U.S. patent application number 12/786782 was filed with the patent office on 2010-09-23 for method for purifying cancer-specific proliferating cell nuclear antigen.
Invention is credited to PAMELA E. BECHTEL, ROBERT J. HICKEY, DEREK J. HOELZ, LINDA H. MALKAS, MIN PARK, LAUREN SCHNAPER, DRAGANA TOMIC.
Application Number | 20100240874 12/786782 |
Document ID | / |
Family ID | 27753317 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240874 |
Kind Code |
A1 |
MALKAS; LINDA H. ; et
al. |
September 23, 2010 |
METHOD FOR PURIFYING CANCER-SPECIFIC PROLIFERATING CELL NUCLEAR
ANTIGEN
Abstract
An isolated antibody that binds a cancer specific Proliferating
Cell Nuclear Antigen (csPCNA) that binds to the amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIle
AspSerPhePheArgLeuAlaGlnGlnGluLysGluAspAlaLysArg (SEQ ID No. 1). An
immunoassay that utilizes an isolated antibody that binds a form of
csPCNA that binds to the SEQ ID No. 1 to identify the presence of
csPCNA in a sample.
Inventors: |
MALKAS; LINDA H.;
(INDIANAPOLIS, IN) ; HICKEY; ROBERT J.;
(INDIANAPOLIS, IN) ; BECHTEL; PAMELA E.; (TEMPE,
AZ) ; PARK; MIN; (LOS ALAMOS, NM) ; HOELZ;
DEREK J.; (INDIANAPOLIS, IN) ; TOMIC; DRAGANA;
(BALTIMORE, MD) ; SCHNAPER; LAUREN; (LUTHERVILLE,
MD) |
Correspondence
Address: |
WHITEFORD, TAYLOR & PRESTON, LLP;ATTN: GREGORY M STONE
SEVEN SAINT PAUL STREET
BALTIMORE
MD
21202-1626
US
|
Family ID: |
27753317 |
Appl. No.: |
12/786782 |
Filed: |
May 25, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11983939 |
Nov 13, 2007 |
7723487 |
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12786782 |
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10083576 |
Feb 27, 2002 |
7294471 |
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11983939 |
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Current U.S.
Class: |
530/387.3 ;
530/387.9 |
Current CPC
Class: |
C07K 14/4738 20130101;
G01N 33/57484 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.9 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under Grant
Nos. CA-83199, CA-57350-04, and CA-74904 awarded by NIH and Grant
No. DAMD-17-97-1-7037 awarded by Department of Defense. The
government has certain rights in the invention.
Claims
1. An antibody that binds to a cancer specific Proliferating Cell
Nuclear Antigen (csPCNA), wherein said csPCNA binds to the amino
acid sequence corresponding to SEQ ID. No. 1.
2. The antibody of claim 1, wherein the antibody is a monoclonal
antibody.
3. The antibody of claim 1, wherein the antibody is a polyclonal
antibody.
4. The antibody of claim 1, wherein the antibody is a recombinant
antibody.
5. The antibody of claim 1, wherein said antibody is used in an
immunoassay.
6. A method for isolating an antibody that binds to a csPCNA,
comprising the steps of: (A) obtaining a sample, (B) contacting
said sample with a peptide comprising SEQ ID No. 1, forming a
csPCNA-peptide complex, (C) isolating said csPCNA from said
csPCNA-peptide complex, (D) producing an antibody that binds to
csPCNA, wherein said csPCNA binds to the aminoacid sequence
corresponding to SEQ ID No. 1, and (E) isolating said antibody.
7. The method of claim 6, wherein said antibody is produced by
challenging a mammal with the isolated csPCNA of step C.
8. The method of claim 7, wherein said mammal is selected from the
group consisting of rabbits, goats, horses, mice, rats, and
sheep.
9. The method of claim 6, wherein said antibody is produced by
challenging a hen with said isolated csPCNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Pat. No.
7,723,487, application Ser. No. 11/983,939, entitled Method for
Purifying Cancer-Specific Proliferating Cell Nuclear Antigen filed
Nov. 13, 2007, and issued on May 25, 2010, which is a continuation
of U.S. Pat. No. 7,294,471, application Ser. No. 10/083,576,
entitled Method for Purifying Cancer-Specific Proliferating Cell
Nuclear Antigen filed Feb. 27, 2002, and issued on Nov. 13, 2007,
both of which are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The present invention is directed to a method for purifying
cancer-specific Proliferating Cell Nuclear Antigen (csPCNA), as
well as to an ELISA for distinguishing csPCNA from
native-proliferating cell nuclear antigen (nPCNA) and diagnosing
cancer.
BACKGROUND OF THE INVENTION
A. Cancer
[0004] One of the least understood and most complex disease
processes is the transformation that occurs as a cell becomes
malignant. This process involves both genetic mutations and
proteomic transformations, the result of which allows the cell to
escape normal controls preventing inappropriate cell division. All
cancer cells are unique and distinct from other cells. Despite this
uniqueness, cancer cells share some common attributes. Most cancer
cells proliferate outside of the normal cell cycle controls,
exhibit morphological changes and exhibit various biochemical
disruptions to cellular processes.
[0005] Cancer is usually diagnosed when a tumor becomes visible
well after the first on-set of cellular changes. Many cancers are
diagnosed after a biopsy sample is examined by histology for
morphologic abnormalities, evidence of cell proliferation and
genetic irregularities. There is a clear need to identify and
characterize new markers for malignancy. Recently there has been an
effort to define markers for the diagnosis and prognosis of
malignancies. Many of the genetic and biochemical changes occur
during the early development of a tumor and these changes should be
exploited for the early diagnosis of cancer.
[0006] Breast cancer is the leading cause of death among women in
the Western world. Recent data suggests that there is a strong
correlation between late detection and poor prognosis of this
disease. Analysis of a thousand clinical cases indicates that there
is extensive genetic damage and a high rate of DNA synthesis in
breast tumors in comparison with normal breast tissue. These data
suggest that an alteration in the DNA replication machinery of
breast cancer cells may contribute to uncontrolled and error-prone
DNA synthesis.
