U.S. patent application number 12/715292 was filed with the patent office on 2010-08-12 for genetic alterations associated with cancer.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Joe W. Gray, Yiling Lu, Gordon B. Mills, Laleh Shayesteh.
Application Number | 20100204125 12/715292 |
Document ID | / |
Family ID | 27419801 |
Filed Date | 2010-08-12 |
United States Patent
Application |
20100204125 |
Kind Code |
A1 |
Shayesteh; Laleh ; et
al. |
August 12, 2010 |
GENETIC ALTERATIONS ASSOCIATED WITH CANCER
Abstract
The present invention provides new probes for the detection of
chromosomal alterations associated with cancer, particularly
ovarian cancer. The probes bind selectively with target nucleic
acid sequences at 3q26.
Inventors: |
Shayesteh; Laleh; (Foster
City, CA) ; Gray; Joe W.; (San Francisco, CA)
; Mills; Gordon B.; (Houston, TX) ; Lu;
Yiling; (Houston, TX) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The Regents of the University of
California
Oakland
CA
Board of Regents, The University of Texas System
Austin
TX
|
Family ID: |
27419801 |
Appl. No.: |
12/715292 |
Filed: |
March 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08905508 |
Aug 4, 1997 |
7670767 |
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12715292 |
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08837046 |
Apr 5, 1997 |
6110673 |
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08905508 |
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08783729 |
Jan 16, 1997 |
6277563 |
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08837046 |
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Current U.S.
Class: |
514/1.1 ;
435/375; 435/6.11; 435/6.14; 435/7.1; 506/9; 514/19.3; 514/44R |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; C12Q 2600/156 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
514/12 ; 435/6;
506/9; 435/7.1; 435/375; 514/44.R |
International
Class: |
A61K 38/16 20060101
A61K038/16; C12Q 1/68 20060101 C12Q001/68; C40B 30/04 20060101
C40B030/04; G01N 33/53 20060101 G01N033/53; C12N 5/02 20060101
C12N005/02; A61K 31/7088 20060101 A61K031/7088; A61P 35/04 20060101
A61P035/04 |
Claims
1. A method of screening for the presence of chromosomal
alterations associated with cancer in a sample, the method
comprising: contacting a nucleic acid sample from a human patient
with a probe which binds selectively to a target nucleic acid
sequence at 3q26.3, wherein the probe is contacted with the sample
under conditions in which the probe binds selectively with the
target nucleic acid sequence to faun a stable hybridization
complex; and detecting the formation of a hybridization
complex.
2. The method of claim 1, wherein the target nucleic acid sequence
is in a PIK3CA gene.
3. The method of claim 1, wherein the nucleic acid sample is from a
ovarian sample from the patient.
4. The method of claim 1, wherein the probe selectively hybridizes
to a region between markers D3S1275 and D3S1548.
5. The method of claim 1, wherein the probe selectively hybridizes
to the same nucleic acid sequence as a YAC clone having coordinates
806D8 or 945H6 in the Genethon/CEPH mega YAC library.
6. The method of claim 1, wherein the probe is a member of an
array.
7. The method of claim 1, further comprising contacting the sample
with a reference probe which binds selectively to a centromeric
DNA.
8. The method of claim 1, wherein the step of detecting the
hybridization complex comprises determining the copy number of the
target sequence.
9. The method of claim 1, further comprising the step of contacting
the sample with a probe which binds selectively to a target nucleic
acid sequence at 19q13.1-19q13.2.
10. The method of claim 9, wherein the target nucleic acid sequence
is in an AKT2 gene.
11. A kit for the detection of a chromosome alterations correlated
with cancer, the kit comprising a compartment which contains a
nucleic acid probe which binds selectively to a target nucleic acid
sequence in 3q26, wherein the probe binds selectively with the
target nucleic acid sequence.
12. The kit of claim 11, wherein the probe selectively hybridizes
to a region between markers D3S1275 and D3S1548.
13. The kit of claim 11, wherein the probe selectively hybridizes
to the same nucleic acid sequence as a YAC clone having coordinates
806D8 or 945H6 in the Genethon/CEPH mega YAC library.
14. A method of screening for the presence of chromosomal
alterations associated with cancer in a sample, the method
comprising: contacting the sample with an antibody specifically
immunoreactive with a protein antigen encoded by a nucleic acid
sequence at 3q26.3; and detecting the formation of an
antigen/antibody complex.
15. A method of inhibiting the pathological proliferation of cancer
cells, the method comprising inhibiting the activity of a gene
product of an endogenous gene at 3q26.3.
16. The method of claim 15, wherein the endogenous gene maps to a
region between markers D3S1275 and D3S1548.
17. The method of claim 15, wherein the endogenous gene maps to a
region defined by YACs having coordinates 806D8 and 945H6 in the
Genethon/CEPH mega YAC library.
18. The method of claim 15, wherein the endogenous gene is
PIK3CA.
19. A method of inhibiting the pathological proliferation of
ovarian cancer cells in a patient, the method comprising
administering a therapeutically effective dose of an inhibitor of
PI kinase to the patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending application
Ser. No. 08/905,508 filed Aug. 4, 1997, which is a
continuation-in-part of application Ser. No. 08/837,046 filed Apr.
5, 1997, now U.S. Pat. No. 6,110,673, which is a continuation of
application Ser. No. 08/783,729 filed Jan. 16, 1997, now U.S. Pat.
No. 6,277,563, all of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Molecular genetic mechanisms responsible for the development
and progression of many cancers remain largely unknown.
Identification of sites of frequent and recurring allelic deletion
or gain is a first step toward identifying some of the important
genes involved in the malignant process. Previous studies in
retinoblastoma (Friend, et al. Nature, 323:643-6 (1986)) and other
cancers (Cawthon, et al., Cell, 62:193-201 (1990); Baker, et al.,
Science, 244:217-21 (1989); Shuin, et al., Cancer Res, 54:2832-5
(1994)) have amply demonstrated that definition of regional
chromosomal deletions occurring in the genomes of human tumors can
serve as useful diagnostic markers for disease and are an important
initial step towards identification of critical genes. Similarly,
regions of common chromosomal gain have been associated with
amplification of specific genes (Visakorpi, et al., Nature
Genetics, 9:401-6 (1995)).
[0003] Comparative genomic hybridization (CGH) is a relatively new
molecular technique used to screen DNA from tumors for regional
chromosomal alterations (Kallioniemi, et al., Science, 258:818-21
(1992) and WO 93/18186). Unlike microsatellite or Southern analysis
allelotyping studies, which typically sample far less than 0.1% of
the total genome, a significant advantage of CGH is that all
chromosome arms are scanned for losses and gains. Moreover, because
CGH does not rely on naturally occurring polymorphisms, all regions
are informative, whereas polymorphism-based techniques are limited
by homozygous (uninformative) alleles among a fraction of tumors
studied at every locus.
[0004] Increases in copy number in the long arm of chromosome 3, in
particular 3q25-3qter, has been associated with cancer. Increases
in copy number in this area have been seen not only in ovarian
tumors (Iwabuchi et al., Cancer Research 55:6172-8180 (1995) and
Arnold et al., Genes Chromosomes Cancer 16:46-54 (1996)) but also
in brain tumors, head and neck cancer, lung cancer, ductal breast
cancer, renal cell and other urinary tract cancers, and cervical
cancer. Ried et al., Genes Chromosomes Cancer 15:234-245 (1996);
Yeatman et al. Clin Exp Metastasis 14:246-252 (1996); Brzoska et
al., Cancer Res 15:3055-3059 (1995); Ried et al., Cancer Res
54:1801-1806 (1994); Cher et al. Cancer Research 56:3091-3102
(1996); Heselmeyer et al., Proc. Natl. Acad. Sci. USA 93:479-484
(1996); Levin et al. Genes Chromosomes Cancer 13:175-185 (1995);
and Speicher et al., Cancer Res 55:1010-3 (1995).
[0005] The identification of narrower regions of genetic alteration
or genes associated with cancers such as ovarian cancer would be
extremely useful in the early diagnosis or prognosis of these
diseases. The present invention addresses these and other
needs.
SUMMARY OF THE INVENTION
[0006] The present invention provides compositions and methods for
detecting genetic alterations correlated with cancer. The invention
can be used to detect alterations in a 2 MB region at 3q26.3 that
are associated with a number of cancers. Examples include ovarian
cancer, brain cancer, lung cancer, head and neck tumors, renal cell
and other urinary tumors, cervical cancer, and ductal breast
cancer. The invention is particularly useful for detecting
alterations associated with ovarian cancer.
[0007] The methods comprise contacting a nucleic acid sample from a
patient with a probe which binds selectively to a target nucleic
acid sequence on 3q26.3 correlated with cancer. The target region
is typically between markers D3S 1275 or D3S 1266 and D3S1548. In
particular, the invention provides sequences from genes encoding
the catalytic subunit of phosphatidylinositol kinase type 3
(PIK3CA) or the glucose transporter, GLUT2. The probes of the
invention are contacted with the sample under conditions in which
the probe binds selectively with the target nucleic acid sequence
to form a hybridization complex. The formation of the hybridization
complex is then detected. Typically, the number of regions of
hybridization are counted. Abnormalities are detected as increases
above normal in the regions of hybridization. In some embodiments,
the methods of the invention further comprise detection of
amplifications at 19q13.1-13.2. This region includes AKT2, a
putative oncogene.
