U.S. patent application number 10/165099 was filed with the patent office on 2003-10-02 for methods and compositions for the diagnosis of cancer susceptibilities and defective dna repair mechanisms and treatment thereof.
This patent application is currently assigned to Dana Farber Cancer Institute. Invention is credited to D'Andrea, Alan D., Fox, Edward A., Grompe, Markus, Taniguchi, Toshiyasu, Timmers, Cynthia.
Application Number | 20030188326 10/165099 |
Document ID | / |
Family ID | 46150144 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030188326 |
Kind Code |
A1 |
D'Andrea, Alan D. ; et
al. |
October 2, 2003 |
Methods and compositions for the diagnosis of cancer
susceptibilities and defective DNA repair mechanisms and treatment
thereof
Abstract
Methods and compositions for the diagnosis of cancer
susceptibilities, defective DNA repair mechanisms and treatments
thereof are provided. Among sequences provided here, the FANCD2
gene has been identified, and probes and primers are provided for
screening patients in genetic-based tests and for diagnosing
Fanconi Anemia and cancer. The FANCD2 gene can be targeted in vivo
for preparing experimental mouse models for use in screening new
therapeutic agents for treating conditions involving defective DNA
repair. The FANCD2 polypeptide has been sequenced and has been
shown to exist in two isoforms identified as FANCD2-S and the
monoubiquinated FANCD-L form. Antibodies including polyclonal and
monoclonal antibodies have been prepared that distinguish the two
isoforms and have been used in diagnostic tests to determine
whether a subject has an intact Fanconi Anemia/BRCA pathway.
Inventors: |
D'Andrea, Alan D.;
(Winchester, MA) ; Taniguchi, Toshiyasu; (Boston,
MA) ; Timmers, Cynthia; (Columbus, OH) ;
Grompe, Markus; (Portland, OR) ; Fox, Edward A.;
(Boston, MA) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Assignee: |
Dana Farber Cancer
Institute
|
Family ID: |
46150144 |
Appl. No.: |
10/165099 |
Filed: |
June 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10165099 |
Jun 6, 2002 |
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09998027 |
Nov 2, 2001 |
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60245756 |
Nov 3, 2000 |
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Current U.S.
Class: |
800/8 ; 435/226;
435/320.1; 435/325; 435/6.12; 435/69.1; 435/7.23; 536/23.2 |
Current CPC
Class: |
C07K 14/47 20130101;
C12Q 1/6886 20130101; A01K 2217/075 20130101; G01N 33/5091
20130101; G01N 2333/47 20130101; G01N 2800/52 20130101; C12Q 1/025
20130101; C12Q 2600/158 20130101; C07K 16/18 20130101; G01N 33/6893
20130101; G01N 33/57484 20130101; G01N 2500/00 20130101; G01N
33/5011 20130101; A01K 2217/05 20130101; C12Q 2600/156 20130101;
G01N 33/574 20130101 |
Class at
Publication: |
800/8 ; 435/6;
435/7.23; 435/69.1; 435/320.1; 435/325; 435/226; 536/23.2 |
International
Class: |
A01K 067/00; C12Q
001/68; G01N 033/574; C07H 021/04; C12N 009/64; C12P 021/02; C12N
005/06 |
Goverment Interests
[0002] The work described herein was supported by the National
Institute of Health, NIH Grant No. Health grants RO1HL52725-04,
RO1DK43889-09, 1PO1HL48546, and PO1HL54785-04. The US Government
has certain rights to the claimed invention.
Claims
We claim:
1. A method of diagnosing or determining if a patient has cancer or
is at increased risk of cancer, the method comprising testing a
Fanconi Anemia/BRCA pathway gene for the presence of a
cancer-associated defect, wherein said presence of one or more
cancer-associated defects is indicative of cancer or an increased
risk of cancer in said patient.
2. The method according to claim 1, wherein said cancer is breast,
ovarian, or prostate cancer.
3. The method according to claim 1, wherein said cancer-associated
defect results in a reduction in the ratio of FANC D2-L relative to
FANC D2-S as compared to said ratio in a patient without one or
more cancer-associated defects in a Fanconi Anemia/BRCA pathway
gene.
4. A method of diagnosing or determining if a patient has cancer or
is at increased risk of cancer, the method comprising testing a
Fanconi Anemia/BRCA pathway protein for the presence of a
cancer-associated defect, wherein said presence of a
cancer-associated defect is indicative of cancer or an increased
risk of cancer in said patient.
5. The method of claim 4, wherein said cancer is breast, ovarian,
or prostate cancer.
6. A method of diagnosing or determining if a patient is at
increased risk of developing cancer, comprising the steps of: (a)
providing a tissue sample from said patient; (b) inducing DNA
damage in the cells of said tissue sample; and (c) assaying for the
presence of FANC D2-S and FANC D2-L proteins in said cells; wherein
a reduction in the ratio of FANC D2-L to FANC D2-S is indicative
that said patient is at increased risk of developing cancer.
7. The method of claim 6, wherein said cancer is breast, ovarian,
or prostate cancer.
8. The method of claim 6, wherein said patient has no known
cancer-associated defects in the BRCA-1 or BRCA-2 genes.
9. The method of claim 6, wherein said patient has one or more
cancer-associated defects in the BRCA-1 or BRCA-2 genes.
10. The method of claim 6, wherein a plurality of said tissue
samples are distributed in an array.
11. A method of determining if a patient has cancer, or is at
increased risk of developing cancer, wherein the patient has no
known cancer causing defect in the BRCA 1 or BRCA-2 genes, said
method comprising the steps of: (a) providing a DNA sample from
said patient; (b) amplifying the FANC D2 gene from said patient
with the FANC D2 gene-specific polynucleotide primers of SEQ ID
NOs: 115-186; (c) sequencing the amplified FANC D2 gene; and (d)
comparing the FANC D2 gene sequence from said patient to a
reference FANC D2 gene sequence, where a discrepancy between the
two gene sequences indicates the presence of a cancer-associated
defect; wherein the presence of one or more cancer-associated
defects indicates said patient has cancer or is at an increased
risk of developing cancer.
12. The method of claim 11, wherein said cancer is breast, ovarian,
or prostate cancer.
13. The method of claim 11, wherein said patient has no known
cancer-associated defects in the BRCA-1 or FANC-D1/BRCA-2
genes.
14. The method of claim 11, wherein said patient has one or more
cancer-associated defects in the BRCA-1 or FANC-D1/BRCA-2
genes.
15. The method of claim 11, wherein a plurality of said DNA samples
are distributed on a microarray.
16. A method of screening for a chemosensitizing agent, said method
comprising the steps of: (a) providing a potential inhibitor of the
Fanconi Anemia/BRCA pathway; (b) providing a tumor cell line that
is resistant to one or more anti-neoplastic agents; (c) contacting
said tumor cell line and said potential inhibitor of the Fanconi
Anemia/BRCA pathway and said one or more anti-neoplastic agents;
and (d) measuring the growth rate of said tumor cell line in the
presence of said inhibitor of the Fanconi Anemia/BRCA pathway and
said anti-neoplastic agent; wherein a reduced growth rate of the
tumor cell line, relative to cells of the tumor cell line in the
presence of the anti-neoplastic agent and the absence of said
inhibitor of the Fanconi Anemia/BRCA pathway, is indicative that
the potential inhibitor is a chemosensitizing agent.
17. The method of claim 16, wherein said potential inhibitors of
the Fanconi Anemia/BRCA pathway are screened on a microarray,
wherein the microarray contains addresses containing one or more
cells that are resistant to one or more anti-neoplastic agents.
18. The method according to claim 16, wherein said potential
inhibitor of the Fanconi Anemia/BRCA pathway is an inhibitor of the
ubiquitination of the FANC D2 protein.
19. The method according to claim 16, wherein said anti-neoplastic
agent is cisplatin.
20. The method according to claim 16, wherein said tumor cell line
is an ovary cancer cell line.
21. A method of treating a patient having a cancer, wherein the
cancer is resistant to a anti-neoplastic agent, comprising the step
of administering a therapeutically effective amount of an inhibitor
of the Fanconi Anemia/BRCA pathway together with said
anti-neoplastic agent.
22. The method according to claim 21, wherein the said
anti-neoplastic agent is cisplatin.
23. The method according to claim 21, wherein said potential
inhibitor of the Fanconi Anemia/BRCA pathway is an inhibitor of the
ubiquitination of the FANC D2 protein.
24. The method according to claim 21, wherein said tumor cell line
is an ovary cancer cell line.
25. A method for screening for a cancer therapeutic, the method
comprising the steps of: (a) providing one or more cells containing
a Fanconi Anemia/BRCA pathway gene having one or more cancer
associated defects; (b) growing said cells in the presence of a
potential cancer therapeutic; and (c) determining the rate of
growth of said cells in the presence of said potential cancer
therapeutic relative to the rate of growth of equivalent cells
grown in the absence of said potential cancer therapeutic; wherein
a reduced rate of growth of said cells in the presence of said
potential cancer therapeutic, relative to the rate of growth of
equivalent cells grown in the absence of said potential cancer
therapeutic, indicates that the potential cancer then is a cancer
therapeutic.
26. The method of claim 26, wherein said cells containing a Fanconi
Anemia/BRCA pathway gene having one or more cancer associated
defects are distributed in a array.
27. A method of predicting the efficacy of a therapeutic agent in a
cancer patient, comprising the steps of: (a) providing a tissue
sample from said cancer patient who is being treated with said
therapeutic agent; (b) inducing DNA damage in the cells of said
tissue sample; (c) detecting the presence of FANC D2-L protein in
said cells; wherein the presence of FANC D2-L is indicative of a
reduced efficacy of said therapeutic agent in said cancer
patient.
28. The method of claim 27, wherein said therapeutic agent is
cisplatin.
29. A method of determining resistance of tumor cells to an
anti-neoplastic agent, comprising the steps of: (a) providing a
tissue sample from a patient who is being treated with an
anti-neoplastic agent; (b) inducing DNA damage in the cells of said
tissue sample; and (c) determining the methylation state of a
Fanconi Anemia/BRCA pathway gene; wherein methylation of a Fanconi
Anemia/BRCA gene is indicative of resistance of the tumor cells to
an anti-neoplastic agent.
30. The method according to claim 29, wherein said Fanconi
Anemia/BRCA gene is the FANC F gene.
31. The method according to claim 29, wherein said anti-neoplastic
agent is cisplatin.
32. A kit for detecting defects in the FANC D2 gene, comprising a
polynucleotide primer pair specific for the FANC D2 gene, a
reference FANC D2 gene sequence and packaging materials
therefore.
33. A kit for detecting the presence of FANC D2-L, comprising a
FANC D2-L-specific antibody and packaging materials therefore.
34. A kit for determining the methylation state of a Fanconi
Anemia/BRCA pathway gene, comprising FANC D2 polynucleotide primer
pairs and probes, a control unmethylated reference FANC D2 gene
sequence and packaging materials therefore.
35. A kit for screening for a chemosensitizing agent, comprising a
tumor cell line that is resistant to one or more anti-neoplastic
agents and packaging materials therefore.
36. The kit of claim 35, wherein said tumor cell line is an ovary
tumor cell line.
37. The kit according to claim 36, wherein said ovary tumor cell
line is a cisplatin resistant ovary tumor cell line.
38. The kit according to claim 36, wherein said anti-neoplastic
agent is cisplatin.
39. A microarray containing one or more nucleic acid sequences from
one or more Fanconi Anemia/BRCA pathway genes.
40. The microarray of claim 39, wherein the genes are selected from
the group consisting of: ATM, FANC A, FANC B, FANC C, FANC D1, FANC
D2, FANC E, FANC F and FANC G.
41. A method of determining if a patient has cancer, or is at
increased risk of developing cancer, said method comprising the
steps of: (a) providing the microarray of claim 39; (b) providing a
nucleic acid sample from said patient; (c) hybridizing said nucleic
acid sample to said nucleic acid sequences from the Fanconi
Anemia/BRCA pathway on said microarray; and (d) detecting the
presence of mutations in the Fanconi Anemia/BRCA pathway genes in
the nucleic acid sample from said patient; wherein said detecting
the presence of mutations is indicative of a patient who has
cancer, or is at increased risk of developing cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
Application Ser. No. 09/998,027, filed Nov. 2, 2001, which in turn
claims priority from provisional application 60/245,756 filed Nov.
3, 2000. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND
[0003] The present invention relates to the diagnosis of cancer
susceptibilities in subjects having a defect in the FANCD2 gene and
the determination of suitable treatment protocols for those
subjects who have developed cancer. Animal models with defects in
the FANCD2 gene can be used to screen for therapeutic agents.
[0004] Fanconi Anemia (FA) is an autosomal recessive cancer
susceptibility syndrome characterized by birth defects, bone marrow
failure and cancer predisposition. Cells from FA patients display a
characteristic hypersensitivity to agents that produce interstrand
DNA crosslinks such as mitomycin C or diepoxybutane. FA patients
develop several types of cancers including acute myeloid leukemias
and cancers of the skin, gastrointestinal; and gynecological
systems. The skin and gastrointestinal tumors are usually squamous
cell carcinomas. At least 20% of patients with FA develop cancers.
The average age of patients who develop cancer is 15 years for
leukemia, 16 years for liver tumors and 23 years for other tumors.
(D'Andrea et al., Blood, (1997) Vol. 90, p. 1725, Garcia-Higuera et
al., Curr. Opin. Hematol., (1999) Vol. 2, pp. 83-88 and Heijna et
al., Am. J. Hum. Genet. Vol. 66, pp. 1540-1551).
[0005] FA is genetically heterogeneous. Somatic cell fusion studies
have identified at least seven distinct complementation groups
(Joenje et al., (1997) Am. J. Hum. Genet., Vol. 61, pp. 940-944 and
Joenje et al., (2000) Am. J. Hum. Genet, Vol. 67, pp. 759-762).
This observation has resulted in the hypothesis that the FA genes
define a multicomponent pathway involved in cellular responses to
DNA cross-links. Five of the FA genes (FANCA, FANCC, FANCE, FANCF
and FANCG) have been cloned and the FANCA, FANCC and FANCG proteins
have been shown to form a molecular complex with primarily nuclear
localization. FANCC also localizes in the cytoplasm. Different FA
proteins have few or no known sequence motifs with no strong
homologs of the FANCA, FANCC, FANCE, FANCF, and FANCG proteins in
non-vertebrate species. FANCF has weak homology of unknown
significance to an E. Coli RNA binding protein. The two most
frequent complementation groups are FA-A and FA-C which together
account for 75%-80% of FA patients. Multiple mutations have been
recognized in the FANCA gene that span 80 kb and consists of at
least 43 exons. FANCC has been found to have 14 exons and spans
approximately 80 kb. A number of mutations in the FANCC gene have
been identified which are correlated with FA of differing degrees
of severity. FA-D has been identified as a distinct but rare
complementation group. Although FA-D patients are phenotypically
distinguishable from patients from other subtypes, the FA protein
complex assembles normally in FA-D cells (Yamashita et al, (1998)
P.N.A.S., Vol. 95, pp. 13085-13090).
[0006] The cloned FA proteins encode orphan proteins with no
sequence similarity to each other or to other proteins in GenBank
and no functional domains are apparent in the protein sequence.
Little is known regarding the cellular or biochemical function of
these proteins.
[0007] Diagnosis of FA is complicated by the wide variability in FA
patient phenotype. Further confounding diagnosis, approximately 33%
of patients with FA have no obvious congenital abnormalities.
Moreover, existing diagnostic tests do not differentiate FA
carriers from the general population. The problems associated with
diagnosis are described in D'Andrea et al., (1997). Many cellular
phenotypes have been reported in FA cells but the most consistent
is hypersensitivity to bifunctional alkylating agents such as
mitomycin C or diepoxybutane. These agents produce interstrand DNA
cross-links (an important class of DNA damage).
[0008] Diagnosing cancer susceptibility is complicated because of
the large number of regulatory genes and biochemical pathways that
have been implicated in the formation of cancers. Different cancers
depending on how they arise and the genetic lesions involved may
determine how a subject responds to any particular therapeutic
treatments. Genetic lesions that are associated with defective
repair mechanisms may give rise to defective cell division and
apoptosis which in turn may increase a patient's susceptibility to
cancer. FA is a disease condition in which multiple pathological
outcomes are associated with defective repair mechanisms in
addition to cancer susceptibility.
[0009] An understanding of the molecular genetics and cell biology
of Fanconi Anemia pathway can provide insights into prognosis,
diagnosis and treatment of particular classes of cancers and
conditions relating to defects in DNA repair mechanisms that arise
in non-FA patients as well as FA patients.
SUMMARY OF THE INVENTION
[0010] The invention features a method of diagnosing or determining
if a patient has cancer or is at increased risk of cancer, where
the method includes testing a Fanconi Anemia/BRCA pathway gene for
the presence of a cancer-associated defect, where said presence of
one or more cancer-associated defects is indicative of cancer or an
increased risk of cancer in said patient. The cancer can be breast,
ovarian, or prostate cancer, or other forms of cancer. The
cancer-associated defect can be one which results in a reduction in
the ratio of FANC D2-L relative to FANC D2-S as compared to the
ratio in a patient without one or more cancer-associated defects in
a Fanconi Anemia/BRCA pathway gene.
[0011] The invention also features a method of diagnosing or
determining if a patient has cancer or is at increased risk of
cancer, where the method includes testing a Fanconi Anemia/BRCA
pathway protein for the presence of a cancer-associated defect,
where said presence of a cancer-associated defect is indicative of
cancer or an increased risk of cancer in said patient. The cancer
can be breast, ovarian, or prostate cancer, or other forms of
cancer.
[0012] An another aspect, the invention features a method of
diagnosing or determining if a patient is at increased risk of
developing cancer, where the method includes the steps of: (a)
providing a tissue sample from said patient; (b)inducing DNA damage
in the cells of said tissue sample; and (c) assaying for the
presence of FANC D2-S and FANC D2-L proteins in said cells; wherein
a reduction in the ratio of FANC D2-L to FANC D2-S is indicative
that said patient is at increased risk of developing cancer. The
cancer can be breast, ovarian, or prostate cancer, or other forms
of cancer. The patient can be known or not known to have any
previously-known cancer-associated defects in the BRCA-1 or BRCA-2
genes. A plurality of such tissue samples can be distributed on or
in an array.
[0013] An another aspect, the invention features a method of
determining if a patient has cancer, or is at increased risk of
developing cancer, where the patient has no known cancer causing
defect in the BRCA 1 or BRCA-2 genes, where the method comprises
the steps of: (a) providing a DNA sample from said patient; (b)
amplifying the FANC D2 gene from said patient with the FANC D2
gene-specific polynucleotide primers of SEQ ID NOs: 115-186; (c)
sequencing the amplified FANC D2 gene; and (d) comparing the FANC
D2 gene sequence from said patient to a reference FANC D2 gene
sequence, where a discrepancy between the two gene sequences
indicates the presence of a cancer-associated defect; where the
presence of one or more cancer-associated defects indicates said
patient has cancer or is at an increased risk of developing cancer.
The cancer can be breast, ovarian, or prostate cancer, or other
forms of cancer. The patient can be known or not known to have any
previously-known cancer-associated defects in the BRCA-1 or
FANC-D1/BRCA-2 genes. A plurality of such tissue samples can be
distributed on or in an array. SEQ ID NOs: 115-186 are matched sets
of primers, as shown in Table 7, with the odd-numbered primers
being forward primers, and the even-numbered primers being reverse
primers. Primers can also be used from different pairs, to make new
pairings of primers, e.g., SEQ ID NO: 115 can be used with SEQ ID
NO: 118, etc. By "discrepancy" is meant a difference between the
two sequences, where the difference is know to be associated with
cancer.
[0014] In a further aspect, the invention features a method of
screening for a chemosensitizing agent, where the method comprises
the steps of: (a) providing a potential inhibitor of the Fanconi
Anemia/BRCA pathway; (b) providing a tumor cell line that is
resistant to one or more anti-neoplastic agents; (c) contacting
said tumor cell line and said potential inhibitor of the Fanconi
Anemia/BRCA pathway and said one or more anti-neoplastic agents;
and (d) measuring the growth rate of said tumor cell line in the
presence of said inhibitor of the Fanconi Anemia/BRCA pathway and
said anti-neoplastic agent; where a reduced growth rate of the
tumor cell line, relative to cells of the tumor cell line in the
presence of the anti-neoplastic agent and the absence of said
inhibitor of the Fanconi Anemia/BRCA pathway, is indicative that
the potential inhibitor is a chemosensitizing agent. The potential
inhibitors of the Fanconi Anemia/BRCA pathway can be screened on a
microarray, where the microarray contains addresses containing one
or more cells that are resistant to one or more anti-neoplastic
agents. The potential inhibitor of the Fanconi Anemia/BRCA pathway
can be an inhibitor of the ubiquitination of the FANC D2 protein.
The anti-neoplastic agent can be cisplatin. The tumor cell line can
be an ovary cancer cell line.
[0015] In another aspect, the invention features a method of
treating a patient having a cancer, where the cancer is resistant
to a anti-neoplastic agent, where the method comprises the step of
administering a therapeutically effective amount of an inhibitor of
the Fanconi Anemia/BRCA pathway together with said anti-neoplastic
agent. The anti-neoplastic agent can be cisplatin. The potential
inhibitor of the Fanconi Anemia/BRCA pathway can be an inhibitor of
the ubiquitination of the FANC D2 protein. The tumor cell line can
be an ovary cancer cell line.
[0016] In an additional aspect, the invetion features a method for
screening for a cancer therapeutic, where the method comprises the
steps of: (a) providing one or more cells containing a Fanconi
Anemia/BRCA pathway gene having one or more cancer associated
defects; (b) growing said cells in the presence of a potential
cancer therapeutic; and (c) determining the rate of growth of said
cells in the presence of said potential cancer therapeutic relative
to the rate of growth of equivalent cells grown in the absence of
said potential cancer therapeutic; where a reduced rate of growth
of said cells in the presence of said potential cancer therapeutic,
relative to the rate of growth of equivalent cells grown in the
absence of said potential cancer therapeutic, indicates that the
potential cancer then is a cancer therapeutic. The cells can
contain a Fanconi Anemia/BRCA pathway gene having one or more
cancer associated defects are distributed in a array, or several
such genes.
[0017] The invention also features a method of predicting the
efficacy of a therapeutic agent in a cancer patient, where the
method comprises the steps of: (a) providing a tissue sample from
said cancer patient who is being treated with said therapeutic
agent; (b) inducing DNA damage in the cells of said tissue sample;
and (c) detecting the presence of FANC D2-L protein in said cells;
where the presence of FANC D2-L is indicative of a reduced efficacy
of said therapeutic agent in said cancer patient. The therapeutic
agen can be an anti-neoplastic agent, e.g., can be cisplatin.
Alternatively, in step (c), one can detect both FANC-D2-S and
FANC-D2-L, where a reduction in the ratio of FANC D2-L relative to
FANC D2-S as compared to the ratio in a non-cancer patient
indicates reduced efficacy.
[0018] The invention also features a method of determining
resistance of tumor cells to an anti-neoplastic agent, comprising
the steps of: (a) providing a tissue sample from a patient who is
being treated with an anti-neoplastic agent; (b) inducing DNA
damage in the cells of said tissue sample; and (c) determining the
methylation state of a Fanconi Anemia/BRCA pathway gene; where
methylation of a Fanconi Anemia/BRCA gene is indicative of
resistance of the tumor cells to an anti-neoplastic agent. The
Fanconi Anemia/BRCA gene can be the FANC F gene. The
anti-neoplastic agent can be cisplatin.
[0019] The invention also features a kit for detecting defects in
the FANC D2 gene, comprising a polynucleotide primer pair specific
for the FANC D2 gene, a reference FANC D2 gene sequence and
packaging materials therefore.
[0020] The invention also features a kit for detecting the presence
of FANC D2-L, comprising a FANC D2-L-specific antibody and
packaging materials therefore.
[0021] The invention also features a kit for determining the
methylation state of a Fanconi Anemia/BRCA pathway gene, comprising
FANC D2 polynucleotide primer pairs and probes, a control
unmethylated reference FANC D2 gene sequence and packaging
materials therefore.
[0022] The invention also features a kit for screening for a
chemosensitizing agent, comprising a tumor cell line that is
resistant to one or more anti-neoplastic agents and packaging
materials therefore. The tumor cell line can be an ovary tumor cell
line, e.g., a cisplatin resistant ovary tumor cell line. The
anti-neoplastic agent can be cisplatin.
[0023] The invention also features a microarray containing one or
more nucleic acid sequences from one or more Fanconi Anemia/BRCA
pathway genes. The genes can be selected from the group consisting
of: ATM, FANC A, FANC B, FANC C, FANC D1, FANC D2, FANC E, FANC F
and FANC G.
[0024] The invention also features the use of such a microarray in
a method of determining if a patient has cancer, or is at increased
risk of developing cancer, where the method comprises the steps of:
(a) providing the microarray; (b) providing a nucleic acid sample
from said patient; (c) hybridizing said nucleic acid sample to said
nucleic acid sequences from the Fanconi Anemia/BRCA pathway on said
microarray; and (d) detecting the presence of mutations in the
Fanconi Anemia/BRCA pathway genes in the nucleic acid sample from
said patient; where detecting the presence of mutations is
indicative of a patient who has cancer, or is at increased risk of
developing cancer.
[0025] In a one embodiment of the invention there is provided an
isolated nucleic acid molecule that includes a polynucleotide
selected from (a) a nucleotide sequence encoding a polypeptide
having an amino acid sequence as shown in SEQ ID NO: 4; (b) a
nucleotide sequence at least 90% identical to the polynucleotide of
(b); (c) a nucleotide sequence complementary to the polynucleotide
of (b); (d) a nucleotide sequence at least 90% identical to the
nucleotide sequence shown in SEQ ID NO: 5-8, 187-188; and (e) a
nucleotide sequence complementary to the nucleotide sequence of
(d). The polynucleotide may be an RNA molecule or a DNA molecule,
such as a cDNA.
[0026] In another embodiment of the invention, an isolated nucleic
acid molecule is provided that consists essentially of a nucleotide
sequence encoding a polypeptide having an amino acid sequence
sufficiently similar to that of SEQ ID NO: 4 to retain the
biological property of conversion from a short form to a long form
of FANCD2 in the nucleus of a cell for facilitating DNA repair.
Alternately, the isolated nucleic acid molecule consists
essentially of a polynucleotide having a nucleotide sequence at
least 90% identical to SEQ ID NO: 9-191 or complementary to a
nucleotide sequence that is at least 90% identical to SEQ ID NO:
9-191.
[0027] In an embodiment, a method is provided for making a
recombinant vector that includes inserting any of the isolated
nucleic acid molecules described above into a vector. A recombinant
vector product may be made by this method and the vector may be
introduced to form a recombinant host cell into a host cell.
[0028] In an embodiment of the invention, a method is provided for
making an FA-D2 cell line, that includes (a) obtaining cells from a
subject having a biallelic mutation in a complementation group
associated with FA-D2; and (b) infecting the cells with a
transforming virus to make the FA-D2 cell line where the cells may
be selected from fibroblasts and lymphocytes and the transforming
virus selected from Epstein Barr virus and retrovirus. The FA-D2
cell line may be characterized by determining the presence of a
defective FANDC2 in the cell line for example by performing a
diagnostic assay selected from (i) a Western blot or nuclear
immunofluorescence using an antibody specific for FANCD2 and (ii) a
DNA hybridization assay.
[0029] In an embodiment of the invention, a recombinant method is
provided for producing a polypeptide, that includes culturing a
recombinant host cell wherein the host cell includes any of the
isolated nucleic acid molecules described above.
[0030] In an embodiment of the invention, an isolated polypeptide,
including an amino acid sequence selected from (a) SEQ ID NO: 4;
(b) an amino acid sequence at least 90% identical to (a); (c) an
amino acid sequence which is encoded by a polynucleotide having a
nucleotide sequence which is at least 90% identical to at least one
of SEQ ID NO: 5-8, 187-188; (d) an amino acid sequence which is
encoded by a polynucleotide having a nucleotide sequence which is
at least 90% identical to a complementary sequence to at least one
of SEQ ID NO: 5-8, 187-188; and (e) a polypeptide fragment of
(a)-(d) wherein the fragment is at least 50 aminoacids in
length.
[0031] The isolated polypeptide may be encoded by a DNA having a
mutation selected from nt 376A to G, nt 3707G to A, nt 904C to T
and nt 958C to T. Alternatively, the polypeptide may be
characterized by a polymorphism in DNA encoding the polypeptide,
the polymorphism being selected from nt 1122A to O, nt 1440T to C,
nt 1509C to T, nt 2141C to T, nt 2259T to C, nt 4098T to G, nt
4453G to A. Alternatively, the polypeptide may be characterized by
a mutation at amino acid 222 or amino acid 561.
[0032] In an embodiment of the invention, an antibody preparation
is described having a binding specificity for a FANCD2 protein
where the antibody may be a monoclonal antibody or a polyclonal
antibody and wherein the FANCD2 may be FANCD2-S or FANCD2-L.