[0007] Human breast cells mediate DNA synthesis using the
multiprotein replication complex termed the DNA synthesome (Coll et
al, Oncology Research, 8:435-447 (1996)). The DNA synthesome is
fully competent to support in vitro DNA replication. The
transformation of non-malignant human breast cells to a malignant
state is accompanied by an alteration to a specific component of
DNA synthesome, Proliferating Cell Nuclear Antigen (PCNA). PCNA is
a well-known cell-cycle marker protein, originally identified as an
antigen for autoimmune disease (Bechtel et al, Cancer Research,
58:3264-3269 (1998)).
B. PCNA in Cancer/Other Cell Processes
[0008] PCNA is currently used in the diagnosis of malignancy, as
well as in evaluating the prognosis of the patient (Schonborn et
al, J. Cancer Res. Clinical Oncology, 121:122 (1995)). PCNA is a
small (36 kD) nuclear protein involved in many cellular processes.
PCNA plays crucial roles in both DNA replication and DNA repair
mechanisms. PCNA has also been associated with transcription
events. PCNA forms a trimer in the nucleus and acts as an accessory
protein to polymerase 6, and also interacts with a variety of other
proteins (Downey et al, Cancer Cells, 6:1211-1218 (1988)). In the
evaluation of malignancy, PCNA is often used as a marker for cell
proliferation. However, PCNA alone does not correlate with the
stage of malignancy or the patient outcome.
[0009] A novel PCNA from breast cancer cells has been identified.
The malignant breast cancer cells express a unique, acidic form of
PCNA protein, i.e., csPCNA, which can clearly be distinguished from
the basic form of this protein found in non-malignant cells, i.e.,
nPCNA. This alteration is most likely the result of a
post-translational modification (Bechtel et al, Cancer Res.,
58:3264-3269 (1998)). However, prior to the present invention an
effective method to purify csPCNA has not been described.
[0010] Recent advances in biochemical and genetic studies strongly
indicate that PCNA may interact with different proteins involved in
DNA mismatch repair, Okazaki fragments ligation, DNA methylation
and chromatin assembly (Balajee et al, Mutat. Res., 404:3-11
(1998); Ceccotti et al, Curr Biol: 6:1528-1531 (1996); Chen et al,
Proc. Natl. Acad. Sci., USA, 93:11597-11602 (1996); Chuang et al,
Science, 277:1996-2000 (1997); Dimitrova et al, J. Cell. Biol.,
146:709-722 (1999); Eki et al, J. Biol. Chem., 266:3087-3100
(1991); Eki et al, J. Biol. Chem., 267:7284-7294 (1992); Greene et
al, Hum. Mol. Genet: 8, 2263-2273 (1999); Gu et al, Nucleic Acids
Res., 26:1173-1178 (1998); Henderson et al, Embo J., 13:1450-1459
(1994); Johnson et al, J. Biol. Chem., 271:27987-27990 (1996);
Kelman, Oncogene, 14:629-640 (1997); Kolodner et al, Curr. Opin.
Genet. Dev., 9:89-96 (1999); Krude, Curr. Biol., 9:R394-R396
(1999); Lee et al, J. Biol. Chem., 266:22707-22717 (1991); Levin et
al, Proc. Natl. Acad. Sci., USA, 94:12863-12868 (1997); Levin et
al, Curr. Biol., 10:919-922 (2000); Martini et al, J. Cell. Biol.,
143:563-575 (1998); Merrill et al, Genetics, 148:611-624 (1998);
Mimura et al, Genes Cells, 5:439-452 (2000); Miura, J. Radiat. Res.
(Tokyo), 40:1-12 (1999); Moggs et al, Mol. Cell. Biol.,
20:1206-1218 (2000); Nishikawa et al, Jpn. J. Cancer Res.,
88:1137-1142 (1997); Otterlei et al, Embo J., 18:3834-3844 (1999);
Pan et al, Proc. Natl. Acad. Sci., USA, 90:6-10 (1993); Schweitzer
et al, Genetics, 152:953-963 (1999); Shibahara et al, Cell,
96:575-85 (1999); Sinicrope et al, Clin. Cancer Res., 4:1251-1261
(1998); Tom et al, J. Biol. Chem., 276:24817-24825 (2001);
Tomkinson et al, Mutat. Res., 407:1-9 (1998); Tsurimoto, Front.
Biosci., 4:D849-D858 (1999); Umar et al, Cell, 87:65-73 (1996); and
Wu et al, Nucleic Acids Res., 24:2036-2043 (1996)).
[0011] Xeroderma Pigmentosum's G protein (XPG) is reported to
interact with PCNA (Gary et al, J. Biol. Chem., 272(39):24522-24529
(1997)). The DNA repair endonuclease XPG binds to proliferating
cell nuclear antigen (PCNA) and shares sequence elements with the
PCNA-binding regions of FEN-1 and cyclin-dependent kinase inhibitor
p21. XPG is a repair endonuclease similar to FEN-1 and required for
nucleotide excision repair. The human XPG endonuclease cuts on the
3' site of a DNA lesion, during nucleotide excision repair.
[0012] In the present invention, XPG protein was unexpectedly found
to be useful in selectively purifying csPCNA, and as a part of an
ELISA system which can distinguish the csPCNA from the nPCNA. The
detection of csPCNA in the ELISA serves as a powerful marker for
early detection of malignancy.
SUMMARY OF THE INVENTION
[0013] An object of the present invention is to provide a method
for selectively purifying csPCNA.
[0014] Another object of the present invention is to provide an
ELISA for detection of csPCNA and early detection of
malignancy.
[0015] The above-described objects, as well as others, which will
be apparent from the detailed description of the invention provided
hereinafter, have been met in one embodiment by a method for
purifying csPCNA comprising the steps of:
[0016] (A) obtaining a tissue or body fluid sample comprising
csPCNA;
[0017] (B) contacting said sample with a peptide comprising the
amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPhePheArgLeuAlaGlnG-
lnGluLysGluAsp AlaLysArg (SEQ ID NO:1), wherein said peptide is
immobilized on a solid support and binds to said csPCNA to form a
peptide-csPCNA complex; and
[0018] (C) isolating csPCNA from said peptide-csPCNA complex so as
to purify said csPCNA.