[0008] Alternatively, sample DNA from the patient can be
fluorescently labeled and competitively hybridized against
fluorescently labeled normal DNA to normal lymphocyte metaphases or
to arrays of nucleic acid molecules which map to 3q26.3.
Alterations in DNA copy number in the sample DNA are then detected
as increases in sample DNA as compared to normal DNA at the 3q26.3
region.
Definitions
[0009] A "nucleic acid sample" as used herein refers to a sample
comprising DNA in a form suitable for hybridization to a probes of
the invention. The nucleic acid may be total genomic DNA, total
mRNA, genomic DNA or mRNA from particular chromosomes, or selected
sequences (e.g. particular promoters, genes, amplification or
restriction fragments, cDNA, etc.) within particular
cancer-associated amplifications. The nucleic acid sample may be
extracted from particular cells or tissues. The tissue sample from
which the nucleic acid sample is prepared is typically taken from a
patient suspected of having the disease associated with the
amplification being detected. The sample may be prepared such that
individual nucleic acids remain substantially intact and typically
comprises interphase nuclei prepared according to standard
techniques. A "nucleic acid sample" as used herein may also refer
to a substantially intact condensed chromosome (e.g. a metaphase
chromosome). Such a condensed chromosome is suitable for use as a
hybridization target in in situ hybridization techniques (e.g.
FISH). The particular usage of the term "nucleic acid sample"
(whether as extracted nucleic acid or intact metaphase chromosome)
will be readily apparent to one of skill in the art from the
context in which the term is used. For instance, the nucleic acid
sample can be a tissue or cell sample prepared for standard in situ
hybridization methods described below. The sample is prepared such
that individual chromosomes remain substantially intact and
typically comprises metaphase spreads or interphase nuclei prepared
according to standard techniques.
[0010] The sample may also be isolated nucleic acids immobilized on
a solid surface (e.g., nitrocellulose) for use in Southern or dot
blot hybridizations and the like. In some embodiments, the probe
may be a member of an array of nucleic acids as described, for
instance, in WO 96/17958. In some cases, the nucleic acids may be
amplified using standard techniques such as PCR, prior to the
hybridization. The sample is typically taken from a patient
suspected of having cancer associated with the abnormality being
detected.
[0011] A "chromosome sample" as used herein refers to a tissue or
cell sample prepared for standard in situ hybridization methods
described below. The sample is prepared such that individual
chromosomes remain substantially intact and typically comprises
metaphase spreads or interphase nuclei prepared according to
standard techniques.
[0012] "Nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, would encompass known analogs of
natural nucleotides that can function in a similar manner as
naturally occurring nucleotides.
[0013] "Subsequence" refers to a sequence of nucleic acids that
comprise a part of a longer sequence of nucleic acids.
[0014] A "probe" or a "nucleic acid probe", as used herein, is
defined to be a collection of one or more nucleic acid fragments
whose hybridization to a target can be detected. The probe is
typically labeled as described below so that its binding to the
target can be detected. In some embodiments, the sample comprising
the target nucleic acid is labeled and the probe is not labeled.
For instance, when the probes are prepared as an array of nucleic
acids which selectively bind a number of desired target
sequences.
[0015] The probe is produced from a source of nucleic acids from
one or more particular (preselected) portions of the genome, for
example one or more clones, an isolated whole chromosome or
chromosome fragment, or a collection of polymerase chain reaction
(PCR) amplification products. The probes of the present invention
are produced from nucleic acids found in the regions of genetic
alteration as described herein. The probe may be processed in some
manner, for example, by blocking or removal of repetitive nucleic
acids or enrichment with unique nucleic acids. Thus the word
"probe" may be used herein to refer not only to the detectable
nucleic acids, but to the detectable nucleic acids in the form in
which they are applied to the target, for example, with the
blocking nucleic acids, etc. The blocking nucleic acid may also be
referred to separately. What "probe" refers to specifically is
clear from the context in which the word is used.
[0016] "Hybridizing" refers the binding of two single stranded
nucleic acids via complementary base pairing.
[0017] "Bind(s) substantially" or "binds specifically" or "binds
selectively" or "hybridizing specifically to" refers to
complementary hybridization between a probe and a target sequence
and embraces minor mismatches that can be accommodated by reducing
the stringency of the hybridization media to achieve the desired
detection of the target nucleic acid sequence. These terms also
refer to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent conditions when
that sequence is present in a complex mixture (e.g., total
cellular) DNA or RNA. The term "stringent conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, but to no other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength and pH. The Tm is
the temperature (under defined ionic strength, pH, and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Typically, stringent conditions will be those in which the salt
concentration is at least about 0.02 Na ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
60.degree. C. Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide.
[0018] One of skill will recognize that the precise sequence of the
particular probes described herein can be modified to a certain
degree to produce probes that are "substantially identical" to the
disclosed probes, but retain the ability to bind substantially to
the target sequences. Such modifications are specifically covered
by reference to the individual probes herein. The term "substantial
identity" of nucleic acid sequences means that a nucleic acid
comprises a sequence that has at least 90% sequence identity, more
preferably at least 95%, compared to a reference sequence using the
methods described below using standard parameters.
[0019] Two nucleic acid sequences are said to be "identical" if the
sequence of nucleotides in the two sequences is the same when
aligned for maximum correspondence as described below. The term
"complementary to" is used herein to mean that the complementary
sequence is identical to all or a portion of a reference nucleic
acid sequence.
[0020] Sequence comparisons between two (or more) nucleic acids are
typically performed by comparing sequences of the two sequences
over a "comparison window" to identify and compare local regions of
sequence similarity. A "comparison window", as used herein, refers
to a segment of at least about 20 contiguous positions, usually
about 50 to about 200, more usually about 100 to about 150 in which
a sequence may be compared to a reference sequence of the same
number of contiguous positions after the two sequences are
optimally aligned.
[0021] Optimal alignment of sequences for comparison may be
conducted by the local homology algorithm of Smith and Waterman
Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm
of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search
for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci.
(U.S.A.) 85: 2444 (1988), by computerized implementations of these
algorithms.
[0022] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the nucleic acid sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0023] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to the same
sequence under stringent conditions. Stringent conditions are
sequence dependent and will be different in different
circumstances. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
Tm is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Typically, stringent conditions will be those as described
above.
[0024] As, used herein, an "antibody" refers to a protein
consisting of one or more polypeptides substantially encoded by
immunoglobulin genes or fragments of immunoglobulin genes. The
recognized immunoglobulin genes include the kappa, lambda, alpha,
gamma, delta, epsilon and mu constant region genes, as well as the
myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0025] The phrase "specifically binds to a protein" or
"specifically immunoreactive with", when referring to an antibody
refers to a binding reaction which is determinative of the presence
of the protein in the presence of a heterogeneous population of
proteins and other biologics. Thus, under designated immunoassay
conditions, the specified antibodies bind to a particular protein
and do not bind in a significant amount to other proteins present
in the sample. Specific binding to a protein under such conditions
may require an antibody that is selected for its specificity for a
particular protein. For example, antibodies can be raised to the
particular proteins disclosed here. Such antibodies will bind the
proteins and not any other proteins present in a biological sample.
A variety of immunoassay formats may be used to select antibodies
specifically immunoreactive with a particular protein. For example,
solid-phase ELISA immunoassays are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold
Spring Harbor Publications, New York, for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a schematic representation of a YAC/P1 physical
map at chromosome 3q26 between D3S1275 and D3S1602. YACs are from
the Genethon/CEPH megaYAC library and are illustrated as horizontal
bars. Each YAC is listed by coordinate name. The size of each is
listed in parentheses. P1 clones are listed by RMC number
information about these probes is available through the Resource
for Molecular Cytogenetics (http://rmc-www.lbl.gov). STS evaluated
in this study are listed above vertical lines showing their
approximate locations. STSs contents of specific clones confirmed
by PCR are listed as open circles. Predicted STS not confirmed by
PCR are shown as open squares. Genes mapped within the YAC/P1
contig in this study or reported elsewhere (Dib et al. Nature
380:152-154 (1996); Gemmill et al. Nature 377:299-319 (1995); and
Nucifora et al. Blood 86:1-14 (1995)) are shown below the clones to
which they map.
[0027] FIGS. 2A-2C show DNA sequence copy number maps generated
using dual color FISH with probes generated from YAC or P1 clones
shown in FIG. 1. All values shown as the ratio of the number of
hybridization signals produced by a test probe to the number of
signals produced by a reference probe at D3S1293. Specific probes
used for each map are indicated in the upper left panel of each set
of maps. FIG. 2A shows maps of ovarian cancer cell lines CAOV433,
OVCAR-3, CAOV-3, CAOV420, CAOV432, CAOV429, OCC1 and SKOV-3. FIG.
2B shows maps of primary ovarian tumors. FIG. 2C shows maps of
melanoma cell lines 355 and 457 and two breast cancer cell lines
ZR-75-03 and MCF-7.
[0028] FIG. 3 shows PI3-kinase activity from p11.alpha. or p85
immunoprecipitates.
