pro Arg Pro Ser Ser Glu Lys
360 365 370 375
ATC GCC GCC ATC GCG GAG AAG CTG GAC CTG AAG AAA AAC GTG GTG CGC 1385
Ile Ala Ala Iie Ala Glu Lys Leu Asp Leu Lys Lys Asn Val Ual Arg
380 385 390
GTC TGG TTC TGC AAC CAG AGG CAG AAA CAG AAA AGA ATG AAA TAT TCC 1433
Ual Trp Phe Cys Asn Gln Arg Gln Lys Gln Lys Arg Met Lys Tyr Ser
395 400 405
GCC GGC ATT TAG AAGACTCTTG GCCTCTCCAG AGACGCCCCT TTCCTCGTCC 1485
Ala Gly Ile
410
GCTCTTTTCT CTCCTCTCTT CTGCCTCTTT TCACT'rfTGG CGACTAGAAA1545
CAATTCCAGT
AAATGTGAAT CTCGACAAAT CGAGGACTGA AGAGGGAGCG AACGAGCGAA1605
CAACTGAGCC
CAAGCCGGTG AGAATGTGAA ACAGTTTCTC AAAGGAAAGA ATAACAAAAG1665
ATGGTATTTG
TCTGTTGTAG CAAAGTTGTC CCTTTGAACC CCACCTCGGC TTCTTCAGAG1725
GAAGTGTGGA
3 5 GATGGCTGTT TGCAGGAAGG CAGACGAGAC AGTGTTTAAA 1785
AAGTCCACAA GAATGATCAA
GTAAGATTTG TfITTATTCT TACAGACATC ACCCGTGTTC AAGTTTAAAA1845
GTACACTiTG
CAACTATTTT TCAGAAATAG AAATTGATTC AGGACTAAAA CTTTAAACTA1905
GAGTTGATGC
TTAATGTGAT AGAGACATCT CTAAAGTATT TTGAATTTTA AAAAAAGATG1965
GCAGATTTTC
SUBSTITUTE SHEET (RULE 26)
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TGCATTTACA CTGTATATTA TATATATATT TTTATTGTGG 2025
TTCTTACCCC CTTTTCCTTC
TCTGAAGTGT TAATGCTTAA GAAAAGAGTT GCGCCTGCTG 2085
TGTTCACTGA TCTTGAAAGC
TATTATTAGA TTATTGCAGA ACAACCCTCT GTAAATTATT 2145
AATTTATCTC TCTAGCAACT
TAATTTTGTG CACATTCTAA TTAATTAAAC TTCTTCCGTC 2205
TAAAAAAAGT GGGGGAAATG
TATAGCTAGT AACGTTCAAA AAATTTTGTT TGATGAGTTT 2265
ACCGAATTTT TACAGCT1TC
CTCCTATACT GTGTTCCTTT TGACCCATTT GTATATTCTC 2325
ACTTGAATGA AGATTGTTTT
TTTCTTTGTT TTTACTGGTA GTGTTCTGAT TTGTGAGTCG 2385
ACACTCAGTA ATGGATGTCT
TAATCGTGTA GACCTGATTC ACTGTCTGAA GTATTGTTTA 2445
CTTCGTTACA TATTTAATGG
GGATTCCCAC ATTGTCCCCA TGACACATGA GC~CTCTCAC 2505
TTACCCTTAC ACACACACAC
ACACACACAC ACACACCTCT AACAGAAGGG AAGAAGCAGT 2565
TGGAAGCATG ACCGATGCAC
CATTTTCTAG TTTTAGGTGC ATTTGCCACT TGGTGTTTGC 2625
CCTTCAGATT TTAGATTTCA
CCAAGGTATT TCAGTCTTCC AGTTTTCAAT TGCTTTGTTG 2685
GCTACATGTT AATATTTATA
GGAATACTTC AGTTTTTCCT TTTGGAGGTT TGTTTGTAGA 2745
AAAACTAATT TGAACTATAA
GAAAGACAGT GCACTGCTTG TAAATTCACA TTGTTTGGAA 2805
AAATTCTTTT GGAACAAAAA
ATTAGGTACA TGATAACTGG TACCTTATCT ACTGTAAATA 2865
TTTCATTAAA AATGATGCAC
ACATAGATAT ATTCTTACAA ATTFTGCTGT ATTGCTGTTC 2925
TCTTTGAGGC TCTCCAAAGT
CTTGAGTTCT GTATATGGCC TGGTTTCTTG TTTTTATTAA 2985
TAGATGGTTT ATTTACTATG
GTAATGTATT AATTTATTTT TGGTGTTGTT CGATTGTCTT 3045
TCATTGAAGA GATAATTTTA
ATGTTTTATT GGCAACGTAT GCTGCTTfTT CATTAAAATA 3105
TGCTATTAAA ATTAAATGGC
TTTTA 3110
SUBSTITUTE SHEET (RULE 26)