[0019] In another embodiment, the above-identified objects have
been met by an immunoassay for detecting csPCNA comprising:
[0020] (1) contacting a test sample with a peptide comprising the
amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPhePheArgLeuAlaGlnGlnGluLysG-
luAsp AlaLysArg (SEQ ID NO:1), which has been immobilized on a
solid support so as to bind csPCNA to said peptide to form a
peptide-csPCNA complex; and
[0021] (2) contacting said peptide-csPCNA complex with an anti-PCNA
antibody and detecting binding of said antibody to said
complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a flow chart depicting steps involved in the
isolation of total PCNA and csPCNA.
[0023] FIG. 2 shows a Western blot of fractions collected during
the isolation of PCNA. The fractions were separated on a 10% (w/v)
SDS-PAGE gel, transferred to nitrocellose and analyzed using
anti-PCNA antibodies. Lane 1: H; Lane 2: S1; Lane 3: S2; Lane 4:
NE/S3; Lane 5: PCFT; Lane 6: PCLS; Lane 7: PCHS; Lane 8: PSFT; Lane
9: PSW; Lane 10: PSE; Lane 11: QS; Lane 12: XPGS; Lane 13:
XPGE.
[0024] FIG. 3 shows a Coomasie stained gel of fractions collected
during the isolation of PCNA. The fractions were separated on a 10%
(w/v) SDS-PAGE gel, and then stained with Coomasie blue. Lane 1: H;
Lane 2: S1; Lane 3: S2; Lane 4: NE/S3; Lane 5: PCFT; Lane 6: PCLS;
Lane 7: PCHS; Lane 8: PSFT; Lane 9: PSW; Lane 10: PSE; Lane 11: QS;
Lane 12: XPGS; Lane 13: XPGE.
[0025] FIG. 4 shows the results of XPGE which was subjected to
2-dimensional SDS-PAGE and Western blot analysis to identify which
form of PCNA was present.
[0026] FIG. 5A shows the results of a validation study on the
viability of the streptavidin surface of streptavidin coated
plates.
[0027] FIG. 5B shows the results of a study for determining the
maximal binding capability of biotinylated XPG-GST protein to
streptavidin-coated plates.
[0028] FIG. 6 shows densitometric analyses of total PCNA in P4
fractions from MCF7 and MCF10A cells.
[0029] FIGS. 7A-7C show the results of ELISAs using MCF7 P4 and
MCF10A P4 proteins.
[0030] FIG. 8 shows the results of an ELISA assay comparing the
abundance of csPCNA in extracts of MCF7 (malignant) cells and MCF
10A (normal) cells.
DETAILED DESCRIPTION OF THE INVENTION
[0031] As discussed above, in one embodiment, the above-described
objects of the present invention have been met by a method for
purifying csPCNA comprising the steps of:
[0032] (A) obtaining a tissue or body fluid sample comprising
csPCNA;
[0033] (B) contacting said sample with a peptide comprising the
amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPhePheArgLeuAlaGlnG-
lnGluLysGluAs pAlaLysArg (SEQ ID NO:1), wherein said peptide is
immobilized on a solid support and binds to said csPCNA to form a
peptide-csPCNA complex; and
[0034] (C) isolating csPCNA from said peptide-csPCNA complex so as
to purify said csPCNA.
[0035] Preferably, prior to step (B), the thus obtained tissue or
body fluid sample of step (A) is subjected to a process comprising
the steps of:
[0036] (1) homogenizing cells constituting said tissue or body
fluid to obtain a homogenate (H);
[0037] (2) separating said H into a nuclear pellet fraction (NP)
and a cytosolic fraction (S1);
[0038] (3) extracting nuclei from said NP to obtain a nuclear
extract (NE);
[0039] (4) subjecting said S1 to centrifugation to obtain a
post-mitochondrial cytosolic supernatant (S2);
[0040] (5) subjecting said S2 to centrifugation to obtain a
post-mitochondrial/post-microsomal cytosolic supernatant (S3);
[0041] (6) combining said NE and said S3 to form an NE/S3 fraction,
applying the resulting NE/S3 fraction to a phosphocellulose (a weak
anion exchange matrix) column and collecting the flow through
(PCFT);
[0042] (7) applying the resulting PCFT to a phenylsepharose (a
hydrophobic chromatography matrix) column, eluting the column with
buffer comprising ethylene glycol and collecting the eluant
(PSE);
[0043] (8) dialyzing out ethylene glycol present in the PSE to
obtain a dialyzate; and
[0044] (9) applying the resulting dialyzate to a Q-Sepharose (a
strong anion exchange matrix) column, eluting with a dialyzate
buffer comprising a salt gradient, and collecting and pooling
PCNA-containing fractions to obtain said sample.
[0045] More preferably, the tissue or body fluid sample of step (A)
further comprises native-PCNA (nPCNA), said nPCNA does not bind to
said peptide in step (B) but flows through the column (XPGS),
whereas csPCNA binds to said peptide in step (B) to form a
peptide-csPCNA complex and in step (C) isolating csPNA is effected
using an elution buffer whereby csPCNA is eluted from said
csPCNA-complex (XPGE).
[0046] The particular tissue or body fluid sample which is employed
in the present invention is not critical thereto. Examples of
tissue which can be employed in the present invention include
cervical, mammary glands, esophageal, glial cells, lung, stomach,
intestine, prostate, and white blood cells. Examples of body fluid
which can be employed in the present invention include urine, serum
and whole blood.
[0047] The source of the tissue or body fluid is from a subject
afflicted with a cancer. The particular cancer is not critical to
the present invention. The cancers can be carcinomas, sarcomas,
lymphomas, or leukemias. Examples of such cancers include cervical
carcinoma, mammary gland carcinoma of ductal or lobular origin,
gliomas, prostate, lung, esophageal, stomach, and ovarian
cancer.
[0048] In step (B) the solid support employed is critical to the
present invention, because the XPG peptide of SEQ ID NO:1 can be
expressed as a fusion protein, e.g., a GST fusion
(GST=glutathione-S-transferase), and the support will depend on the
fusion partner. An example of the solid support which can be
employed in this case include Glutathione Sepharose (Pharmacia).
However, expression of the XPG-fusion protein in a Calmodulin or
6.times.His (oligo (6.times.) histidine) format can also be used in
the present invention.
[0049] In step (C), csPCNA is isolated from the complex by, for
example, elution with buffer comprising 50 mM Tris-HCl (pH 8.0).