[0029] FIGS. 4A and 4B show the effect of inhibition of PI3-kinase
activity with LY294002 on proliferation and cell viability. NOE,
MCF10F or OVCAR3 (as indicated) were starved of serum overnight and
then cultured in 0.5% serum. FIG. 4A shows .sup.3H-thymidine
incorporation measured by a 18 hour pulse following initiation of
culture. FIG. 4B shows MTT dye conversion measured 96 hours
following initiation of culture. Similar results were obtained with
cells cultured in serum-free media or in the presence of 10 ng/ml
EGF.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0030] Identification of Chromosomal Regions and Genes Associated
with Cancer
[0031] The present invention is based on a comprehensive molecular
cytogenetic analysis of the genomes of ovarian cancer cells using
comparative genomic hybridization (CGH). CGH studies on epithelial
ovarian cancer have revealed several regions that are present in
increased or decreased copy number. More than 40% of these tumors
show an increase in copy number on the long arm of chromosome 3, in
particular in the region of 3q25-3qter (Iwabuchi et al., supra).
This increase in copy number seems to be an early event for ovarian
cancer. Increases in copy number in this region have also been
observed in brain tumors, lung cancer, head and neck tumors, renal
cell and other urinary tumors, cervical cancer, and ductal breast
cancer.
[0032] Genomic regions that are found to be sites of increased DNA
copy number in a large fraction of the cell lines and primary tumor
cells are likely to include oncogenes that are present at increased
copy number and hence overexpressed. Gene amplification is one
method by which cells escape from normal controls of proliferation.
The resulting overexpression or altered expression of these genes
and their products is believed to play an important role in the
development of a variety of human cancers (Weinberg, Cancer
61:1963-1968 (1988); Bishop, Cell 64: 235-248 (1991)).
[0033] The present invention is based in part on the discovery of
specific cloned genomic DNA sequences showing increased copy number
in a 2 MB region at 3q26.3 region. The region generally corresponds
to region defined by markers D3S1275 or D3S1266 and D3S1548.
Increased copy number was assessed using FISH and a number of P1,
YAC, and cosmid clones known to map to this region. As shown below,
probes associated with the region have shown an increase in copy
number in ovarian cancer cell lines and ovarian tumor samples. The
P1 was picked using PCR primers specific to the Glucose transporter
gene, GLUT2. This gene is responsible for glucose signaling for
beta cell insulin release. Its RNA product is found mostly in adult
liver and pancreas, specifically in insulin-producing beta cells
(Fukumoto et al., Proc. Nat. Acad. Sci. 85:5434-5438 (1988)). The
sequence of cDNA from the gene is described in Fukumoto et al. This
gene has been associated with is non-insulin-dependent diabetes
mellitus (NIDDM). In NIDDM the highly conserved regions of this
gene have been found mutated, resulting in abolished transport
activity of the gene (Mueckler et al., J. Biol. Chem.
269:17765-17767 (1994)).
[0034] The 3q26 region also harbors the sequences for another gene,
the catalytic subunit of phosphatidylinositol kinase type 3 (PI3K).
The cloning of cDNA and genomic DNA encoding the catalytic subunit
is described in Volinia et al. Genomics 24:472-477 (1994) and
WO93/21328.
[0035] PIK3CA (also referred to as p110.alpha.), the 110 kD
catalytic subunit of PI3-K binds to several isoforms of p85, a
tyrosine kinase receptor adaptor protein, to form heterodimer
proteins with PI3-kinase activity upon binding to activated
tyrosine kinase receptors such as platelet derived growth factor
(PDGF), insulin-like growth factor I (IGF-1), nerve growth factor
(NGF), colony stimulating growth factor 1 (CSF-1) and epidermal
growth factor (EGF). PI3-kinase activity also has been found to be
increased in cells transformed with polyoma middle T, v-src, vlms
and v-ahl (Kapeller et al. Bioessays 16:565-576 (1994)). The
PI3-kinase heterodimer is postulated to bind to phosphorylated
transmembrane tyrosine kinase receptor dimers and associated
proteins (e.g. ras-GAP, PLCg) through SH2 domains in the p85
adaptor subunit after which the p110 catalytic subunit, PIK3CA,
phosphorylates phosphoinositides and possibly serine/threonine
proteins as part of a signaling response. The mechanism of signal
transduction for PI3-kinase is not completely understood. However,
two protein kinases, the serine-threonine kinase, Akt (also known
as protein kinase B and Rac) and the p70 ribosomal protein S6
kinase (p70S6K) have been placed downstream of PI3-kinase (see,
Burgening and Coffer, Nature 376:599-602 (1995); Franke et al.,
Cell 81:727-736 (1995)). Akt activity appears to be regulated by
binding of phosphatidylinositol-3,4-biphosphate (Ptdins-3,4-P2) to
a pleckstrin homology domain (Franke et al, Cell 88:435-437
(1997)).
[0036] In addition, PI3K is required to maintain basal and insulin
stimulated glucose and amino acid transport (Tsakiridis et al.,
Endocrinology 136:4315-4322 (1995)). It is therefore likely that an
increased expression in PI3K levels could also upregulate the
nearby GLUT2 gene. As explained below, compounds that inhibit
expression of these genes or inhibit activity of the encoded
protein have therapeutic potential in cancers, such as ovarian
cancer.
[0037] A number of high molecular weight kinases have been cloned
that have sequence similarities to PIK3CA. These kinases have a
range of cellular functions such as meiotic and V(D)j
recombination, chromosome maintenance and repair, cell cycle
progression, and cell cycle checkpoints, and with dysfunctions
resulting in medical disorders ranging from a loss of immunological
function to cancer. Therefore, increases in copy number in the
PIK3CA in ovarian tumor samples may have implications in the level
of tumor aggressiveness or patient prognosis, and the analysis of
this gene at the tumor level could improve early diagnosis, and
assist in better patient therapy and survival for this disease.
[0038] In some embodiments of the invention, probes specific to
19q13.1-13.2 can be used in the methods, as well. Amplification of
this region has been correlated with ovarian cancer using FISH and
molecular studies (see, e.g., Thompson et al. Cancer Genet
Cytogenet 87: 55-62 (1996)). The AKT2 gene, discussed above, is
located in this region. AKT2 encodes a member of a subfamily of
protein-serine/threonine kinases and is thought to be a human
homologue of an oncogene isolated from the retrovirus, AKT8. Staal,
Proc Natl Acad Sci USA 84:5034-7 (1987). A cDNA encoding the
protein is described by Cheng et al. Proc Natl Acad Sci USA 89:
9267-71 (1992). Amplification of 19q13.1-13.2 region and
overexpression of the AKT2 gene have been identified in ovarian and
pancreatic cancer (see, e.g., Bellacosa et al., supra, Thompson et
al. supra, and Miwa et al. Biochem Biophys Res Commun 225:968-74
(1996)). Inhibition of AKT2 expression and tumorigenicity has been
demonstrated using antisense RNA. Cheng et al. Proc Natl Acad Sci
USA 93:3636-41 (1996).
[0039] AKT activity appears to be regulated by binding of
phosphatidylinositol-3,4-biphosphate (Ptdins-3,4-P.sub.2) to a
pleckstrin homology domain. The activation of AKT has been
associated with increased cell survival through a reduction in
apoptosis. Without wishing to be bound by theory, it is believed
that amplification of PIK3CA in ovarian cancer contributes to
cancer progression and/or initiation by reducing apoptotic death
and increasing cell proliferation rate. The possible decrease in
apoptosis is relevant since apoptosis likely plays an important
role removal of epithelial cells that become detached from the
stroma during ovulation. Reduced apoptosis in these cells might
lead to malignancy since severed studies now suggest that
disruption of the stroma (e.g. by overexpression of
metalloproteinases) causes cancer in murine mammary cells.
[0040] In the present invention it has been found that both the
PIK3CA and AKT2 genes are amplified in cancers, such as ovarian
cancer. Thus, detection of amplification and/or overexpression of
these genes is useful in the early diagnosis of cancers.
[0041] In addition, in some embodiments, the expression of other
genes associated with cancer (e.g., tumor suppressor genes or
oncogenes) can be monitored in the present invention. For instance,
expression of wild-type p53 can be monitored according to known
techniques. Mutation or loss of the p53 gene is the most common
genetic alteration in human cancers (Bartek et al. (1991) Oncogene,
6: 1699-1703, Holistein et al. (1991) Science, 253: 49-53).
Preparation of Probes of the Invention
[0042] A number of methods can be used to identify probes which
hybridize specifically to the 3q26 region other than those
exemplified here. For instance, probes can be generated by the
random selection of clones from a chromosome specific library, and
then mapped to each chromosome or region by digital imaging
microscopy. This procedure is described in U.S. Pat. No. 5,472,842.
Briefly, a genomic or chromosome specific DNA is digested with
restriction enzymes or mechanically sheared to give DNA sequences
of at least about 20 kb and more preferably about 40 kb to 300 kb.
Techniques of partial sequence digestion are well known in the art.
See, for example Perbal, A Practical Guide to Molecular Cloning 2nd
Ed., Wiley N.Y. (1988). The resulting sequences are ligated with a
vector and introduced into the appropriate host. Exemplary vectors
suitable for this purpose include cosmids, yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1
phage. Typically, cosmid libraries are prepared. Various libraries
spanning entire chromosomes are also available commercially from
for instance Genome Systems.
[0043] Once a probe library is constructed, a subset of the probes
is physically mapped on the selected chromosome. FISH and digital
image analysis can be used to localize clones along the desired
chromosome. Briefly, the clones are mapped by FISH to metaphase
spreads from normal cells using e.g., FITC as the fluorophore. The
chromosomes may be counterstained by a stain which stains DNA
irrespective of base composition (e.g., propidium iodide), to
define the outlining of the chromosome. The stained metaphases are
imaged in a fluorescence microscope with a polychromatic
beam-splitter to avoid color-dependent image shifts. The different
color images are acquired with a CCD camera and the digitized
images are stored in a computer. A computer program is then used to
calculate the chromosome axis, project the two (for single copy
sequences) FITC signals perpendicularly onto this axis, and
calculate the average fractional length from a defined position,
typically the p-telomere. This approach is described, for instance,
in U.S. Pat. No. 5,472,842.