[0033] In an embodiment of the invention, a diagnostic method is
provided for measuring FANCD2 isoforms in a biological sample where
the method includes (a) exposing the sample to a first antibody for
forming a first complex with FANCD2-L and optionally a second
antibody for forming a second complex with FANCD2-S; and (b)
detecting with a marker, the amount of the first complex and the
second complex in the sample. The sample may be intact cells or
lysed cells in a lysate. The biological sample may be from a human
subject with a susceptibility to cancer or having the initial
stages of cancer. The sample may be from a cancer in a human
subject, wherein the cancer is selected from melanoma, leukemia,
astocytoma, glioblastoma, lymphoma, glioma, Hodgkins lymphoma,
chronic lymphocyte leukemia and cancer of the pancreas, breast,
thyroid, ovary, uterus, testis, pituitary, kidney, stomach,
esophagus and rectum. The biological sample may be from a human
fetus or from an adult human and may be derived from any of a blood
sample, a biopsy sample of tissue from the subject and a cell line.
The biological sample may be derived from heart, brain, placenta,
liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,
testis, uterus, small intestine, colon, peripheral blood or
lymphocytes. The marker may be a fluorescent marker, the
fluorescent marker optionally conjugated to the FANCD2-L antibody,
a chemiluminescent marker optionally conjugated to the FANCD2-L
antibody and may bind the first and the second complex to a third
antibody conjugated to a substrate. Where the sample is a lysate,
it may be subjected to a separation procedure to separate FANCD2
isoforms and the separated isoforms may be identified by
determining binding to the first or the second FANCD2 antibody.
[0034] In an embodiment of the invention, a diagnostic test is
provided for identifying a defect in the Fanconi Anemia pathway in
a cell population from a subject, that includes selecting an
antibody to FANCD2 protein and determining whether the amount of an
FAND2-L isoform is reduced in the cell population compared with
amounts, in a wild type cell population; such that if the amount of
the FANCD2-L protein is reduced, then determining whether an amount
of any of FANCA, FANCB, FANCC, FANCD1, FANCE, FANCF or FANCG
protein is altered in the cell population compared with the wild
type so as to identify the defect in the Fanconi Anemia pathway in
the cell population. In one example, the amount of an isoform
relies on a separation of the FANCD2-L and FANCD2-S isoforms where
the separation may be achieved by gel electrophoresis or by a
migration binding banded test strip.
[0035] In an embodiment of the invention, a screening assay for
identifying a therapeutic agent, is provided that includes
selecting a cell population in which FAND2-L is made in reduced
amounts; exposing the cell population to individual members of a
library of candidate therapeutic molecules; and identifying those
individual member molecules that cause the amount of FANCD2-L to be
increased in the cell population. In one example, the cell
population is an in vitro cell population. In another example, the
cell population is an in vivo cell population, the in vivo
population being within an experimental animal, the experimental
animal having a mutant FANCD2 gene. In a further example, the
experimental animal is a knock-out mouse in which the mouse FAND2
gene has been replaced by a human mutant FANCD2 gene. In another
example, a chemical carcinogen is added to the cell population in
which FANCD2 is made in reduced amounts, to determine if any member
molecules can cause the amount of FANCD2-L to be increased so as to
protect the cells form the harmful effects of the chemical
carcinogen.
[0036] In an embodiment of the invention, an experimental animal
model is provided in which the animal FANCD2 gene has been removed
and optionally replaced by any of the nucleic acid molecules
described above.
[0037] In an embodiment of the invention, a method is provided for
identifying in a cell sample from a subject, a mutant FANCD2
nucleotide sequence in a suspected mutant FANCD2 allele which
comprises comparing the nucleotide sequence of the suspected mutant
FANCD2 allele with the wild type FANCD2 nucleotide sequence wherein
a difference between the suspected mutant and the wild type
sequence identifies a mutant FANCD2 nucleotide sequence in the cell
sample. In one example, the suspected mutant allele is a germline
allele. In another example, identification of a mutant FANCD2
nucleotide sequence is diagnostic for a predisposition for a cancer
in the subject or for an increased risk of the subject bearing an
offspring with Fanconi Anemia. In another example, the suspected
mutant allele is a somatic allele in a tumor type and identifying a
mutant FANCD2 nucleotide sequence is diagnostic for the tumor type.
In another example, the nucleotide sequence of the wild type and
the suspected mutant FANCD2 nucleotide sequence is selected from a
gene, a mRNA and a cDNA made from a mRNA. In another example,
comparing the polynucleotide sequence of the suspected mutant
FANCD2 allele with the wild type FANCD2 polynucleotide sequence,
further includes selecting a FANCD2 probe which specifically
hybridizes to the mutant FANCD2 nucleotide sequence, and detecting
the presence of the mutant sequence by hybridization with the
probe. In another example, comparing the polynucleotide sequence of
the suspected mutant FANCD2 allele with the wild type FANCD2
polynucleotide sequence, further comprises amplifying all or part
of the FANCD2 gene using a set of primers specific for wild type
FANCD2 DNA to produce amplified FANCD2 DNA and sequencing the
FANCD2 DNA so as to identify the mutant sequence. In another
example, where the mutant FANCD2 nucleotide sequence is a germline
alteration in the FANCD2 allele of the human subject, the
alteration is selected from the alterations set forth in Table 3
and where the mutant FANCD2 nucleotide sequence is a somatic
alteration, in the FANCD2 allele of the human subject, the
alteration is selected from the alterations set forth in Table
3.
[0038] In an embodiment of the invention, a method is provided for
diagnosing a susceptibility to cancer in a subject which comprises
comparing the germline sequence of the FANCD2 gene or the sequence
of its mRNA in a tissue sample from the subject with the germline
sequence of the FANCD2 gene or the sequence of its mRNA wherein an
alteration in the germline sequence of the FANCD2 gene or the
sequence of its mRNA of the subject indicates the susceptibility to
the cancer. An alteration may be detected in a regulatory region of
the FANCD2 gene. An alteration in the germline sequence may be
determined by an assay selected from the group consisting of (a)
observing shifts in electrophoretic mobility of single-stranded DNA
on non-denaturing polyacrylamide gels, (b) hybridizing a FANCD2
gene probe to genomic DNA isolated from the tissue sample, (c)
hybridizing an allele-specific probe to genomic DNA of the tissue
sample, (d) amplifying all or part of the FANCD2 gene from the
tissue sample to produce an amplified sequence and sequencing the
amplified sequence, (e) amplifying all or part of the FANCD2 gene
from the tissue sample using primers for a specific FANCD2 mutant
allele, (f) molecularly cloning all or part of the FANCD2 gene from
the tissue sample to produce a cloned sequence and sequencing the
cloned sequence, (g) identifying a mismatch between (i) a FANCD2
gene or a FANCD2 mRNA isolated from the tissue sample, and (ii) a
nucleic acid probe complementary to the human wild-type FANCD2 gene
sequence, when molecules (i) and (ii) are hybridized to each other
to form a duplex, (h) amplification of FANCD2 gene sequences in the
tissue sample and hybridization of the amplified sequences to
nucleic acid probes which comprise wild-type FANCD2 gene sequences,
(i) amplification of FANCD2 gene sequences in the tissue sample and
hybridization of the amplified sequences to nucleic acid probes
which comprise mutant FANCD2 gene sequences, (j) screening for a
deletion mutation in the tissue sample, (k) screening for a point
mutation in the tissue sample, (l) screening for an insertion
mutation in the tissue sample, and (m) in situ hybridization of the
FANCD2 gene of said tissue sample with nucleic acid probes which
comprise the FANCD2 gene.
[0039] In an embodiment of the invention, a method is provided for
diagnosing a susceptibility for cancer in a subject, includes: (a)
accessing genetic material from the subject so as to determine
defective DNA repair; (b) determining the presence of mutations in
a set of genes, the set comprising FAND2 and at least one of FANCA,
FANCB, FANCC, FANCD1, FANCDE, FANDF, FANDG, BRACA1 and ATM; and (c)
diagnosing susceptibility for cancer from the presence of
mutations, in the set of genes.
[0040] In an embodiment of the invention, a method is provided for
detecting a mutation in a neoplastic lesion at the FANCD2 gene in a
human subject which includes: comparing the sequence of the FANCD2
gene or the sequence of its mRNA in a tissue sample from a lesion
of the subject with the sequence of the wild-type FANCD2 gene or
the sequence of its mRNA, wherein an alteration in the sequence of
the FANCD2 gene or the sequence of its mRNA of the subject
indicates a mutation at the FANCD2 gene of the neoplastic lesion. A
therapeutic protocol may be provided for treating the neoplastic
lesion according to the mutation at the FANCD2 gene of the
neoplastic lesion.
[0041] In an embodiment of the invention, a method is provided for
confirming the lack of a FANCD2 mutation in a neoplastic lesion
from a human subject which comprises comparing the sequence of the
FANCD2 gene or the sequence of its mRNA in a tissue sample from a
lesion of said subject with the sequence of the wild-type FANCD2
gene or the sequence of its RNA, wherein the presence of the
wild-type sequence in the tissue sample indicates the lack of a
mutation at the FANCD2 gene.
[0042] In an embodiment of the invention, a method is provided for
determining a therapeutic protocol for a subject having a cancer,
that includes (a) determining if a deficiency in FANCD2-L occurs in
a cell sample from the subject by measuring FANCD2 isoforms using
specific antibodies; (b) if a deficiency is detected in (a), then
determining whether the deficiency is a result of genetic defect in
non-cancer cells; and (c) if (b) is positive, reducing the use of a
therapeutic protocol that causes increased DNA damage so as to
protect normal tissue in the subject and if (b) is negative, and
the deficiency is contained within a genetic defect in cancer cells
only, then increasing the use of a therapeutic protocol that causes
increased DNA damage so as to adversely affect the cancer
cells.
[0043] In an embodiment of the invention, a method of treating a FA
pathway defect in a cell target is provided that includes:
administering an effective amount of FANCD2 protein or an exogenous
nucleic acid to the target. The FA pathway defect may be a
defective FANCD2 gene and the exogenous nucleic acid vector may
further include introducing a vector according to those described
above. The vector may be selected from a mutant herpes virus, a
E1/E4 deleted recombinant adenovirus, a mutant retrovirus, the
viral vector being defective in respect of a viral gene essential
for production of infectious new virus particles. The vector may be
contained in a lipid micelle.
[0044] In an embodiment of the invention, a method is provided for
treating a patient with a defective FANCD2 gene, that includes
providing a polypeptide described in SEQ ID NO: 4, for functionally
correcting a defect arising from a condition arising from the
defective FANCD2 gene.
[0045] In an embodiment of the invention, a cell based assay for
detecting a FA pathway defect is provided that includes obtaining a
cell sample from a subject; exposing the cell sample to DNA
damaging agents; and detecting whether FANCD2-L is upregulated, the
absence of upregulation being indicative of the FA pathway defect.
In the cell-based assay, amounts of FANCD2 may be measured by an
analysis technique selected from: immunoblotting for detecting
nuclear foci; Western blots to detect amounts of FANCD2 isoforms
and quantifying mRNA by hybridizing with DNA probes.
[0046] In an embodiment of the invention, a kit is provided for use
in detecting a cancer cell in a biological sample, that includes
(a) primer pair which binds under high stringency conditions to a
sequence in the FANCD2 gene, the primer pair being selected to
specifically amplify an altered nucleic acid sequence described in
Table 7; and containers for each of the primers.
[0047] As used herein, the "Fanconi Anemia/BRCA pathway" or
"Fanconi Anemia Pathway" refers to the genes within the 7
complementation groups (FA-A to FA-G), the BRCA-1 gene and the ATM
gene and their respective proteins that interact in a pathway
referred herein as the Fanconi Anemia/BRCA pathway and regulate the
cellular response to DNA damage (see FIG. 22).
[0048] The genes of the Fanconi Anemia/BRCA pathway are:
1 1) FANC-A (e.g., Genbank Accession No.: NM_000135) 2) FANC-B (not
yet cloned) 3) FANC-C (e.g., Genbank Accession No.: NM_000136) 4)
FANC-D1/ (e.g., Genbank Accession No.: U43746) BRCA-2 5) FANC-D2
(e.g., Genbank Accession No.: NM_033084) 6) FANC-E (e.g., Genbank
Accession No.: NM_021922) 7) FANC-F (e.g., Genbank Accession No.:
NM_022725) 8) FANC-G (e.g., Genbank Accession No.: BC000032) 9)
BRCA-1 (e.g., Genbank Accession No.: U14680) 10) ATM (e.g., Genbank
Accession No.: U33841)
[0049] As used herein, "testing a Fanconi Anemia/BRCA pathway
protein for the presence of a cancer-associated defect" refers to
the method of determining if a protein encoded by a Fanconi
Anemia/BRCA pathway gene, as defined herein, harbors a defect, as
defined herein, that can cause or is associated with a cancer in a
patient.
[0050] As used herein, the term "defect" refers to any alteration
of a gene or protein within the Fanconi Anemia BRCA pathway, and/or
proteins, with respect to any unaltered gene or protein within the
Fanconi Anemia/BRCA pathway.
[0051] "Alteration" of a gene includes, but is not limited to: a)
alteration of the DNA sequence itself, i.e., DNA mutations,
deletions, insertions, substitutions; b) DNA modifications
affecting the regulation of gene expression such as regulatory
region mutations, modification in associated chromatin, modications
of intron sequences affecting mRNA splicing, modification affecting
the methylation/demethylation state of the gene sequence; c) mRNA
medications affecting protein translation or mRNA transport or mRNA
splicing.
[0052] "Alteration" of a protein includes, but is not limited to,
amino acid deletions, insertions, substitutions; modification
affecting protein phosphorylation or glycosylation; modifications
affecting protein transport or localization; modifications
affecting the ability to form protein complexes with one or more
associated proteins or changes in the amino acid sequence caused by
changes in the DNA sequence encoding the amino acid.
[0053] As used herein, the term "increased risk" or "elevated risk"
refers to the greater incidence of cancer in those patients having
altered Fanconi Anemia/BRCA genes or proteins as compared to those
patients without alterations in the Fanconi Anemia/BRCA pathway
genes or proteins. "Increased risk" also refers to patients who are
already diagnosed with cancer and may have an increased incidence
of a different cancer form. According to the invention, "increased
risk" of cancer refers to cancer-associated defects in a Fanconi
Anemia/BRCA pathway gene that contributes to a 50%, preferably 90%,
more preferably 99% or more increase in the probability of
acquiring cancer relative to patients who do not have a
cancer-associated defect in a Fanconi Anemia/BRCA pathway gene.
[0054] As used herein, an "inhibitor of the Fanconi Anemia/BRCA
pathway", according to the invention, refers to any compound that
disrupts FANC D2-L protein function either directly or indirectly.
Disruption of FANC D2-L protein function can be achieved either
through disruption of any of the other FANC proteins upstream of
the FANC D2 protein within the pathway, inhibition of the
ubiquitination of the FANC D2-S to the FANC D2-L isoform,
inhibition of subsequent nuclear transport of the FANC D2-L protein
or disrupton of the association of the FANC D2-L protein with the
nuclear BRCA DNA repair protein complex. An "inhibitor" according
to the invention can be nucleic acids (anti-sense RNA or DNA
oligonucleotides), proteins (humanized antibodies), peptides or
small molecule drugs that specifically bind to FANC D2-L and
disrupt FANC D2-L protein function. In a most preferred embodiment,
the inhibitor of the Fanconi Anemia/BRCA pathway is a small
molecule inhibitor of the mono-ubiquitination of the FANC D2
protein.
[0055] As used herein, a "reduction in the ratio of FANC D2-L
relative to FANC D2-S" refers to a decrease in the percentage of
the total amount of FANC D2 protein that is in the FANC D2-L
isoform. In a preferred embodiment, the total amount of FANC D2
protein that is in the FANC D2-L isoform is at most 25%, preferably
10%, more preferably 1% and most preferably 0%. Such a reduction
indicates a defect in one or more genes or proteins of the Fanconi
Anemia/BRCA pathway, as defined herein.
[0056] As used herein, "testing a Fanconi Anemia/BRCA pathway
protein for the presence of a cancer-associated defect" refers to
the method of determining if a protein encoded within the 7
complementation groups (A, B, C, D, E, F and G) that comprise the
Fanconi Anemia/BRCA gene pathway, harbor a defect or other
mutation, as defined herein, that can cause or contribute to a
cancer in a patient.
[0057] As used herein, the term "inducing DNA damage" refers to
both chemical and physical methods of damaging DNA. Chemicals that
damage DNA include, but are not limited to, acids/bases and various
mutagens, such as ethidium bromide, acridine orange, as well as
free radicals. Physical methods include, but are not limited to,
ionizing radiation, such as X rays and gamma rays, and ultraviolet
(UV) radiation. Both methods of "inducing DNA damage" can result in
DNA mutations that typically include, but are not limited to,
single-strand breaks, double-strand breaks, alterations of bases,
insertions, deletions or the cross-linking of DNA strands.
[0058] As used herein, the term "tissue biopsy" refers to a
biological material, which is isolated from a patient. The term
"tissue", as used herein, is an aggregate of cells that perform a
particular function in an organism and encompasses cell lines and
other sources of cellular material including, but not limited to, a
biological fluid for example, blood, plasma, sputum, urine,
cerebrospinal fluid, lavages, and leukophoresis samples.
[0059] As used herein, the term "amplifying", when applied to a
nucleic acid sequence, refers to a process whereby one or more
copies of a particular nucleic acid sequence is generated from a
template nucleic acid, preferably by the method of polymerase chain
reaction (Mullis and Faloona, 1987, Methods Enzymol., 155:335).
"Polymerase chain reaction" or "PCR" refers to an in vitro method
for amplifying a specific nucleic acid template sequence. The PCR
reaction involves a repetitive series of temperature cycles and is
typically performed in a volume of 50-100 .mu.l. The reaction mix
comprises dNTPs (each of the four deoxynucleotides dATP, dCTP,
dGTP, and dTTP), primers, buffers, DNA polymerase, and nucleic acid
template. The PCR reaction comprises providing a set of
polynucleotide primers wherein a first primer contains a sequence
complementary to a region in one strand of the nucleic acid
template sequence and primes the synthesis of a complementary DNA
strand, and a second primer contains a sequence complementary to a
region in a second strand of the target nucleic acid sequence and
primes the synthesis of a complementary DNA strand, and amplifying
the nucleic acid template sequence employing a nucleic acid
polymerase as a template-dependent polymerizing agent under
conditions which are permissive for PCR cycling steps of (i)
annealing of primers required for amplification to a target nucleic
acid sequence contained within the template sequence, (ii)
extending the primers wherein the nucleic acid polymerase
synthesizes a primer extension product. "A set of polynucleotide
primers" or "a set of PCR primers" can comprise two, three, four or
more primers.
[0060] Other methods of amplification include, but are not limited
to, ligase chain reaction (LCR), polynucleotide-specific base
amplification (NSBA), or any other method known in the art.
[0061] As used herein, the term "polynucleotide primer" refers to a
DNA or RNA molecule capable of hybridizing to a nucleic acid
template and acting as a substrate for enzymatic synthesis under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid template is catalyzed to produce
a primer extension product which is complementary to the target
nucleic acid template. The conditions for initiation and extension
include the presence of four different deoxyribonucleoside
triphosphates and a polymerization-inducing agent such as DNA
polymerase or reverse transcriptase, in a suitable buffer ("buffer"
includes substituents which are cofactors, or which affect pH,
ionic strength, etc.) and at a suitable temperature. The primer is
preferably single-stranded for maximum efficiency in amplification.
"Primers" useful in the present invention are generally between
about 10 and 35 nucleotides in length, preferably between about 15
and 30 nucleotides in length, and most preferably between about 18
and 25 nucleotides in length.
[0062] As defined herein, "a tumor" is a neoplasm that may either
be malignant or non-malignant. Tumors of the same tissue type
originate in the same tissue, and may be divided into different
subtypes based on their biological characteristics.
[0063] As used herein, the term "cancer" refers to a malignant
disease caused or characterized by the proliferation of cells which
have lost susceptibility to normal growth control. "Malignant
disease" refers to a disease caused by cells that have gained the
ability to invade either the tissue of origin or to travel to sites
removed from the tissue of origin.
[0064] As used herein, the term "antibody" refers to an
immunoglobulin having the capacity to specifically bind a given
antigen. The term "antibody" as used herein is intended to include
whole antibodies of any isotype (IgG, IgA, IgM, IgE, etc), and
fragments thereof which are also specifically reactive with a
vertebrate, e.g., mammalian, protein. Antibodies can be fragmented
using conventional techniques and the fragments screened for
utility in the same manner as whole antibodies. Thus, the term
includes segments of proteolytically-cleaved or
recombinantly-prepared portions of an antibody molecule that are
capable of selectively reacting with a certain protein.
Non-limiting examples of such proteolytic and/or recombinant
fragments include Fab, F(ab')2, Fab, Fv, and single chain
antibodies (scFv) containing a V[L] and/or V[H] domain joined by a
peptide linker. The scFv's may be covalently or non-covalently
linked to form antibodies having two or more binding sites.
Antibodies may be labeled with detectable moieties by one of skill
in the art. In some embodiments, the antibody that binds to an
entity one wishes to measure (the primary antibody) is not labeled,
but is instead detected by binding of a labeled secondary antibody
that specifically binds to the primary antibody.
[0065] A patient is "treated" according to the invention if one or
preferably more symptoms of cancer as described herein are
eliminated or reduced in severity, or prevented from progressing or
developing further.
[0066] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, i.e., treatment, healing, prevention or
amelioration of the relevant medical condition, or an increase in
rate of treatment, healing, prevention or amelioration of such
conditions.
[0067] As used herein, the term "cancer therapeutic" refers to a
compound that prevents the onset or progression of cancer or
prevents cancer metastasis or reduces, delays, or eliminates the
symptoms of cancer.
[0068] As used herein, the term "inhibitor of the
mono-ubiquitination " refers to a compound that prevents or
inhibits the ubiquitination of the FANC D2 gene. "Ubiquitination"
is defined as the covalent linkage of ubiquitin to a protein by a
E3 mono-ubiquitin ligase. In a preferred embodiment, the "inhibitor
of the mono-ubiquitination" refers to any inhibitor of a FANC
protein complex with E3 FANC D2 monoubiquitin ligase activity such
that FANC D2 monoubiquitin ligase activity is inhibited.
[0069] As used herein, the term "cisplatin" refers to an agent with
the following chemical structure: 1
[0070] Cisplatin, also called cis-diamminedichloroplatinum(II), is
one of the most frequently used anticancer drugs. It is an
effective component of several different combination drug protocols
used to treat a variety of solid tumors. These drugs are used in
the treatment of testicular cancer (with bleomycin and
vinblastine), bladder cancer, head and neck cancer (with bleomycin
and fluorouracil), ovarian cancer (with cyclophosphamide or
doxorubicin) and lung cancer (with etoposide). Cisplatin has been
found to be the most active single agent against most of these
tumors. Cisplatin is commercially available as `Platinol` from
Bristol Myers Squibb Co. Cisplatin, is one of a number of platinum
coordination complexes with antitumor activity. The platinum
compounds are DNA cross-linking agents similar to but not identical
to the alkylating agents. The platinum compounds exchange chloride
ions for nucleophilic groups of various kinds. Both the cis and
trans isomers do this but the trans isomer is known to be
bioligically inactive for reasons not completely understood. To
possess antitumor activity a platinum compound must have two
relatively labile cis-oriented leaving groups. The principal sites
of reaction are the N7 atoms of guanine and adenine. The main
interaction is formation of intrastrand cross links between the
drug and neighboring guanines. Intrastrand cross linking has been
shown to correlate with clinical response to cisplatin therapy.
DNA/protein cross linking also occurs but this does not correlate
with cytotoxicity. Cross-resistance between the two groups of drugs
is usually not seen indicating that the mechanisms of action are
not identical. The types of cross linking with DNA may differ
between the platinum compounds and the typical alkylating
agents.
[0071] As used herein, "resistance to one or more anti-neoplastic
agents" refers the ability of cancer cells to develop resistance to
anticancer drugs. Mechanisms of drug resistance include decreased
intracellular drug levels caused by an increased drug efflux or
decreased inward transport, increased drug inactivation, decreased
conversion of drug to an active form, altered amount of target
enzyme or receptor (gene amplification), decreased affinity of
target enzyme or receptor for drug, enhanced repair of the
drug-induced defect, decreased activity of an enzyme required for
the killing effect (topoisomerase II). In a preferred embodiment of
the invention, drug resistance refers to the enhanced repair of DNA
damage induced by one or more anti-neoplastic agents. In another
preferred embodiment of the invention, the enhanced repair of DNA
damage induced by one or more anti-neoplastic agents is due to a
constitutively active Fanconi Anemia/BRCA DNA repair pathway.
[0072] As used herein, the term "anti-neoplastic agent" refers to a
compound that is used to treat cancer. According to the invention,
an "anti-neoplastic agent" encompasses chemotherapy compounds as
well as other anti-cancer agents known in the art. In a preferred
embodiment, the "anti-neoplastic agent" is cisplatin.
Anti-neoplastic agents according to the invention also include
cancer therapy protocols using chemotherapy compounds in
conjunction with radiation therapy and/or surgery. Radiation
therapy relies on the local destruction of cancer cells through
ionizing radiation that disrupts cellular DNA. Radiation therapy
can be externally or internally originated, high or low dose, and
delivered with computer-assisted accuracy to the site of the tumor.
Brachytherapy, or interstitial radiation therapy, places the source
of radiation directly into the tumor as implanted "seeds."
[0073] As used herein, the term "a reduced growth rate" refers to a
decrease of 50%, preferably 90%, more preferably 99% and most
preferably 100% in the rate of cellular proliferation of a tumor
cell line that is being treated with a potential inhibitor of the
Fanconi Anemia/BRCA pathway and one or more chemotherapy compounds
relative to cells of a tumor cell line that is not being treated
with a potential inhibitor of the Fanconi Anemia/BRCA pathway and
one or more chemotherapy compounds.
[0074] As used herein, the term "chemosensitizing agent" refers to
any compound that renders a cell or cell population sensitive to a
chemotherapy compound and results in a "reduced growth rate" as
defined herein. A chemosensitizing agent is a compound that is
generally not cytotoxic in itself, but modifies the host or tumor
cells to enhance anticancer therapy. According to the invention,
cellular resistance to a chemotherapy compound is reversed in the
presence of a chemosensitizing agent. In a preferred embodiment,
the chemosensitizing agent is an inhibitor of the Fanconi
Anemia/BRCA pathway. In a most preferred embodiment, the
chemosensitizing agent is an inhibitor of the mono-ubiquitination
of the FANC D2 protein.
[0075] As used herein, the "methylation state of a Fanconi
Anemia/BRCA pathway gene" refers to the presence of one or more
methylated cytosines (5 m-C) within a Fanconi Anemia/BRCA pathway
gene and results in a decrease or inhibition of gene expression of
90%, 99% or preferably 100% relative to a gene that is not
methylated. In a preferred embodiment, the methylated cytosines
reside within CpG islands. According to the invention, a gene is
said to be "methylated" when one or more of CpG residues is
methylated.
[0076] As used herein, "microarray", or "array", refers to a
plurality of unique biomolecules attached to one surface of a solid
support. Preferably, a biomolecule of the invention a potential
inhibitor of the Fanconi Anemia/BRCA pathway as described herein.
In this embodiment, the microarray of the invention comprises
nucleic acids, proteins, polypeptides, peptides, fusion proteins or
small molecules that are immobilised on a solid support, generally
at high density. Each of the biomolecules is attached to the
surface of the solid support in a pre-selected region. Suitable
solid supports are available commercially, and will be apparent to
the skilled person. The supports may be manufactured from materials
such as glass, ceramics, silica and silicon. The supports usually
comprise a flat (planar) surface, or at least an array in which the
molecules to be interrogated are in the same plane. In one
embodiment, the array is on microbeads. In one embodiment, the
array comprises at least 10, 500, 1000, 10,000 different
biomolecules attached to one surface of the solid support.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] The foregoing features of the invention will be more readily
understood by reference to the following detailed description,
taken with reference to the accompanying drawings, in which:
[0078] FIG. 1A provides a Western blot demonstrating that the
Fanconi Anemia protein complex is required for the
monoubiquitination of FANCD2. Normal (WT) cells (lane 1) express
two isoforms of the FANCD2 protein, a low molecular weight isoform
(FANCD2-S) (155 kD) and a high molecular weight isoform (FANCD2-L)
(162 kD). Lanes 3, 7, 9, 11 show that FA cell lines derived from
type A, C, G, and F patients only express the FANCD2-S isoform.
Lanes 4, 8, 10, 12 show the restoration of the high molecular
weight isoform FANCD2-L following transfection of cell lines with
corresponding FAcDNA.