Again, nPCNA does not bind to the column matrix, but flows through
the column and appears in the flow-through liquid exiting the
column while the complex and contaminating proteins are being
loaded onto the column.
[0050] The peptide represented by SEQ ID NO:1 may be synthesized
chemically or by recombinant DNA techniques, as described by Gary
et al, J. Biol. Chem., 232:24522-24529 (1997). Further, as noted
above, the peptide may be in the form of a fusion protein. The
partner of the fusion protein is not critical to the present
invention. Examples of such partners include
Glutathione-S-Transferase (GST), Calmodulin Binding protein, and
oligo(6.times.) histidine.
[0051] The resulting purified csPCNA can be used to produce
antibodies (monoclonal or polyclonal) specific for csPCNA by
conventional techniques. The resulting purified csPCNA can also be
used as standards for diagnostic kits, as well as enabling
development of specific inhibitors for csPCNA (not nPCNA), the
identification of the site(s) on the PCNA polypeptide that is (are)
modified in nPCNA, provide the baseline for comparison to identify
the type of modification sustained by nPCNA and lacking from
csPCNA, the identification of specific metabolic pathways that
mediate the addition or removal of this (these) post-translational
modifications.
[0052] Products of antibodies specific for csPCNA can be produced
by challenging mammals (e.g., rabbits, goats, horses, etc) with the
peptide sequence which is post-translationally modified in nPCNA,
but not in csPCNA. All commercially available antibodies to PCNA
that exist to date (.about.10) recognize the interdomain connector
loop. They cannot distinguish csPCNA from nPCNA. Identification of
the amino acid sequence of PCNA that is bound by the XPG peptide
only in the csPCNA form of PCNA provides the target for preparing
selective antibodies recognizing only csPCNA.
[0053] Specific inhibitors of csPCNA's interaction with their
target proteins can be produced by construction or expression of
peptides identical to the interacting domains of csPCNA and by
computational chemistry methods. Through computational chemistry,
the sites of interactions can be modeled and searches of existing
3-D chemical library structures or the design of new compounds can
be made to disrupt and/or promote interaction between csPCNA and
the proteins.
[0054] Using mass spectrometry peptide analysis of tryptic
fragments of csPCNA and nPCNA will identify the fragments
(peptides) that are unique to csPCNA. Sequence identification of
these peptides using LC/MS-TDF mass spectrometry will identify the
sequence, and thus the position of the modified peptides or amino
acids within nPCNA that are modified in csPCNA (or that are
modified in csPCNA and are not modified in nPCNA).
[0055] Furthermore, molecular weight and sequence analysis of these
peptides by mass spectroscopy will indicate what types of
post-translational modifications have been sustained by csPCNA
and/or nPCNA. The identification of these post-translational
modifications then leads to the pathways and/or enzymes and/or
other molecules responsible for either the addition of the
modification or the loss of the modification from the csPCNA. In
addition, computer analysis of the amino acid domains of the csPCNA
that interact with the XPG peptide can reveal the presence of
specific consensus sequences for a particular type of
post-translational modification.
[0056] csPCNA is believed to be a better diagnostic/prognostic
indicator for cancer than current methodologies which measure total
PCNA. This is because total PCNA of malignant tissue/cells takes
into account nPCNA and csPCNA, while this method of
diagnostic/prognostic indication detects a form of PCNA only found
in malignant cells/tissue. Based upon the abundance of the cancer
specific form of csPCNA relative to the abundance of nPCNA the
degree or extent of malignancy can be estimated. Using an ELISA
assay, e.g., one whose results are shown in FIG. 8, the abundance
of PCNA can be measured relative to a set of standards of csPCNA of
varying abundance. Comparison of the experimentally determined
absorbance (using the ELISA) for the sample to the absorbance of
the standards can be used to indicate the abundance of the csPCNA
in the sample.
[0057] Accordingly, in another embodiment, the above described
objects of the present invention have been met by an immunoassay
for detecting csPCNA comprising:
[0058] (1) contacting a test sample with a peptide comprising the
amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPhePheArgLeuAlaGlnGlnGluLysG-
lu AspAlaLysArg (SEQ ID NO:1), which has been immobilized on a
solid support so as to bind csPCNA to said peptide to form a
peptide csPCNA complex; and
[0059] (2) contacting said peptide-csPCNA complex with an anti-PCNA
antibody and detecting binding of said antibody to said
complex.
[0060] The particular test sample employed is not critical to the
present invention and may include any of the tissue or body fluid
samples discussed above.
[0061] The particular format of the immunoassay of the present
invention is not critical to the present invention. Examples of
such formats include an ELISA, radio-immuno assay, dot blot assay,
slot blot assay, immunoprecipitation and protein quantification,
immuno-PCR, and Western blot.
[0062] Antibodies to PCNA can be prepared by challenging mammals
(e.g., rabbits, goats, horses, etc) with the peptide sequence which
is post-translationally modified in nPCNA, but not in csPCNA. All
commercially available antibodies to PCNA that exist to date
(.about.10) recognize the interdomain connector loop. They cannot
distinguish csPCNA from nPCNA. Identification of the amino acid
sequence of PCNA that is bound by the XPG peptide only in the
csPCNA form of PCNA provides the target for preparing selective
antibodies recognizing only csPCNA.
[0063] The detectable enzyme employed in the ELISA is not critical
to the present invention. Examples of such detection enzymes
include horse radish peroxidase and alkaline phosphatase.
[0064] As discussed above, the partner of the fusion protein is not
critical to the present invention and examples of such partners
include GST, Calmodulin Binding Protein and
oligo(6.times.)histidine.
[0065] The particular mode of immobilization of the fusion protein
on the solid support is not critical to the present invention.
[0066] It is preferable that the fusion protein is immobilized on
the solid support via biotin-streptavidin conjugation.
[0067] The following examples are provided for illustrative
purposes only, and are in no way intended to limit the scope of the
present invention.
Example 1
Isolation of Total PCNA
[0068] Total PCNA was isolated using a series of centrifugation and
chromatographic steps as shown in FIG. 1, and discussed in detail
below.