[0044] Sequence information of the genes identified here permits
the design of highly specific hybridization probes or amplification
primers suitable for detection of target sequences from these
genes. As noted above, the complete sequence of these genes is
known. Means for detecting specific DNA sequences within genes are
well known to those of skill in the art. For instance,
oligonucleotide probes chosen to be complementary to a selected
subsequence within the gene can be used. Alternatively, sequences
or subsequences may be amplified by a variety of DNA amplification
techniques (for example via polymerase chain reaction, ligase chain
reaction, transcription amplification, etc.) prior to detection
using a probe. Amplification of DNA increases sensitivity of the
assay by providing more copies of possible target subsequences. In
addition, by using labeled primers in the amplification process,
the DNA sequences may be labeled as they are amplified.
Labeling Probes
[0045] Methods of labeling nucleic acids are well known to those of
skill in the art. Preferred labels are those that are suitable for
use in in situ hybridization. The nucleic acid probes may be
detectably labeled prior to the hybridization reaction.
Alternatively, a detectable label which binds to the hybridization
product may be used. Such detectable labels include any material
having a detectable physical or chemical property and have been
well-developed in the field of immunoassays.
[0046] As used herein, a "label" is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. Useful labels in the present invention include
radioactive labels (e.g. .sup.32P, .sup.125I, .sup.14C, .sup.3H and
.sup.35S), fluorescent dyes (e.g. fluorescein, rhodamine, Texas
Red, etc.), electron-dense reagents (e.g. gold), enzymes (as
commonly used in an ELISA), colorimetric labels (e.g. colloidal
gold), magnetic labels (e.g. Dynabeads.TM.), and the like. Examples
of labels which are not directly detected but are detected through
the use of directly detectable label include biotin and dioxigenin
as well as haptens and proteins for which labeled antisera or
monoclonal antibodies are available.
[0047] The particular label used is not critical to the present
invention, so long as it does not interfere with the in situ
hybridization of the probe. However, probes directly labeled with
fluorescent molecules (e.g. fluorescein-12-dUTP, Texas Red-5-dUTP,
etc.) are preferred for chromosome hybridization.
[0048] A direct labeled probe, as used herein, is a probe to which
a detectable label is attached. Because the detectable label is
already attached to the probe, no subsequent steps are required to
associate the probe with the detectable label. In contrast, an
indirect labeled probe is one which bears a moiety to which a
detectable label is subsequently bound, typically after the probe
is hybridized with the target nucleic acid.
[0049] In addition the label must be detectible in as low copy
number as possible thereby maximizing the sensitivity of the assay
and yet be detectible above any background signal. Finally, a label
must be chosen that provides a highly localized signal thereby
providing a high degree of spatial resolution when physically
mapping the stain against the chromosome. Particularly preferred
fluorescent labels include fluorescein-12-dUTP and Texas
Red-5-dUTP.
[0050] The labels may be coupled to the probes in a variety of
means known to those of skill in the art. In some embodiments the
nucleic acid probes are labeled using nick translation or random
primer extension (Rigby, et al. J. Mol. Biol., 113: 237 (1977) or
Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989)).
Particularly preferred methods for labeling probes are described in
U.S. Pat. No. 5,491,224. These methods involve direct labeling the
probes by chemical modification of cytosine residues.
Use of Nucleic Acids of the Invention to Detect Chromosomal
Alterations
[0051] Using the results provided here, one of skill can prepare
nucleic acid probes specific to the 3q26 region of genetic
alteration that is associated with ovarian and other cancer. In
particular, nucleic acid sequences from the GLUT2 gene or the
PIK3CA gene can be used to detect copy number increase of these
genes. The probes can be used in a variety of nucleic acid
hybridization assays to detect the presence (in particular
increased copy number) of the target gene. Thus, the probes are
useful, for example, in the early diagnosis or prognosis of cancer.
As noted above, the probes are particularly useful for detecting
alteration associated with ovarian cancer. The regions can also be
used for a large number of other cancers as described above.
[0052] The genetic alterations are detected through the
hybridization of a probe of this invention to a nucleic acid sample
in which it is desired to screen for the alteration. Suitable
hybridization formats are well known to those of skill in the art
and include, but are not limited to, variations of Southern Blots,
northern blots, CGH, in situ hybridization and quantitative
amplification methods such as quantitative PCR (see, e.g. Sambrook
et al., Kallioniemi et al., Proc. Natl Acad Sci USA, 89: 5321-5325
(1992), and PCR Protocols, A Guide to Methods and Applications,
Innis et al., Academic Press, Inc. N.Y., (1990)).
[0053] The sample used in the methods will, of course, depend upon
the particular method used to detect the target. For instance, the
nucleic acid sample can be a tissue or cell sample prepared for
standard in situ hybridization methods. The sample or probes may
also be isolated nucleic acids immobilized on a solid surface
(e.g., nitrocellulose) for use in Southern or dot blot
hybridizations and the like. In some embodiments, the probes of the
invention may comprise an array of nucleic acids as described, for
instance, in WO 96/17958).
[0054] In a preferred embodiment, the regions disclosed here are
identified using in situ hybridization. Generally, in situ
hybridization comprises the following major steps: (1) fixation of
tissue or biological structure to analyzed; (2) prehybridization
treatment of the biological structure to increase accessibility of
target DNA, and to reduce nonspecific binding; (3) hybridization of
the mixture of nucleic acids to the nucleic acid in the biological
structure or tissue; (4) posthybridization washes to remove nucleic
acid fragments not bound in the hybridization and (5) detection of
the hybridized nucleic acid fragments. The reagent used in each of
these steps and their conditions for use vary depending on the
particular application.
[0055] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. In this case, human
genomic DNA or Cot1 DNA is used as an agent to block such
hybridization. The preferred size range is from about 200 by to
about 1000 bases, more preferably between about 400 to about 800 by
for double stranded, nick translated nucleic acids.
[0056] Hybridization protocols for the particular applications
disclosed here are described in Pinkel et al. Proc. Natl. Acad.
Sci. USA, 85: 9138-9142 (1988) and in EPO Pub. No. 430,402.
Suitable hybridization protocols can also be found in Methods in
Molecular Biology Vol. 33: In Situ Hybridization Protocols, K. H.
A. Choo, ed., Humana Press, Totowa, N.J., (1994). In a particularly
preferred embodiment, the hybridization protocol of Kallioniemi et
al., Proc. Natl Acad Sci USA, 89: 5321-5325 (1992) is used.
[0057] Typically, it is desirable to use dual color FISH, in which
two probes are utilized, each labeled by a different fluorescent
dye. A test probe that hybridizes to the region of interest is
labeled with one dye, and a control probe that hybridizes to a
different region is labeled with a second dye. A nucleic acid that
hybridizes to a stable portion of the chromosome of interest, such
as the centromere region, is often most useful as the control
probe. In this way, differences between efficiency of hybridization
from sample to sample can be accounted for.
[0058] The FISH methods for detecting chromosomal abnormalities can
be performed on nanogram quantities of the subject nucleic acids.
Paraffin embedded tumor sections can be used, as can fresh or
frozen material. Because FISH can be applied to the limited
material, touch preparations prepared from uncultured primary
tumors can also be used (see, e.g., Kallioniemi, A. et al.,
Cytogenet. Cell Genet. 60: 190-193 (1992)). For instance, small
biopsy tissue samples from tumors can be used for touch
preparations (see, e.g., Kallioniemi, A. et al., Cytogenet. Cell
Genet. 60: 190-193 (1992)). Small numbers of cells obtained from
aspiration biopsy or cells in bodily fluids (e.g., blood, urine,
sputum and the like) can also be analyzed.
[0059] In various blot formats (e.g., dot blots, Southern blots,
and Northern blots) nucleic acids (e.g., genomic DNA, cDNA or RNA)
are hybridized to a probe specific for the target region. Either
the probe or the target can be immobilized on the solid surface.
Comparison of the intensity of the hybridization signal from the
probe for the target region with the signal from a probe directed
to a control (non amplified or deleted) such as centromeric DNA,
provides an estimate of the relative copy number of the target
nucleic acid. Procedures for carrying out Southern hybridizations
are well known to those of skill in the art. see, e.g., Sambrook et
al., supra.
[0060] Other hybridization formats use arrays of probes or targets
to which nucleic acid samples are hybridized as described, for
instance, in WO 96/17958. Techniques capable of producing high
density arrays can also be used for this purpose (see, e.g., Fodor
et al. Science 767-773 (1991) and U.S. Pat. No. 5,143,854). As used
herein, a "nucleic acid array" is a plurality of target elements,
each comprising one or more target nucleic acid molecules
immobilized on a solid surface to which probe nucleic acids are
hybridized. Target nucleic acids of a target element typically have
their origin in the 3q26 region disclosed here. The target nucleic
acids of a target element may, for example, contain sequence from
specific genes or clones disclosed here. Target elements of various
dimensions can be used in the arrays of the invention. Generally,
smaller, target elements are preferred. Typically, a target element
will be less than about lcm in diameter. Generally element sizes
are from 1 .mu.m to about 3 mm, preferably between about 5 .mu.m
and about 1 mm.