[0079] FIG. 1B shows a Western blot obtained after HeLa cells were
transfected with a cDNA encoding HA-ubiquitin. After transfection,
cells were treated with the indicated dose of mitomycin C (MMC).
Cellular proteins were immunoprecipitated with a polyclonal
antibody (E35) to FANCD2, as indicated. FANCD2 was
immunoprecipitated, and immune complexes were blotted with
anti-FANCD2 or anti-HA monoclonal antibody.
[0080] FIG. 1C shows a Western blot obtained after HeLa cells were
transfected with a cDNA encoding HA-ubiquitin. After transfection,
cells were treated with the indicated dose of ionizing radiation
(IR). FANCD2 was immunoprecipitated, and immune complexes were
blotted with anti-FANCD2 or anti-HA monoclonal antibody.
[0081] FIG. 1D shows a Western blot obtained after PA-G fibroblast
line (FAG326SV) or corrected cells (FAG326SV plus FANCG cDNA) were
transfected with the HA-Ub cDNA, FANCD2 was immunoprecipitated, and
immune complexes were blotted with anti-FANCD2 or anti-HA
antisera.
[0082] FIG. 1E shows a Western blot obtained after treatment of
HeLa cells with 1 mM hydroxyurea for 24 hours. HeLa cell lysates
were extracted and incubated at the indicated temperature for the
indicated time period with or without 2.5 .mu.M ubiquitin aldehyde.
The FANCD2 protein was detected by immunoblot with monoclonal
anti-FANCD2 (F117).
[0083] FIG. 2 demonstrates that the Fanconi Anemia pathway is
required for the formation of FANCD2 nuclear foci. Top panel shows
anti-FANCD2 immunoblots of SV40 transformed fibroblasts prepared as
whole cell extracts. Panels a-h show immunofluorescence with the
affinity-purified anti-FANCD2 antiserum. The uncorrected (mutant,
M) FA fibroblasts were FA-A (GM6914), FA-G (FAG326SV), FA-C
(PD426), and FA-D (PD20F). The FA-A, FA-G, and FA-C fibroblasts
were functionally complemented with the corresponding FA cDNA. The
FA-D cells were complemented with neomycin-tagged human chromosome
3p (Whitney et al., 1995).
[0084] FIG. 3 shows the cell cycle dependent expression of the two
isoforms of the FANCD2 protein. (a) HeLa cells, SV40 transformed
fibroblasts from an FA-A patient (GM6914), and GM6914 cells
corrected with FANCA cDNA were synchronized by the double thymidine
block method. Cells corresponding to the indicated phase of the
cell cycle were lysed, and processed for FANCD2 immunoblotting (b)
Synchrony by nocodazole block (c) Synchrony by mimosine block (d)
HeLa cells were synchronized in the cell cycle using nocodazole or
(e) mimosine, and cells corresponding to the indicated phase of the
cell cycle were immunostained with the anti-FANCD2 antibody and
analyzed by immunofluorescence.
[0085] FIG. 4 shows the formation of activated FANCD2 nuclear foci
following cellular exposure to MMC, Ionizing Radiation, or
Ultraviolet Light. Exponentially-growing HeLa cells were either
untreated or exposed to the indicated DNA damaging agents, (a)
Mitomycin C (MMC), (b) .gamma.-irradiation (IR), or (c) Ultraviolet
Light (WV), and processed for FANCD2 immunoblotting or FANCD2
immunostaining. (a) Cells were continuously exposed to 40 ng/ml MMC
for 0-72 hours as indicated, or treated for 24 hours and fixed for
immunofluorescence. (b) and (c) Cells were exposed to
.gamma.-irradiation (10 Gy, B) or UV light (60 J/m2 C) and
collected after the indicated time (upper panels) or irradiated
with the indicated doses and harvested one hour later (lower
panels). For immunofluorescence analysis cells were fixed 8 hours
after treatment (B, 10 Gy, C, 60 J/m2). (d) The indicated
EBV-transformed lymphoblast lines from a normal individual (PD7) or
from various Fanconi Anemia patients were either treated with 40
ng/ml of Mitomycin C continuously (lanes 1-21) or exposed to 15 Gy
of .gamma.-irradiation (lanes 22-33) and processed for FANCD2
immunoblotting. The upregulation of FANCD-L after MMC or IR
treatment was seen in PD7 (lanes 2-5) and in the corrected FA-A
cells (lanes 28-33), but was not observed in any of the mutant
Fanconi Anemia cell lines. Similarly, IR-induced FANCD2 nuclear
foci were not detected in PA fibroblasts (FA-G+IR) but were
restored after functional complementation (PA-G+FANCG).
[0086] FIG. 5 shows co-localization of activated FANCD2 and BRCA1
in Discrete Nuclear Foci following DNA damage. HeLa cells were
untreated or exposed to Ionizing Radiation (10 Gy) as indicated,
and fixed 8 hours later. (a) Cells were double-stained with the D-9
monoclonal anti-BRCA1 antibody (green, panels a, d, g, h) and the
rabbit polyclonal anti-FANCD2 antibody (red, panels b, e, h, k),
and stained cells were analyzed by immunofluorescence. Where green
and red signals overlap (Merge, panels c, f, i, l) a yellow pattern
is seen, indicating co-localization of BRCA1 and FANCD2. (b)
Co-immunoprecipitation of FANCD2 and BRCA1. HeLa cells were
untreated (IR) or exposed to 15 Gy of .gamma.-irradiation (+IR) and
collected 12 hours later. Cell lysates were prepared, and cellular
proteins were immunoprecipitated with either the monoclonal FANCD2
antibody (FI-17, lanes 9-10), or any one of three
independently-derived monoclonal antibodies to human BRCA1 (lanes
3-8): D-9 (Santa Cruz), Ab-1 and Ab-3 (Oncogene Research Products).
The same amount of purified mouse IgG (Sigma) was used in control
samples (lanes 1-2). Immune complexes were resolved by SDS-PAGE and
were immunoblotted with anti-FANCD2 or anti-BRCA1 antisera. The
FANCD-L isoform preferentially coimmunoprecipitated with BRCA1.
[0087] FIG. 6 shows the co-localization of activated FANCD2 and
BRCA1 in discrete nuclear foci during S phase. (a) HeLa cells were
synchronized in late G1 with mimosine and released into S phase. S
phase cells were double-stained with the monoclonal anti-BRCA1
antibody (green, panels a, d) and the rabbit polyclonal anti-FANCD2
antibody (red, panels b, e), and stained cells were analyzed by
immunofluorescence. Where green and red signals overlap (merge,
panels c, f), a yellow pattern is seen, indicating co-localization
of BRCA1 and FANCD2. (b) HeLa cells synchronized in S phase were
either untreated (a, b, k, l) or exposed to IR (50 Gy, panels c, d,
m, n), MMC (20 .mu.g/ml, panels c, f, o, p), or UV (100 j/m2,
panels g, h, q, r) as indicated and fixed 1 hour later. Cells were
subsequently immunostained with an antibody specific for FANCD2 or
BRCA1.
[0088] FIG. 7 shows that FANCD2 forms foci on synaptonemal
complexes that can co-localize with BRCA1 during meiosis I in mouse
spermatocytes. (a) Anti-SCP3 (white) and anti-FANCD2 (red) staining
of synaptonemal complexes in a late pachytene mouse nucleus. (b)
SCP3 staining of late pachytene chromosomes. (c) Staining of this
spread with preinmmune serum for the anti-FANCD2 E35 antibody. (d)
Anti-SCP3 staining of synaptonemal complexes in a mouse diplotene
nucleus. (e) Costaining of this spread with E35 anti-FANCD2
antibody. Note staining of both the unpaired sex chromosomes and
the telomeres of the autosomes with anti-FANCD2. (f) Costaining of
this spread with anti-BRCA1 antibody. The sex chromosomes are
preferentially stained. (g) Anti-FANCD2 staining of late pachytene
sex chromosome synaptonemal complexes. (h) Anti-BRCA1 staining of
the same complexes. (i) Anti-FANCD2 (red) and anti-BRCA1 (green)
co-staining (co-localization reflected by yellow areas).
[0089] FIG. 8 provides a schematic interaction of the FA proteins
in a cellular pathway. The FA proteins (A, C, and G) bind in a
functional nuclear complex. Upon activation of this complex, by
either S phase entry or DNA damage, this complex enzymatically
modifies (monoubiquitinates) the D protein. According to this
model, the activated D protein is subsequently targeted to nuclear
foci where it interacts with the BRCA1 protein and other proteins
involved in DNA repair.
[0090] FIG. 9 shows a Northern blot of cells from heart, brain,
placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus,
prostate, testis, uterus, small intestine, colon and peripheral
blood lymphocytes from a human adult and brain, lung, liver and
kidney from a human fetus probed with a full-length FANCD2 cDNA and
exposed for 24 hours.
[0091] FIG. 10 shows allele specific assays for mutation analysis
of 2 FANCD2 families where the family pedigrees (a, d) and panels
b, c, e and f are vertically aligned such that the corresponding
mutation analysis is below the individual in question. Panels a-c
depict the PD20 and panels d-f the VU008 family. Panels b and e
show the segregation of the maternal mutations as detected by the
creation of a new MspI site (PD20) or DdeI site (VU008). The
paternally inherited mutations in both families were detected with
allele specific oligonucleotide hybridization (panels c and f).
[0092] FIG. 11 shows a Western blot analysis of the FANCD2 protein
in human Fanconi Anemia cell lines. Whole cell lysates were
generated from the indicated fibroblast and lymphoblast lines.
Protein lysates (70 g) were probed directly by immunoblotting with
the anti-FANCD2 antiserum. The FANCD2 proteins (155 kD and 162 kD)
are indicated by arrows. Other bands in the immunoblot are
non-specific. (a) Cell lines tested included wild-type cells (lanes
1,7), PD20 Fibroblasts (lane 2), PD20 lymphoblasts (lane 4),
revertant MMC-resistant PD20 lymphoblasts (lane 5, 6), and
chromosome 3p complemented PD20 fibroblasts (lane 3). Several other
FA group D cell lines were analyzed including HSC62 (lane 8) and
VU008 (lane 9). FA-A cells were HSC72 (lane 10), FA-C cells were
PD4 (lane 11), and FA-G cells were EUFA316 (lane 12). (b)
Identification of a third FANCD2 patient. FANCD2 protein was
readily detectable in wild-type and FA group G cells but not in
PD733 cells. (c) Specificity of the antibody. PD20i cells
transduced with a retroviral FANCD2 expression vector displayed
both isoforms of the FANCD2 protein (lane 4) in contrast to empty
vector controls (lane 3) and untransfected PD20i cells (lane 2). In
wild-type cells the endogenous FANCD2 protein (two isoforms) was
also immunoreactive with the antibody (lane 1).
[0093] FIG. 12 shows functional complementation of FA-D2 cells with
the cloned FANCD2 cDNA. The SV40-transformed FA-D2 fibroblast line,
PD20i, was transduced with pMMP-puro (PD20+vector) or pMMP-FANCD2
(PD20+FANCD2 wt). Puromycin-selected cells were subjected to MMC
sensitivity analysis. Cells analyzed were the parental PD20F cells
(.DELTA.), PD20 corrected with human chromosome 3p (.largecircle.),
and PD20 cells transduced with either pMMP-puro (.quadrature.) or
pMMP-FANCD2(wt)-puro (.diamond-solid.).
[0094] FIG. 13 shows a molecular basis for the reversion of PD20
Lymphoblasts. (a) PCR primers to exons 5 and 6 were used to amplify
cDNA. Control samples (right lane) yielded a single band of 114 bp,
whereas PD20 cDNA (left lane) showed 2 bands, the larger reflecting
the insertion of 13 bp of intronic sequence into the maternal
allele. Reverted, MMC resistant lymphoblasts (middle lane) from
PD20 revealed a third, inframe splice variant of 114+36 bp (b)
Schematic representation of splicing at the PANCD2 exon 5/intron 5
boundary. In wild-type cDNA 100% of splice events occur at the
proper exon/intron boundary, whereas the maternal A->G mutation
(indicated by arrow) leads to aberrant splicing, also in 100%. In
the reverted cells all cDNAs with the maternal mutation also had a
second sequence change (fat arrow) and showed a mixed splicing
pattern with insertion of either 13 bp (.about.40% of mRNA) or 36
bp (.about.60% of mRNA).
[0095] FIG. 14 shows an FANCD2 Western blot of cancer cell lines
derived from patients with ovarian cancer.
[0096] FIG. 15 shows a sequence listing for amino acid sequence of
human FANCD2 and alignment with fly and plant homologues using the
BEAUTY algorithm (Worley et al., (1995) Genome Res. Vol. 5, pp.
173-184). (SEQ. ID. NO: 1-3) Black boxes indicate amino acid
identity and gray similarity. The best alignment scores were
observed with hypothetical proteins in D melanogaster
(p=8.4.times.10-58, accession number AAF55806) and A thaliana
(p=9.4.times.10-45, accession number B71413).
[0097] FIG. 16 is the FANCD cDNA sequence-63 to 5127 nucleotides
(SEQ ID NO: 5) and polypeptide encoded by this sequence from amino
acid 1 to 1472 (SEQ ID NO: 4).
[0098] FIG. 17 is the nucleotide sequence for FANCD-S.ORF (SEQ ID
NO: 187) compared with FANCD cDNA (SEQ ID NO: 188).
[0099] FIG. 18 is the nucleotide sequence for human FANCD2-L (SEQ
ID NO: 6).
[0100] FIG. 19 is the nucleotide sequence for human FANCD2-S (SEQ
ID NO: 7).
[0101] FIG. 20 is the nucleotide sequence for mouse FANCD2 (SEQ ID
NO: 8).
[0102] FIG. 21 depicts protocol used to analyze the methylation
state of the FANC F gene.
[0103] FIG. 22 depicts the Fanconi Anemia/BRCA pathway.
DETAILED DESCRIPTION
[0104] "FANCD2-L therapeutic agent" shall mean any of a protein
isoform, and includes a peptide, a peptide derivative, analogue or
isomer of the FANCD2-L protein and further include any of a small
molecule derivative, analog, isomer or agonist that is functionally
equivalent to FANCD2-L. Also included in the definition is a
nucleic acid encoding FANCD2 which may be a full length or partial
length gene sequence or cDNA or may be a gene activating nucleic
acid or a nucleic acid binding molecule including an aptamer of
antisense molecule which may act to modulate gene expression.
[0105] "Nucleic acid encoding FANCD-2" shall include the complete
cDNA or genomic sequence of FANCD2 or portions thereof for
expressing FANCD2-L protein as defined above. The nucleic acid may
further be included in a nucleic acid carrier or vector and
includes nucleic acid that has been suitably modified for effective
delivery to the target site.
[0106] "Stringent conditions of hybridization" will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and preferably in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1000 mM,
typically less than 500 mM, and preferably less than 200 mM.
[0107] "Substantial homology or similarity" for a nucleic acid is
when a nucleic acid or fragment thereof is "substantially
homologous" (or "substantially similar") to another if, when
optimally aligned (with appropriate nucleotide insertions or
deletions) with the other nucleic acid (or its complementary
strand), there is nucleotide sequence identity in at least about
60% of the nucleotide bases, usually at least about 70%, more
usually at least about 80.
[0108] "Antibodies" includes polyclonal and/or monoclonal
antibodies and fragments thereof including single chain antibodies
and including single chain antibodies and Fab fragments, and
immunologic binding equivalents thereof, which have a binding
specificity sufficient to differentiate isoforms of a protein.
These antibodies will be useful in assays as well as
pharmaceuticals.
[0109] "Isolated" is used to describe a protein, polypeptide or
nucleic acid which has been separated from components which
accompany it in its natural state. An "isolated" protein or nucleic
acid is substantially pure when at least about 60 to 75% of a
sample exhibits a single amino acid or nucleotide sequence.
[0110] "Regulatory sequences" refers to those sequences normally
within 100 kb of the coding region of a locus, but they may also be
more distant from the coding region, which affect the expression of
the gene (including transcription of the gene, and translation,
splicing, stability or the like of the messenger RNA).
[0111] "Polynucleotide" includes RNA, cDNA, genomic DNA, synthetic
forms, and mixed polymers, both sense and antisense strands, and
may be chemically or biochemically modified or may contain
non-natural or derivatized nucleotide bases, as will be readily
appreciated by those skilled in the art. Such modifications
include, for example, labels, methylation, substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.), charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),
and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
Also included are synthetic molecules that mimic nucleic acids in
their ability to bind to a designated sequence via hydrogen bonding
and other chemical interactions.
[0112] "Mutation" is a change in nucleotide sequence within a gene,
or outside the gene in a regulatory sequence compared to wild type.
The change may be a deletion, substitution, point mutation,
mutation of multiple nucleotides, transposition, inversion, frame
shift, nonsense mutation or other forms of aberration that
differentiate the nucleic acid or protein sequence from that of a
normally expressed gene in a functional cell where expression and
functionality are within the normally occurring range.
[0113] "Subject" refers to an animal including mammal, including
human.
[0114] "Wild type FANCD2" refers to a gene that encodes a protein
or an expressed protein capable of being monoubiquinated to form
FANCD2-L from FANCD-S within a cell.
[0115] We have found that some Fanconi Anemia has similarities with
a group of syndromes including ataxia telangiectasia (AT),
Xeroderma pigmentosum (XP), Cockayne syndrome (CS), Bloom's
syndrome, myelodysplastic syndrome, aplastic anemia, cancer
susceptibility syndromes and HNPCC (see Table 2). These syndromes
have an underlying defect in DNA repair and are associated with
defects in maintenance of chromosomal integrity. Defects in
pathways associated with DNA repair and maintenance of chromosomal
integrity result in genomic instability, and cellular sensitivity
to DNA damaging agents such as bifunctional alkylating agents that
cause intrastrand crosslinking. Moreover, deficiencies in DNA
repair mechanisms appear to substantially increase the probability
of initiating a range of cancers through genetic rearrangements.
This observation is pertinent with regard to the clinical use of
DNA cross-linking drugs including mitomycin C, cisplatin,
cyclophosphamide, psoralen and UVA irradiation.
[0116] Although Fanconi Anemia is a rare disease, the pleiotropic
effects of FA indicate the importance of the wild type function of
FA proteins in the pathway for diverse cellular processes including
genome stability, apoptosis, cell cycle control and resistance to
DNA crosslinks. The cellular abnormalities in FA include
sensitivity to cross-linking agents, prolongation of G2 phase of
cell cycle, sensitivity to oxygen including poor growth at ambient
O2, overproduction of O2 radicals, deficient O2 radical defense,
deficiency in superoxide dismutase; sensitivity to ionizing
radiation (G2 specific); overproduction of tumor necrosis factor,
direct defects in DNA repair including accumulations of DNA
adducts, and defects in repair of DNA cross-links, genomic
instability including spontaneous chromosome breakage, and
hypermutability by deletion mechanism, increased aptosis, defective
p53 induction, intrinsic stem cell defect, including decreased
colony growth in vitro; and decreased gonadal stem cell
survival.
[0117] These features are reflective of the involvement of FA in
maintenance of hematopoietic and gonadal stem cells, as well as the
normal embryonic development of many different structures,
including the skeleton and urogenital systems. Cell samples from
patients were analyzed to determine defects in the FA
complementation group D. Lymphoblasts from one patient gave rise to
the PD20 cell line which was found to be mutated in a different
gene from HSC62 derived from another patient with a defect in the D
complementation group Mutations from both patients mapped to the D
complementation group but to different genes hence the naming of
two FANCD proteins-FANCD1 (HSC62) and FANCD2 (PD20) (Timmers et
al., (2001) Molecular Cell, Vol. 7, pp. 241-248). We have shown
that FANCD2 is the endpoint of the FA pathway and is not part of
the FA nuclear complex nor required for its assembly or stability
and that FANCD2 exists in two isoforms, FANCD2-S and FANCD2-L. We
have also shown that transformation of the protein short form
(FAND2-S) to the protein long form (FANCD2-L) occurs in response to
the FA complex (FIG. 8). Defects in particular proteins associated
with the FA pathway result in failure to make an important post
translationally modified form of FANCD2 identified as FANCD2-L. The
two isoforms of FANCD2 are identified as the short form and the
long form.
[0118] Failure to make FANCD2-L correlates with errors in DNA
repair and cell cycle abnormalities associated with diseases listed
above.
[0119] To understand more about the role of FANCD2 in the
aforementioned syndromes, we cloned the FANCD2 gene and determined
the protein sequence. The FANCD2 gene has an open reading frame of
4,353 base pairs and forty four exons which encodes a novel 1451
amino acid nuclear protein, with a predicted molecular weight of
166 kD. Western blot analysis revealed the existence of 2 protein
isoforms of 162 and 155 kD. The sequence corresponding to the 44
Intron/Exon Junctions are provided in Table 6 (SEQ ID NO:
9-94).
[0120] Unlike previously cloned FA proteins, FANCD2 proteins from
several nonvertebrate eukaryotes showed highly significant
alignment scores with proteins in D. melanogaster, A. thaliana, and
C. elegans. The drosophila homologue, has 28% amino acid identity
and 50% similarity to FANCD2 (Figure and SEQ ID NO: 1-3) and no
functional studies have been carried out in the respective species.
No proteins similar to FANCD2 were found in E. coli or S.
cerevisiae.
[0121] We obtained the FANCD2 DNA sequence (SEQ ID NO: 5) by
analyzing the chromosome 3p locus in PD20 and VU008, two FA cell
lines having biallelic mutations in the FANCD2 gene (FIG. 10). The
cell lines were assigned as complementation group D because
lymphoblasts from the patients failed to complement HSC62, the
reference cell line for group D. FANCD2 mutations were not detected
in this group D reference cell line which indicates that the gene
mutated in HSC62 is the gene encoding FANCD1 and in PD20 and VU008
is FANCD2 (FIG. 11). Microcell mediated chromosome transfer was
used to identify the mutations (Whitney et al., Blood, (1995) Vol.
88, 49-58). Detailed analysis of five microcell hybrids containing
small overlapping deletions encompassing the locus narrowed the
candidate region of the FANCD2 gene to 200 kb. The FANCD2 gene was
isolated as follows: Three candidate ESTs were localized in or near
this FANCD2 critical region. Using 5' and 3' RACE to obtain
full-length cDNAs, the genes were sequenced, and the expression
pattern of each was analyzed by northern blot. EST SCC34603 had
ubiquitous and low level expression of a 5 kb and 7 kb mRNA similar
to previously cloned FA genes. Open reading frames were found for
TIGR-A004X28, AA609512 and SGC34603 and were 234, 531 and 4413 bp
in length respectively. All 3 were analyzed for mutations in PD20
cells by sequencing cloned RT-PCR products. Whereas no sequence
changes were detected in TIGR-A004X28 and AA609512, five sequence
changes were found in SGC34603. Next, we determined the structure
of the SGC34603 gene by using cDNA sequencing primers on BAC 177N7
from the critical region.
[0122] Based on the genomic sequence information, PCR primer pairs
were designed (Table 7), the exons containing putative mutations
were amplified, and allele-specific assays were developed to screen
the PD20 family as well as 568 control chromosomes. Three of the
alleles were common polymorphisms; however, 2 changes were not
found in the controls and thus represented potential mutations
(Table 3). The first was a maternally inherited A->G change at
nt 376. In addition to changing an amino acid (S126G), this
alteration was associated with mis-splicing and insertion of 13 bp
from intron 5 into the mRNA. 43/43 (100%) independently cloned
RT-PCR products with the maternal mutation contained this
insertion, whereas only 3% (1/31) of control cDNA clones displayed
mis-spliced mRNA. The 13 bp insertion generated a frame-shift and
predicts a severely truncated protein only 180 aminoacids in
length. The second alteration was a paternally inherited missense
change at position 1236 (R1236H). The segregation of the mutations
in the PD20 core family is depicted in FIG. 10. Because the
SGC34603 gene of PD20 contained both a maternal and a paternal
allele not present on 568 control chromosomes and because the
maternal mutation was associated with mis-splicing in 100% of cDNAs
analyzed, we concluded that SGC34603 is the FANCD2 gene.
[0123] The protein encoded by FANCD2 is absent in PD20: To further
confirm the identity of SGC34603 as FANCD2, an antibody was raised
against the protein, and Western blot analysis was performed (FIG.
11). The specificity of the antibody was shown by retroviral
transduction and stable expression FANCD2 in PD20 cells (FIG. 11).
In wild-type cells this antibody detected two bands (155 and 162
kD) which we call FANCD2-S and -L (best seen in FIG. 11). FANCD2
protein levels were markedly diminished in all MMC-sensitive cell
lines from patient PD20 (FIG. 11a, lanes 2, 4) but present in all
wild-type cell lines and FA cells from other complementation
groups. Furthermore, PD20 cells corrected by microcell-mediated
transfer of chromosome 3 also made normal amounts of protein (FIG.
11a, lane 3).
[0124] Functional-complementation of FA-D2 cells with the FANCD2
cDNA: We next assessed the ability of the cloned FANCD2 cDNA to
complement the MMC sensitivity of FA-D2 cells (FIG. 12). The full
length FANCD2 cDNA was subcloned into the retroviral expression
vector, pMMP-puro, as previously described (Pulsipher et al.
(1998), Mol. Med., Vol. 4, pp. 468-479). The transduced PD-20 cells
expressed both isoforms of the FANCD2 protein, FANCD2-S and
FANCD2-L (FIG. 12c). Transduction of PA-D2 (PD20) cells with
pMMP-FANCD2 corrected the MMC sensitivity of the cells. These
results further show that the cloned FANCD2 cDNA encodes the
FANCD2-S protein, which can be post-translationally-modified to the
FANCD2-L isoform. This important modification is discussed in
greater detail below.
[0125] Analysis of a phenotypically reverted PD20 clone: We next
generated additional evidence demonstrating that the sequence
variations in PD20 cells were not functionally neutral
polymorphisms. Towards this end we performed a molecular analysis
of a revertant lymphoblast clone (PD20-cl.1) from patient PD20
which was no longer sensitive to MMC. Phenotypic reversion and
somatic mosaicism are frequent findings in FA and have been
associated with intragenic events such as mitotic recombination or
compensatory frame-shifts. Indeed, -60% of maternally derived
SGC34603 cDNAs had a novel splice variant inserting 36 bp of intron
5 sequence rather than the usually observed 13 bp (FIG. 13). The
appearance of this in-frame splice variant correlated with a de
novo base change at position IVS5+6 from G to A (FIG. 13) and
restoration of the correct reading frame was confirmed by Western
blot analysis. In contrast to all MMC sensitive fibroblasts and
lymphoblasts from patient PD20, PD20-cl.1 produced readily
detectable amounts of FANCD2 protein of slightly higher molecular
weight than the normal protein.
[0126] Analysis of cell lines from other "FANCD" patients: The
antibody was also used to screen additional FA patient cell lines,
including the reference cell line for FA group D, HSC and 2 other
cell lines identified as group D by the European Fanconi Anemia
Registry (EUFAR). VU008 did not express the FANCD2 protein and was
found to be a compound heterozygote, with a missense and nonsense
mutation, both in exon 12, and not found on 370 control chromosomes
(Table 3, FIG. 11). The missense mutation appears to destabilize
the FANCD2 protein, as there is no detectable FANCD2 protein in
lysates from VU008 cells. A third patient PD733 also lacked FANCD2
protein (FIG. 11b, lane 3) and a splice mutation leading to absence
of exon 17 and an internal deletion of the protein was found. The
correlation of the mutations with the absence of FANCD2 protein in
cell lysates derived from these patients substantiates the identity
of FANCD2 as a FA gene. In contrast, readily detectable amounts of
both isoforms of the FANCD2 protein were found in HSC62 (FIG. 11a,
lane 8) and VU423 cDNA and genomic DNA from both cell lines were
extensively analyzed for mutations, and none were found. In
addition, a whole cell fusion between VU423 and PD20 fibroblasts
showed complementation of the chromosome breakage phenotype (Table
5). Taken together these data show that FA group D are genetically
heterogeneous and that the gene(s) defective in HSC62 and VU423 are
distinct from FANCD2.
[0127] The identification and sequencing of the FANCD2 gene and
protein provides a novel target for therapeutic development,
diagnostic tests and screening assays for diseases associated with
failure of DNA repair and cell cycle abnormalities including but
not limited to those listed in Table 2.
[0128] The following description provides novel and useful insights
into the biological role of FANCD2 in the FA pathway which provides
a basis for diagnosis and treatment of the aforementioned
syndromes.
[0129] Evidence that FA cells have an underlying molecular defect
in cell cycle regulation include the following: (a) FA cells
display a cell cycle delay with 4N DNA content which is enhanced by
treatment with chemical crosslinking agents, (b) the cell cycle
arrest and reduced proliferation of FA cells can be partially
corrected by overexpression of a protein, SPHAR, a member of the
cyclin family of proteins and (c) caffeine abrogates the G2 arrest
of FA cells. Consistent with these results, caffeine constitutively
activates cdc2 and may override a normal G2 cell cycle checkpoint
in FA cells. Finally, the FANCC protein binds to the cyclin
dependent kinase, cdc2. We propose that the FA complex may be a
substrate or modulator of the cyclinB/cdc2 complex.