[0069] A. Nuclear Extract (NE)
[0070] Human breast cancer cells, MCF7 cells (ATCC No. HTB-22),
were grown in DMEM which was supplemented with 10% (v/v) fetal
bovine serum, 1.0% (w/w) penicillin/streptomycin, and non-essential
amino acids, and then frozen until use. 13.9 g of MCF7 cells were
resuspended in 1 volume of homogenization buffer comprising 200 mM
sucrose, 50 mM HEPES (pH 7.5), 5.0 mM KCl, 2.0 mM DTT and 0.1 mM
PMSF, and bounce homogenized for 30 strokes. The homogenate (H) was
then centrifuged at 3000 rpm for 10 min, the cytosolic supernatant
removed (S1) and 10 ml of nuclear extraction buffer comprising 50
mM KCl, 50 mM HEPES (pH 7.5), 5.0 mM MgCl.sub.2, 5.0 mM EDTA, 5.0
mM EGTA, 1.0 mM DTT and 0.1 mM PMSF, was added to the nuclear
pellet.
[0071] The resulting nuclear pellet was rocked at 4.degree. C. for
2 hour. The nuclear pellet was centrifuged at 2,500 rpm for 10 min.
The resulting supernatant was removed and centrifuged at
100,000.times.g in a Ti50.2 rotor for 1 hr. The resulting
supernatant, i.e., nuclear extract (NE), was collected.
[0072] EDTA and EGTA were added to S1 to 5.0 mM, and the resulting
fraction was centrifuged in an SS34 rotor at 17,000.times.g for 15
min. The resulting post-mitochondrial supernatant (S2) was
collected, and centrifuged at 100,000.times.g for 1 hr in a Ti50.2
rotor. The resulting post mitochondrial and post-microsomal
cytosolic supernatant (S3), was collected.
[0073] The NE was then combined with the S3 to obtain an NE/S3
fraction.
[0074] B. Phosphocellulose Column
[0075] Phosphocellulose (PC) was resuspended in a low salt buffer
(LS) comprising 200 mM KCl, 50 mM HEPES (pH 7.5), 5.0 mM
MgCl.sub.2, 1.0 mM DTT and 0.1 mM PMSF. A 20 ml column was poured
and attached to a BioRad Biologic system. 25 ml of the NE/S3
fraction was loaded onto the column, and the flow through (PCFT)
collected. A low salt fraction (PCLS) was then collected by washing
the column with 150 ml of LS buffer. A high salt fraction (PCHS)
was then by washing the column collected with 150 ml of high salt
buffer (HS) comprising 1.0 M KCl, 50 mM HEPES (pH 7.5), 5.0 mM
MgCl.sub.2, 1.0 mM DTT and 0.1 mM PMSF. The fractions were analyzed
by Western blot analysis as described below, to determine which
fractions contained PCNA.
[0076] C. Phenylsepharose Column
[0077] A phenylsepharose column (PS) was prepared as directed by
the manufacturer (Sigma Chemical Co.) and a 6.0 ml column was
poured. The column was incubated with 40 ml pre-equilibration
buffer comprising 20 mM potassium phosphate (pH 7.0), 0.5 mM EDTA,
0.1 mM EGTA, 10% (v/v) glycerol, 1.0 M ammonium sulfate and 1.0 mM
DTT, then the identified PCNA-containing fraction from the PC
column was adjusted to 1.0 M ammonium sulfate and applied to the PS
column. The PS column was washed with 40 ml of wash buffer
comprising 20 mM potassium phosphate, 0.5 mM EDTA, 0.1 mM EGTA, 10%
(v/v) glycerol, 0.5 M ammonium sulfate and 1.0 mM DTT (PSW), and
the flow through collected (PSFT). Fractions were eluted with 40 ml
of elution buffer comprising 20 mM potassium phosphate (pH 7.0),
0.5 mM EDTA, 0.1 mM EGTA, 20% (v/v) glycerol, 10% (v/v) ethylene
glycol and 1.0 mM DTT (PSE).
[0078] The PSE fractions were dialyzed in a 0.5 M KCl buffer
comprising 0.5 M KCl, 50 mM HEPES (pH 7.5), 1.0 mM of protease
inhibitor cocktail (Sigma Chemical and/or Behringer-Mannheim and/or
Calbio-Chem), 5.0 mM MgCl.sub.2 and 1.0 mM DTT; a 0.2 M KCl buffer
comprising 0.2M KCl, 50 mM HEPES (pH 7.5), 1.0 mM of a protease
inhibitor cocktail (Sigma Chemical and/or Behringer-Mannheim and/or
Calbio-Chem), 5.0 mM MgCl.sub.2 and 1.0 mM DTT; and TDEG buffer
comprising 50 mM Tris (pH 7.5), 1.0 mM DTT, 1.0 mM EDTA, and 10%
(v/v) glycerol, and containing 100 mM KCl. The fractions were then
analyzed by Western Blot, as described below, to determine which
fractions contained PCNA, and the PCNA-containing fractions were
combined.
[0079] D. Q-Sepharose Column
[0080] A 5.0 ml Q-Sepharose column (BioRad) was attached to a
BioRad Biologic system. The combined PCNA-containing fractions from
the PS column were concentrated to 12.5 ml, and then applied to the
column. Fractions (1.0 ml) were eluted using TDEG buffer with a
salt gradient of 0.1 M KCl to 0.6 M KCl. The resulting fractions
(QS) were analyzed by Western blot, as described below, and the
PCNA-containing fractions were combined.
[0081] E. Western Blot Analysis
[0082] Western blot analysis was performed as described by Bechtel
et al, Cancer Res., 58:3264-3269 (1998). Specifically, Western blot
analysis was performed using an antibody against PCNA (Amersham)
1:1000, anti-mouse 1:3000 (Amersham) and detection using
chemiluminesence (Pharmacia).
[0083] The fractions collected at all of the steps of the
purifications process were run on a 10% (w/v) SDS-PAGE gel,
transferred to nitrocellulose and examined by Western blot analysis
(FIG. 2) using an antibody against PCNA or stained using Coomasie
blue (FIG. 3).
[0084] As shown in FIG. 2, PCNA was present in all of the fractions
through the phosphocellulose flow through (PCFT). Low levels of
PCNA were found in phosphocellulose low salt (PCLS) and high salt
(PCHS) fractions, but were undetectable in the 10 .mu.g/ml samples
loaded onto the gel. PCNA becomes concentrated in the
phenylsepharose eluant (PSE) and Q-sepharose (QS) fractions. The
stained gels (FIG. 3) illustrate that at each step, PCNA was
further purified through the Q-sepharose column.