[0061] The target elements of the arrays may be arranged on the
solid surface at different densities. The target element densities
will depend upon a number of factors, such as the nature of the
label, the solid support, and the like. One of skill will recognize
that each target element may comprise a mixture of target nucleic
acids of different lengths and sequences. Thus, for example, a
target element may contain more than one copy of a cloned piece of
DNA, and each copy may be broken into fragments of different
lengths. The length and complexity of the target sequences of the
invention is not critical to the invention. One of skill can adjust
these factors to provide optimum hybridization and signal
production for a given hybridization procedure, and to provide the
required resolution among different genes or genomic locations.
Typically, the target sequences will have a complexity less than
about 1 Mb, sometimes 10 kb and about 500 kb, and usually from
about 50 kb to about 150 kb.
Detection of Proteins of the Invention
[0062] The gene products described here (GLUT2 and PIK3CA) as well
as gene products (e.g., AKT2) whose expression is effected by GLUT2
and PIK3CA can be detected and quantified by any of a number of
means well known to those of skill in the art. These may include
analytic biochemical methods such as electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography, and
the like, or various immunological methods such as fluid or gel
precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis, radioimmunoassay(RIA), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays, western
blotting, and the like.
[0063] In a preferred embodiment, the proteins are detected using
an immunoassay. As used herein, an immunoassay is an assay that
utilizes an antibody to specifically bind to the analyte (e.g.,
GLUT2, PIK3CA or AKT2 proteins). The immunoassay is thus
characterized by detection of specific binding of the protein to an
antibody raised against it as opposed to the use of other physical
or chemical properties to isolate, target, and quantify the analyte
(see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and
4,837,168). For a review of the general immunoassays, see also
Methods in Cell Biology Volume 37: Antibodies in Cell Biology,
Asai, ed. Academic Press, Inc. New.York (1993); Basic and Clinical
Immunology 7th Edition, Stites & Terr, eds. (1991).
[0064] The proteins are preferably quantified in a biological
sample derived from a mammal, more preferably from a human patient.
As used herein, a biological sample is a sample of biological
tissue or fluid that contains a protein concentration that may be
correlated with amplification of the 3q regions disclosed here.
Particularly preferred biological samples include, but are not
limited to biological fluids such as whole blood, serum, or urine,
or tissue samples including, but not limited to tissue biopsy
(e.g., needle biopsy) samples.
[0065] The antibody (e.g., anti-GLUT2, anti-PIK3CA or anti-AKT2)
may be produced by any of a number of means well known to those of
skill in the art (see, e.g. Methods in Cell Biology Volume 37:
Antibodies in. Cell. Biology, Asai, ed. Academic Press, Inc. New
York (1993); and Basic and Clinical Immunology 7th Edition, Stites
& Terr, eds. (1991)). The antibody may be a whole antibody or
an antibody fragment. It may be polyclonal or monoclonal, and it
may be produced by challenging an organism (e.g. mouse, rat,
rabbit, etc.) with one of these proteins or an epitope derived
therefrom. Alternatively, the antibody may be produced de novo
using recombinant DNA methodology. The antibody can also be
selected from a phage display library screened against the protein
(see, e.g. Vaughan et al. (1996) Nature Biotechnology, 14: 309-314
and references therein).
[0066] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled protein or a labeled antibody.
Alternatively, the labeling agent may be a third moiety, such as
another antibody, that specifically binds to the antibody/protein
complex.
[0067] In a preferred embodiment, the labeling agent is a second
antibody bearing a label. Alternatively, the second antibody may
lack a label, but it may, in turn, be bound by a labeled third
antibody specific to antibodies of the species from which the
second antibody is derived. The second can be modified with a
detectable moiety, such as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0068] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G may
also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species. See, generally Kronval,
et al., J. Immunol., 111:1401-1406 (1973), and Akerstrom, et al.,
J. Immunol., 135:2589-2542 (1985).
[0069] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures, such as 10.degree. C. to 40.degree. C.
[0070] Immunoassays for detecting the proteins may be either
competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of captured analyte (e.g., PIK3CA) is
directly measured. In one preferred "sandwich" assay, for example,
the capture agent (anti-PIK3CA antibodies) can be bound directly to
a solid substrate where they are immobilized. These immobilized
antibodies then capture protein present in the test sample. The
PIK3CA protein thus immobilized is then bound by a labeling agent,
such as a second PIK3CA antibody bearing a label. Alternatively,
the second PIK3CA antibody may lack a label, but it may, in turn,
be bound by a labeled third antibody specific to antibodies of the
species from which the second antibody is derived. The second can
be modified with a detectable moiety, such as biotin, to which a
third labeled molecule can specifically bind, such as
enzyme-labeled streptavidin.
[0071] In competitive assays, the amount of analyte (e.g., PIK3CA)
present in the sample is measured indirectly by measuring the
amount of an added (exogenous) analyte displaced (or competed away)
from a capture agent (e.g., anti-PIK3CA antibody) by the analyte
present in the sample. In one competitive assay, a known amount of,
for instance, PIK3CA, is added to the sample and the sample is then
contacted with a capture agent such as an antibody that
specifically binds PIK3CA protein. The amount of PIK3CA protein
bound to the antibody is inversely proportional to the
concentration of PIK3CA protein present in the sample.
[0072] In another embodiment, the antibody (e.g., anti-PIK3CA) is
immobilized on a solid substrate. The amount of PIK3CA protein
bound to the antibody may be determined either by measuring the
amount of PIK3CA present in an protein/antibody complex, or
alternatively by measuring the amount of remaining uncomplexed
PIK3CA protein. The amount of protein may be detected by providing
a labeled PIK3CA protein.
[0073] A hapten inhibition assay is another preferred competitive
assay. In this assay a known analyte (e.g., PIK3CA protein) is
immobilized on a solid substrate. A known amount of anti-PIK3CA
antibody is added to the sample, and the sample is then contacted
with the immobilized PIK3CA protein. In this case, the amount of
anti-PIK3CA antibody bound to the immobilized protein is inversely
proportional to the amount of PIK3CA present in the sample. Again
the amount of immobilized antibody may be detected by detecting
either the immobilized fraction of antibody or the fraction of the
antibody that remains in solution. Detection may be direct where
the antibody is labeled or indirect by the subsequent addition of a
labeled moiety that specifically binds to the antibody as described
above.
[0074] In other embodiments, Western blot (immunoblot) analysis is
used to detect and/or quantify the presence of the proteins in the
sample. The technique generally comprises separating sample
proteins by gel electrophoresis on the basis of molecular weight,
transferring the separated proteins to a suitable solid support,
(such as a nitrocellulose filter, a nylon filter, or derivatized
nylon filter), and incubating the sample with the antibodies that
specifically bind the desired protein. Other assay formats include
liposome immunoassays (LIA), which use liposomes designed to bind
specific molecules (e.g., antibodies) and release encapsulated
reagents or markers. The released chemicals are then detected
according to standard techniques (see, Monroe et al. (1986) Amer.
Clin. Prod. Rev. 5:34-41).
[0075] The particular label or detectable group used in an
immunoassay is not a critical aspect of the invention, so long as
it does not significantly interfere with the specific binding of
the antibody used in the assay. The detectable group can be any
material having a detectable physical or chemical property. Such
detectable labels have been well-developed in the field of
immunoassays and, in general, most any label useful in such methods
can be applied to the present invention. Thus, a label is any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include magnetic beads (e.g.
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
texas red, rhodamine, and the like), radiolabels (e.g., .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P), enzymes (e.g., horse
radish peroxidase, alkaline phosphatase and others commonly used in
an ELISA), and colorimetric labels such as colloidal gold or
colored glass or plastic (e.g. polystyrene, polypropylene, latex,
etc.) beads.
Kits of the Invention
[0076] This invention also provides diagnostic kits for the
detection of chromosomal abnormalities at the regions disclosed
here. For instance, the kits may include one or more nucleic acid
probes to the regions described herein. The kits can additionally
include blocking probes, instructional materials describing how to
use the kit contents in detecting the alterations. The kits may
also include one or more of the following: various labels or
labeling agents to facilitate the detection of the probes, reagents
for the hybridization including buffers, a metaphase spread, bovine
serum albumin (BSA) and other blocking agents, sampling devices
including fine needles, swabs, aspirators and the like, positive
and negative hybridization controls and so forth.
[0077] Alternatively, the kits can be used for detection of the
gene products disclosed here. In these embodiments, the kits will
contain reagents for detecting proteins (e.g., GLUT2, PIK3CA or
AKT2) or antibodies against them in serum or other biological
fluids. Such a kit includes antibodies which specifically recognize
the target proteins and a labeling system, including enzyme
substrates and the like, suitable for detecting the immune
complexes formed by the target antigens and antibodies. The kits
also include appropriate washing solutions, dilution buffers, and
the like for preparation and analysis of biological samples.
Therapeutic Uses of Genes and Their Gene Products
[0078] The genes identified here (e.g., PIK3CA and GLUT2) and their
polypeptide products can be used to modulate the activity of the
gene products of endogenous genes. Alternatively, the activity of
gene products in biochemical pathways controlled by PIK3CA or GLUT2
expression (e.g., AKT2) can be modulated. By modulating activity of
the gene products, pathological conditions associated with their
overexpression or lack of expression can be treated. Any of a
number of techniques well known to those of skill in the art can be
used for this purpose.