[0130] Additionally, evidence that FA cells have an underlying
defect in DNA repair is suggested by (a) FA cells that are
sensitive to DNA cross-linking agents and ionizing radiation (IR),
suggesting a specific defect in the repair of cross-linked DNA or
double strand breaks; (b) DNA damage of FA cells which results in a
hyperactive p53 response, suggesting the presence of defective
repair yet intact checkpoint activities; and (c) FA cells with a
defect in the fidelity of non-homologous end joining and an
increased rate of homologous recombination (Garcia-Higuera et al.,
Mol. Cell., (2001) Vol. 7, pp. 249-262), (Grompe et al., Hum. Mol.
Genet., (2001) Vol. 10, pp. 1-7).
[0131] Despite these general abnormalities in cell cycle and DNA
repair, the mechanism by which FA pathway regulates these
activities has remained elusive. Here we show that the FANCD2
protein functions downstream of the FA protein complex. In the
presence of the assembled FA protein complex, the FANCD2 protein is
activated to a high molecular weight, monoubiquitinated isoform
which appears to modulate an S phase specific DNA repair response.
The activated FANCD2 protein accumulates in nuclear foci in
response to DNA damaging agents and co-localizes and
coimmunoprecipitates with a known DNA repair protein, BRCA1. These
results resolve previous conflicting models of the FA pathway
(D'Andrea et al., 1997) and demonstrate that the FA proteins
cooperate in a cellular response to DNA damage.
[0132] The FA pathway includes the formation of the FA multisubunit
nuclear complex which in addition to A/C/G, we have shown also
includes FANCF as a subunit of the complex (FIG. 8). The FA pathway
becomes "active" during the S phase to provide S phase specific
repair response or checkpoint response. The normal activation of
the FA pathway which relies on the FA multisubunit complex results
in the regulated monoubiquitination of the phosphoprotein-FANCD2
via a phosphorylation step to a high molecular weight activated
isoform identified as FANCD-2L (FIG. 1). Monoubiquitination is
associated with cell trafficking. FANCD2-L appears to modulate an S
phase specific DNA repair response (FIG. 3). The failure of FA
cells to activate the S phase specific activation of FANCD2 is
associated with cell cycle specific abnormalities. The activated
FANCD2 protein accumulates in nuclear foci in response to the DNA
damaging agents, MMC and IR, and co-localizes and
co-immunoprecipitates with a known DNA repair protein, BRCA1 (FIGS.
4-6). These results resolve previous conflicting models of FA
protein function (D'Andrea et al., 1997) and strongly support a
role of the FA pathway in DNA repair.
[0133] We have identified for the first time, an association
between FANCD2 isoforms with respect to the FA pathway and proteins
that are known diagnostic molecules for various cancers. A similar
pathway with respect to DNA damage for the BRCA1 protein which is
activated to a high molecular weight, post-translationally-modified
isoform in S phase or in response to DNA damage suggests that
activated FANCD2 protein interacts with BRCA1. More particularly,
the regulated monoubiquitination of FANCD2 appears to target the
FANCD2 protein to nuclear foci containing BRCA1. FANCD2
co-immunoprecipitates with BRCA1, and may further bind with other
"dot" proteins, such as RAD50, Mre11, NBS, or RAD51. Recent studies
demonstrate that BRCA1 foci are composed of a large (2 Megadalton)
multi-protein complex (Wang et al., Genes Dev., (2001) Vol. 14, pp.
927-939). This complex includes ATM, ATM substrates involved in DNA
repair functions (BRCA1), and ATM substrates involved in checkpoint
functions (NBS). It is further suggested that damage recognition
and activation of the FA pathway involve kinases which respond to
DNA damage including ATM, ATR, CHK1, or CHK2.
[0134] We have found that the DNA damaging reagents, IR and MMC,
activate independent post-translational modifications of FANCD2
result in distinct functional consequences. IR activates the
ATM-dependent phosphorylation of FANCD2 at Serine 222 resulting in
an S phase checkpoint response. MMC activates the BRACA-1 dependent
and FA pathway dependent monoubiquitination of FANCD2 at lysine
561, resulting in the assembly of FANCD2/BRCA1 nuclear foci and MMC
resistance. FANCD2 therefore has two independent functional roles
in the maintenance of chromosomal stability resulting from two
discrete post-translational modifications provide a link between
two additional cancer susceptibility genes (ATM and BRCA1) in a
common pathway. Several additional lines of evidence support an
interaction between FANCD2 and BRCA1. First, the BRCA1 (-/-) cell
line, HCC1937 (Scully et al., Mol. Cell, (1999) Vol. 4, pp.
1093-1099) has a "Fanconi Anemia-like" phenotype, with chromosome
instability and increased tri-radial and tetra-radial chromosome
formations. Second, although FA cells form BRCA1 foci (and RAD51
foci) normally in response to IR, BRCA1 (-/-) cells have no
detectable BRCA1 foci and a greatly decreased number of FANCD2 foci
compared to normal cells. Functional complementation of BRCA1 (-/-)
cells restored BRCA1 foci and FANCD2 foci to normal levels, and
restored normal MMC resistance.
[0135] The amount of FANCD2-L is determined in part by the amount
of FAND2-S that is synthesized from the fancd2 gene and in part by
the availability of the FA complex to monoubiquinated FANCD2-S to
form FANCD2-L. The association of FANCD2-L with nuclear foci
including BRCA and ATM and determining the role of FANCD2-L in DNA
repair make this protein a powerful target for looking at potential
cancer development in patients for a wide range of cancers. Such
cancers include those that arise through lesions on chromosome 3p
as well as cancers on other chromosomes such that mutations result
in interfering with production of upstream members of the FA
pathway such as FANCG, FANCC or FANCA. Cancer lines and primary
cells from cancer patients including tumor biopsies are being
screened for FANCD-L and abnormal levels of this protein is
expected to correlate with early diagnosis of disease. Because
FANCD2 protein is a final step in a pathway to DNA repair, it is
envisaged that any abnormality in a protein in the one or more
pathways that lead to the conversion of FANCD2-S to FANCD-L will be
readily detected by measuring levels of FANCD2. Moreover, levels of
FANCD2 affect how other proteins such as BRCA and ATM functionally
interact in the nucleus with consequences for the patient. Analysis
of levels of FANCD2 in a patient is expected to aid a physician in
a clinical decision with respect to understanding the class of
cancer presented by the patient. For instance, if a cancer cell
fails to generate the monoubiquinated FANCD2-L isoform, the cell
may have increased chromosome instability and perhaps increased
sensitivity to irradiation or chemotherapeutic agents. This
information will assist the physician in procedure improved
treatment for the patient.
[0136] Fanconi Anemia is associated not only with a broad spectrum
of different cancers but also with congenital abnormalities.
Development of the fetus is a complex but orderly process. Certain
proteins have a particularly broad spectrum of effects because they
disrupt this orderly progression of development. The FA pathway
plays a significant role in development and disruption of the FA
pathway results in a multitude of adverse effects. Errors in the FA
pathway are detectable through the analysis of the FAND2-L protein
from fetal cells. FANCD2 represents a diagnostic marker for normal
fetal development and a possible target for therapeutic
intervention.
[0137] Consistent with the above, we have shown that FANCD2 plays a
role in the production of viable sperm. FANCD2 forms foci on the
unpaired axes of chromosomes XY bivalents in late pachytene and in
diplotene murine spermatocytes (FIG. 7). Interestingly, FANCD2 foci
are also seen at the autosomal telomeres in diplonema. Taken
together with the known fertility defects in FA patients and FA-C
knockout mice, our observations suggest that activated FANCD2
protein is required for normal progression of spermatocytes through
meiosis I. Most of the FANCD2 foci seen on the XY axes were found
to co-localize with BRCA1 foci, suggesting that the two proteins
may function together in meiotic cells. Like BRCA1, FANCD2 was
detected on the axial (unsynapsed) elements of developing
synaptonemal complexes. Since recombination occurs in synapsed
regions, FANCD2 may function prior to the initiation of
recombination, perhaps to help prepare chromosomes for synapsis or
to regulate subsequent recombinational events. The relatively
synchronous manner in which FANCD2 assembles on meiotic
chromosomes, and forms dot structures in mitotic cells, suggests a
role of FANCD2 in both mitotic and meiotic cell cycle control.
[0138] Embodiments of the invention are directed to the use of the
post translationally modified isoform: FANCD-2L as a diagnostic
target for determining the integrity of the FA pathway.
Ubiquitination of FANCD2 and the formation of FANCD2 nuclear foci
are downstream events in the FA pathway, requiring the function of
several FA genes. We have found that biallelic mutations of any of
the upstream FA genes (FANCA, FANCB, FANCC, FANCE, FANCF and FANCG)
block the posttranslational modification of FANCD2 the
unubiquitinated FANCD2 (FANCD2-S) form to the ubiquitinated
(FANCD2-L). Any of these upstream defects can be overridden by
transfecting cells with FANCD2 cDNA (FIG. 1a).
[0139] We have demonstrated for the first time the existence of
FANCD2 and its role in the FA pathway. We have shown that FANCD2
accumulates in nuclear foci in response to DNA damaging agents
where it is associated with other DNA repair proteins such as BRCA1
and ATM. We have also demonstrated that FANCD2 exists in two
isoforms in cells where a reduction in one of the two isoforns,
FANCD2-L is correlated with Fanconi Anemia and with increased
cancer susceptibility. We have used these findings to propose a
number of diagnostic tests for use in the clinic that will assist
with patient care.
[0140] These tests include: (a) genetic and prenatal counseling for
parents concerned about inherited Fanconi Anemia in a future
offspring or in an existing pregnancy; (b) genetic counseling and
immunodiagnostic tests for adult humans to determine increased
susceptibility to a cancer correlated with a defective FA pathway;
and (c) diagnosing an already existing cancer in a subject to
provide an opportunity for developing treatment protocols that are
maximally effective for the subject while minimizing side
effects.
[0141] The diagnostic tests described herein rely on standard
protocols known in the art for which we have provided novel
reagents to test for FANCD2 proteins and nucleotide sequences.
These reagents include antibodies specific for FANCD2 isoforms,
nucleotide sequences from which vectors, probes and primers have
been derived for detecting genetic alterations in the FANCD2 gene
and cells lines and recombinant cells for preserving and testing
defects in the FA pathway.
[0142] We have prepared monoclonal and polyclonal antibody
preparations as described in Example 1 that are specific for
FANCD2-L and FANCD2-S proteins. In addition, FANCD2 isoform
specific antibody fragments and single chain antibodies may be
prepared using standard techniques. We have used these antibodies
in wet chemistry assays such as immnunoprecipitation assays, for
example Western blots, to identify FANCD2 isoforms in biological
samples (FIG. 1). Conventional immunoassays including enzyme linked
immunosorbent assays (ELISA), radioimmune assays (RIA),
immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA)
and further including sandwich assays may also be used. Other
immunoassays may utilize a sample of whole cells or lysed cells
that are reacted with antibody in solution and optionally analyzed
in a liquid state within a reservoir. Isoforms of FANCD2 can be
identified in situ in intact cells including cell lines, tissue
biopsies and blood by immunological techniques using for example
fluorescent activated cell sorting, and laser or light microscopy
to detect immunofluorescent cells (FIGS. 1-7, 9-14). For example,
biopsies of tissues or cell monolayers, prepared on a slide in a
preserved state such as embedded in paraffin or as frozen tissue
sections can be exposed to antibody for detecting FANCD2-L and then
examined by fluorescent microscopy.
[0143] In an embodiment of the invention, patient-derived cell
lines or cancer cell lines are analyzed by immunoblotting and
immunofluorescence to provide a novel simple diagnostic test for
detecting altered amounts of FANCD2 isoforms. The diagnostic test
also provides a means to screen for upstream defects in the FA
pathway and a practical alternative to the currently employed
DEB/MMC chromosome breakage test for FA, because individuals with
upstream defects in the FA pathway are unable to ubiquitinate
FANCD2. Other assays may be used including assays that combine
retroviral gene transfer to form transformed patient derived cell
lines (Pulsipher et al., Mol. Med., (1998) Vol. 4, pp. 468-79)
together with FANCD2 immunoblotting to provide a rapid subtyping
analysis of newly diagnosed patients with any of the syndromes
described in Table 2, in particular, that of FA.
[0144] The above assays may be performed by diagnostic
laboratories, or, alternatively, diagnostic kits may be
manufactured and sold to health care providers or to private
individuals for self-diagnosis. The results of these tests and
interpretive information are useful for the healthcare provider in
diagnosis and treatment of a patient's condition.
[0145] Genetic tests can provide for a subject, a rapid reliable
risk analysis for a particular condition against an epidemiological
baseline. Our data suggests that genetic heterogeneity occurs in
patients with FA within the FANCD2 complementation group. We have
found a correlation between genetic heterogeneity and disease as
well as genetic heterogeneity and abnormal post-translational
modifications that result in the presence or absence of FANCD2-L.
This correlation provides the basis for prognostic tests as well as
diagnostic tests and treatments for any of the syndromes
characterized by abnormal DNA repair. For example, nucleic acid
from a cell sample obtained from drawn blood or from other cells
derived from a subject can be analyzed for mutations in the FANCD2
gene and the subject may be diagnosed to have an increased
susceptibility to cancer.
[0146] We have located the FANCD2 gene at 3p25.3 on chromosome 3p
in a region which correlates to a high frequency of cancer.
Cytogenetic and loss of heterozygosity (LOH) studies have
demonstrated that deletions of chromosome 3p occur at a high
frequency in all forms of lung cancer (Todd et al., Cancer Res.
Vol. 57, pp. 1344-52). For example, homnozygous deletions were
found in three squamous cell lines within a region of 3p21.
Homozygous deletions were also found in a small cell tumor at 3p12
and a 3p14.2. (Franklin et al., Cancer Res. (1997), Vol. 57, pp.
1344-52). The present mapping of FANCD2 is supportive of the theory
that this chromosomal region contains important tumor suppressor
genes. Further support for this has been provided by a recent
publication of Sekine et al., Human Molecular Genetics, (2001) Vol.
10, pp. 1421-1429, who reported localization of a novel
susceptibility gene for familial ovarian cancer to chromosome
3p22-p25. The reduction or absence of FANCD2-L is here proposed to
be diagnostic for increased risk of tumors resulting from mutations
not only at the FANCD2 site (3p25.3) but also at other sites in the
chromosomes possibly arising from defects in DNA repair following
cell damage arising from exposure to environmental agents and
normal aging processes.
[0147] As more individuals and families are screened for genetic
defects in the FANCD2 gene, a data base will be developed in which
population frequencies for different mutations will be gathered and
correlations made between these mutations and health profile for
the individuals so that the predictive value of genetic analysis
will continually improve. An example of an allele specific pedigree
analysis for FANCD2 is provided in FIG. 10 for two families.
[0148] Diagnosis of a mutation in the FANCD2 gene may initially be
detected by a rapid immunological assay for detecting reduced
amounts of FANCD2-L proteins. Positive samples may then be screened
with available probes and primers for defects in any of the genes
in the PA pathway. Where a defect in the FANCD2 gene is implicated,
primers or probes such as provided in Table 7 may be used to detect
a mutation. In those samples, where a mutation is not detected by
such primers or probes, the entire FANCD2 gene may be sequenced to
determine the presence and location of the mutation in the
gene.
[0149] Nucleic acid screening assays for use in identifying a
genetic defect in the FANCD2 gene locus may include PCR and non PCR
based assays to detect mutations. There are many approaches to
analyzing cell genomes for the presence of mutations in a
particular allele. Alteration of a wild-type FANCD2 allele,
whether, for example, by point mutation, deletion or insertions can
be detected using standard methods employing probes (U.S. Pat. No.
6,033,857). Standard methods include: (a) fluorescent in situ
hybridization (FISH) which may be used on whole intact cells; and
(b) allele specific oligonucleotides (ASO) may be used to detect
mutations using hybridization techniques on isolated nucleic acid
(Conner et al., Hum. Genet., (1989) Vol. 85, pp. 55-74). Other
techniques include (a) observing shifts in electrophoretic mobility
of single-stranded DNA on non-denaturing polyacrylamide gels, (b)
hybridizing a FANCD2 gene probe to genomic DNA isolated from the
tissue sample, (c) hybridizing an allele-specific probe to genomic
DNA of the tissue sample, (d) amplifying all or part of the FANCD2
gene from the tissue sample to produce an amplified sequence and
sequencing the amplified sequence, (e) amplifying all or pant of
the FANCD2 gene from the tissue sample using primers for a specific
FANCD2 mutant allele, (f) molecular cloning all or part of the
FANCD2 gene from the tissue sample to produce a cloned sequence and
sequencing the cloned sequence, (g) identifying a mismatch between
(i) a FANCD2 gene or a FANCD2 mRNA isolated from the tissue sample,
and (ii) a nucleic acid probe complementary to the human wild-type
FANCD2 gene sequence, when molecules (i) and (ii) are hybridized to
each other to form a duplex, (h) amplification of FANCD2 gene
sequences in the tissue sample and hybridization of the amplified
sequences to nucleic acid probes which comprise wild-type FANCD2
gene sequences, (i) amplification of FANCD2 gene sequences in the
tissue sample and hybridization of the amplified sequences to
nucleic acid probes which comprise mutant FANCD2 gene sequences,
(j) screening for a deletion mutation in the tissue sample, (k)
screening for a point mutation in the tissue sample, (l) screening
for an insertion mutation in the tissue sample, and (m) in situ
hybridization of the FANCD2 gene of the tissue sample with nucleic
acid probes which comprise the FANCD2 gene.
[0150] It is often desirable to scan a relatively short region of a
gene or genome for point mutations: The large numbers of
oligonucleotides needed to examine all potential sites in the
sequence can be made by efficient combinatorial methods (Southern,
E. M et al., Nucleic Acids Res., (1994) Vol. 22, pp. 1368-1373).
Arrays may be used in conjunction with ligase or polymerase to look
for mutations at all sites in the target sequence (U.S. Pat. No.
6,307,039). Analysis of mutations by hybridization can be performed
for example by means of gels, arrays or dot blots.
[0151] The entire gene may be sequenced to identify mutations (U.S.
Pat. No. 6,033,857). Sequencing of the FANCD2 locus can be achieved
using oligonucleotide tags from a minimally cross hybridizing set
which become attached to their complements on solid phase supports
when attached to target sequence (U.S. Pat. No. 6,280,935).
[0152] Other approaches to detecting mutations in the FANCD2 gene
include those described in U.S. Pat. Nos. 6,297,010, 6,287,772 and
6,300,076. It is further contemplated that the assays may employ
nucleic acid microchip technology or analysis of multiple samples
using laboratories on chips. Correlation of these mutations with
the results of genetic studies on breast, ovarian or prostate
cancer patients can then be used to determine if an identified
defect within the FANC D2 gene is a cancer-associated defect
according to the invention.
[0153] A subject who has developed a tumor maybe screened using
nucleic acid diagnostic tests or antibody based tests to detect a
FANCD2 gene mutation or a deficiency in FANCD2-L protein. On the
basis of such screening samples may be obtained from subjects
having a wide range of cancers including melanoma, leukemia,
astocytoma, glioblastoma, lymphoma, glioma, Hodgkins lymphoma,
chronic lymphocyte leukemia and cancer of the pancreas, breast,
thyroid, ovary, uterus, testis, pituitary, kidney, stomach,
esophagus and rectum. The clinician has an improved ability to
select a suitable treatment protocol for maximizing the treatment
benefit for the patient. In particular, the presence of a genetic
lesion or a deficiency in FANCD2-L protein may be correlated with
responsiveness to various existing chemotherapeutic drugs and
radiation therapies.
[0154] New therapeutic treatments may be developed by screening for
molecules that modulate the monoubiquitination of FANCD2-S to give
rise to FANCD2-L in cell assays (Examples 11-12) and in knock-out
mouse models (Example 10). Such molecules may include those that
bind directly to FANCD2 or to molecules such as BRACA-2 that
appears to interact with BRACA-1 which in turn appears to be
activated by FANCD2.
[0155] In addition to screening assays that rely on defects in the
FANCD2 gene or protein, an observed failure of the ubiquitination
reaction that is necessary for the formation of FANCD2-L may result
from a defect in the FA pathway at any point preceding the post
translational modification of FANCD2 including FANCD2-S itself.
Knowing the terminal step in the reactions, enables a screening
assay to be formulated in which small molecules are screened in
cells containing "broken FA pathway" or in vitro until a molecule
is found to repair the broken pathway. This molecule can then be
utilized as a probe to identify the nature of the defect. It may
further be used as a therapeutic agent to repair the defect. For
example, we have shown that cell cycle arrest and reduced
proliferation of FA cells can be partially corrected by
overexpression of a protein, SPHAR, a member of the cyclin family
of proteins. This can form the basis of an assay which is suitable
as a screen for identifying therapeutic small molecules.
[0156] Cells which are deficient in the posttranslational modified
FANCD2 are particularly sensitive to DNA damage. These cells may
serve as a sensitive screen for determining whether a compound
(including toxic molecules) has the capability for damaging DNA.
Conversely, these cells also serve as a sensitive screen for
determining whether a compound can protect cells against DNA
damage.
[0157] FA patients and patients suffering from syndromes associated
with DNA repair defects die from complications of bone marrow
failure. Gene transfer is a therapeutic option to correct the
defect. Multiple defects may occur throughout the FA pathway. We
have shown that the terminal step is critical to proper functioning
of the cell and the organism. In an embodiment of the invention,
correction of defects anywhere in the FA pathway may be
satisfactorily achieved by gene therapy or by therapeutic agents
that target the transformation of FANCD2-S to FANCD2-L so that this
transformation is successfully achieved.
[0158] Gene therapy may be carried out according to generally
accepted methods, for example, as described by Friedman in "Therapy
for Genetic Disease," T. Friedman, ed., Oxford University Press
(1991), pp. 105-121. Targeted tissues for ex vivo or in vivo gene
therapy include bone marrow for example, hematopoietic stem cells
prior to onset of anemia and fetal tissues involved in
developmental abnormalities. Gene therapy can provide wild-type
FAND2-L function to cells which carry mutant FANCD2 alleles.
Supplying such a function should suppress neoplastic growth of the
recipient cells or ameliorate the symptoms of Fanconi Anemia.
[0159] The wild-type FANCD-2 gene or a part of the gene may be
introduced into the cell in a vector such that the gene remains
extrachromosomal. In such a situation, the gene may be expressed by
the cell from the extrachromosomal location. If a gene portion is
introduced and expressed in a cell carrying a mutant FANCD-2
allele, the gene portion may encode a part of the FANCD-2 protein
which is required for non-neoplastic growth of the cell.
Alternatively, the wild-type FANCD-2 gene or a part thereof may be
introduced into the mutant cell in such a way that it recombines
with the endogenous mutant FANCD-2 gene present in the cell.
[0160] Viral vectors are one class of vectors for achieving gene
therapy. Viral-mediated gene transfer can be combined with direct
in vivo gene transfer using liposome delivery, allowing one to
direct the viral vectors to the tumor cells and not into the
surrounding nondividing cells. Alternatively, a viral vector
producer cell line can be injected into tumors (Culver et al.,
1992). Injection of producer cells would then provide a continuous
source of vector particles. This technique has been approved for
use in humans with inoperable brain tumors.
[0161] The vector may be injected into the patient, either locally
at the site of the tumor or systemically (in order to reach any
tumor cells that may have metastasized to other sites). If the
transfected gene is not permanently incorporated into the genome of
each of the targeted tumor cells, the treatment may have to be
repeated periodically.
[0162] Vectors for introduction of genes both for recombination and
for extrachromosomal maintenance are known in the art (for example
as disclosed in U.S. Pat. No. 5,252,479 and PCT 93/07282, and U.S.
Pat. No. 6,303,379) and include viral vectors such as retroviruses,
herpes viruses (U.S. Pat. No. 6,287,557) or adenoviruses (U.S. Pat.
No. 6,281,010) or a plasmid vector containing the FANCD2-L.
[0163] A vector carrying the therapeutic gene sequence or the DNA
encoding the gene or piece of the gene may be injected into the
patient either locally at the site of a tumor or systemically so as
to reach metastasized tumor cells. Targeting may be achieved
without further manipulation of the vector or the vector may be
coupled to a molecule having a specificity of binding for a tumor
where such molecule may be a receptor agonist or antagonist and may
further include a peptide, lipid (including liposomes) or
saccharide including an oligopolysaccharide or polysaccharide) as
well as synthetic targeting molecules. The DNA may be conjugated
via polylysine to a binding ligand. If the transfected gene is not
permanently incorporated into the genome of each of the targeted
tumor cells, the treatment may have to be repeated
periodically.
[0164] Methods for introducing DNA into cells prior to introduction
into the patient may be accomplished using techniques such as
electroporation, calcium phosphate coprecipitation and viral
transduction as described in the art (U.S. Pat. No. 6,033,857), and
the choice of method is within the competence of the routine
experimenter.
[0165] Cells transformed with the wild-type FANCD2 gene or mutant
FANCD2 gene can be used as model systems to study remission of
diseases resulting from defective DNA repair and drug treatments
which promote such remission.
[0166] As generally discussed above, the FANCD2 gene or fragment,
where applicable, may be employed in gene therapy methods in order
to increase the amount of the expression products of such genes in
abnormal cells. Such gene therapy is particularly appropriate for
use in pre-cancerous cells, where the level of FANCD2-L polypeptide
may be absent or diminished compared to normal cells and where
enhancing the levels of FANCD2-L may slow the accumulation of
defects arising from defective DNA repair and hence postpone
initiation of a cancer state. It may also be useful to increase the
level of expression of the FANCD2 gene even in those cells in which
the mutant gene is expressed at a "normal" level, but there is a
reduced level of the FANCD2-L isoform. The critical role of
FANCD2-L in normal DNA repair provides an opportunity for
developing therapeutic agents to correct a defect that causes a
reduction in levels of FANCD2-L. One approach to developing novel
therapeutic agents is through rational drug design. Rational drug
design can provide structural analogs of biologically active
polypeptides of interest or of small molecules with which they
interact (e.g., agonists, antagonists, inhibitors or enhancers) in
order to fashion more active or stable forms of the polypeptide, or
to design small molecules which enhance or interfere with the
function of a polypeptide in vivo (Hodgson, 1991). Rational drug
design may provide small molecules or modified polypeptides which
have improved FANCD2-L activity or stability or which act as
enhancers, inhibitors, agonists or antagonists of FANCD2-L
activity. By virtue of the availability of cloned FANCD2 sequences,
sufficient amounts of the FANCD2-L polypeptide may be made
available to perform such analytical studies as x-ray
crystallography. In addition, the knowledge of the FANCD2-L protein
sequence provided herein will guide those employing computer
modeling techniques in place of, or in addition to x-ray
crystallography.
[0167] Peptides or other molecules which have FANCD2-L activity can
be supplied to cells which are deficient in the protein in a
therapeutic formulation. The sequence of the FANCD2-L protein is
disclosed for several organisms (human, fly and plant) (SEQ ID NO:
1-3). FANCD2 could be produced by expression of the cDNA sequence
in bacteria, for example, using known expression vectors with
additional posttranslational modifications. Alternatively, FANCD2-L
polypeptide can be extracted from FANCD2-L-producing mammalian
cells. In addition, the techniques of synthetic chemistry can be
employed to synthesize FANCD2-L protein. Other molecules with
FANCD2-L activity (for example, peptides, drugs or organic
compounds) may also be used as a therapeutic agent. Modified
polypeptides having substantially similar function are also used
for peptide therapy.
[0168] Similarly, cells and animals which carry a mutant FANCD2
allele or make insufficient levels of FANCD2-L can be used as model
systems to study and test for substances which have potential as
therapeutic agents. The cells which may be either somatic or
germline can be isolated from individuals with reduced levels of
FANCD2-L. Alternatively, the cell line can be engineered to have a
reduced levels of FANCD2-L, as described above. After a test
substance is applied to the cells, the DNA repair impaired
transformed phenotype of the cell is determined.
[0169] The efficacy of novel candidate therapeutic molecules can be
tested in experimental animals for efficacy and lack of toxicity.
Using standard techniques, animals can be selected after
mutagenesis of whole animals or after genetic engineering of
germline cells or zygotes to form transgenic animals. Such
treatments include insertion of mutant FANCD2 alleles, usually from
a second animal species, as well as insertion of disrupted
homologous genes. Alternatively, the endogenous FANCD2 gene of the
animals may be disrupted by insertion or deletion mutation or other
genetic alterations using conventional techniques (Capecchi,
Science, (1989) Vol. 244, pp. 1288-1292) (Valancius and Smithies,
1991). After test substances have been administered to the animals,
the growth of tumors must be assessed. If the test substance
prevents or suppresses pathologies arising from defective DNA
repair, then the test substance is a candidate therapeutic agent
for the treatment of the diseases identified herein.