Example 2
Isolation of csPCNA
[0085] The purified total PCNA from the Q-sepharose column was
subjected to extraction using a 29 amino acid peptide (SEQ ID NO:1)
derived from protein XPG, which had been fused to a GST tag. This
portion of the XPG protein, i.e., amino acids 981-1009, has been
shown to bind PCNA (Gary et al, supra).
[0086] A. Isolation of GST-XPG Fusion Protein
[0087] A peptide fragment of the protein XPG was cloned into E.
coli as a GST fusion protein, as described by Gary et al,
supra.
[0088] The transformed bacteria were grown overnight in a shaker at
37.degree. C. in 500 ml of Terrific broth comprising 23.5 g
Terrific broth powder (Life Technologies Inc., Gaithersburg, Md.),
2.0 ml glycerol and 100 .mu.g/ml Ampicillin. GST expression was
induced by adding 0.1 IPTG to the culture and incubating for 4 hrs.
The bacteria were collected by centrifugation in a GS lite rotor at
5000 rpm for 10 min. The pellet was resuspended in PBS and
centrifuged in a SS34 rotor at 5000 rpm for 10 min. The pellet was
resuspended in B-Per (Pierce, Rockford, Ill.), rocked at room
temperature for 10 min and centrifuged at 27,000.times.g for 10 min
in a SS34 rotor. The supernatant, i.e., bacterial lysate, was
collected.
[0089] Glutathione Sepharose 4B matrix beads were resuspended in
the storage buffer supplied by the manufacturer, 2.0 ml of the
resuspended beads were washed 10 volumes of 4.degree. C. PBS,
pelleted, and then the beads were resuspended in 1.0 ml of cold
PBS. The resulting GST beads were incubated with the bacterial
lysate for 30 min at room temperature and centrifuged in a table
top centrifuge at 2500 rpm to obtain GST-XPG-glutathione beads.
[0090] B. Purification of PCNA
[0091] The resulting GST-XPG-glutathione beads were equilibriated
in T.sub.50K.sub.300/P.sub.100 buffer comprising 50 mM Tris (pH
7.5), 300 mM KCl and 100 mM potassium phosphate (pH 7.4). The beads
were then incubated with the PCNA containing fractions from the
Q-Sepharose column for 30 min at 4.degree. C. and centrifuged at
2500 rpm in a table top centrifuge. The supernatant was decanted
and the beads were washed with equilibriation buffer comprising 50
mM Tris (pH 7.5), 300 mM KCl and 100 mM potassium phosphate (pH
7.4) (XPGS). PCNA was eluted by incubating the beads with elution
buffer comprising 0.50 mM Tris (pH 7.5) for 30 min at 4.degree. C.,
centrifuging in a microfuge and collecting the supernatant (XPGE).
The wash (XPGS) and eluant (XPGE) were examined by Western blot, as
described above, for the presence of PCNA, and by Coomasie
staining. The results are shown in FIGS. 2 and 3.
[0092] As shown in FIG. 2, both XPGE and XPGS fractions contained
PCNA. Further, as shown in FIG. 3, only a single protein was
present in the XPGE fraction and only a few proteins were present
in the XPGS fractions.
[0093] C. 2D Page
[0094] The XPGE was further analyzed by 2D PAGE as described below,
to determine which form of PCNA was present.
[0095] 2D PAGE was performed as described by Bechtel et al, supra.
Specifically, XPGE (20-40 .mu.g of protein) was loaded onto a
first-dimension tube gel comprising 9.2 M urea, 4.0% (w/v)
acrylamide, 2.0% (v/v) ampholytes (pH 3-10), and 20% (v/v) Triton
X-100. The polypeptides were separated along a pH gradient created
using 100 mM NaOH and 10 mM H.sub.3PO.sub.4. The tube gels were
then placed onto an 8.0% (w/v) acrylamide-SDS gel, and the
polypeptides were resolved by molecular weight. The proteins were
then examined by Western blot as described above. The results are
shown in FIG. 4.
[0096] As shown in FIG. 4, csPNCA is the only form of PCNA present
in the XPGE fraction.
[0097] D. Polymerase Assays
[0098] DNA polymerase .delta. activity was measured as described by
Han et al, Biochem. Pharm., 60:403-411 (2000). csPCNA has the
ability to stimulate the processivity of pol 6, as measured using
the assay conditions described by Han et al, supra. More
specifically, poly[dG-dC]/[dG-dC] was used as the template, at a
concentration of 0.2 OD.sub.260 units/ml, and the reaction mixture
contained 1.0-2.0 .mu.g of purified DNA synthesome protein, 10 mM
MgCl.sub.2, 10 mM dCTP, 25 mM HEPES (pH 5.9), 200 .mu.g/ml of
bovine serum albumin, 100 .mu.Ci/ml of [.sup.32P]dGTP, and 5.0%
(v/v) glycerol. The poly[dG-dC]/[dG-dC] template was boiled for 5
minutes and chilled on ice prior to use in the assay. The reaction
mixture containing these components was incubated at 37.degree. C.
for 15 minutes, and then spotted onto Whatman DE81 filters (Whatman
International Ltd, Maidstone, England). The amount of radiolabelled
nucleotide bound to the filters was quantified after washing the
filters with 10 ml per filter of 0.3 M NaPPi (pH 7.5); followed by
washing the filters three times with 10 ml per filter of 0.1 M
NH.sub.4 formate (pH 7.4). Afterwards, the filters were given a
final wash in 95% (v/v) ethanol and then air dried, placed in
scintillation vials, covered with 3 ml of scintillation fluid, and
placed in a Packard TriCarb 2100TR scintillation counter (Packard
Instruments Co., Meriden, Conn.).
[0099] The results of this assay demonstrate that addition of PCNA
to purified polymerase .delta. increases the processivity of
polymerase .delta. (See Bravo et al, Nature, 326:515-517 (1987);
Downey et al, Cancer Cells, 6:1211-1218 (1988); and Tan et al,
Proc. Natl. Acad. Sci., USA, 90:11014 (1986)). The addition of
csPCNA to purified polymerase .delta. was found to increase its
processitivity as reported (Tan et al, supra). The addition of
csPCNA to the synthesome preparation did little to increase
processitivity, presumably because the synthesome contains bound
PCNA.