[0079] The genes of the invention are particularly used for the
treatment of various cancers such as cancers of the ovaries. Other
diseases may also be treated with the sequences of the
invention.
[0080] The polypeptides encoded by the genes can be used as
immunogens to raise antibodies either polyclonal or monoclonal. The
antibodies can be used to detect the polypeptides as therapeutic
agents to inhibit the polypeptides, or as targeting moieties in
immunotoxins. The production of monoclonal antibodies against a
desired antigen is well known to those of skill in the art and is
not reviewed in detail here.
[0081] The PIK3CA or GLUT2 genes are particularly useful for gene
therapy techniques well known to those skilled in the art. Gene
therapy as used herein refers to the multitude of techniques by
which gene expression may be altered in cells. Such methods
include, for instance, introduction of DNA encoding ribozymes or
antisense nucleic acids to inhibit expression as well as
introduction of functional wild-type genes to replace mutant genes.
A number of suitable viral vectors are known. Such vectors include
retroviral vectors (see Miller, Curr. Top. Microbiol. Immunol. 158:
1-24 (1992); Salmons and Gunzburg, Human Gene Therapy 4: 129-141
(1993); Miller et al., Methods in Enzymology 217: 581-599, (1994))
and adeno-associated vectors (reviewed in Carter, Curr. Opinion
Biotech. 3: 533-539 (1992); Muzcyzka, Curr. Top. Microbiol.
Immunol. 158: 97-129 (1992)). Other viral vectors that may be used
within the methods include adenoviral vectors, herpes viral vectors
and Sindbis viral vectors, as generally described in, e.g., Jolly,
Cancer Gene Therapy 1:51-64 (1994); Latchman, Molec. Biotechnol.
2:179-195 (1994); and Johanning et al., Nucl. Acids Res.
23:1495-1501 (1995).
[0082] Delivery of nucleic acids linked to a heterologous
promoter-enhancer element via liposomes is also known (see, e.g.,
Brigham, et al. (1989) Am. J. Med. Sci., 298:278-281; Nabel, et al.
(1990) Science, 249:1285-1288; Hazinski, et al. (1991) Am. J. Resp.
Cell Molec. Biol., 4:206-209; and Wang and Huang (1987) Proc. Natl.
Acad. Sci. (USA), 84:7851-7855); coupled to ligand-specific,
cation-based transport systems (Wu and Wu (1988) J. Biol. Chem.,
263:14621-14624). Naked DNA expression vectors have also been
described (Nabel et al. (1990), supra); Wolff et al. (1990)
Science, 247:1465-1468).
[0083] The nucleic acids and encoded polypeptides of the invention
can be used directly to inhibit the endogenous genes or their gene
products. For instance, inhibitory nucleic acids may be used to
specifically bind to a complementary nucleic acid sequence. By
binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA,
or RNA-DNA duplex is formed. These nucleic acids are often termed
"antisense" because they are usually complementary to the sense or
coding strand of the gene, although approaches for use of "sense"
nucleic acids have also been developed. The term "inhibitory
nucleic acids" as used herein, refers to both "sense" and
"antisense" nucleic acids. Inhibitory nucleic acid methods
encompass a number of different approaches to altering expression
of specific genes that operate by different mechanisms.
[0084] In brief, inhibitory nucleic acid therapy approaches can be
classified into those that target DNA sequences, those that target
RNA sequences (including pre-mRNA and mRNA), those that target
proteins (sense strand approaches), and those that cause cleavage
or chemical modification of the target nucleic acids (ribozymes).
These different types of inhibitory nucleic acid technology are
described, for instance, in Helene, C. and Toulme, J. (1990)
Biochim. Biophys. Acta., 1049:99-125. Inhibitory nucleic acid
complementary to regions of c-myc mRNA has been shown to inhibit
c-myc protein expression in a human promyelocytic leukemia cell
line, HL60, which overexpresses the c-myc proto-oncogene. See
Wickstrom E. L., et al., (1988) PROC. ACAD. SCI. USA (USA),
85:1028-1032 and Harel-Bellan, A., et al., (1988) Exp. Med.,
168:2309-2318.
[0085] The encoded polypeptides of the invention can also be used
to design molecules (peptidic or nonpeptidic) that inhibit the
endogenous proteins by, for instance, inhibiting interaction
between the protein and a second molecule specifically recognized
by the protein. Methods for designing such molecules are well known
to those skilled in the art.
[0086] For instance, polypeptides can be designed which
have-sequence identity with the encoded proteins or may comprise
modifications (conservative or non-conservative) of the sequences.
The modifications can be selected, for example, to alter their in
vivo stability. For instance, inclusion of one or more D-amino
acids in the peptide typically increases stability, particularly if
the D-amino acid residues are substituted at one or both termini of
the peptide sequence.
[0087] The polypeptides can also be modified by linkage to other
molecules. For example, different N- or C-terminal groups may be
introduced to alter the molecule's physical and/or chemical
properties. Such alterations may be utilized to affect, for
example, adhesion, stability; bio-availability, localization or
detection of the molecules.
[0088] Alternatively, other non-peptidic compounds which inhibit
the activity of the gene products described here can be used to
treat cancer. For instance, compounds which inhibit PIK3CA activity
(e.g., Y294002, Wortmannin, Rapamycin) can be used therapeutically.
A number of glucose transport inhibitors are available. Exemplary
glucose transport inhibitors include cytochalasin B and ethanol
(see, e.g., Colville et al. Biochemical Journal 290:701-706 (1993)
and Nagamatsu et al., Bioch. Molec. Biol. Int. 37:675-680
(1995)).
[0089] Pharmaceutical compositions containing the inhibitory
compounds of the invention (e.g., polypeptides, nucleic acids,
non-peptidic inhibitors of enzyme activity) are suitable for use in
a variety of drug delivery systems. Suitable formulations for use
in the present invention are found in Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed.
(1985).
[0090] The subject compounds, by themselves or as conjugates, may
be prepared as formulations in pharmaceutically acceptable media,
for example saline, PBS, and glucose, generally at a
therapeutically effective dose, the concentrations of which will be
determined empirically in accordance with conventional procedures
for the particular purpose. The additives may include bactericidal
agents, stabilizers, buffers, or the like.
[0091] In order to enhance serum half-life, polypeptides may be
encapsulated, introduced into the lumen of liposomes, prepared as a
colloid, or other conventional technique may be employed which
provides an extended serum half-life of the peptides. A variety of
methods are available for preparing liposomes, as described in,
e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.
Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028, all of which are
incorporated herein by reference.
[0092] The amount of inhibitory compound administered to the
patient will vary depending upon what is being administered, the
purpose of the administration, such as the state of the patient,
the manner of administration, and the like. In therapeutic
applications, compositions are administered to a patient already
suffering from a disease in an amount sufficient to cure or at
least partially arrest the symptoms of the disease and its
complications. An amount adequate to accomplish this is defined as
"therapeutically effective dose." Amounts effective for this use
will depend on the severity of the disease and the weight and
general state of the patient.
[0093] The pharmaceutical compositions are intended for parenteral,
topical, oral or local administration, such as by aerosol or
transdermally, for prophylactic and/or therapeutic treatment.
Commonly, the pharmaceutical compositions are administered
parenterally, e.g., intravenously or intraperitonealy.
[0094] Two or more inhibitory compounds of the invention may be
combined to form a "cocktail" under certain circumstances for
increased efficacy. The compounds of the invention may also be used
in conjunction with other pharmaceutically active agents.
EXAMPLES
Example 1
[0095] This example describes the identification of genes in a 2 MB
region at 3q26.3 region. Increased copy number of this region is
correlated with ovarian cancer.
[0096] CGH studies have identified DNA copy number abnormalities at
3q25-26 associated with ovarian tumors (Iwabuchi et al., Cancer
Research 55:6172-8180 (1995)). To physically map the 3q26 region, a
number of yeast artificial chromosome (YAC), P1, and cosmid clones
that were known to genetically map to the region were physically
mapped to the 3q region. Several yeast artificial chromosome (YAC)
and P1 clones known to genetically map to this region were
physically mapped using FISH and fractional length analysis. Clone
positions were reconfirmed by PCR using STSs specific to the
region. Clones were then picked according to their physical map
position and hybridized onto interphase cells of ovarian cancer,
breast cancer, and melanoma cell lines, as well as nuclei of
paraffin-embedded ovarian tumors. One of the P1 clones and its 5
associated YAC clones have shown increases in copy number in 8
ovarian cancer cell lines and 6 primary ovarian tumor samples,
small increases in copy number in the breast cancer lines, and no
increases in copy number in the melanoma lines. Based on these
results, the region of increased copy number was narrowed to a 2 MB
region at 3q26.3.
Materials and Methods
[0097] Probes. Yeast artificial chromosome (YAC) clones were
obtained from Genethon/CEPH of France. YAC clones were chosen based
on their genetic map along 3q24-3qter. Each YAC was grown and
checked for chimerism by FISH. P1 clones were obtained by screening
a human genomic P1 library (DuPont, Boston, Mass.) using PCR with
primers specific to chromosome 3. Those P1 clones mapping to
3q25-3qter were used for further study. A P1 clone mapping to the
3p region was used throughout the experiments as a reference
marker.
[0098] Nonchimeric YAC clones as well as all the P1 clones were
mapped onto chromosome 3 by digital image analysis of their
physical distance from the terminus of the p arm (Flpter analysis)
generally as described in Mascio, et al. Cytometry 19:51-9 (1995)
and Sakamoto et al, Cytometry 19:60-9 (1995)).