[0170] The subject invention provides for Fanconi Anemia/BRCA-based
diagnostic assays to determine if a patient has cancer or is at an
increased risk of cancer. The invention also features screening
methods for the discovery of novel cancer therapeutics that are
inhibitors of the Fanconi Anemia/BRCA pathway. Finally, the
invention provides methods for the chemosensitization of tumor
cells that have become resistant to one or more chemotherapy
compounds as well as assays to determine the efficacy of
chemotherapy drugs.
[0171] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
cell biology, microbiology and recombinant DNA techniques, which
are within the skill of the art. Such techniques are explained
fully in the literature. See, e.g., Sambrook, Fritsch &
Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second
Edition; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic
Acid Hybridization (B.D. Harnes & S. J. Higgins, eds., 1984); A
Practical Guide to Molecular Cloning (B. Perbal, 1984); (Harlow, E.
and Lane, D.) Using Antibodies: A Laboratory Manual (1999) Cold
Spring Harbor Laboratory Press; and a series, Methods in Enzymology
(Academic Press, Inc.); Short Protocols In Molecular Biology,
(Ausubel et al., ed., 1995). All patents, patent applications, and
publications mentioned herein, both supra and infra, are hereby
incorporated by reference in their entirety.
[0172] Tissue Biopsies
[0173] The invention provides for the preparation of cellular
extracts from tissue biopsies of patients including, but not
limited to brain, heart, lung, lymph nodes, eyes, joints, skin and
neoplasms associated with these organs. "Tissue biopsy" also
encompasses the collection of biological fluids including but not
limited to blood, plasma, sputum, urine, cerebrospinal fluid,
lavages, and leukophoresis samples. In a preferred embodiment,
"tissue biopsies" according to the invention are taken from tumors
of the breast, ovary or prostate. "Tissue biopsies" are obtained
using techniques well known in the art including needle aspiration
and punch biopsy of the skin.
[0174] Cisplatin
[0175] Cisplatin has been widely used to treat cancers such as
metastatic testicular or ovarian carcinoma, advanced bladder
cancer, head or neck cancer, cervical cancer, lung cancer or other
tumors. Cisplatin can be used alone or in combination with other
agents, with efficacious doses used in clinical applications of
15-20 mg/m2 for 5 days every three weeks for a total of three
courses. Exemplary doses may be 0.50 mg/m2, 1.0 mg/m2, 1.50 mg/m2,
1.75 mg/m2, 2.0 mg/m2, 3.0 mg/m2, 4.0 mg/m2, 5.0 mg/m2, 10 mg/m2.
Of course, all of these dosages are exemplary, and any dosage
in-between these points is also expected to be of use in the
invention. Cisplatin is not absorbed orally and must therefore be
delivered via injection intravenously, subcutaneously,
intratumorally or intraperitoneally. Procedures for proper handling
and disposal of anticancer drugs should be considered. Several
guidelines on this subject have been published and are known by
those in the art.
[0176] For example, PLATINOL-AQ, (cisplatin injection) NDC
0015-3220-22 (Bristol Myers Squibb) is supplied as a sterile,
multidose vial without preservatives. Each multidose vial contains
50 mg of cisplatin NDC 0015-3221-22 and should be stored at
15.degree. C.-25.degree. C. and protected from light. The cisplatin
remaining in the amber vial following initial entry is stable for
28 days protected from light or for 7 days under fluorescent room
light.
[0177] The prescribing information for PLATINOL-AQ, (cisplatin
injection) NDC 0015-3220-22 is available from Bristol Myers Squibb.
The plasma concentrations of cisplatin decay monoexponentially with
a half-life of about 20 to 30 minutes following bolus
administrations of 50 or 100 mg/m2 doses. Monoexponential decay and
plasma half-lives of about 0.5 hour are also seen following two
hour or seven hour infusions of 100 mg/m2. After the latter, the
total-body clearances and volumes of distribution at steady-state
for cisplatin are about 15 to 16 L/h/m2 and 11 to 12 L/m2.
[0178] Dosage and Administration of Cisplatin
[0179] The dosage and administration of cisplatin for the treatment
of cancer is known in the art. The prescribing information of
PLATINOL-AQ (Bristol Myers Squibb) recommends the following
guidelines for dosage and administration:"Needles or intravenous
sets containing aluminum parts that may come in contact with
PLATINOL-AQ should not de used for preparation or administration.
Aluminum reacts with PLATINOL-AQ, causing precipitate formation and
a loss of potency".
[0180] Metastatic Testicular Tumors: The usual PLATINOL-AQ dose for
the treatment of testicular cancer in combination with other
approved chemotherapeutic agents is 20 mg/m2 I.V. daily for 5 days
per cycle.
[0181] Metastatic Ovarian Tumors: The usual PLATINOL-AQ dose for
the treatment of metastatic ovarian tumors in combination with
CYTOXAN (cy-clophosphamide) is 75-100 mg/m2 I.V. per cycle once
every 4 weeks, (Day 1). The dose of CYTOXAN when used in
combination with PLATINOL-AQ is 600 mg/m2 I.V. once every 4 weeks,
(Day 1). For directions for the administration of CYTOXAN, refer to
the CYTOXAN package insert. In combination therapy, PLATINOL-AQ and
CYTOXAN are administered sequentially. As a single agent,
PLATINOL-AQ should be administered at a dose of 100 mg/m2 I.V. per
cycle once every 4 weeks.
[0182] Advanced Bladder Cancer: PLATINOL-AQ (cisplatin injection)
should be administered as a single agent at a dose of 50-70 mg/m2
I.V. per cycle once every 3 to 4 weeks depending on the extent of
prior exposure to radiation therapy and/or prior chemotherapy. For
heavily pretreated patients an initial dose of 50 mg/m2 per cycle
repeated every four weeks is recommended. Pretreatment hydration
with 1 to 2 liters of fluid infused for 8 to 12 hours prior to a
PLATINOL-AQ dose is recommended. The drug is then diluted in 2
liters of 5% Dextrose in 1/2 or 1/3 normal saline containing 37.5 g
of mannitol, and infused over a 6- to 8-hour period. If diluted
solution is not to be used within 6 hours, protect solution from
light. Do not dilute PLATINOL-AQ in just 5% Dextrose Injection.
Adequate hydration and urinary output must be maintained during the
following 24 hours. A repeat course of PLATINOL-AQ should not be
given until the serum creatinine is below 1.5 mg/100 mL, and/or the
BUN is below 25 mg/100 mL. A repeat course should not be given
until circulating blood elements are at an acceptable level
(platelets>100,000/mm2, WBC>4,000/mm2). Subsequent doses of
PLATINOL-AQ should not be given until an audiometric analysis
indicates that auditory acuity is within normal limits. As with
other potentially toxic compounds, caution should be exercised in
handling the aqueous solution. Skin reactions associated with
accidental exposure to cisplatin may occur. The use of gloves is
recommended. The aqueous solution should be used intravenously only
and should be administered by I.V. infusion over a 6- to 8-hour
period.
[0183] Dosage and Administration of a Chemosensitizing Agent
[0184] Methods of cancer chemosensitization are reported in U.S.
Pat. No. 5,776,925, which is incorporated herein in its entirety.
Cancer treatment according to the present invention envisions the
use of one or more anti-neoplastic agents in conjunction with
compounds that are not necessarily cytotoxic in themselves, but
modify the host or tumor so as to enhance anticancer therapy. Such
agents are called chemosensitizers.
[0185] Treatment with a chemosensitizing agent is therapeutically
effective in a cancer patient, according to the invention, if tumor
size is decreased by 10%, preferably 25%, preferably 50%, more
preferably 75%, most preferably 100% in the presence of an
antineoplastic agent and corresponding chemosensitizing agent as
compared to tumor size after treatment with the anti-neoplastic
agent but in the absence of the corresponding chemosenziting
agent.
[0186] The present invention provides for pharmaceutical
compositions comprising a therapeutically effective amount of a
chemosensitizing agent, as disclosed herein, in combination with a
pharmaceutically acceptable carrier or excipient. The
chemosensitizers in accordance with the invention, may be
administered to a patient locally or in any systemic fashion,
whether intravenous, subcutaneous, intramuscular, parenteral,
intraperitoneal or oral. Preferably, administration will be
systemic in conjunction with or before the administration of one or
more anti-neoplastic agents. In a preferred embodiment, the
anti-neoplastic agent is cisplatin that is administered according
to protocols well known in the art and as described herein.
[0187] For oral administration, the chemosensitizing agents useful
in the invention will generally be provided in the form of tablets
or capsules, as a powder or granules, or as an aqueous solution or
suspension. Tablets for oral use may include the active ingredients
mixed with pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavouring agents, colouring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0188] Capsules for oral use include hard gelatin capsules in which
the active ingredient is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredients is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil.
[0189] For subcutaneous and intravenous use, the chemosensitizing
agents of the invention will generally be provided in sterile
aqueous solutions or suspensions, buffered to an appropriate pH and
isotonicity. Suitable aqueous vehicles include Ringer's solution
and isotonic sodium chloride. Aqueous suspensions according to the
invention may include suspending agents such as cellulose
derivatives, sodium alginate, polyvinyl-pyrrolidone and gum
tragacanth, and a wetting agent such as lecithin. Suitable
preservatives for aqueous suspensions include ethyl and n-propyl
p-hydroxybenzoate.
[0190] The chemosensitizing agents useful according to the
invention may also be presented as liposome formulations.
[0191] In general a suitable dose will be in the range of 0.01 to
100 mg per kilogram body weight of the recipient per day,
preferably in the range of 0.2 to 10 mg per kilogram body weight
per day. The desired dose is preferably presented once daily, but
may be dosed as two, three, four, five, six or more sub-doses
administered at appropriate intervals throughout the day. These
sub-doses may be administered in unit dosage forms, for example,
containing 10 to 1500 mg, preferably 20 to 1000 mg, and most
preferably 50 to 700 mg of active ingredient per unit dosage form.
Dosages of chemosensitizing agents useful according to the
invention will vary depending upon the condition to be treated or
prevented and on the identity of the chemosensitizing agent being
used. Estimates of effective dosages and in vivo half-lives for the
individual compounds encompassed by the invention can be made on
the basis of in vivo testing using an animal model, such as the
mouse model described herein or an adaptation of such method to
larger mammals.
[0192] In addition to their administration singly, the compounds
useful according to the invention can be administered in
combination with other known chemosensitizing agents and
anti-neoplastic agents, as described herein. In any event, the
administering physician can adjust the amount and timing of drug
administration on the basis of results observed using standard
measures of cancer activity known in the art.
[0193] Anti-neoplastic Agents
[0194] Nonlimiting examples of anti-neoplastic agents include,
e.g., antimicrotubule agents, topoisomerase inhibitors,
antimetabolites, mitotic inhibitors, alkylating agents,
intercalating agents, agents capable of interfering with a signal
transduction pathway, agents that promote apoptosis, radiation, and
antibodies against other tumor-associated antigens (including naked
antibodies, immunotoxins and radioconjugates). Examples of the
particular classes of anti-cancer agents are provided in detail as
follows: antitubulin/antimicrotubule, e.g., paclitaxel,
vincristine, vinblastine, vindesine, vinorelbin, taxotere;
topoisomerase I inhibitors, e.g., topotecan, camptothecin,
doxorubicin, etoposide, mitoxantrone, daunorubicin, idarubicin,
teniposide, amsacrine, epirubicin, merbarone, piroxantrone
hydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU),
methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine
phosphate, cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin,
alanosine, pyrazofurin, N-Phosphoracetyl-L-Asparate, i.e., PALA,
pentostatin, 5-azacitidine, 5-Aza 2'-deoxycytidine, ara-A,
cladribine, 5-fluorouridine, FUDR, tiazofairin,
N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N--
methylamino]-2-thenoyl]-L-glutamic acid; alkylating agents, e.g.,
cisplatin, carboplatin, mitomycin C, BCNU, i.e., Carmustine,
melphalan, thiotepa, busulfan, chlorambucil, plicamycin,
dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen
mustard, uracil mustard, pipobroman, 4-ipomeanol; agents acting via
other mechanisms of action, e.g., dihydrolenperone, spiromustine,
and desipeptide; biological response modifiers, e.g., to enhance
anti-tumor responses, such as interferon; apoptotic agents, such as
actinomycin D; and anti-hormones, for example anti-estrogens such
as tamoxifen or, for example antiandrogens such as
4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-me-
thyl-3'-(trifluoromethyl) propionanilide.
[0195] An anti-neoplastic agent is therapeutic in a cancer patient,
according to the invention, if tumor size is decreased by 10%,
preferably 25%, preferably 50%, more preferably 75%, most
preferably 100% when compared to tumor size prior to the initiation
of treatment with an anti-neoplastic agent.
[0196] In a further embodiment, an anti-neoplastic agent, according
to the invention, is therapeutically effective if the cancer
patient remains cancer free, i.e., without any detectable tumors,
for preferably 6 months, preferably 1 year, more preferably 2 years
and most preferably 5 years or more after initiation of cancer
therapy.
[0197] Inhibitors of the Fanconi Anemia/BRCA Pathway According to
the Invention
[0198] Potential inhibitors of the Fanconi Anemia/BRCA pathway
include, but are not limited to, biomolecules that disrupt the
expression or function of Fanconi Anemia/BRCA pathway genes or
proteins as defined herein. Potential inhibitors of the Fanconi
Anemia/BRCA pathway include, but are not limited, to Fanconi
Anemia/BRCA pathway gene antisense nucleic acids (antisense Fanconi
Anemia/BRCA pathway gene RNAs, oligonucleotides, modified
oligonucleotides, RNAi), dominant negative mutants of the Fanconi
Anemia/BRCA pathway gene pathway as well as inhibitors of Fanconi
Anemia/BRCA pathway gene transcription, mRNA processing, mRNA
transport, protein translation, protein modification, protein
transport, nuclear transport and Fanconi Anemia/BRCA protein
complex formation.
[0199] In a most preferred embodiment, the present invention
provides for small molecule inhibitors of the FANC-D2 ubiquitin E3
ligase.
[0200] Microarrays According to the Invention
[0201] To identify cancer therapeutics or chemosensitizing agents,
the invention provides for the use of microarrays.
[0202] In one embodiment, the microarray of the invention is used
to identify chemosensitizing agents.
[0203] In another embodiment, the microarray of the invention is
used to test tissue biopsy samples for the presence of
cancer-associated defects within the Fanconi Anemia/BRCA pathway
genes.
[0204] In another embodiment, the microarrays of the invention are
used to screen for inhibitors of the Fanconi Anemia/BRCA gene
pathway.
[0205] In another embodiment, the microarrays of the invention are
to be used to screen for inhibitors of the FANC-D2 ubiquitin E3
ligase.
[0206] In another embodiment, the invention provides for tissue
microarrays comprising tissue biopsy samples from patients who have
a cancer or who may be at risk of cancer that are screening for the
presence of cancer associated defects within Fanconi Anemia/BRCA
gene pathway as defined herein. In a preferred embodiment, the
tissue microarrays of the present invention are used to screen for
the presence of mon-ubiqutinated FANC D2-L.
[0207] In another embodiment, the invention provides for tissue
microarrays comprising tissue biopsy samples from patients having
BRCA-1 and BRCA-2/FANC D-1 cancer-associated defects.
[0208] In another embodiment, the invention provides for tissue
microarrays comprising tissue biopsy samples from patients that do
not have BRCA-1 and BRCA-2/FANC D-1 cancer-associated defects.
[0209] A "sequencing array" contains regions of the entire open
reading frame of the genes in question, in order to look for
mutations in the clincial sample. A "transcriptional profiling
array" can have sequences from the 3' end of the genes in
questions, in order to determine the expression of mRNAs in the
clinical sample.
[0210] A transcriptional profiling array will be used to look at
mRNA levels corresponding to each of the genes in the pathway. For
instance, a breast or ovarian cancer which has a decrease in one of
the transcripts, e.g., corresponding to FANC F would show that
there is a defect in the Fanconi Anemia/BRCA pathway, due to
decreased FANCF expression.
[0211] Construction of a Microarray
[0212] Substrate of the Microarray
[0213] In one embodiment of the invention, the microarray or array
comprises a substrate to facilitate handling of the microarray
through a variety of molecular procedures. As used herein,
"molecular procedure" refers to contact of the microarray with a
test reagent or molecular probe such as an antibody, nucleic acid
probe, enzyme, chromagen, label, and the like. In one embodiment, a
molecular procedure comprises a plurality of hybridizations,
incubations, fixation steps, changes of temperature (from
-4.degree. C. to 100.degree. C.), exposures to solvents, and/or
wash steps.
[0214] In a further embodiment of the invention, the microarray
comprises a substrate to facilitate exposure of tissue biopsy
samples to different potential inhibitors of the Fanconi
Anemia/BRCA pathway, cancer therapeutics or chemosensitizing
agents.
[0215] In one embodiment of the invention, the microarray substrate
is solvent resistant. In another embodiment of the invention, the
substrate is transparent. The substrate may be biological,
non-biological, organic, inorganic, or a combination of any of
these, existing as particles, strands, precipitates, gels, sheets,
tubing, spheres, beads, containers, capillaries, pads, slices,
films, plates, slides, chips, etc. The substrate is preferably flat
or planar but may take on a variety of alternative surface
configurations. The substrate may be a polymerized Langmuir
Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SIN4,
modified silicon, or other nonporous substrate, plastic, such as
polyolefin, polyamide, polyacarylamide, polyester, polyacrylic
ester, polycarbonate, polytetrafluoroethylene, polyvinyl acetate,
and a plastic composition containing fillers (such as glass
fillers), extenders, stabilizers, and/or antioxidants; celluloid,
cellophane or urea formaldehyde resins or other synthetic resins
such as cellulose acetate ethylcellulose, or other transparent
polymer. Other substrate materials will be readily apparent to
those of skill in the art upon review of this disclosure.
[0216] In one embodiment, the microarray substrate is rigid;
however, in another embodiment, the profile array substrate is
semi-rigid or flexible (e.g., a flexible plastic comprising
polycarbonate, cellular acetate, polyvinyl chloride, and the like).
In a further embodiment, the array substrate is optically opaque
and substantially non-fluorescent. Nylon or nitrocellulose
membranes can also be used as array substrates and include
materials such as polycarbonate, polyvinylidene fluoride (PVDF),
polysulfone, mixed esters of cellulose and nitrocellulose, and the
like.
[0217] The size and shape of the substrate may generally be varied.
The substrate may have any convenient shape, such as a disc,
square, sphere, circle, etc. However, preferably, the substrate
fits entirely on the stage of a microscope. In one embodiment, the
profile array substrate is planar. In one embodiment of the
invention, the microarray substrate is 1 inch by 3 inches,
77.times.50 mm, or 22.times.50 mm. In another embodiment of the
invention, the microarray substrate is at least 10-200
mm.times.10-200 mm.
[0218] Additional Features of the Substrate
[0219] In one embodiment of the invention, the substrate comprises
a location for placing an identifier (e.g., a wax pencil or crayon
mark, an etched mark, a label, a bar code, a microchip for
transmitting radio or electronic signals, and the like). In one
embodiment, the location comprises frosted glass. In one
embodiment, the microchip communicates with a processor which
comprises or can access stored information relating to the identity
and address of sublocations on the array, and/or including
information regarding the individual from whom the tissue was
taken, e.g., prognosis, diagnosis, medical history, family medical
history, drug treatment, age of death and cause of death, and the
like.
[0220] Sublocations
[0221] The microarray comprises a plurality of sublocations. Each
sublocation comprises a tissue stably associated therewith (e.g.,
able to retain its position relative to another sublocation after
exposure to at least one molecular procedure). In one embodiment,
the tissue is a tissue which has morphological features
substantially intact which can be at least viewed under a
microscope to distinguish subcellular features (e.g., such as a
nucleus, an intact cell membrane, organells,and/or other
cytological features), i.e., the tissue is not lysed.
[0222] In one embodiment of the invention, the microarray comprises
from 2-1000 sublocations. In another embodiment, the microarray
comprises 2, 5, 10, 20, 25, 30, 45, 50, 55, 60, 65, 75, 100, 150,
200, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000
or more sublocations. In one embodiment of the invention, each
sublocation is from 2-10 mm apart. In another embodiment of the
invention, each sublocation comprises at least one dimension which
is 20-600 mm. The sublocations can be organized in any pattern, and
each sublocation can be generally any shape (square, circular,
oval, elliptical, disc shaped, rectangular, triangular, and the
like).
[0223] In a preferred embodiment, the sublocations are positioned
in a regular repeating pattern (e.g., rows and columns) such that
the identification of each sublocation as to tissue type can be
ascertained by the use of an array locator. In one embodiment, the
array locator is a template having a plurality of shapes, each
shape corresponding to the shape of each sublocation in the array,
and maintaining the same relationships as each sublocation on the
array. The array locator is marked by coordinates, allowing the
user to readily identify a sublocation on the array by virtue of
unique coordinates. In one embodiment of the invention, the array
locator is a transparent sheet (e.g., plastic, acetate, and the
like). In another embodiment of the invention, the array locator is
a sheet comprising a plurality of holes, each hole corresponding in
shape and location to each sublocation on the array.
[0224] In one aspect, the invention provides for arrays wherein the
compounds comprising the array are spotted onto a solid support,
e.g., spotted using a robotic GMS 417 arrayer (Affymetrix, Calif.).
Alternatively, spotting may be carried out using contact printing
technology or other methods known in the art.
[0225] Types of Microarrays According to the Invention
[0226] Small Molecule Arrays
[0227] In the small molecule microarrays or arrays of the
invention, the small molecules are stably associated with the
surface of a solid support, wherein the support may be a flexible
or rigid solid support. By "stably associated" is meant that each
small molecule maintains a unique position relative to the solid
support under binding and washing conditions. As such, the samples
are non-covalently or covalently stably associated with the support
surface. Examples of non-covalent association include non-specific
adsorption, binding based on electrostatic interactions (e.g., ion
pair interactions), hydrophobic interactions, hydrogen bonding
interactions, specific binding through a specific binding pair
member covalently attached to the support surface, and the like.
Examples of covalent binding include covalent bonds formed between
the small molecules and a functional group present on the surface
of the rigid support (e.g., --OH), where the functional group may
be naturally occurring. The surface of the substrate can be
preferably provided with a layer of linker molecules, although it
will be understood that the linker molecules are not required
elements of the invention. The linker molecules are preferably of
sufficient length to permit small molecules of the invention and on
a substrate to bind to small molecules and to interact freely with
molecules exposed to the substrate.
[0228] The amount of small molecule present in each composition
will be sufficient to provide for adequate binding and detection of
target small molecules during the assay in which the array is
employed. Generally, the amount of each small molecule stably
associated with the solid support of the array is at least about
0.1 pg, preferably at least about 0.5 pg and more preferably at
least about 1 pg, where the amount may be as high as 1000 pg or
higher, but will usually not exceed about 100 pg. In a preferred
embodiment, the microarray has a density exceeding 1, 2, 5, 7, 10,
15 or 20 or more small molecules/cm2.
[0229] Tissue Microarrays
[0230] In a preferred embodiment of the invention, the microarrays
or arrays comprise human tissue samples. The microarrays according
to the invention comprise a plurality of sublocations, each
sublocation comprising a tissue sample having at least one known
biological characteristic (e.g., such as tissue type). In a
preferred embodiment of the invention, the plurality of
sublocations comprise cancerous tissue at different neoplastic
stages.
[0231] In one embodiment of the invention, the cancerous cells at
individual sublocations are from an individual with an underlying
cancer or predisposition to having a cancer.
[0232] In one embodiment of the invention, the cancerous cells at
individual sublocations are from an individual with
cancer-associated defects in the BRCA-1 and/or FANC D1/BRCA-2
genes.
[0233] In one embodiment, the microarray comprises at least one
sublocation comprising cancerous cells from a single patient and
comprises a plurality of sublocations comprising cells from other
tissues and organs from the same patient. In a different
embodiment, a microarray is provided comprising cells from a
plurality of individuals who have all died from the same pathology,
or from individuals being treated with the same drug (including
those who recovered from the disease and/or those who did not).
[0234] In another embodiment of the invention, the microarray
comprises a plurality of sublocations comprising cells from
individuals sharing a trait in addition to cancer. In one
embodiment of the invention, the trait shared is gender, age, a
pathology, predisposition to a pathology, exposure to an infectious
disease (e.g., HIV), kinship, death from the same illness,
treatment with the same drug, exposure to chemotherapy or
radiotherapy, exposure to hormone therapy, exposure to surgery,
exposure to the same environmental condition, the same genetic
alteration or group of alterations, expression of the same gene or
sets of genes.
[0235] In a further embodiment of the invention, each sublocation
of the microarray comprises cells from different members of a
pedigree sharing a family history of cancer (e.g., selected from
the group consisting of sibs, twins, cousins, mothers, fathers,
grandmothers, grandfathers, uncles, aunts, and the like). In
another embodiment of the invention, the "pedigree microarray"
comprises environment-matched controls (e.g., husbands, wives,
adopted children, stepparents, and the like). In still a further
embodiment of the invention, the microarray is a reflection of a
plurality of traits representing a particular patient demographic
group of interest, e.g., overweight smokers, diabetics with
peripheral vascular disease, individuals having a particular
predisposition to disease (e.g., sickle cell Anemia, Tay Sachs,
severe combined immunodeficiency), wherein individuals in each
group have cancer.
[0236] In a preferred embodiment of the invention, the microarrays
comprise human tissue biopsies.
[0237] FANC D2 -/- as disclosed herein. In one embodiment, the
microarray comprises multiple tissues from such a mouse. In another
embodiment of the invention, the microarray comprises tissues from
mice that are FANC D2 -/- as disclosed herein, and which have been
treated with a cancer therapy (e.g., drugs, antibodies, protein
therapies, gene therapies, antisense therapies, and the like).
[0238] Screening of Chemosensitizing Agents and Novel Cancer
Therapeutics
[0239] The microarrays of the invention are used to screen for
chemosensitizing agents and cancer therapeutics. The screening
procedures used are disclosed in Examples 15 and 16.
[0240] Measurement of Resistance to a Chemotherapy Agent
[0241] Methylation of the FANC F gene within tumor cells that are
treated with cisplatin results in the repression of FANC F gene
expression and thereby causes a disruption in the tumor cell's DNA
damage repair mechanisms and resulting in resistance to cisplatin.
The invention therefore provides for the determination of the
methylation state of any of the Fanconi Anemia/BRCA pathway genes
(see Example 19). In a preferred embodiment, the invention provides
microarrays of tissue biopsy samples from patients being treated
with one or more chemotherapy compounds for the determination of
the methylation state of the Fanconi Anemia/BRCA genes as a
measurement of the degree of a tumor's resistance to one or more
chemotherapy compounds. Methods of measuring DNA methylation of
genes are well known in the art (see U.S. Pat. Nos. 6,200,756;
6,331,393; 6,251,594).
[0242] Kits According to the Invention
[0243] The invention provides for kits useful for screening for
chemosensitizers and cancer therapeutics, as well as kits useful
for diagnosis of cancer or predisposition toward cancer involving
cancer-associated defects in the Fanconi Anemia/BRCA gene pathway.
Kits useful according to the invention include isolated FANC D2
polynucleotide primer pairs, probes, inhibitors of the Fanconi
Anemia/BRCA pathway and a FANC D2-specific antibody. In addition,
kits can contain control unmethylated FANC D2 genes. In a further
embodiment, a kit according to the invention can contain an ovary
cancer tumor cell line. All kits according to the invention will
comprise the stated items or combinations of items and packaging
materials therefore. Kits will also include instructions for
use.
[0244] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
EXAMPLES
Example 1
[0245] Experimental Protocols used in Examples 2-8.
[0246] Cell Lines and Culture Conditions. Epstein-Barr virus (EBV)
transformed lymphoblasts were maintained in RPMI media supplemented
with 15% heat-inactivated fetal calf serum (FCS) and grown in a
humidified 5% CO2-containing atmosphere at 37.degree. C. A control
lymphoblast line (PD7) and FA lymphoblast lines (FA-A (HSC72), FA-C
(PD-4), FA-D (PD-20), FA-F (EUFA121), and FA-G (EUFA316)) have been
previously described (de Winter et al., Nat. Genet., (1998) Vol.
20, pp. 281-283) (Whitney et al., Nat. Genet., (1995) Vol. 11, pp.
341-343) (Yamashita et al., P.N.A.S., (1994) Vol. 91, pp.
6712-6716) (de Winter et al., Am. J. Hum. Genet., (2001), Vol. 57,
pp. 1306-1308). PD81 is a lymphoblast cell line from an FA-A
patient. The SV40-transformed FA fibroblasts, GM6914, PD426,
FAG326SV and PD20F, as well as HeLa cells, were grown in DMEM
supplemented with 15% FCS. FA cells (both lymphoblasts and
fibroblasts) were functionally complemented with pMMP retroviral
vectors containing the corresponding FANC cDNAs, and functional
complementation was confirmed by the MMC assay (Garcia-Higuera et
al., Mol. Cell. Biol., (1999) Vol. 19, pp. 4866-4873) (Kuang et
al., Blood, (2000), Vol. 96, pp. 1625-1632).