Example 3
ELISA Assay
[0100] A. Isolation of GST-XPG Fusion Protein
[0101] The PCNA binding domain of XPG (SEQ ID NO:1) was ligated
into a pGEX-4T-1 expression vector (Amersham Pharmacia Biotech,
Inc., Piscataway, N.J.). Recombinant protein was expressed in BL21
(DE3) Escherichia coli using 0.8 mM IPTG. Cells were lysed using
B-Per reagents (Pierce, Rockford, Ill.); the lysate was incubated
with glutathione-agarose beads for 2 hours at 4.degree. C. and
subsequently centrifuged for 10 minutes at 3000 rpm. The lysate was
washed twice with PBS followed by elution with 10 mM reduced
glutathione (Sigma, Co., St. Louis, Mo.) in 50 mM Tris-HCl (pH
7.4).
[0102] The resulting purified XPG-GST was biotinylated using a
commercial ECL protein biotinylation kit (Amersham Pharmacia
Biotech. Inc., Piscataway, N.J.). Briefly, 1.0 mg of protein was
diluted in 1.0 ml of biocarbonate buffer (pH 8.6), and incubated
for 1 hour at room temperature, in 30 .mu.l of biotinylation
reagent per mg of protein. After incubation, the protein sample was
applied to a Sephadex G25 column and eluted with 5.0 ml of PBS (pH
7.4). Fractions of biotinylated XPG-GST protein were then
collected.
[0103] The protein profile of the biotinylated XPG-GST protein was
analyzed by 12% (w/v) SDS-PAGE and Silver Stain procedure (Bio-Rad
silver stained plus kit, Bio-Rad, Hercules, Calif.). To determine
the presence of XPG-GST protein, an anti-GST antibody (Amersham
Pharmacia Biotech, Inc., Piscataway, N.J.) was used at a 1:1000
dilution, followed by HRP-labeled anti-goat IgG (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) at a 1:12,000 dilution in
10% (v/v) blocking buffer. The presence of biotinylated fraction of
XPG-GST protein was detected using streptavidin-horse radish
peroxidase conjugated protein (Amersham Pharmacia Biotech, Inc.,
Piscataway, N.J.) at dilution of 1:6,000 in PBST. Immunodetection
was performed using ECL Western Blotting Detection Kit (Amersham
Pharmacia Biotech, Inc., Piscataway, N.J.). The biotinylated
XPG-GST was seen as a 32 kDa discrete protein band.
[0104] To determine a suitable concentration of biotinylated
XPG-GST protein which may be enough to saturate all
streptavidin-binding sites on wells of a 96-well
streptavidin-coated plate (Pierce, Rockford, Ill.), a validation of
the streptavidin surface of streptavidin-coated plate was first
carried out using biotinylated-HRP enzyme. Specifically, different
dilutions of biotinylated enzyme HRP (biotin-HRP) (Sigma, Co., St.
Louis, Mo.) stock solution were incubated for 1 hour at room
temperature, washed 5 times with PBS (pH 7.4) containing 0.1% (v/v)
Tween 20. Then, 100 .mu.l of substrate solution for HRP enzyme
(Sigma Co., St. Louis, Mo.) was added, followed by 15 minutes of
incubation at 25.degree. C. To stop the reaction, 50 .mu.l of stop
solution (0.5 M H.sub.2SO.sub.4) was added. Absorbance was measured
at 450 nm. Streptavidin-HRP (Sigma Co., St. Louis, Mo.) was used at
different dilutions in 0.1% (v/v) polyoxyethylenesorbitoan
Monolaurate in PBS (pH 7.4) (PBST) as a control to show
non-specific protein-protein interactions. The results are shown in
FIG. 5A.
[0105] As shown in FIG. 5A, streptavidin-coated plates were found
to be completely saturated by biotin-HRP even at 1:150,000 dilution
per well.
[0106] Since the streptavidin-coated plate was completely saturated
by biotin-HRP at 1:150,000 dilution, the same dilution of this
enzyme was used for determination of a suitable concentration of
biotinylated XPG-GST protein to use. Specifically,
streptavidin-coated plate was first incubated for 1 hour with
different concentrations of biotinylated XPG-GST protein (10-150
.mu.g/ml) at 25.degree. C. After washing 5 times with PBST,
biotin-HRP was added in a 1:150,000 dilution. HRP-linked enzyme was
detected by adding 3,3', 5,5'-tetramethylbenzidin liquid substrate
system for ELISA (Sigma) of substrate-reacting solution, and
absorbance was measured at 450 nm. The results are shown in FIG.
5B.
[0107] As shown in FIG. 5B, the optimal concentration of
biotinylated XPG-GST protein that was required for saturation of
all of the streptavidin-binding sites on the plate was 100
.mu.g/ml.
[0108] B. ELISA
[0109] A streptavidin-coated plate was incubated with 100 .mu.g/ml
of biotinylated XPG-GST protein for 1 hour at 25.degree. C. Then,
the wells of the plate were washed with PBST and incubated with
different dilutions of protein samples, isolated from MCF7 (ATCC
No. HTB-22) (Michigan Cancer Foundation, Detroit, Mich.) and MCF10A
(Michigan Cancer Foundation, Detroit, Mich.) breast cell lines. The
protein samples were obtained as follows.
[0110] MCF7 and MCF10A were cultured according to the protocols
provided by the American Type Culture Collection (ATCC). Briefly,
MCF7 cell cultures were maintained in Dulbecco's Modified Essential
Media (DMEM) supplemented with 5.0% (v/v) fetal bovine serum (FBS),
100 units/ml of penicillin, 100 .mu.g/ml of streptomycin, and 1.0%
(w/v) non-essential amino acids. MCF10A cultures were maintained in
DMEM/F12 supplemented with 5.0% (v/v) Ca.sup.++ horse serum, 10 mM
HEPES, 10 .mu.g/ml of insulin, 20 ng/ml of epidermal growth factor
(EGF), 100 ng/ml of cholera enterotoxin, 0.5 .mu.g/ml of
hydrocortisone, 100 units/ml of penicillin, and 100 .mu.g/ml of
streptomycin. Both cell types were grown as monolayers at
37.degree. C., in 5.0% CO.sub.2 atmosphere. Semiconfluent (50-75%)
cell cultures were harvested and washed three times with
phosphate-buffered saline (PBS), and then pelleted using low-speed
centrifugation, i.e., 200.times.g for 5 min at 4.degree. C. The
cell pellets were stored at -80.degree. C. until use. Next, the DNA
synthesome-enriched fraction, i.e., the P4 faction, was isolated
from the pellets of the non-malignant (MCF10A) and malignant (MCF7)
breast cell lines as described by Coll et al, Oncol. Res., 8(10,
11):435-447 (1996), and the protein concentration in the P4
fraction was determined by a colorimetric assay.