[0099] All probes were labeled for hybridization by random priming
(BioPrime kit, BRL). The 3q region probes were labeled with
digoxigenin-11-dUTP (Boehringer-Mannheim) and detected using
Fluorescein-antidigoxigenin. The reference Pi probe on 3p was
directly labeled with Texas-Red dUTP (NEN DuPont).
[0100] Normal human metaphase spreads, cell lines, and
paraffin-embedded tumor samples. Normal human metaphase spreads
were prepared as previously described (Kallioniemi et al., supra).
Slides were denatured in 70% formamide/2.times.SSC at 72 degrees
for 3 to 10 minutes (depending on the slide batch) and then
serially dried in 70%, 85%, and 100% ethanol.
[0101] Ovarian cancer cell lines SKOV3, CAOV3, CAOV433, CAOV420,
CAOV432, CAOV429, OCC1 and OVCAR3 and breast cancer cell lines
MCF-7 and MDA-MDB-453 were obtained from ATCC. Melanoma cell lines
355 and 457 were kindly provided by Dr. Taetle (University of
Arizona). All cells were resuspended in 2 ml of 0.075M KCl
hypotonic solution, incubated at 37 degrees for 20 minutes, fixed,
and dropped onto slides.
[0102] Paraffin-embedded epithelial ovarian tumor samples were
provided by Dr. Teresa Yang-Feng (Yale University). All samples
were checked to contain >60% tumor cells. These samples were
deparaffinized, using xylene, washed with ethanol, then with water,
digested with pepsin, and cytospun onto slides in order to
concentrate the cell population.
[0103] FISH, physical mapping, and slide scorings. Cell line and
normal metaphase slides were denatured in 70% formamide/2.times.SSC
for 5 minutes at 72 degrees, followed by drying through 70%, 85%,
and 100% ethanol. Paraffin-embedded tumor materials were fixed for
10 minutes in methanol-acetic acid (3:1) prior to denaturation in
70% formamide/2.times.SSC for 10 minutes at 80 degrees, then
digested with 5 ug/MI proteinase K for 10 minutes at 37 degrees,
followed by drying through 70%, 85%, and 100% ethanol for 2 minutes
each.
[0104] 40 ng of each probe was placed on each slide along with 5 ug
Cot1 DNA (to suppress repetitive sequences) in a total of 10 ul of
50% formamide/2.times.SSC/10% dextran sulfate, and slides were
coverslipped and sealed. After an overnight incubation at 37
degrees, slides were washed to remove unbound probes, stained
immunochemically with fluoresceinantidigoxigenin, counterstained
with 0.2 uM 4,6-diamino-2-phenylindole in antifade solution for
chromosome identification, and visualized under fluorescent
microscope. For physical mapping, multicolor images of metaphase
chromosomes and their associated probes were acquired using the
QUIPS (quantitative image processing system). Analysis of the
hybridization signals is completely automated, and carried out
using the Xquips software (Mascio, et al. Cytometry 19:51-9 (1995)
and Sakamoto et al, Cytometry 19:60-9 (1995)). Briefly, analysis
consisted of chromosome segmentation, medial axis calculation,
hybridization domain segmentation, center of mass calculation, and
contrast enhancement. Fractional location of a domain was
determined from the end of the short arm to the valid hybridization
signal (Flpter analysis). On average, 20 Flpter measurements were
made for each probe, and probe location on a chromosome was
reported as the mean +/- one standard error of the mean of the
measurements. Probe order was determined from the mean Flpter
values.
[0105] For interphase cells, simultaneous Texas Red and Fluorescein
signals were visualized using a double bandpass filter on the X63
objective of a Zeiss Axioscope camera. At least 100 cells were
counted for each probe set.
Results
[0106] Physical mapping. FIG. 1 shows the genomic organization of
the clones used in this study. In addition, the order of the clones
for the region of interest was also checked by PCR using several
STSs known to map to this region and confirmed by comparing these
results to the recently published YAC maps in the Genome Directory
Naylor et al., Cytogenet. Cell Genet. 72: 255-70 (1995) and the
Whitehead institute's integrated map Dib et al., Nature 380:152-4
(1996).
[0107] Copy number abnormalities in cancer cell lines for the 3q26
region. CGH studies delineated the region of increase in copy
number at best to about 10 Megabases. FISH with well-mapped clones
specific to the region was used to refine the region of increase in
copy number on 3q26 in ovarian cancer to 2 megabases. FIGS. 2A-2C
show the data from hybridization experiments onto ovarian, breast,
and melanoma cell lines. The graphs are relative copy numbers of
the pfobes in the cell lines as a function of probe distance along
the chromosome. One P1 clone, Glut2, and 5 YAC clones that share
sequences with this P1 (683F10, 784H12, 806D8, 822G9, 945H6)
consistently show increases in copy number in all ovarian cell
lines. A minimal region of increased copy number was defined by
YACs 806D6 and 945H6. FISH to the ovarian cell lines using these
and other overlapping YAC and P1 clones produced 2 to 4 times more
hybridization signals than a reference probe at 3p25 carrying the
STS D3S1293. The region of highest copy number typically did not
extend outside the region defined by these two YACs (FIG. 2A).
OVCAR3 shows a larger region of amplification throughout the
region, with the aforementioned clones still manifesting the
largest increase in copy number. Breast cancer lines ZR75-30 and
MCF-7 showed a smaller increase in copy number for the same clones
in the region, and melanoma lines 355 and 457 failed to show
increases in copy number in this region (FIG. 2C).
[0108] Copy number abnormalities in ovarian tumor samples. All
paraffin-embedded ovarian tumor samples also show the same regions
of increase in copy umber that were seen in the ovarian cancer cell
lines. As seen from Figure, the region of increase in copy number
is better defined in the tumor samples, with a sharp increase in
the relative copy number for the P1 and its associated YACS.
Increases in copy number for tumor sample 595-7615 seem to involve
a larger amplicon, as all the probes tested in this tumor show a
relatively elevated copy number.
[0109] Based on the FISH results of tumor samples and cell lines,
we have delineated the critical region of increase in copy number
in ovarian cancer on the long arm of chromosome 3 to the region of
3q26.3, spanning one P1 and 5 YACs that share sequences with this
P1.
[0110] A search of the Unigene and Genome Databases in the critical
region revealed 2 known genes, glucose transporter 2 (GLUT2) and
phosphatidylinositol 3-kinase, catalytic alpha polypeptide
(PIK3CA). Other genes including, protein L22 (RPL22), ectropic
viral integration site 1 (EVI1), Cornelia De Lange Syndrome (CDL),
butyrylcholinesterase (BCHE), epithelial cell transforming sequence
2 (ECT2), Friend murine leukemia virus integration site 1 homologue
(FIM1), and myelodysplasia syndrome 1 (MDS1) map near to but
outside the critical region of increased copy number (FIG. 1). The
RNA component of the telomerase, HTR, maps to the distal edge of
the critical and has been suggested as a candidate gene selected
for by copy number increase in this area (Soder et al. Oncogene
14:1013-1021 (1997). This gene was mapped to YAC 821G8 by PCR using
primers to HTR. The region targeted by this YAC is present in
increased copy number in one of the six tumors ans in 4 of 8
ovarian cancer cell lines.
Example 2
[0111] This example shows that PIK3CA expression is increased in
ovarian cancers as a result of the PIK3CA copy number increase.
[0112] As noted above. PIK3CA is an attractive as a candidate
oncogene in ovarian cancer because of the broad range of cellular
functions that are modulated by increased PI3-kinase activity
(Cantley et al. J. Am. Soc. Nephrol. 5:1872-1881 (1995), Fry
Biochi. Biophys. Ada 1226:237-268 (1994)). These include increased
including cell proliferation, accelarated glucose transport and
catabolism (Frevert et al. Mol Cell Biol 117:190-198
(1997),Tsakiridis et al., Endocrinology 136:4315-4322 (1995),
altered cell adhesion (Chen et al. J. Biol Chem. 269:31229-31233
(1994), Kinashi et al. Blood 86:2086-2090 (1995)) and altered
vesicle transport (Joly et al. J. Biol. Chem. 270:13225-13230
(1995) and Dudek et al. Science 275:661-665 (1997)).
[0113] Increased PI-kinase activity also is implicated in
abrogating apoptosis. For example, neuronal survival is increased
after activation of PI-kinase by treatment with IGF1 ((Franke et
al, Cell 88:435-437 (1997), c-myc induced apoptosis is decreased in
fibroblasts after activation of the downstream Akt (Kauffinann-Zeh
et al. Nature 385:544-548 (1997)), survival is decreased in
cisplatin-treated ovarian cancer cells after treatment with
rapamycin (Shi et al. Cancer Research 55:982-1988 (1995)) and
survival is increased in MDCK cells detached from the extracellular
matrix after activation of Akt (Khwaja et al. EMBO J. 16:2783-2793
(1997)). Observations linking PI-kinase activity more directly to
cancer include association of the PI-kinase pathway with the ras
and wnt signaling pathways (both known to be disregulated by
genetic aberrations in human cancers) and demonstration that c-p3k,
an avian homolog of PIK3CA is a potent transforming gene in
cultured chicken embryo fibroblasts (Chang et al. Science
276:1848-1850 (1997)). In addition, Akt2, a homolog of the
downstream PI-kinase effector Akt, has been found to be amplified
in -15% of ovarian cancers (Cheng et al. Proc Natl Acad Sci USA 89:
9267-71 (1992)).