[0247] Cell Cycle Synchronization. HeLa cells, GM6914 cells, and
GM6914 cells corrected with the pMMP-FANCA retrovirus were
synchronized by the double thymidine block method as previously
described, with minor modifications (Kupfer et al., Blood, (1997)
Vol. 90, pp. 1047-1054). Briefly, cells were treated with 2 mM
thymidine for 18 hours, thymidine-free media for 10 hours, and
additional 2 mM thymidine for 18 hours to arrest the cell cycle at
the G1/S boundary. Cells were washed twice with PBS and then
released in DMEM+15% FCS and analyzed at various time
intervals.
[0248] Alternatively, HeLa cells were treated with 0.5 mM mimosine
(Sigma) for 24 hours for synchronization in late G1 phase (Krude,
1999), washed twice with PBS, and released into DMEM+15% FCS. For
synchronization in M phase, a nocodazole block was used (Ruffner et
al., Mol. Cell. Biol., (1999) Vol. 19, pp. 4843-4854). Cells were
treated with 0.1 .mu.g/ml nocodazole (Sigma) for 15 hours, and the
non-adherent cells were washed twice with PBS and replated in
DMEM+15%.
[0249] Cell Cycle Analysis. Trypsinized cells were resuspended in
0.5 ml of PBS and fixed by adding 5 ml of ice-cold ethanol. Cells
were next washed twice with PBS with 1% bovine serum albumin
fractionV (1% BSA/PBS) (Sigma), and resuspended in 0.24 ml of 1%
BSA/PBS. After adding 30 .mu.l of 500 .mu.t/ml propidium iodide
(Sigma) in 38 mM sodium citrate (pH7.0) and 30 .mu.l of 10 mg/ml
DNase free RNaseA (Sigma), samples were incubated at 37.degree. C.
for 30 min. DNA content was measured by FACScan (Beckton
Dickinson), and data were analyzed by the CellQuest and Modfit LT
program (Becton Dickinson).
[0250] Generation of an anti-FANCD2 antiserum. A rabbit polyclonal
antiserum against FANCD2 was generated using a GST-FANCD2
(N-terminal) fusion protein as an antigen source. A 5' fragment was
amplified by polymerase chain reaction (PCR) from the full length
FANCD2 cDNA with the primers (SEQ ID NO:95) DF4EcoRI
(5'AGCCTCgaattcGTTTCCAA AAGAAGACTGTCA-3') and (SEQ ID NO:96)
DR816Xh (5'-GGTATCctcgagTCAAGACGA CAACTTATCCATCA-3'). The resulting
PCR product of 841 bp, encoding the amino-terminal 272 amino acids
of the FANCD2 polypeptide was digested with EcoRI/XhoI and
subcloned into the EcoRI/Xhol sites of the plasmid pGEX4T-1
(Pharmacia). A GST-FANCD2 (N-terminal) fusion protein of the
expected size (54 kD) was expressed in E. coli strain DH5.gamma.,
purified over glutathione-S-sepharose, and used to immunize a New
Zealand White rabbit. An FANCD2-specific immune antiserum was
affinity-purified by passage over an AminoLink Plus column (Pierce)
loaded with GST protein and by passage over an AminoLink Plus
column loaded with the GST-FANCD2 (N-terminal) fusion protein.
[0251] Generation of anti-FANCD2 MoAbs. Two anti-FANCD2 monoclonal
antibodies were generated as follows. Balb/c mice were immunized
with a GST-FANCD2 (N-terminal) fusion protein, which was the same
fusion protein used for the generation of the rabbit polyclonal
antiserum (E35) against FANCD2. Animals were boosted with immunogen
for the four days before fusion, splenocytes were harvested, and
hybridization with myeloma cells was performed. Hybridoma
supernatants were collected and assayed using standard ELISA assay
as the initial screen and immunoblot analysis of FANCD2 as the
secondary screen. Two anti-human FANCD2 monoclonal antibodies
(MoAbs) (FI17 and FI14) were selected for further study. Hybridoma
supernatants from the two positive cell lines were clarified by
centrifugation. Supernatants were used as MoAbs for western
blotting. MoAbs were purified using an affinity column for IgG.
MoAbs were stored as 0.5 mg/ml stocks in phosphate buffered saline
(PBS). Anti-HA antibody (HA.11) was from Babco.
[0252] Immunoblotting. Cells were lysed with 1.times. sample buffer
(50 mM Tris-HCl pH6.8, 86 mM 2-mercaptoethanol, 2% sodium dodecyl
sulfate (SDS), boiled for 5 min, and subjected to 7.5%
polyacrylamide SDS gel electrophoresis. After electrophoresis,
proteins were transferred to nitrocellulose using a submerged
transfer apparatus (BioRad) filled with 25 mM Tris base, 200 mM
glycine, 20% methanol. After blocking with 5% non-fat dried milk in
TBS-T (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween 20) the
membrane was incubated with the primary antibody diluted in TBS-T
(1:1000 dilution for the affinity-purified anti-FANCD2 polyclonal
antibody (E35) or anti-HA (HA. l 1), 1:200 dilution for the
anti-FANCD2 mouse monoclonal antibody FI17), washed extensively and
incubated with the appropriate horseradish peroxidase-linked
secondary antibody (Amersham). Chemiluminescence was used for
detection.
[0253] Generation of DNA Damage. Gamma irradiation was delivered
using a Gamma cell 40 apparatus. UV exposure was achieved using a
Stratalinker (Stratagene) after gently aspirating the culture
medium. For Mitomycin C treatment cells were continuously exposed
to the drug for the indicated time. Hydroxyurea (Sigma) was added
to a final concentration of 1 mM for 24 hours.
[0254] Detection of Monoubiquitinated FANCD2. HeLa cells (or the
FA-G fibroblasts, FAG326SV) were transfected using FuGENE6 (Roche),
following the manufacturer's protocol. HeLa cells were plated onto
15 cm tissue culture dishes and were transfected with 15 .mu.g of a
HA-tagged ubiquitin expression vector (pMT 123) (Treier et al.,
Cell, (1994) Vol. 78, pp. 787-798) per dish. Twelve hours following
transfection, cells were treated with the indicated concentration
of MMC (0, 10, 40, 160 ng/ml) or the indicated dose of IR (0, 5,
10, 10, 20 Gy). After 24 hour-incubation with MMC, or two hours
after IR treatment, whole cell extracts were prepared in Lysis
Buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100)
supplemented with protease inhibitors (1 .mu.g/ml leupeptin and
pepstatin, 2 .mu.g/ml aprotinin, 1 mM phenylmethylsulfonylfluoride)
and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium
fluoride). Using the polyclonal antibody to FANCD2 (E35),
immunoprecipitation (IP) was performed essentially as described
(Kupfer et al., 1997) except that each IP was normalized to contain
4 mg of protein. As a negative control, preimmune serum from the
same rabbit was used in IP reaction. Immunoblotting was done using
anti-HA (HA.11), or anti-FANCD2 (FI17) monoclonal antibody.
[0255] Ubiquitin Aldehyde Treatment. HeLa cells were treated with 1
mM hydroxyurea for 24 hours, and whole cell extracts were prepared
in Lysis Buffer supplemented with protease inhibitors and
phosphatase inhibitors. 200 .mu.g of cell lysate in 67 .mu.l of
reaction with 6.7 .mu.l of 25 .mu.M ubiquitin aldehyde
(BostonBiochem) in DMSO or with 6.7 .mu.l of DMSO were incubated at
30.degree. C. or at 37.degree. C. for the indicated periods.
Sixty-seven microliters of 2.times. sample buffer was added to each
sample, and the samples were boiled for 5 min, separated by 7.5%
SDS-PAGE, and immunoblotted for FANCD2 using the FI17 monoclonal
anti-human FANCD2 antibody.
[0256] Immunofluorescence Microscopy. Cells were fixed with 2%
paraformaldehyde in PBS for 20 min, followed by permeabilization
with 0.3% Triton-X-100 in PBS (10 min). After blocking in 10% goat
serum, 0.1% NP-40 in PBS (blocking buffer), specific antibodies
were added at the appropriate dilution in blocking buffer and
incubated for 2-4 hours at room temperature. FANCD2 was detected
using the affinity-purified E35 polyclonal antibody (1/100). For
BRCA1 detection, we used a commercial monoclonal antibody (D-9,
Santa Cruz) at 2 .mu.g/ml. Cells were subsequently washed three
times in PBS+0.1% NP-40 (10-15 min each wash) and species-specific
fluorescein or Texas red-conjugated secondary antibodies (Jackson
lmmunoresearch) were diluted in blocking buffer (anti-mouse 1/200,
anti-rabbit 1/1000) and added. After 1 hour at room temperature
three more 10-15 min washes were applied and the slides were
mounted in Vectashield (Vector laboratories). Images were captured
on a Nikon microscope and processed using Adobe Photoshop
software.
[0257] Meiotic Chromosome Staining. Surface spreads of pachytene
and diplotene spermatocytes from male mice between the ages of 16
and 28 days old were prepared as described by (Peters et al.,
1997). A polyclonal goat antibody to the mouse SCP3 protein was
used to visualize axial elements and synaptonemal complexes in the
meiotic preparations. The M118 mouse monoclonal antibody against
mouse BRCA1 was generated by standard techniques, by immunizing
mice with murine BRCA1 protein. The affinity-purified E35 rabbit
polyclonal antibody was used in 1:200 dilution to detect FANCD.
Antibody incubation and detection procedures were a modification of
the protocol of (Moens et al., J. Cell. Biol., (1987) Vol. 105, pp.
93-103) as described by (Keegan et al., Genes Dev., (1996) Vol. 10,
pp. 2423-2437). Combinations of donkey-anti mouse
IgG-FITC-congugated, Donkey-anti rabbit IgG-TRITC-congugated, and
Donkey-anti goat IgGCy5-congugated secondary antibodies were used
for detection (Jackson ImmunoResearch Laboratories). All
preparations were counterstained with 4', 6' diamino-2-phenylindole
(DAPI, Sigma) and mounted in a DABCO (Sigma) antifade solution. The
preparations were examined on a Nikon E1000 microscope
(60.times.CFI Plan Apochromat and 100.times.CR Plan Fluor
oil-immersion objectives). Each fluorochrome (FITC, TRITC, Cy5 and
DAPI) image was captured separately as an 800.times.1000 pixel
12-bit source image via IPLab software (Scanalytics) controlling a
cooled-CCD camera (Princeton Instruments MicroMax) and the separate
12 bit grey scale images were resampled, 24-bit pseudocolored and
merged using Adobe Photoshop.
Example 2
[0258] The FA Genes Interact in a Common Cellular Pathway.
[0259] Normal lymphoblasts express two isoforms of the FANCD2
protein, a short form (FANCD2-S, 155 kD) and a long form (FANCD2-L,
162 kD). FIG. 1 shows what happened when whole cell extracts were
prepared from a lymphoblast line and cellular proteins were
immunoprecipitated with an anti-FANCD2 antiserum. Normal wild type
cells expressed two isoforms of the FANCD2 protein--a low molecular
weight isoform FANCD2-S (155 kD isoform) and a high molecular
weight isoform (FANCD2-L) (162 kD isoform). FANCD2-S is the primary
translation product of the cloned FANCD2 cDNA. We next evaluated a
large series of FA lymphoblasts and fibroblasts for expression of
the FANCD2 isoforms (Table 5). Correction of these FA cell lines
with the corresponding FA cDNA resulted in functional
complementation and restoration of the high molecular weight
isoform, FANCD2-L.
[0260] As previously described, FA cells are sensitive to the DNA
crosslinking agent, MMC, and in some cases, to ionizing radiation
(IR). Interestingly, FA cells from multiple complementation groups
(A, C, G, and F) only expressed the FANCD2-S isoform (FIG. 1A,
lanes 3, 7, 9, 11). FA cells from complementation groups B and E
also express only the FANCD2-S. Functional correction of the MMC
and IR sensitivity of these FA cells with the corresponding FANC
cDNA restored the FA protein complex (Garcia-Higuera et al., 1999)
and restored the high molecular weight isoform (FANCD2-L) (FIG. 1A,
lanes 4, 8, 10, 12). Taken together, these results demonstrate that
the FA protein complex, containing FANCA, FANCC, FANCF, and FANCG,
directly or indirectly regulates the expression of the two isoforms
of FANCD2. The six cloned FA genes therefore appear to interact in
a common pathway.
Example 3
[0261] The FA Protein Complex is Required for the
Monoubiquitination of FANCD2.
[0262] The high molecular weight isoform of FANCD2 could result
from one or more mechanisms, including alternative splicing of the
FANCD2 mRNA or post-translational modification(s) of the FANCD2
protein. Treatment with phosphatase did not convert FANCD2-L to
FANCD2-S, demonstrating that phosphorylation alone does not account
for the observed difference in their molecular mass.
[0263] In order to identify other possible post-translational
modifications of FANCD2, we initially sought cellular conditions
which regulate the conversion of FANCD2-S to FANCD2-L (FIGS. 1B,
C). Since FA cells are sensitive to MMC and IR, we reasoned that
these agents might regulate the conversion of FANCD2-S to FANCD2-L
in normal cells. Interestingly, HeLa cells treated with MMC (FIG.
1B, lanes 1-6) or IR (FIG. 1C, lanes 1-6) demonstrated a
dose-dependent increase in the expression of the FANCD2-L
isoform.
[0264] To determine whether FANCD2-L is a ubiquitinated isoform of
FANCD2-S, we transfected HeLa cells with a cDNA encoding
HA-ubiquitin (Treier et al., 1994). Cellular exposure to MMC (FIG.
1B, lanes 7-10) or IR (FIG. 1C, lanes 7-10) resulted in a
dose-dependent increase in the HA-ubiquitin conjugation of FANCD2.
Only the FANCD2-L isoform, and not the FANCD2-S isoform, was
immunoreactive with an anti-HA antibody. Although FANCD2 was not
ubiquinated in FA cells, FANCD2 ubiquination was restored upon
functional complementation of these cells. Although FANCD2 was not
ubiquitinated in FA cells, FANCD2 ubiquitination was restored upon
functional complementation of these cells. Since the FANCD2-S and
FANCD2-L isoforms differ by 7 kD, the FANCD2-L probably contains a
single ubiquitin moiety (76 amino acids) covalently bound by an
amide linkage to an internal lysine residue of FANCD2.
[0265] To confirm the monoubiquitination, we isolated FANCD2-L
protein from HeLa cells and analyzed its tryptic fragments by mass
spectrometry (Wu et al., Science, (2000), Vol. 289, p. 11a).
Ubiquitin tryptic fragments were unambiguously identified, and a
site of monoubiquitination (K561 of FANCD2) was also identified.
Interestingly, this lysine residue is conserved among FANCD2
sequences from human, Drosophila, and C. elegans, suggesting that
the ubiquitination of this site is critical to the FA pathway in
multiple organisms. Mutation of this lysine residue, FANCD2
(K561R), resulted in loss of FANCD2 monoubiquitination.
Example 4
[0266] Formation of Nuclear Foci Containing FANCD2 Requires an
Intact FA Pathway.
[0267] We examined the immunofluorescence pattern of the FANCD2
protein in uncorrected, MMC-sensitive FA fibroblasts and
functionally-complemented fibroblasts (FIG. 2).
[0268] The corrected FA cells expressed both the FANCD2-S and
FANCD2-L isoforms (FIG. 2A, lanes 2, 4, 6, 8). The endogenous
FANCD2 protein was observed exclusively in the nucleus of human
cells, and no cytoplasmic staining was evident (FIG. 2B, a-h). The
PD-20 (FA-D) cells have decreased nuclear immunofluorescence (FIG.
2B, d), consistent with the decreased expression of FANCD2 protein
in these cells by immunoblot (FIG. 2A, lane 7). In PD20 cells
functionally-corrected with the FANCD2 gene by chromosome transfer,
the FANCD2 protein stained in two nuclear patterns. Most corrected
cells had a diffuse nuclear pattern of staining, and a minor
fraction of cells stained for nuclear foci (see dots, panel h).
Both nuclear patterns were observed with three
independently-derived anti-FANCD2 antisera (1 polyclonal, 2
monoclonal antisera). FA fibroblasts from subtypes A, G, and C
showed only the diffuse pattern of FANCD2 nuclear
immunofluorescence. Functional complementation of these cells with
the FANCA, FANCG, or FANCC cDNA, respectively, restored the MMC
resistance of these cells (Table 6), and restored the nuclear foci
in some cells. The presence of the high molecular weight FANCD2-L
isoform therefore correlates with the presence of FANCD2 nuclear
foci, suggesting that only the monoubiquitinated FANCD2-L isoform
is selectively localized to these foci.
Example 5
[0269] The FANCD2 Protein is Localized to Nuclear Foci During S
Phase of the Cell Cycle.
[0270] Since only a fraction of the asynchronous
functionally-complemented cells contained FANCD2 nuclear foci, we
reasoned that these foci might assemble at discrete times during
the cell cycle. To test this hypothesis, we examined the formation
of the FANCD2-L isoform and FANCD2 nuclear foci in synchronized
cells (FIG. 3). HeLa cells were synchronized at the G1/S boundary,
released into S phase, and analyzed for formation of the FANCD2-L
isoform (FIG. 3A). The FANCD2-L isoform was expressed specifically
during late GI phase and throughout S phase. Synchronized,
uncomplemented FA cells (FA-A fibroblasts, GM6914) expressed normal
to increased levels of FANCD2-S protein but failed to express
FANCD2-L at any time during the cell cycle. Functional
complementation of these FA-A cells by stable transfection with the
FANCA cDNA restored S phase-specific expression of FANCD2-L. The S
phase specific expression of the FANCD2-L isoform was confirmed
when HeLa cells were synchronized by other methods, such as
nocodazole arrest (FIG. 3B) or mimosine exposure (FIG. 3B). Cells
arrested in mitosis did not express FANCD2-L, suggesting that the
FANCD2-L isoform is removed or degraded prior to cell division
(FIG. 3B, mitosis). Taken together, these results demonstrate that
the monoubiquitination of the FANCD2 protein is highly regulated
during the cell cycle, and that this modification requires an
intact FA pathway.
[0271] The cell cycle dependent expression of the FANCD2-L isoform
also correlated with the formation of FANCD2 nuclear foci (FIG.
3C). Nocodazole arrested (mitotic) cells express no FANCD2-L
isoform and exhibit no FANCD2 nuclear foci (FIG. 3C, 0 hour). When
these synchronized cells were allowed to traverse S phase (15 to 18
hours), an increase in FANCD2 nuclear foci was observed.
Example 6
[0272] The FANCD2 Protein is Localized to Nuclear Foci in Response
to DNA Damage.
[0273] We examined the accumulation of the FANCD2-L isoform and
FANCD2 nuclear foci in response to DNA damage (FIG. 4). Previous
studies have shown that FA cells are sensitive to agents which
cause DNA interstrand crosslinks (MMC) or double strand breaks (IR)
but are relatively resistant to ultraviolet light (UV) and
monofunctional alkylating agents. MMC activated the conversion of
FANCD2-S to FANCD2-L in asynchronous HeLa cells (FIG. 4A). Maximal
conversion to FANCD2-L occurred 12-24 hours after MMC exposure,
correlating with the time of maximal FANCD2 nuclear focus
formation. There was an increase in FANCD2 nuclear foci
corresponding to the increase in FANCD2-L. Ionizing radiation also
activated a time-dependent and dose-dependent increase in FANCD2-L
in HeLa cells, with a corresponding increase in FANCD2 foci (FIG.
4B). Surprisingly, ultraviolet (UV) light activated a
time-dependent and dose-dependent conversion of FANCD2-S to
FANCD2-L, with a corresponding increase in FANCD2 foci (FIG.
4C).
[0274] We tested the effect of DNA damage on FA cells (FIG. 4D). FA
cells from multiple complementation groups (A, C, and G) failed to
activate the FANCD2-L isoform and failed to activate FANCD2 nuclear
foci in response to MMC or IR exposure. These data suggest that the
cellular sensitivity of FA cells results, at least in part, from
their failure to activate FANCD2-L and FANCD2 nuclear foci.
Example 7
Co-Localization of Activated FANCD2 and BRCA1 Protein.
[0275] Like FANCD2, the breast cancer susceptibility protein,
BRCA1, is upregulated in proliferating cells and is activated by
post-translational modifications during S phase or in response to
DNA damage. BRCA has a carboxy terminus 20 amino acids which
contain a highly acidic HMG-like domain suggesting a possible
mechanism for chromatin repair. The BRCA1 protein co-localizes in
IR-inducible foci (IRIFs) with other proteins implicated in DNA
repair, such as RAD51 or the NBS/Mre11/RAD50 complex. Cells with
biallelic mutations in BRCA1 have a defect in DNA repair and are
sensitive to DNA damaging agents such as IR and MMC (Table 5).
Taken together, these data suggest a possible functional
interaction between the FANCD2 and BRCA1 proteins. BRCA foci are
large (2 mDa) multiprotein complexes including ATM and ATM
substrates involved in DNA repair (BRCA1) and checkpoint functions
(NBS).
[0276] In order to determine whether the activated FANCD2 protein
co-localizes with the BRCA1 protein, we performed double
immunolabeling of HeLa cells (FIG. 5). In the absence of ionizing
radiation, approximately 30-50% of cells contained BRCA1 nuclear
foci (FIG. 5A). In contrast, only rare cells traversing S phase
contained FANCD2 dots (b, e). These nuclear foci were also
immunoreactive with antisera to both BRCA1 and FANCD2 (c, f).
Following IR exposure, there was an increase in the number of cells
containing nuclear foci and the number of foci per cell. These
nuclear foci were larger and more fluorescent than foci observed in
the absence of IR. Again, these foci contained both BRCA1 and
FANCD2 protein (i, 1). An interaction of FANCD2-L and BRCA1 was
further confirmed by coimmunoprecipitation of the proteins (FIG.
5B) from exponentially growing HeLa cells exposed to IR.
[0277] We examined the effect of BRCA1 expression on the formation
of FANCD2-L and nuclear foci (FIG. 6). The BRCA1 (-/-) cell line,
HCC1937, expresses a mutant form of the BRCA1 protein with a
carboxy terminal truncation. Although these cells expressed a low
level of FANCD2-L (FIG. 6A), IR failed to activate an increase in
FANCD2-L levels. Also, these cells had a decreased number of
IR-inducible FANCD2 foci (FIG. 6B, panels c, d). Correction of
these BRCA1 (-/-) cells by stable transfection with the BRCA1 cDNA
restored IR-inducible FANCD2 ubiquitination and nuclear foci (FIG.
6B, panels k, 1). These data suggest that the wild-type BRCA1
protein is required as an"organizer" for IR-inducible FANCD2 dot
formation and further suggests a functional interaction between the
proteins.
Example 8
[0278] Co-Localization of FANCD2 And BRCA1 on Meiotic
Chromosomes.
[0279] The association of FANCD2 and BRCA1 in mitotic cells
suggested that these proteins might also co-localize during meiotic
prophase. Previous studies have demonstrated that the BRCA1 protein
is concentrated on the unsynapsed/axial elements of human
synaptonemal complexes in zygotene and pachytene spermatocytes. To
test for a possible colocalization of FANCD2 and BRCA1 in meiotic
cells, we examined surface spreads of late pachytene and early
diplotene mouse spermatocytes for the presence of FANCD2 and BRCA1
protein (FIG. 7). We found that the rabbit polyclonal anti-FANCD2
antibody E35 specifically stained the unpaired axes of the X and Y
chromosomes in late pachynema (FIG. 7a) and in diplonema (FIGS. 7d,
7e and 7g). Under the same experimental conditions, preimmune serum
did not stain synaptonemal complexes (FIGS. 7b and 7c). The M118
anti-BRCA1 antibody stained the unpaired sex chromosomes in mouse
pachytene and diplotene spermatocytes (FIGS. 7f and 7h). FANCD2 Ab
staining of the unsynapsed axes of the sex chromosomes was
interrupted, giving a beads-on-a-string appearance (FIG. 7g). A
consecutive examination of 20 pachytene nuclei indicated that most
(.about.65%) of these anti-FANCD2 foci co-localized with regions of
intense anti-BRCAI staining, further supporting an interaction
between these proteins (FIGS. 7g, 7h, and 7i). These results
provide the first example of a FANC protein (activated FANCD2)
which binds to chromatin.
Example 9
[0280] Experimental Protocols for Obtaining and Analyzing the DNA
and Protein Sequence for FANCD2.
[0281] Northern Hybridizations. Human adult and fetal multi-tissue
mRNA blots were purchased from Clontech (Palo Alto, Calif.). Blots
were probed with 32P labeled DNA from EST clone SGC34603. Standard
hybridization and washing conditions were used. Equal loading was
confirmed by re-hybridizing the blot with an actin cDNA probe.
[0282] Mutation Analysis. Total cellular RNA was reverse
transcribed using a commercial kit (Gibco/BRL). The 5' end section
of FANCD2 was amplified from the resulting patient and control cDNA
with a nested PCR protocol. The first round was performed with
primers (SEQ ID NO: 97) MG471 5'-AATCGAAAACTACGGGCG-3' and (SEQ ID
NO: 98) MG457 5'-GAGAACACATGAATGAACGC-3'. The PCR product from this
round was diluted 1:50 for a subsequent round using primers (SEQ ID
NO: 99) MG492 5'-GGCGACGGCTTCTCGG AAGTAATTTAAG-3' and (SEQ ID NO:
100) MG472 5'-AGCGGCAGGAGGTTTATG-3'. The PCR conditions were as
follows: 94.degree. C. for 3 min, 25 cycles of 94.degree. C. for 45
sec, 50.degree. C. for 45 sec, 72.degree. C. for 3 min and 5 min of
72.degree. C. at the end. The 3' portion of the gene was amplified
as described above but with primers, (SEQ ID NO: 101) MG474
5'-TGGCGGCAGACAGAAG TG-3' and (SEQ ID NO: 102) MG475
5'-TGGCGGCAGACAGAAGTG-3'. The second round of PCR was performed
with (SEQ ID NO: 103) MG491 5'-AGAGAGCCAACCTGAGCGA TG-3' and (SEQ
ID NO: 104) MG476 5'-GTGCCAGACTCTGGTGGG-3'. The PCR products were
gel-purified, cloned into the pT-Adv vector (Clontech) and
sequenced using internal primers.
[0283] Allele specific assays. Allele specific assays were
performed in the PD20 family and 290 control samples (=580
chromosomes). The PD20 family is of mixed Northern European descent
and VU008 is a Dutch family. Control DNA samples were from
unrelated individuals in CEPH families (n=95), samples from
unrelated North American families with either ectodermal dysplasia
(n=95) or Fanconi Anemia (n=94). The maternal nt376a.fwdarw.g
mutation in the PD20 family created a novel MspI restriction site.
For genomic DNA, the assay involved amplifying genomic DNA using
the primers (SEQ ID NO: 105) MG792 5'-AGGAGACACCCTTCCTA TCC-3'
located in exon 4 and (SEQ ID NO: 106) M0803 5'-GAAGTTGGCAAAACAGAC
TG-3' which is in intron 5. The size of the PCR product was 340 bp,
yielding two fragments of 283 bp and 57 bp upon MspI digestion if
the mutation was present. For analysis of the reverted cDNA clones,
PCR was performed using primers (SEQ ID NO: 107) MG924
5'-TGTCTTGTGA GCGTCTGCAGG-3' and (SEQ ID NO: 108) MG753 5'-AGGTT
TTGATAATGGCAGGC-3'. The paternal exon 37 mutation (R1236H) in PD20
and exon 12 missense mutation (R302W) in VU008 were tested by
allele specific oligonucleotide (ASO) hybridization (Wu et al.,
DNA, (1989) Vol. 8, pp. 135-142). For the exon 12 assay, genomic
DNA was amplified with primers (SEQ ID NO: 109) MG979 5
'-ACTGGACTGTGCCTACCCACTATG-3' and (SEQ ID NO: 110) MG984
5'-CCTGTGTGAGGATGAGCTCT-3'. Primers (SEQ ID NO: 171) MG818
5'-AGAGGTAGGGAAGGAAGCTAC-3' and (SEQ ID NO: 172) MG813
5'-CCAAAGTCCA CTTCTTGAAG-3' were used for exon 37. Wild-type (SEQ
ID NO:l ll) (5'-TTCTCCCGAAG CTCAG-3' for R302W and (SEQ ID NO:
112)5'-TTTCTTCCGTGTGATGA-3' for R1236H) and mutant SEQ ID NO: 111
(5'-TTCTCCCAAAGCTGAG-3'R302W and SEQ ID NO: 112
(5'-TYTCTTCCATGTGATGA-3' for R1236H) oligonucleotides were
end-labeled with .gamma.32P-[ATP] and hybridized to dot-blotted
target PCR products as previously ss novel DdeI site. The wild-type
PCR product digests into a 117 and 71 bp product, whereas the
mutant allele yields three fragments of 56, 61 and 71 bps in
length. PCR in all of the above assays was performed with 50 ng of
genomic DNA for 37 cycles of 94.degree. C. for 25 sec, 50.degree.