[0111] The protein profile of the P4 fraction isolated from MCF7
and MCF10A cells was analyzed by 12% (w/v) SDS-PAGE, Western blots
using PCNA antibody, and densitometric analysis. More specifically,
5-300 .mu.g of the samples were resolved in 12% (w/v) SDS-PAGE and
transferred to nitrocellulose membrane. Western blot analysis was
performed using a monoclonal PCNA antibody (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.). The values after scanning
Western blot fluorograms, were expressed in Arbitary units (Au).
Discrete protein bands were quantified using BIORAD GS 710 Imaging
Densitometer. The results of the densitometric analyses are shown
in FIG. 6, which reflects the average of three independent assays,
expressed as means.+-.SE.
[0112] As shown in FIG. 6, the results demonstrate that the P4
fraction isolated from MCF7 cells contains twice the PCNA protein
than that found in the P4 fraction isolated from MCF10A cells. Even
when this result was taken in consideration as discussed below, XPG
peptide was still able to distinguish csPCNA in the two cell
lines.
[0113] Thereafter, dilutions of the P4 fraction from MCF7 and
MCF10A cells were added to the biotinylated XPG-GST coated plates.
Incubation was performed overnight at 4.degree. C. in buffer
comprising 20 mM Tris-HCl (pH 7.4), 60 mM NaCl, 300 mM KCl, and 100
mM KPO.sub.4, as described by Gary et al, supra. After washing with
PBST, the wells were incubated with PCNA antibody labeled with HRP
enzyme, for 1 hour at room temperature with constant agitation. The
HRP-conjugated monoclonal PCNA antibody was obtained from Santa
Cruz Biotechnology, Inc., Santa Cruz, Calif., and was used at
dilution of 1:500 in 10% (v/v) blocking buffer comprising PBS (pH
7.4), 0.1% (w/v) BSA and 0.05% (v/v) Tween 20. HRP enzyme was
detected with TMB (3,3',5,5'-tetramethylbenzidin) (Sigma) substrate
and absorbency was read at 450 nm. The results, which are shown in
FIG. 7A, reflect the average of three independent assays, expressed
as means.+-.SE.
[0114] As shown in FIG. 7A, XPG peptide is capable of
distinguishing two forms of PCNA in the ELISA assay.
[0115] Next, serial dilutions of the P4 proteins were tested in
duplicate in the ELISA and the mean value of absorbency was
calculated and used for comparisons. Standard curves, representing
the correlation between absorbency and the abundance of the
malignant and non-malignant form of PCNA, were prepared and
compared to each other. The results, which are shown in FIG. 7B
(MCF7) and FIG. 7C (MCF10A), represented the mean values from three
independent experiments.
[0116] As shown in FIGS. 7B and 7C, the XPG peptide was found to
have a higher binding affinity for csPCNA present in MCF7 breast
cell lines.
[0117] Next, MCF10A and MCF7 cells were grown as described above.
Semi-confluent (50-75%) cell cultures were harvested and washed
three times with PBS, and then pelleted using low-speed
centrifugation, i.e., 200.times.g for 5 min at 4.degree. C. The
cell pellets were stored at -80.degree. C. until use. Next, the DNA
synthesome-enriched fraction, i.e., the P4 faction, was isolated
from the pellets of the non-malignant (MCF10A) and malignant (MCF7)
breast cell lines as described by Coll et al, Oncol. Res., 8(10,
11):435-447 (1996).
[0118] The P4 fraction isolated from MCF7 and MCF10A cells was
analyzed by ELISA. Specifically, the P4 fraction was added to
biotinylated XPG-GST coated plates. Incubation was performed
overnight at 4.degree. C. in the buffer comprising 20 mM Tris-HCl
(pH 7.4), 60 mM NaCl, 300 mM KCl, 100 mM KPO.sub.4 as described
above. After washing with PBST, the wells were incubated with PCNA
antibody (Santa Cruz Biotechnology, Inc.), labeled with HRP enzyme,
for 1 hour at room temperature with constant agitation. The PCNA
antibody was used at dilution of 1:500 in 10% (v/v) blocking buffer
comprising PBS (pH 7.4), 0.1% (w/v) BSA and 0.05% (v/v) Tween 20.
HRP enzyme was detected with TMB (3,3',5,5'-tetramethylbenzidin)
(Sigma) substrate and absorbency was read at 450 nm. The amount of
PCNA is indicated in arbitrary units. The results are shown in FIG.
8.
[0119] As shown in FIG. 8, and similar to the results in FIG. 6,
the P4 fraction isolated from MCF7 cells contains twice the PCNA
protein than that found in the P4 fraction isolated from the MCF10A
cells. Even when this result was taken in consideration (where 10
.mu.g of the P4 fraction isolated from MCF10 cells=1 arbitrary unit
PCNA; and 5.0 .mu.g of the P4 fraction isolated from MCF7 cells=1
arbitrary unit PCNA), XPG peptide was still able to distinguish
csPCNA in the two cell lines.
[0120] While the invention has been described in detail and with
reference to specific embodiments thereof, it will be apparent to
one skilled in the art that various changes and modifications can
be made therein without departing from the spirit and scope
thereof.
Sequence CWU 1
1
1129PRTHomo Sapiens 1Leu Lys Gln Leu Asp Ala Gln Gln Thr Gln Leu
Arg Ile Asp Ser Phe1 5 10 15Phe Arg Leu Ala Gln Gln Glu Lys Glu Asp
Ala Lys Arg 20 25
* * * * *