[0114] Experiments were performed to determine whether PIK3CA
expression and OI3-kinase activity are increased in ovarian cancers
as a result of the PIK3CA copy number increase. Expression of the
PIK3CA subunit of PI3-kinase was assessed by Western blot analysis
in cancer cell lines and normal epithelia cells. Ovarian cancer
cell lines and breast cancer cell lines were cultured in RPMI 1640
medium with 10% fetal bovine serum (FBS). Normal ovarian surface
epithelia cells (NOE) were isolated from fresh normal ovarian
biopsy specimens and cultured in medium 199 with Earle's salt and
MCDB105 (1:1 ratio) plus 15% FBS and 10 ng/ml of EGF. Tumor cells
(ASC) were purified from ascites of ovarian cancer patients using
density gradients and negative selection with CD45 immunomagnetic
beads. Cells were lysed in 1% NP-40 lysis buffer (50 mM Hepes,
pH7.4, 150 mM NaCl, 50 mM ZnC12, 50 mM NaH2PO4, 50 mM NaF, 2 mM
EDTA, 1 mM Na3VO4, 2 mM PMSF, 10 mg/ml of aprotinin). Protein
concentration of the cell lysates were determined by BCA protein
assay subjected to immunoprecipitation with 2 mg of goat polyclonal
antibody against the amino terminus of PIK3CA (Santa Cruz
Biotechnology) and protein G-conjugated sepharose 4B (Pharmacia)
for total 3 hour incubation. Immunoprecipitated proteins were
separated by 8% SDS PAGE and immunoblotted with a goat antibody to
the carboxy terminus of PIK3CA (Santa Cruz Biotechnology).
HRP-conjugated donkey anti-goat IgG was used as secondary reagent.
The membranes were developed by ECL and exposed to X-ray films.
[0115] PIK3CA expression relative to normal ovarian epithelial
(NOE) cells was assessed in three ovarian cancer cell lines
(OVCAR3, OCC1 and SKOV3) and Was significantly overexpressed in all
three lines. Tumor cells purified from ovarian ascites fluid also
overexpressed PIK3CA in 5 of 6 cases. However, PIK3CA was not
highly expressed in the breast cancer cell line MDA-MB-453 that did
not have increased PIK3CA copy number nor in normal breast
epithelial cell lines MCF10F or several other breast cancer cell
lines. Immunodepletion experiments with an monoclonal antibody
against p85 showed that essentially all PIK3CA protein precipitates
with p85 protein in cells in which PIK3CA is overexpressed. The
reverse experiment; immunodepletion with an antibody against PIK3CA
showed depletion of only about half of all p85 protein. Thus, it is
reasonable to expect that overexpression of PIK3CA will lead to
increased heterodimer formation and increased PI-kinase activity.
This was confirmed by demonstrating that protein immunoprecipitated
with an antibody against PIK3CA had increased lipid kinase activity
in the three ovarian cancer cell lines relative to NOE (FIG. 3).
PI3-kinase activity was assessed in NOE and ovarian cancer cell
lines as follows. NOE and ovarian cancer cells were lysed in 1%
NP-40 lysis buffer. PI3-kinase was immunoprecipitated from each
cell lysate (1 mg of cellular protein) by goat anti-PIK3CA (amino
terminus) antibody or rabbit antibody to p85 regulatory subunit of
PI3-kinase (upstate Biotechnology, Inc.). The immunoprecipitates
were washed sequentially in: a) PBS, 100 mM Na3VO4, 1% Triton
X-100; b) 100 mM Tris, pH7.6, 0.5M LiCl, 100 mM Na3VO4; c) 100 mM
Tris, pH7.6, 100 mM NaCl, 1 mM EDTA, 100 mM Na3VO4; d) 20 mM Hepes,
pH7.5, 50 mM NaCl, 5 mM EDTA, 30 mM NaPPi, 200 mM Na3VO4, 1 mM
PMSF, 0.03% Triton X-100. Immunoprecipitates were resuspended in 30
ml kinase reaction buffer (33 mM Tris, pH7.6, 125 mM NaCl, 15 mM
MgC12, 200 mM adenosine, 20 mM ATP, 30 mCi [g-32P]ATP).
Phosphatidylinositol (PI) was resuspended in 20 mM Hepes, pH7.5 at
2 mg/ml and sonicated on ice for 10 min. PI3-kinase reaction was
initiated by addition of 10 ml of the PI suspension. The reaction
proceeded for 30 min at room temperature and was terminated by 200
ml of IN HCl. Lipids were extracted by 600 ml of chloroform:
methanol (1:1). The organic phase was washed with H2O, collected
and dried by vacuum centrifugation. The lipids were resuspended in
20 ml of chloroform: methanol (1:1) and resolved on Silica gel G60
thin-layer chromatography (TLC) plates in chloroform: methanol:
NH4OH : H2O (60:47:2; 11.3). Radiolabeled phosphatidylinositol
phosphate was visualized by autoradiography and then scraped off
the plates and quantitated by .beta.-scintillation counting.
[0116] Increased PI-kinase activity might contribute to tumor
progression by increasing the rate of cell proliferation and/or
increasing cell survival. Incubation of the OVCAR3 ovarian cancer
cell line with the specific PI-kinase inhibitor LY294002 induced a
marked decrease in cellular proliferation as indicated by thymidine
incorporation (FIG. 4a).
[0117] Cell proliferation and viability assay was assessed before
and after treatment with the PI3-kinase inhibitor LY294002 as
follows. Cells were cultured in 96-well plates (15.times.103
cells/well) and serum starved overnight prior to the addition of
LY294002 (Calbiochem) at various concentrations. For thymidine
incorporation assay, cells were incubated with LY294002 in the
presence of 0.5% DMSO and 0.5% fetal bovine serum for 48 hours
followed by another 18 hour incubation with [3H]thymidine (1
mCi/well, Amersham). Cells were harvested by 5% TCA and 0.25N NaOH.
[3H]thymidine incorporation was measured by b-scintillation
counting. Percent Maximum was converted from cpm as: % Maximum=cpm
(with LY294002)/cpm (without LY294002).times.100. For viability
assay, cells were incubated with LY294002 as described above for 96
hours. MIT (25 ml of 5 ml/ml in PBS) was added to each well and
incubated at 37.degree. C. for 2 hours. Cells were then lysed by
addition of 100 ml lysis buffer (20% SDS in 50%
N,N-dimethylformamide, pH4.7). The cultures were set at 37.degree.
C. overnight and measured by microplate reader at 570 nm
wavelength. Percent Maximum was converted from OD570 as indicated
above.
[0118] OVCAR3 cells were more sensitive to the effect of LY294002
than NOE (FIG. 5A) or the MCF10A or MCF10F cell lines which had
normal PIK3CA copy number and low p110.alpha. levels (see above).
Strikingly, inhibition of PI-kinase with LY294002 resulted in a
marked decrease in viability of OVCAR3 cell as compared to NOE or
MCF10F cells as assessed by the ability to convert the MIT dye
(FIG. 5B). In fact, OVCAR3 cells were 10 (in the presence of 0.5%
FCS) to 100 (in serum free media) times more sensitive to the
effect of LY294002. This decrease in viability (FIG. 5B) was
associated with an increased rate of programmed cell death at 24
(24% vs 51%) and 48 hour (22% vs 52%) as assessed by a fluorescence
based measurement of free DNA ends Apo direct, Phoenix Flow
Systems.
[0119] Taken together, these studies suggest that increased copy
number at 3q26.3 contributes to ovarian cancer genesis and/or
progression by increasing PI-kinase activity. The exact mechanism
by which this occurs remains unknown since the level of p110.alpha.
protein increases more than can be accounted for by the 2 to 4-fold
increase in copy number. One possibility is that the genomic
changes disregulate gene activity by altering regulatory sequences
or by disrupting other feed back control mechanisms. However it
occurs, PI-kinase activity seems to be increased dramatically
relative to NOE in ovarian cancer cell lines and tumors showing
increased PIK3CA copy number.
[0120] Increasing PI-kinase activity might contribute to tumor
development or progression by increasing the rate of cell
proliferation (e.g. by activating ras and/or by altering
transcription of wnt-1 responsive gene by inhibiting the production
of GSK-3 needed for proteolytic degradation of .beta.-catenin
(Hopkin The Journal Of NIH Research 9:21-23 (1997)). The influence
of PI-kinase activity on apoptosis in cells separated from the
extracellular matrix may be significant in ovarian cancer because
of the strong association between ovarian cancer incidence and
number of cycles of ovulation. The disruption of the
stromal-epithelial structure, such as occurs during ovulation, has
been found to induce genetic aberrations and to be tumorigenic in
other model systems. Thus, abrogation of apoptosis by activating
PI-kinase might allow genetically damaged cells to survive and to
evolve toward a malignant phenotype. This is consistent with our
finding that increased copy number at 3q26 is an early event in
ovarian cancer.
[0121] These associations and the strong inhibition by LY294002 of
cell proliferation and survival suggest that therapeutic agents
targeting the PI3-kinase pathway may be effective against ovarian
cancers. In addition, the presence in the serum of one or more of
the proteins in the PI3-kinase pathway also might be diagnostic for
the disease since increased copy number at 3q26.2 has been
described as an early event in ovarian cancer. This is important
since earlier detection of this disease is likely to have a
significant impact on patient survival.
[0122] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference for all purposes.
* * * * *
References