C. for 25 sec and 72.degree. C. for 35 sec.
[0284] Generation of an anti-FANCD2 antiserum. A rabbit polyclonal
antiserum against FANCD2 was generated using a GST-FANCD2
(N-terminal) fusion protein as an antigen source. A 5' fragment was
amplified by polymerase chain reaction (PCR) from the full length
FANCD2 cDNA with the primers (SEQ ID NO: 113) DF4EcoRJ
(5'-AGCCTCgaattcGUTCC AAAAGAAGACTGTCA-3') and (SEQ ID NO: 114)
DR816Xh (5'-GGTATCctcgagTCAAGA CGACAACTTATCCATCA-3'). The resulting
PCR product of 841 bp, encoding the amino-terminal 272 amino acids
of the FANCD2 polypeptide was digested with EcoRI/Xhol and
subcloned into the EcoRI/XhoI sites of the plasmid pGEX4T-1
(Pharmacia). A GST-FANCD2 (N-terminal) fusion protein of the
expected size (54 kD) was expressed in E. coli strain
DH5.quadrature., purified over glutathione-S-sepharose, and used to
immunize a New Zealand White rabbit. An FANCD2-specific immune
antiserum was affinity-purified over an AminoLink Plus column
(Pierce) loaded with GST protein and over an AminoLink Plus column
loaded with the GST-FANCD2 (N-terminal) fusion protein.
[0285] Immunoblotting is as in Example 1.
[0286] Cell Lines and Transfections. PD20i is an immortalized and
PD733 a primary FA fibroblast cell line generated by the Oregon
Health Sciences Fanconi Anemia cell repository (Jakobs et al.,
Somet. Cell. Mol. Genet., (1996), Vol. 22, pp. 151-157). PD20
lymphoblasts were derived from bone marrow samples. VU008 is a
lymphoblast and VU423 a fibroblast line generated by the European
Fanconi Anemia Registry (EUFAR). VU423i was an immortalized line
derived by transfection with SV40 T-antigen (Jakobs et al., 1996)
and telomerase (Bodnar et al., Science, (1998) Vol. 279, pp.
349-352). The other FA cell lines have been previously described.
Human fibroblasts were cultured in MEM and 20% fetal calf serum.
Transformed lymphoblasts were cultured in RPMI 1640 supplemented
with 15% heat-inactivated fetal calf serum.
[0287] To generate FANCD2 expression constructs, the full-length
cDNA was assembled from cloned RT-PCR products in pBluescript and
the absence of PCR induced mutations was confirmed by sequencing.
The expression vectors pIRES-Neo, pEGFP-N1, pRevTRE and pRevTet-off
were from ClonTech (Palo Alto, Calif.). The FANCD2 was inserted
into the appropriate multi-cloning site of these vectors.
Expression constructs were electroporated into cell line PD20 and a
normal control fibroblast cell line, GM639 using standard
conditions (van den Hoff et al., 1992). Neomycin selection was
carried out with 400 .mu.g/ml active G418 (Gibco).
[0288] Whole cell fusions. For the whole cell fusion experiments, a
PD20 cell line (PD20i) resistant to hygromycin B and deleted for
the HPRT locus was used (Jakobs et al., Somat. Cell. Mol. Genet.,
(1997) Vol. 23, pp. 1-7). Controls included PD24 (primary
fibroblasts from affected sibling of PD20) and PD319i (Jakobs et
al., 1997) (immortal fibroblasts from a non-A, C, D or G FA
patient). 2.5.times.105 cells from each cell line were mixed in a
T25 flask and allowed to recover for 24 hours. The cells were
washed with serum-free medium and then fused with 50% PEG for 1
min. After removal of the PEG, the cells were washed 3.times. with
serum-free medium and allowed to recover overnight in complete
medium without selection. The next day, cells were split 1:10 into
selective medium containing 400 .mu.g/ml hygromycin B (Roche
Molecular) and 1X HAT. After the selection was complete, hybrids
were passaged once and then analyzed as described below.
[0289] Retroviral Transduction of FA-D2 cells and complementation
analysis. The fall length FANCD2 cDNA was subcloned into the
vector, pMMP-puro (Pulsipher et al., 1998). Retroviral supernatants
were used to transduce PD20F, and puromycin resistant cells were
selected. Cells were analyzed for MMC sensitivity by the crystal
violet assay (Naf et al., 1998).
[0290] Chromosome Breakage Analysis. Chromosome breakage analysis
was performed by the Cytogenetics Core Lab at OHSU (Portland,
Oreg.). For the analysis (Cohen et al., 1982) cells were plated
into T25 flasks, allowed to recover and then treated with 300 ng/ml
of DEB for two days. After treatment, the cells were exposed to
colcemid for 3 hours and harvested using 0.075 M KC1 and 3:1
methanol:acetic acid. Slides were stained with Wright's stain and
50-100 metaphases were scored for radials.
Example 10
[0291] Mouse Models for FA For Use in Screening Potential
Therapeutic Agents.
[0292] Murine models of FANCD2 can be made using homologous
recombination in embryonic stem cells or targeted disruption as
described in D'Andrea et al., (1997) 90:1725-1736, and Yang et al,
Blood, (2001) Vol. 98, pp. 1-6. The knockout of FANCD2 locus in
mice is not a lethal mutation. These knock-out animals have
increased susceptibility to cancer and furthermore display other
symptoms characteristic of FA. It is expected that administering
certain therapeutic agents to the knock-out mice will reduce their
susceptibility to cancer. Moreover, it is expected that certain
established chemotherapeutic agents will be identified that are
more effective for treating knock-out mice who have developed
cancers as a result of the particular genetic defect and this will
also be useful in treating human subjects with susceptibility to
cancer or who have developed cancers as a result of a mutation in
the FANCD2 locus.
[0293] We can generate experimental mice models with targeted
disruptions of FANCD2 using for example the approach described by
Chen et al, Nat. Genet., (1996) Vol. 12, pp. 448-451, for FANCC who
created a disruption in an exon of the gene, and by Whitney et al.,
(1996) Vol. 88, pp. 49-58, who used homologous recombination to
create a disruption of an exon of the gene. In both animal models,
spontaneous chromosome breakage and an increase in chromosome
breaks in splenic lymphocytes in response to bifunctional
alkylating agents are observed. In both models, FANCD2-/-mice have
germ cell defects and decreased fertility. The FANCD2 murine
knockout model is useful in examining (1) the role of the FANCD2
gene in the physiologic response of hematopoietic cells to DNA
damage, (2) the in vivo effects of inhibitory cytokines on FA
marrow cells, and (3) the efficacy of gene therapy and (4) for
screening candidate therapeutic molecules.
[0294] The availability of other FA gene disruptions will allow the
generation and characterization of mice with multiple FA gene
knockouts. For instance, if 2 FA genes function exclusively in the
same cellular pathway, a double knockout should have the same
phenotype as the single FA gene knockout.
[0295] The murine FANCD2 gene can be disrupted by replacing exons
with an FRT-flanked neomycin cassette via homologous recombination
in 129/SvJae embryonic stem cells. Mice homozygous for the FANCD2
mutation within a mixed genetic background of 129/Sv and C57BL can
be generated following standard protocols. Mouse tail genomic DMA
can be prepared as previously described and used as a template for
polymerase chain reaction (PCR) genotyping.
[0296] Splenocytes can be prepared from 6-week-old mice of known
FANCD2 genotype. The spleen is dissected, crushed in RPMI medium
into a single-cell suspension, and filtered through a 70 .mu.m
filter. Red cells are lysed in hypotonic ammonium chloride. The
remaining splenic lymphocytes are washed in phosphate-buffered
saline and resuspended in RPMI/10% fetal bovine serum plus
phytohemagglutinin. Cells are tested for viability by the trypan
blue exclusion assay. Cells are cultured for 24 hours in media and
exposed to MMC or DEB for an additional 48 hours. Alternatively,
cells are cultured for 50 hours, exposed to IR (2 or 4 Gy, as
indicated), and allowed to recover for 12 hours before chromosome
breakage or trypan blue exclusion (viability) analysis.
[0297] Mononuclear cells can be isolated from the femurs and tibiae
of 4- to 6-week-old FANCD2+/- or FANCD2-/-mice, as previously
described. A total of 2.times.104 cells were cultured in 1 mL of
MethoCult M343 media (StemCell Technologies, Vancouver, BC) with or
without MMC treatment. Colonies are scored at day 7, when most of
the colonies belong to the granulocyte-macrophage colony-forming
unit or erythroid burst-forming unit lineages. Each number are
averaged from duplicate plates, and the data derived from 2
independent experiments.
[0298] Lymphocytes isolated from thymus, spleen, and peripheral
lymph nodes are stained for T- or B-lymphocyte surface molecules
with fluorescein isothiocyanate-conjugated anti-CD3, CD4, and CD19
and PE-conjugated anti-CD8, CD44, CD 45B, immunoglobulin M, and
B220 (BD PharMingen, Calif.). Stained cells were analyzed on a
Counter Epics XL flow cytometry system.
[0299] Mice ovaries and testes were isolated and fixed in 4%
paraformaldehyde and further processed by the core facility of the
Department of Pathology at Massachusetts General Hospital.
Example 11
[0300] Screening Assays Using Antibody Reagents for Detecting
Increased Cancer Susceptibility in Human Subjects.
[0301] Blood samples or tissue samples can be taken from subjects
for testing for the relative amounts of FANCD2-S compared to
FANCD2-L and the presence or absence of FANCD2-L. Using antibody
reagents specific for FANCD2-S and FANCD2-L proteins (Example 1),
positive samples can be identified on Western blots as shown in
FIG. 14. Other antibody assays may be utilized such as, for
example, one step migration binding banded assays described in
5,654,162 and 5,073,484. Enzyme linked immunosorbent assays
(ELISA), sandwich assays, radioimmune assays and other
immunodiagnostic assays known in the art may be used to determine
relative binding concentrations of FANCD2-S and FANCD2-L.
[0302] The Feasibility of this Approach is Illustrated by the
Following:
[0303] FANCD2 Diagnostic Western Blot for Screening Human Cancer
Cell Lines
[0304] Human cancer cell lines were treated with or without
ionizing radiation (as indicated in FIG. 14) and total cell
proteins were electrophoresed, transferred to nitrocellulose and
immunoblotted with the anti-FANCD2 monoclonal antibody of Example
1. Ovarian cancer cell line (TOV21 G) expressed FANCD2-S but not
FANCD2-L (see lanes 9, 10). This cell line has a deletion of human
chromosome 3p overlapping the FANCD2 gene and is hemizygous for
FANCD2 and is predicted to have a mutation in the second FANCD2
allele which therefore fails to be monoubiquinated by the PA
complex hence no FANCD2-L (lanes 9, 10). This example demonstrates
that antibody based tests are suited for determining lesions in the
FANCD2 gene which lead to increased cancer susceptibility.
Example 12
[0305] Screening Assays Using Nucleic Acid Reagents for Detecting
Increased Cancer Susceptibility in Human Subjects.
[0306] Blood samples or tissue samples can be taken from subjects
and screened using sequencing techniques or nucleic acid probes to
determine the size and location of the genetic lesion if any in the
genome of the subject. The screening method may include sequencing
the entire gene or by using sets of probes or single probes to
identify lesions. It is expected that a single lesion may
predominant in the population but that other lesions may arise
throughout the gene with low frequency as is the case for other
genetic conditions such as cystic fibrosis and the P53 tumor
suppressor gene.
[0307] The Feasibility of this Approach is Illustrated by the
Following:
[0308] Peripheral blood lymphocytes are isolated from the patient
using standard Ficoll-Hypaque gradients and genomic DNA is isolated
from these lymphocytes. We use genomic PCR to amplify 44 exons of
the human FANCD2 gene (see primer Table 7) and sequence the two
FANCD2 alleles to identify mutations. Where such mutations are
found, we distinguish these from benign polymorphisms by their
ability to ablate the functional complementation of an FA-D2
indicator cell line.
Example 13
[0309] Measurement of Mono-ubiquitinated FANC D2-L in Tissue
Biopsies
[0310] Tissue biopsies were obtained by needle aspiration or skin
punch biopsy. Cells, resuspended in appropriate culture media in
microtiter plates are then treated with the indicated concentration
of MMC (0, 10, 40, 160 ng/ml) or the indicated dose of IR (0, 5,
10, 10, 20 Gy). After 24 hour-incubation with MMC, or two hours
after IR treatment, whole cell extracts were prepared in Lysis
Buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 1% (v/v) Triton X-100)
supplemented with protease inhibitors (1 .mu.g/ml leupeptin and
pepstatin, 2 .mu.g/ml aprotinin, 1 mM phenylmethylsulfonylfluoride)
and phosphatase inhibitors (1 mM sodium orthovanadate, 10 mM sodium
fluoride). Samples are then tested for the presence of the FANC
D2-L isoform using the anti-FANCD2-L -specific monoclonal antibody,
as disclosed herein, and conventional immunoassays such as the
enzyme linked immunosorbent assay (ELISA) that are commonly used to
quantitate the levels of proteins in cell samples (see Harlow, E.
and Lane, D. Using Antibodies: A Laboratory Manual (1999) Cold
Spring Harbor Laboratory Press).
Example 14
[0311] Diagnosis of Cancer Associated Defects in a Fanconi
Anemia/BRCA Gene or Protein
[0312] PCR Amplification and Sequencing of the Human FANCD2
gene-cDNA and Genomic DNA Templates
[0313] Genomic DNA Sequencing
[0314] In the course of sequencing the FANCD2 gene, it became
apparent that there are at least eight pseudogene sequences for
FANCD2 in the human genome, all located on human chromosome 3p (see
attached Table 8). Accordingly, it was important to design a
specific genomic PCR assay, designed to specifically amplify the
FANCD2 sequence and to exclude the pseudogenes. It is not possible
to design PCR primers close to exons 1, 2, 3, 7-14, 19-22, 23-29,
30-32, 33-36 and 43-44 of the functional FANCD2 gene that do not
also amplify one or more of the non-functional copies of those
exons. By first generating large PCR products that are unique to
these regions of the functional gene, then using those unique
products as templates in subsequent amplification reactions to
produce exonic PCR products with primers that are not unique to the
functional gene, a vast excess of the PCR products from the
functional gene over the PCR products from the copies was
generated. In this manner, mutations in the functional gene are
made detectable.
[0315] Superamplicon PCR
[0316] As indicated above, the purpose of these PCR reactions is to
generate large amplicons (superamplicons) that are unique to
certain regions of the functional FANCD2 gene. The components of
the PCR are: 60 mM Tris-SO.sub.4 (pH8.9), 18 mM
(NH.sub.4)2SO.sub.4, 2.0 mM MgSO.sub.4, 0.2 mM in each of DATP,
dCTP, dGTP, TTP, 0.1 .mu.M of each primer, 5 ng/.mu.l DNA,
0.05units/.mu.l Platinum Taq DNA Polymerase High Fidelity (GIBCO
BRL, Gaithersburg, Md.).
[0317] The thermocycling conditions are: 94.degree. C., 4 min,
followed by 11 cycles, each with a denaturing step at 94.degree. C.
for 20 seconds and an extension step at 72.degree. C. for 300
seconds, and with a 20 second annealing step that decreased
1.degree. C./cycle, beginning at 64.degree. C. in the first cycle
and decreasing to 54.degree. C. In the eleventh cycle; the eleventh
cycle was then repeated 25 times; a 6 minute incubation at
72.degree. C. followed by a 4.degree. C. soak completed the
program.
[0318] The primer identities are as follows (the primer sequences
are in the table 9):.
2 Amplicon Amplicon Exons FwdPrimer RevPrimer Length Name x1-x2
exon 2 F super-1-2 R 2097 1 super exon 1 F super-1-2R 4346 2 super
x3 super-3-F exon 3 R 2323 3 super x7-x14 exon-10-F super-7-14-R
5635 4 super super-7-14-F exon-9-R 4595 5 super x19-x22 exon-21-F
super-19-22 R 1015 6 super super-19-22-F exon-20-R 2749 7 super
x23-x29 exon-27 F super-23-29 R 3371 9 super super-23-29 F exon 26
R 3252 10 super x30-x32 exon 31 F super-30-32 R 2895 11 super
super-30-32 F exon 30 R 299 12 super x33-36 exon 35 F super-33-36 R
2186 13 super super-33-36 F exon 34 R 3457 14 super x43-x44 exon 44
F super-43-44 R 464 15 super super-43-44 F exon 43a R 2040 16
super
[0319] Exonic PCR
[0320] These PCR's are of 2 types: (1) the superamplicon PCR is
used as the DNA template; exons 1-3, 7-14, 19-22, 23-29, 30-32,
33-36 and 43-44 are in this group, and (2) unamplified genomic DNA
is used as the DNA template; exons 4-6, 15-18 and 37-42 are in this
group.
[0321] One primer (designated "-F") in each pair was synthesized
with an 18base M13-21 forward sequence (TGTAAAACGACGGCCAGT) at its
5' end, and the other primer (designated "-R") was synthesized with
an 18 base M13-28 reverse sequence(CAGGAAACAGCTATGACC) at its 5'
end. For exon 15, two overlapping amplicons were designed.
[0322] The components of the 10 ul PCR reaction are: 20 mM
Tris-HCl(pH8.4), 50mM KC1, 1.5 mM MgC12, 0.1 mM in each of dATP,
dCTP, dGTP, TTP, 0.1 .mu.uM of each primer, either lul of a 1:100
dilution of the superamplicon PCR or 5 ng/.mu.l of unamplified
genomic DNA, 0.05 units/.mu.l Taq polymerase (Taq Platinum, GIBCO
BRL, Gaithersburg, Md.). The thermocycling conditions are:
94.degree. C., 4 min, followed by 11 cycles, each with a denaturing
step at 94.degree. C. for 30 seconds and an extension step at
72.degree. C. for 20 seconds, and with a 20 second annealing step
that decreased 1.degree. C./cycle, beginning at 60.degree. C. in
the first cycle and decreasing to 50.degree. C. In the eleventh
cycle; the eleventh cycle was then repeated 25 times; a 6 minute
incubation at 72.degree. C. followed by a 4.degree. C. soak
completed the program.
[0323] cDNA Sequencing
[0324] Two micrograms of total RNA is converted into cDNA using
Superscript First-Strand Synthesis System for RT-PCR (GIBCO/BRL)
according to the manufacturer's instructions. One twentieth of the
RT-PCR reaction is used as the DNA template in each of 18 PCR
reactions; these PCR reactions amplify the coding region of the
cDNA in overlapping fragments. The primers are shown in the table
below.
[0325] One primer (designated "-F") in each pair was synthesized
with an 18base M13-21 forward sequence (TGTAAAACGACGGCCAGT) at its
5' end, and the other primer (designated "-R") was synthesized with
an 18 base M13-28 reverse sequence(CAGGAAACAGCTATGACC) at its 5'
end.
[0326] The components of the 10 ul PCR reaction are: 20 mM
Tris-HCl(pH8.4), 50 mM KC1, 1.5 mM MgCl.sub.2, 0.1 mM in each of
dATP, dCTP, dGTP, TTP, 0.1 .mu.M of each primer, either 1 ul of a
1:100 dilution of the superamplicon PCR or 5ng/.mu.l of unamplified
genomic DNA, 0.05units/.mu.l Taq polymerase (Taq Platinum, GIBCO
BRL, Gaithersburg, Md.).
[0327] The thermocycling conditions are: 94.degree. C., 4 min,
followed by 11 cycles, each with a denaturing step at 94.degree. C.
for 30 seconds and an extension step at 72.degree. C. for 20
seconds, and with a 20 second annealing step that decreased
1.degree. C./cycle, beginning at 60.degree. C. in the first cycle
and decreasing to 50.degree. C. in the eleventh cycle; the eleventh
cycle was then repeated 25 times; a 6 minute incubation at
72.degree. C. followed by a 4.degree. C. soak completed the
program.
[0328] Primer 5' Position Sequence (5' to 3') Length (bp)
3 D1F 24 TGTAAAACGACGGCCAGT CGACGGCTTCTCGGAAGTAA D1R 408
AGGAAACAGCTATGACCAT GCAGACGCTCACAAGACAAA 407 D2F 322
TGTAAAACGACGGCCAGT GACACCCTTCCTATCCCAAAA D2R 689
AGGAAACAGCTATGACCAT CAGGTTCTCTGGAGCAATAC 368 D3F 612
TGTAAAACGACGGCCAGT TGGCTTGACAGAGTTGTGGAT D3R 1019
AGGAAACAGCTATGACCAT CTGTAACCGTGATGGCAAAAC 408 D4F 855
TGTAAAACGACGGCCAGT CGCCAGTTGGTGATGGATAAG D4R 1223
AGGAAACAGCTATGACCAT AAGCATCACCAGGTCAAACAC 369 D5F 1081
TGTAAAACGACGGCCAGT GCGGTCAGAGCTGTATTATTC D5R 1461
AGGAAACAGCTATGACCAT CTGCTGGCAGTACGTGTCAA 401 D6F 1377
TGTAAAACGACGGCCAGT TCGCTGGCTCAGAGTTTGCTT D6R 1765
AGGAAACAGCTATGACCAT GTGCTAGAGAGCTGCTTTCTT 389 D7F 1641
TGTAAAACGACGGCCAGT CCCCTCAGCAAATACGAAAAC D7R 2065
AGGAAACAGCTATGACCAT ACTACGAAGGCATCCTGGAAA 424 D8F 1947
TGTAAAACGACGGCCAGT GCCTCTGCACTTTACTATGATG D8R 2301
AGGAAACAGCTATGACCAT CTCCTCCAAGTTTCCGTTATG 375 D9F 2210
TGTAAAACGACGGCCAGT GGTGACCTCACAGGAATCAG D9R 2573
AGGAAACAGCTATGACCAT TTTCCAAGAGGAGGGACATAG 384 D10F 2438
TGTAAAACGACGGCCAGT CAACTGGTTCCGAGAGATTGT D10R 2859
AGGAAACAGCTATGACCAT CAATGTCCAGCTCTCGGAAAAA 422 D11F 2746
TGTAAAACGACGGCCAGT GTGACCCTACGCCATCTCATA D11R 3138
AGGAAACAGCTATGACCAT ACATTGGGGTCAGCAGTTGAA 393 D12F 3027
TGTAAAACGACGGCCAGT AGAGTCCCCTTTCTCAAGAACA D12R 3413
AGGAAACAGCTATGACCAT GACGCTCTGGCTGAGTAGTT 387 D13F 3334
TGTAAAACGACGGCCAGT CAGCCCTCCATGTCCTTAGT D13R 3742
AGGAAACAGCTATGACCAT AGGGAATGTGGAGGAAGATG 407 D14F 3637
TGTAAAACGACGGCCAGT TGGAGCACACAGAGAGCATT D14R 4010
AGGAAACAGCTATGACCAT GTCTAGGAGCGGCATACATT 374 D15F 3830
TGTAAAACGACGGCCAGT AGCAGACTCGCAGCAGATTCA D15R 4225
AGGAAACAGCTATGACCAT AGCCAGAAAGCCTCTCTACA 396 D16F 4117/4112
TGTAAAACGACGGCCAGT ACAGGAGACTCACCCAACAT D16R-L 4477
AGGAAACAGCTATGACCAT GGGAATGGAAATGGGCATAGA 361 D16R-S 4451
AGGAAACAGCTATGACCAT GACACAGAAGCAGGCAACAA 340 D17F-(L) 4333
TGTAAAACGACGGCCAGT AGAGCAAAGCCACTGAGGTAT D17R-(L) 4768
AGGAAACAGCTATGACCAT GACTCTGTGCTTTGGCTTTCA 436
[0329] DNA sequencing
[0330] An aliquot of each PCR reaction was diluted 1:10 with water.
The diluted PCR product was sequenced on both strands using an M13
Forward and an M13 Reverse Big Dye Primer kit (Applied Biosystems,
Foster City, Calif.) according to the manufacturer's
recommendations. The sequencing products were separated on a
fluorescent sequencer (model 377 from Applied Biosystems, Foster
City, Calif.). Base calls were made by the instrument software, and
reviewed by visual inspection. Each sequence was compared to the
corresponding normal sequence using Sequencher 3.0 software
(LifeCodes).
Example 15
[0331] Method of Screening for A Chemosensitizing Agent
[0332] As shown in the model of the FA/BRCA pathway, the enzymatic
monoubiquitination of FANCD2 is a critical regulatory event. This
event requires an intact FA protein complex (A/C/E/F/G complex) and
requires BRCA1 and BRCA2. While the actual catalytic subunit
required for FANCD2 monoubiquitination remains unknown, it still
remains possible to screen for antagonists of monoubiquitination.
As described elsewhere in this text, an inhibitor of the FA pathway
could, in principal, function as a chemosensitizer of cisplatin in
the treatment of ovarian cancer or other cancers. The screening of
an inhibitor of FANCD2 monoubiquitination can be performed as a
simple mammalian cell-based screen. A mammalian tissue culture cell
line, e.g., Hela calls are first preincubated with random candidate
small molecules. Cell clones are then screened using anti-FANCD2
western blots. An inhibitor (antagonist) of the FA pathway will
block FANCD2 monoubiquitination.
[0333] As described in Garcia-Higuera et al, 2001, BRCA1 may in
fact be the enzyme which monoubiquitinates FANCD2. Accordingly,
BRCA1 has a ubiquitin ligase (Ring Finger) catalytic domain.
Therefore, an in vitro assay will be devised to screen for
BRCA1-mediated monoubiquitination of FANCD2. An inhibitor will be
screened directly for its ability to inhibit this in vitro
reaction. Once inhibitors are identified, such drugs could be used
in animal studies or phase 1 human studies to determine their
functions as cisplatin sensitizers.
Example 16
[0334] Method of Screening for A Potential Cancer Therapeutic.
[0335] Cells and animals which carry a Fanconi Anemia/BRCA pathway
gene having one or more cancer associated defects can be used as
model systems to study and test for substances which have potential
as therapeutic agents. The cells are typically cultured epithelial
cells. These may be isolated from individuals with Fanconi
Anemia/BRCA pathway gene having one or more cancer associated
defects, either somatic or germline. Alternatively, the cell line
can be engineered to carry the mutation in a gene of the Fanconi
Anemia/BRCA pathway gene having one or more cancer associated
defects.
[0336] After a test substance is applied to the cells, the
neoplastically transformed phenotype of the cell is determined. Any
trait of neoplastically transformed cells can be assessed,
including anchorage-independent growth, tumorigenicity in nude
mice, invasiveness of cells, and growth factor dependence. Assays
for each of these traits are known in the art.
[0337] Animals for testing therapeutic agents can be selected after
mutagenesis of whole animals or after treatment of germline cells
or zygotes. Such treatments include insertion of mutant Fanconi
Anemia/BRCA pathway genes having one or more cancer associated
defects, usually from a second animal species, as well as insertion
of disrupted homologous genes. Alternatively, the endogenous
Fanconi Anemia/BRCA pathway gene(s) of the animals may be disrupted
by insertion or deletion mutation or other genetic alterations
using conventional techniques (Capecchi, 1989; Valancius and
Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts
et al., 1992; Philpott et al, 1992; Snouwaert et al., 1992;
Donehower et al, 1992) as outlined in Example 10. After test
substances have been administered to the animals, the growth of
tumors must be assessed. If the test substance prevents or
suppresses the growth of tumors, then the test substance is a
candidate therapeutic agent for the treatment of the cancers
identified herein.
Example 17
[0338] Method of Treatment of a Cancer that is Resistant to an
Anti-neoplastic Agent
[0339] The present example describes the treatment of a patient
with a cancer that is resistant to an anti-neoplastic agent such as
cisplatin. The protocol provides for the administration of
cisplatin as described herein with an increasing dosage of an
inhibitor of the ubiquitination of the FANC D2 protein as a
chemosensitizing agent. Cisplatin and the chemosensitizing agent
can be administered intravenously, subcutaneously, intratumorally
or intraperitoneally. The administering physician can adjust the
amount and timing of drug administration on the basis of results
observed using standard measures of cancer activity known in the
art. Suppression of tumor growth and metastasis is indicative of
effective treatment of the cancer.
Example 18
[0340] A Method of Measuring the Future Efficacy of a Therapeutic
Agent
[0341] Tissue biopsies of neoplasms from cancer patients being
treated with a therapeutic agent are obtained by needle aspiration
or skin punch biopsy. Cells, resuspended in appropriate culture
media in microtiter plates are then treated with the indicated
concentration of MMC (0, 10, 40, 160 ng/ml) or the indicated dose
of IR (0, 5, 10, 10, 20 Gy). After 24 hour-incubation with MMC, or
two hours after IR treatment, it induce DNA damage, whole cell
extracts were prepared in Lysis Buffer (50 mM TrisHCl pH 7.4, 150
mM NaCl, 1% (v/v) Triton X-100) supplemented with protease
inhibitors (1 .mu.g/ml leupeptin and pepstatin, 2 .mu.g/ml
aprotinin, 1 mM phenylmethylsulfonylfluoride) and phosphatase
inhibitors (1 mM sodium orthovanadate, 10 mM sodium fluoride).
Samples are then tested for the presence of the FANC D2-L isoform
using the anti-FANCD2-L -specific monoclonal antibody, as disclosed
herein, and conventional immunoassays such as the enzyme linked
immunosorbent assay (ELISA) that are commonly used to quantitate
the levels of proteins in cell samples (see Harlow, E. and Lane, D.
Using Antibodies: A Laboratory Manual (1999) Cold Spring Harbor
Laboratory Press). Detection of the mono-ubiquitinated FANC D2-L
isoform is considered indicative of a reduced efficacy of the
therapeutic agent being used to treat the cancer patient.
Example 19
[0342] A Method of Determining Resistance to a Chemotherapy
Agent
[0343] A flow chart describing the protocol used to determine the
methylation state of the Fanconi Anemia/BRCA pathway genes is
depicted in FIG. 21.
[0344] Analysis of FANCF methylation.
[0345] DNA methylation patterns in FANCF gene were determined by
methylation specific PCR or PCR-based Hpall restriction enzyme
assay. Genomic DNA was isolated from indicated cell lines using
QIAamp DNA Blood Mini Kit (QIAGEN).
[0346] PCR-based HpaII Restriction Enzyme Assay
[0347] 250 ng of genomic DNA was digested with 30 unit of HpaII or
MspI for 12 hr at 37 .degree. C. 12.5 ng of DNA from each digest
was analyzed by PCR in 10 .mu.l reactions containing 1.times.PCR
buffer, 200 .mu.M each of the four deoxynucleotide triphosphates,
0.5units of AmpliTaq DNA polymerase (Roche), and 0.2 .mu.M of each
primer. PCR was run for 33 cycles, and each cycle constituted
denaturation (45 sec at 94 .degree. C., first cycle 4 min 45 sec),
annealing (1 min at 61.degree. C.), and extension (2 min at 72
.degree. C., last cycle 9 min). PCR reaction was subjected to
electrophoresis on a 1.2% agarose gel containing ethidium bromide.
Primers used were FPF6 (5'-GCACCTCATGGAATCCCTTC-3') (forward) and
FR343 (5'-GTTGCTGCACCAGGTGGTAA-3') (reverse). These primers were
designed using nt -6-14 for the forward primer and nt 403-432 for
the reverse primer.
[0348] Methylation-specific PCR.
[0349] Bisulfite modification of genomic DNA was performed as
previously described (Herman JG et al. Proc Natl Acad Sci USA 93
(18) 9821-6 (1996)). The bisulfite-treated DNA was amplified with
either a methylation-specific or unmethylation-specific primer set.
PCR was run for 40 cycles, and each cycle constituted denaturation
(45 sec at 94 .degree. C., first cycle 4 min 45 sec), annealing (1
min at 65.degree. C.), and extension (2 min at 72.degree. C., last
cycle 9 min). PCR reaction was subjected to electrophoresis on a 3%
Separide (Gibco) gel containing ethidium bromide. The
methylation-specific primers were FF280M
(5'-TTTTTGCGTTTGTTGGAGAATCGGGTTTTC -3') (forward) and
FR432M(5'-ATACACCGCAAACCGCCGACGAACAAAACG-3') (reverse). The
unmethylation-specific primer s were FF280U
(5'-TTTTTGTGTTTGTTGGAGAATTGGG- TTTTT -3') (forward) and FR432U
(5'-ATACACCACAAACCACCAACAAACAAAACA-3')(rev- erse). These primers
were designed using nt 280-309 for the forward primers and nt
403-432 for the reverse primers.
4TABLE 1 Complementation Groups and Responsible Genes of Fanconi
Anemia Estimated percen- Respon- Number Sub- tage of sible
Chromosome of Protein type patients gene location exons product A
66% FANCA 16q24.3 43 163 Kd B 4.3% FANCB -- -- -- C .sup.
12.7%.sup. FANCC 9q22.3 14 63 Kd D1 rare FANCD1 -- -- -- D2 rare
FANCD2 3p25.3 44 155,162 kD E .sup. 12.7%.sup. FANCE 6p21.2-21.3 10
60 kD F rare FANCF 11p15 1 42 kD G rare FANCG 9p13 14 68 kD
(XRCC9)
[0350]
5TABLE 2 Diseases of Genomic Instability Disease Damaging Agent
Neoplasm Function FA Cross-linking Acute Unknown agents leukemia,
myeloblastic hepatic, gastroinstestinal, and gynecological tumors
XP UV light Squamous cell Excision carcinomas repair AT Ionizing
Lymphoma Afferent radiation pathway to p53 Bloom's Alkylating Acute
Cell-cycle Syndrome agents lymphoblastic regulation leukemia
Cockayne's UV light Basal cell Transcription Syndrome carcinoma
coupled repair Hereditary non- Unknown Adenocarcinoma DNA mismatch
polyposis colon of colon, repair cancer (HNPCC) ovarian cancer
[0351]
6TABLE 3 FANCD2 Sequence Alterations Mutations PD20 nt376a.fwdarw.g
S126G/splice nt3707g.fwdarw.a R1236H VU008 nt904c.fwdarw.t R302W
nt958c.fwdarw.t Q320X PD733 deletion of exon 17 Polymorphisms
nt1122a.fwdarw.g V374V nt1440t.fwdarw.c* H480H
nt1509c.fwdarw.t.dagger. N503N nt2141c.fwdarw.t*.dagger. L714P
nt2259t.fwdarw.c D753D nt4098t.fwdarw.g*.dagger. L1366L
nt4453g.fwdarw.a.dagger. 3UTR *PD20 is heterozygous; .dagger.VU008
is heterozygous.
[0352]
7TABLE 4 Chromosome Breakage Analysis of Whole-cell Fusions Cell
DEB MMC % of Cells line/hybrids (ng/ml) (ng/ml) with radials
Phenotype PD20i 300 58 S PD24p 300 na* S VU423p 300 na* S PD319i
300 52 S PD20i/VU423p 300 6 R PD20i/PD24p 300 30 S PD20i/PD319i 300
0 R PD20i 40 48 S VU4231 40 78 S PD20i/VU423i 40 10 R VU423i + chr.
3, 40 74 S clone 1 VU423i + chr. 3, 40 68 S clone 2 VU4231 + chr.
3, 40 88 S clone 3 PD20i + empty 0 0 2 vector 40 24 S 200 62 S
PD20i + 0 0 0 FANCD2 vector 40 2 R 200 10 R Groups of experiments
are separated by line spaces. S, cross-linker sensitive; R,
cross-linker-resistant; i = immortal fibroblast line; p = primary
fibroblasts. *Cell viability at this concentration was too low to
score for radial formation, indicating the exquisite sensitivity of
primary fibroblasts to interstrand DNA-crosslinks.
[0353]
8TABLE 5 IR/Bleo- FA MMC mycin protein sensi- sensi- FA complex
tivity tivity Cell line/plasmid Group (1) (2) (3) Lympho- PD7 Wt +
R R blasts HSC72 A - S HSC72 + A A + R PD4 C - S PD4 + C C + R
EUFA316 G - S EUFA316 + G G + R EUFA121 F - S S EUFA121 + F F + R R
PD20 D + S S PD20(R) D + R R Fibro- GM0637 Wt + R R blasts GM6914 A
- S S GM694 + A A + R R PD426 C - S PDF426 + C C + R FAG326SV G - S
FAG326SV + G G + R PD20F D + S S 20-3-15(+D) D + R R NBS (-/-) NBS
+ S S ATM (-/-) ATM + S S BRCA1 (-/-) BRCA1 + S S (1) The presence
of the FA protein complex (FANCA/FANCG/FANCC) was determined as
previously described (Garcia-Higuera et al, MCB 19:4866-4873, 1999)
(2) MMC sensitivity for determined by the XTT assay for
lymphoblasts or by the crystal violet assay for fibroblasts. (3)
IR/Bleomycin sensitivity was determined by analysis of chromosome
breakage. (See Materials and Methods).
[0354]
9TABLE 6 The Intron/Exon Junctions of FANCD SEQ SEQ ID 5'-Donor ID
3'-Acceptor Exon Size NO. site Score Intron NO. site Score Exon 1
30 9 TCG 87 52 gtttcccgattttg 85 2 gtgagtaag ctctag GAA tg 2 97 10
CCA 83 53 gaaaatttttctat 83 3 gtaagtact tttcag AAA cta 3 141 11 TAG
78 54 ctcttcttttttctg 88 4 gtaatatttta catag CTG 4 68 12 AAA 81 159
55 attttttaaatctcc 78 5 gtatgtatttt ttaag ATA 5 104 13 GAG 86 375
56 gatttctttttttttt 91 6 gtgtggaga acag TAT gg 6 61 14 GAG 89 57
ccctatgtcttctt 86 7 gtaagactg ttttag CCT tc 7 53 15 AAA 87 58
ttctcttcctaaca 80 8 gtaagtggc ttttag CAA gt 8 79 16 AAG 83 364 59
aatagtgtcttcta 85 9 gtaggcttat ctgcag GAC g 9 125 17 GAG 80 60
tctttttctaccatt 86 10 gtggataaa cacag TGA cc 10 88 18 AAG 76 61
tctgtgcttttaatt 85 11 gtagaaaag tttag GTT ac 11 105 19 GAG 80 387
62 ctaatatttactttc 87 12 gtatgctctt tgcag GTA a 12 101 20 AAG 85
342 63 ttcctctctgctac 84 13 gtaaagagc ttgtag TTC tc 13 101 21 AAG
89 237 64 actctctcctgttt 92 14 gtgagatctt tttcag GCA t 14 36 22 AAG
82 65 tgcatatttattga 73 15 gtaatgttca caatag GTG t 15 144 23 TTA 80
66 tctactcttcccc 86 16 gtaagtgtc actcaag GTT ag 16 135 24 CAG 85 67
gttgactctcccc 84 17 gtatgttgaa tgtatag GAA a 17 132 25 AAG 77 68
tggcatcatttttt 89 18 gtatcttattg ccacag GGC 18 111 26 CAG 83 69
tcttcatcatctca 87 19 gttagaggc ttgcag GAT aa 19 110 27 CAG 82 70
aaaaaattctttgt 79 20 gtacacgtg ttttag AAG ga 20 61 28 CAG 93 71
attcttcctctttg 93 21 gtgagttctt ctccag GTG t 21 120 29 CTG 81 445
72 tgtttgtttgcttcc 85 22 gtaaagcca tgaag GAA at 22 74 30 AGG 84 300
73 attctggtttttctc 88 23 gtaggtattg cgcag TGA t 23 147 31 AAA 73 74
aatttatttctcctt 89 24 gtcagtata ctcag ATT gt 24 101 32 TAG 84 370
75 aaatgtttgttctc 86 25 gtatgggat tctcag ATT ga 25 116 33 GAG 88 76
atgtaatttgtact 82 26 gtgagcag ttgcag ATT agt 26 109 34 CAG 89 77
cagcctgctgttt 81 27 gtaagagaa gtttcag TCA gt 27 111 35 TAG 90 272
78 ttctctttttaatat 73 29 gtaagtatgt aaaag AAA t 28 110 36 AAG 78 79
ttgctgtgacttc 85 29 gtattggaat cccatag g GAG 29 144 37 GAA 85 80
tcctttcctccatg 84 30 gtaagtgac tgacag GCT ag 30 117 38 AAG 86 81
taactctgcattta 80 31 gttagtgtag ttat6ag AAC g 31 129 39 CAG 82 118
82 aaaatcatttttatt 79 32 gtcagaagc tttag TGT ct 32 119 40 TTG 85 83
tcttaccttgactt 85 33 gtaagtatgt ccttag GAG g 33 111 41 CAG 90 84
tttttcttgtctcctt 91 34 gtgagtcat acag CCA aa 34 131 42 TTG 73 85
tttgtcttcttttcta 89 35 gtgatgggc acag CTT ct 35 94 43 CTG 84 286 86
atatttgactctca 78 36 gtgagatgtt atgcag TAT t 36 123 44 CAG 92 87
atgcttttcccgtc 88 37 gtaaggga ttctag GCA gtt 37 94 45 CAG 92 88
catatatttggct 81 38 gtgagtaag gccccag at ATT 38 72 46 AAG 93 89
cttgtctttcacct 93 39 gtgagtatg ctccag GTA ga 39 39 47 AAG 89 90
agtgtgtctctctt 86 40 gtgagagat cttcag TAT tt 40 75 48 CGG 86 91
tataaacttattgg 77 41 gtaagagct ttatag GAA aa 41 75 49 AAG 91 92
tgttatttatttcca 86 42 gtaagaag ttcag ATT ggg 42 147 50 CAG 91 93
cttggtccattca 80 43 gtaagcctt catttag GGT gg 43 228 CCA taa + 94
attattctttgccc 44 3'UTR cttag GAT 96 51 GAG gtatctctac a 44 72 GAT
tag + 3'UTR
[0355]
10TABLE 7 PCR Primers to Amplify the 44 Exons of FANCD Primer SEQ
ID Product Annealing Exon Name NO. Primer Sequence (5'-.fwdarw.3')
Size (bp) Temp 1 MG914 115 F: CTAGCACAGAACTCTGCTGC 372 54 MG837 116
R: CTAGCACAGAACTCTGCTGC 2 MG746 117 F: CTTCAGCAACAGCGAAGTA- 422 50
GTCTG MG747 118 R: ATTCTCAGCACTTGAAAAGC- AGG 3 MG773 119 F:
GGACACATCAGTTTTCCTCTC 309 50 MG789 120 R: GAAAACCCATGATTCAGTCC 4-5
MG816 121 F: TCATCAGGCAAGAAACTTGG 467 50 MG803 122 R:
GAAGTTGGCAAAACAGACTG 6 MG804 123 F: GAGCCATCTGCTCATTTCTG 283 50
MG812 124 R: CCCGCTATTTAGACTTGAGC 7 MG775 125 F:
CAAAGTGTTTATTCCAGGAGC 343 50 MG802 126 R: CATCAGGGTACTTTGAACA- TTC
8-9 MG727 127 F: TTGACCAGAAAGGCTCAGT- 640 50 TCC MG915 128 R:
AGATGATGCCAGAGGGTTTA- TCC 10 MG790 129 F: TGCCCAGCTCTGTTCAAACC 222
50 MG774 130 R: AGGCAATGACTGACTGACAC 11 MG805 131 F:
TGCCCGTCTATTTTTGATGA- 392 50 AGC MG791 132 R: TCTCAGTTAGTCTGGGGACAG
12 MG751 133 F: TCATGGTAGAGAGACTGGAC- 432 50 TGTGC MG972 134 R:
ACCCTGGAGCAAATGACAACC 13-14 MG973 135 F: ATTTGCTCCAGGGTACATGGC 555
50 MG974 136 R: GAAAGACAGTGGGAAGGCA- AGC 15 MG975 137 F:
GGGAGTGTGTGGAACAAAT- 513 50 GAGC MG976 138 R: AGTTTCTACAGGCTGGTCCT-
ATTCC 16 MG755 139 F: AACGTGGAATCCCATTGATGC 379 48 MG730 140 R:
TTTCTGTGTTCCCTCCTTGC 17 MG794 141 F: GATGGTCAAGTTACACTGGC 382 50
MG778 142 R: CACCTCCCACCAATTATAGT- ATTC 18 MG808 143 F:
CTATGTGTGTCTCTTTTACA- 234 48 GGG MG817 144 R: AATCTTTCCCACCATATTGC
19 MG779 145 F: CATACCTTCTTTTGCTGTGC 199 48 MG795 146 R:
CCACAGAAGTCAGAATCTC- CACG 20 MG731 147 F: TGTAACAAACCTGCACGTTG 632
56 MG732 148 R: TGCTACCCAAGCCAGTAGTT- TCC 21 MG788 149 F:
GAGTTTGGGAAAGATTGGC- 232 50 AGC MG772 150 R: TGTAGTAAAGCAGCTCTCA-
TGC 22-23 MG733 151 F: CAAGTACACTCTGCACTGCC 652 50 MG758 152 R:
TGACTCAACTTCCCCACCAA- GAG 24-25 MG736 153 F: CTCCCTATGTACGTGGAGT-
732 50 AATAC MG737 154 R: GGGAGTCTTGTGGGAACTAAG 26 MG780 155 F:
TTCATAGACATCTCTCAGC- 284 50 TCTG MG759 156 R: GTTTTGGTATCAGGGAAAGC
27-28 MG760 157 F: AGCCATGCTTGGAATTTTGG 653 50 MG781 158 R:
CTCACTGGGATGTCACAAAC 29 MG740 159 F: GGTCTTGATGTGTGACTTGT- 447 50
ATCCC MG741 160 R: CCTCAGTGTCACAGTGTTCTT- TGTG 30 MG809 161 F:
CATGAAATGACTAGGACAT- 281 48 TCC MG797 162 R: CTACCCAGTGACCCAAACAC
31-32 MG761 163 F: CGAACCCTTAGTTTCTGAGA- 503 50 CGC MG742 164 R:
TCAGTGCCTTGGTGACTGTC 33 MG916 165 F: TTGATGGTACAGACTGGAGGC 274 50
MG810 166 R: AAGAAAGTTGCCAATCCTG- TTCC 34 MG762 167 F:
AGCACCTGAAAATAAGGAGG 343 50 MG743 168 R: GCCCAAAGTTTGTAAGTGT- GAG
35-36 MG787 169 F: AGCAAGAATGAGGTCAAGTTC 590 50 MG806 170 R:
GGGAAAAACTGGAGGAAAG- AACTC 37 MG818 171 F: AGAGGTAGGGAAGGAAGCTAC
233 50 MG813 172 R: CCAAAGTCCACTTCTTGAAG 38 MG834 173 F:
GATGCACTGGTTGCTACATC 275 50 MG836 174 R: CCAGGACACTTGGTTTCTGC 39
MG839 175 F: ACACTCCCAGTTGGAATCAG 370 50 MG871 176 R:
CTTGTGGGCAAGAAATTGAG 40 MG829 177 F: TGGGCTGGATGAGACTATTC 223 50
MG870 178 R: CCAAGGSVSYSYVYYVYHS- HVSSC 41 MG820 179 F:
TGATTATCAGCATAGGCTGG 271 50 MG811 180 R: GATCCCCCAATAGGAACTGC 42
MG763 181 F: CATTCAGATTCACCAGGACAC 227 50 MG782 182 R:
CCTTACATGCCATCTGATGC 43 MG764 183 F: AACCTTCTCCCCTATTACCC 435 50
3'UTR MG835 184 R: GGAAAATGAGAGGCTATA- ATGC 44 MG1006 185 F:
TGTATTCCAGAGGTCACCC- 234 50 AGAGC 3'UTR MG1005 186 R:
CCAGTAAGAAAGGCAAACA- GCG
[0356]
11TABLE 8 FANCD2 LOCI on Human Chromosome 3p Exon Copy Region 1
Copy Region 2 Copy Region 3 FANCD2 Copy Region 4 Copy Region 5 1
201,110 344,395 8,170,539 2 3 4 5 6 7 8 9 10 11 12 6,126,244
8,209,073 13 14 15 16 8,202,791 17 18 19 20 21 22 23 24 18,201,854
25 26 27 28 6,094,448 29 30 31 32 186,164 33 34 35 36 18,178,589 37
38 39 40 41 42 43 44 8,095,780
[0357]
12TABLE 9 Primer Name Sequence Length of Product
hFANCD2_super_1_2_R GGCCCACAGTTTCCGTTTCT -- hFANCD2_super_1_2_F
CAAGGAAGCTAGAAATGAAGAAC hFANCD2_super_3_3_R CTGGGACTACAGACACGTTTT
-- hFANCD2_super_3_3_F GTGTCACGTGTCTGTAATCTC hFANCD2_super_7_14_R
TTAAGACCCAGCGAGGTATTC -- hFANCD2_super_7_14_F TGGGTTTGGTAGGGTAATGTC
hFANCD2_super_19_22_R TGGAAAGTCACTGCGGAGAAA --
hFANCD2_super_19_22_F ACGTAATCACCCCTGTAATCC hFANCD2_super_23_29_R
CACTGCAAACTGCTCACTCAA -- hFANCD2_super_23_29_F
GGCCTTGTGCTAAGTGCTTTT hFANCD2_super_30_32_R ACCCTGGTGGACATACCTTTT
-- hFANCD2_super_30_32_F CCAAAGTACTGGGAGTTTGAG
hFANCD2_super_33_36_R TCTGGGCAACAGAACAAGCAA --
hFANCD2_super_33_36_F GAGCAATTTAGCCTGTGGTTTT hFANCD2_super_43_44_R
ACCATCTGGCCGACATGGTA -- hFANCD2_super_43_44_F AGGGTCCTGAGACTATATACC
hFANCD2_exon1_R TCCCATCTCAGGGCAGATGA 324 hFANCD2_exon1_F
TATGCCCGGCTAGCACAGAA hFANCD2_exon2_R TCTCTCACATGCCTCACACAT 258
hFANCD2_exon2_F CCCCTCTGATTTTGGATAGAG hFANCD2_exon3_R
AAGATGGATGGCCCTCTGATT 354 hFANCD2_exon3_F GACACATCAGTTTTCCTCTCAT
hFANCD2_exon4_R AATCATTCTAGCCCACTCAACT 253 hFANCD2_exon4_F
TGGTTTCATCAGGCAAGAAACT hFANCD2_exon5_R AGCCCCATGAAGTTGGCAAAA 298
hFANCD2_exon5_F GCTTGTGCCAGCATAACTCTA hFANCD2_exon6_R
GCTGTGCTAAAGCTGCTACAA 341 hFANCD2_exon6_F GAGCCATCTGCTCATTTCTGT
hFANCD2_exon7_R CAGAGAAACCAATAGTTTTCAG 280 hFANCD2_exon7_F
AATCTCGGCTCACTGCAATCT hFANCD2_exon8_R AGCTAATGGATGGATGGAAAAG 333
hFANCD2_exon8_F TAGTGCAGTGCCGAATGCATA hFANCD2_exon9_R
TACTCATGAAGGGGGGTATCA 323 hFANCD2_exon9_F TTCACACGTAGGTAGTCTTTCT
hFANCD2_exon10_R CATTACTCCCAAGGCAATGAC 229 hFANCD2_exon10_F
GCCCAGCTCTGTTCAAACCA hFANCD2_exon11_R AGCTCCATTCTCTCCTCTGAA 341
hFANCD2_exon11_F GTGGGAAGATGGAGTAAGAGA hFANCD2_exon12_R
TCTGACAGTGGGATGTCAGAA 211 hFANCD2_exon12_F TGCCTACCCACTATGAATGAG
hFANCD2_exon13_R ATGTGTCCATCTGGCAACCAT 321 hFANCD2_exon13_F
CAGGAACTCCGATCTTGTAAG hFANCD2_exon14_R TGGAGGGGGGAGAAAGAAAG 186
hFANCD2_exon14_F CGTGTTTCGCTGATGTGTCAT hFANCD2_exon15a_R
GGAAGGCCAGTTTGTCAAAGT 325 hFANCD2_exon15a_F GTGTTTGACCTGGTGATGCTT
hFANCD2_exon15b_R CTTATTTCTTAGCACCCTGTCAA 204 hFANCD2_exon15b_F
GTGGAACAAATGAGCATTATCC hFANCD2_exon16_R TTCCCCTTCAGTGAGTTCCAA 332
hFANCD2_exon16_F AGGGAGGAGAAGTCTGACATT hFANCD2_exon17_R
GATTAGCCTGTAGGTTAGGTAT 422 hFANCD2_exon17_F GATGGGTTTGGGTTGATTGTG
hFANCD2_exon18_R CCAGTCTAGGAGACAGAGCT 282 hFANCD2_exon18_F
GGCTATCTATGTGTGTCTCTTT hFANCD2_exon19_R ACGATTAGAAGGGAACATGGAA 328
hFANCD2_exon19_F CGATATCCATACCTTCTTTTGC hFANCD2_exon20_R
TGACAGAGCGAGACTCTCTAA 239 hFANCD2_exon20_F CACACCAACATGGCACATGTA
hFANCD2_exon21_R GAGACAGGGTAGGGCAGAAA 339 hFANCD2_exon21_F
AAAGGGGCGAGTGGAGTTTG hFANCD2_exon22_R GTAACTTCACCAGTGCAACCAA 279
hFANCD2_exon22_F ATGCACTCTCTCTTTTCTACTT hFANCD2_exon23_R
ACAAGGAATCTGCCCCATTCT 356 hFANCD2_exon23_F TTCCCTGTAGCCTTGCGTATT
hFANCD2_exon24_R CCCCACATACACCATGTATTG 258 hFANCD2_exon24_F
CTCCCTATGTACGTGGAGTAA hFANCD2_exon25_R GTGGGACATAACAGCTAGAGA 350
hFANCD2_exon25_F AGGGGAAAGTAAATAGCAAGGA hFANCD2_exon26_R
TCAGGGATATTGGCCTGAGAT 324 hFANCD2_exon26_F GACATCTCTCAGCTCTGGATA
hFANCD2_exon27_R CCAATTACTGATGCCATGATAC 324 hFANCD2_exon27_F
GCATTCAGCCATGCTTGGTAA hFANCD2_exon28_R GATTACTCCAACGCCTAAGAG 354
hFANCD2_exon28_F TCTACCTCTAGGCAGTTTCCA hFANCD2_exon29_R
TCTCCTCAGTGTCACAGTGTT 384 hFANCD2_exon29_F CTTGGGCTAGAGGAAGTTGTT
hFANCD2_exon30_R TACCCAGTGACCCAAACACAA 348 hFANCD2_exon30_F
GAGTTCAAGGCTGGAATAGCT hFANCD2_exon3l_R ACCGTGATTCTCAGCAGCTAA 341
hFANCD2_exon3l_F CCATTGCGAACCCTTAGTTTC hFANCD2_exon32_R
AGTGCCTTGGTGACTGTCAAA 336 hFANCD2_exon32_F CCACCTGGAGAACATTCACAA
hFANCD2_exon33_R TACTGAAAGACACCCAGGTTAT 340 hFANCD2_exon33_F
CACGCCCGACCTCTCAATTC hFANCD2_exon34_R TATAGCAAGAGGGCCTATCCA 349
hFANCD2_exon34_F TTGGGCACGTCATGTGGATTT hFANCD2_exon35_R
GTCCAGTCTCTGACAAACAAC 300 hFANCD2_exon35_F TTAGACCGGGAACGTCTTAGT
hFANCD2_exon36_R GGCCAAGTGGGTCTCAAAAC 398 hFANCD2_exon36_F
CCTCTGGTTCTGTTTTATACTG hFANCD2_exon37_R TCTGGGCAACAGAACAAGCAA 277
hFANCD2_exon37_F CTTCCCAGGTAGTTCTAAGCA hFANCD2_exon38_R
AAGCCAGGACACTTGGTTTCT 274 hFANCD2_exon38_F GCACTGGTTGCTACATCTAAG
hFANCD2_exon39_R GCATCCATTGCCTTCCCTAAA 236 hFANCD2_exon39_F
TGCTCAAAGGAGCAGATCTCA hFANCD2_exon40_R CAGTCCAATTTGGGGATCTCT 309
hFANCD2_exon40_F CCTTGGGCTGGATGAGACTA hFANCD2_exon41_R
CCCCAATAGCAACTGCAGATT 214 hFANCD2_exon41_F GATTGCAAGGGTATCTTGAATC
hFANCD2_exon42_R GCTTAGGTGACCTTCCTTACA 356 hFANCD2_exon42_F
AACATACCGTTGGCCCATACT hFANCD2_exon43a_R AGCATGATCTCGGCTCACCA 366
hFANCD2_exon43a_F GTGGCTCATGCTTGTAATCCT hFANCD2_exon43b_R
TCAGTAGAGATGGGGTTTCAC 358 hFANCD2_exon43b_F CTGCCACCTTAGAGAACTGAA
hFANCD2_exon43c_R CTCAAGCAATCCTCCTACCTT 405 hFANCD2_exon43c_F
TAGAATCACTCCTGAGTATCTC hFANCD2_exon43d_R CAGCTTCTGACTCTGTGCTTT 367
hFANCD2_exon43d_F AGTTGGTGGAGCAGAACTTTG hFANCD2_exon43e_R
CTCGAGATACTCAGGAGTGAT 381 hFANCD2_exon43e_F TCAACCTTCTCCCCTATTACC
hFANCD2_exon43f_R AGTTCTGCTCCACCAACTTAG 306 hFANCD2_exon43f_F
GGTATCCATGTTTGCTGTGTTT hFANCD2_exon44_R GAAAGGCAAACAGCGGATTTC 213
hFANCD2_exon44_F CACCCAGAGCAGTAACCTAAA
[0358] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
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