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Cancer Patent Abstract
A method for purifying cancer-specific Proliferating Cell Nuclear
Antigen (csPCNA) is described, as well as an immunoassay based thereon.
Cancer Patent Claims
What is claimed is:
1. A method for purifying cancer-specific Proliferating Cell Nuclear
Antigen (csPCNA) comprising the steps of: (A) obtaining a tissue
or body fluid sample comprising csPCNA; (B) contacting said sample
with a peptide comprising the amino acid sequence LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPhePheArgLeu
AlaGlnGlnGluLysGluAspAlaLysArg (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 (C) isolating csPCNA from said peptide-csPCNA
complex so as to purify said csPCNA.
2. The method of claim 1, wherein prior to step (B) the thus obtained
tissue or body fluid sample of step (A) is subjected to a process
comprising: (1) homogenizing cells constituting said tissue or body
fluid to obtain a homogenate (H); (2) separating said H into a nuclear
pellet fraction (NP) and a cytosolic fraction (S1); (3) extracting
nuclei from said NP to obtain a nuclear extract (NE); (4) subjecting
said S1 to centrifugation to obtain a post-mitochondrial cytosolic
supernatant (S2); (5) subjecting said S2 to centrifiigation to obtain
a post-mitochondrial/post-microsomal cytosolic supematant (S3);
(6) combining said NE and said S3 to form an NE/S3 fraction, applying
the resulting NE/S3 fraction to a weak anion exchange matrix column
and collecting the flow through (PCFT); (7) applying the resulting
PCFT to a hydrophobic chromatography matrix column, eluting the
column with buffer comprising ethylene glycol and collecting the
eluant (PSE); (8) dialyzing out ethylene glycol present in the PSE
to obtain a dialyzate; and (9) applying the resulting dialyzate
to 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.
3. The method of claim 1 or 2, wherein said tissue or body fluid
sample of step (A) further comprises native PCNA (nPCNA), said nPCNA
does not bind to said peptide in step (B), whereas said csPCNA binds
to said peptide in step (B) to form a peptide-csPCNA complex and
in step (C) isolating csPCNA is effected using an elution buffer
whereby csPCNA is eluted from said csPCNA-complex.
4. An immunoassay for detecting csPCNA comprising: (1) contacting
a test sample with a peptide comprising the amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIleAspSerPheP heArgLeuAlaGlnGlnGluLysGluAspAlaLysArg
(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 (2) contacting said peptide-csPCNA complex with an anti-PCNA
antibody and detecting binding of said antibody to said complex.
5. The immunoassay of claim 4, wherein said assay is an ELISA and
said antibody is labeled with a detectable enzyme.
6. The imniunoassay of claim 5, wherein said enzyme is horse radish
peroxidase.
7. The immunoassay of claim 5, wherein said peptide is a fusion
protein comprising said peptide and Glutathione-S-Transferase.
8. The inimunoassay of claim 7, wherein said fusion protein is
biotinylated and immobilized on said solid support via streptavidin-coated
on said solid support.
9. The method of claim 1, wherein the tissue or body fluid sample
obtained is afflicted with a cancer selected from the group consisting
of carcinomas, sarcomas, lymphomas, and leukemias.
10. The method of claim 9, wherein the tissue or body fluid sample
obtained is selected from the group consisting of cervical, mammary
glands, esophageal, glial cells, lung, stomach, intestine, prostate,
white blood cells, urine, serum, and whole blood.
11. The method of claim 1, wherein said peptide is a fusion protein
comprising said peptide and at least one of Glutathione-S-Transferase,
Calmodulin Binding protein, or oligo (6X) histidine.
12. The immunoassay of claim 4, wherein the test sample is afflicted
with a cancer selected from the group consisting of carcinaomas,
sarcomas, lymphomas, and leukemias.
13. The immunoassay of claim 12, wherein the test sample is obtained
by selecting from the group consisting of cervical, mammary glands,
esophageal, glial cells, lung, stomach, intestine, prostate, white
blood cells, urine, serum, and whole blood.
14. The immunoassay of claim 4, wherein said immunoassay is selected
from the group consisting of radio-immunoassay, dot blot assay,
slot blot assay, immunoprecipitation, protein quantification, imunno-PCR,
and Western blot.
15. The method of claim 1, further comprising the step of producing
antibodies (monoclonal or polyclonal) specific for csPCNA from said
isolated and purified csPCNA.
Cancer Patent Description
FIELD OF THE INVENTION
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
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 cancers are unique and distinct
from other cells, as well as other cancers. 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.
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.
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.
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
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
.delta., 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.
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.
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)).
Xeroderma Pigmentosum (XP)G protein 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' since
of a DNA lesion, during nucleotide excision repair.
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
An object of the present invention is to provide a method for selectively
purifying csPCNA.
Another object of the present invention is to provide an ELISA
for detection of csPCNA and early detection of malignancy.
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: (A) obtaining a tissue or body fluid sample
comprising csPCNA; (B) contacting said sample with a peptide comprising
the amino acid sequence LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArg IleAspSerPhePheArgLeuAlaGlnGlnGluLys
GluAspAlaLysArg (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 (C) isolating csPCNA from said peptide-csPCNA complex
so as to purify said csCNA.
In another embodiment, the above-identified objects have been met
by an immunoassay for detecting csPCNA comprising: (1) contacting
a test sample with a peptide comprising the amino acid sequence
LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIle AspSerPhePheArgLeuAlaGlnGlnGluLysGluAsp
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 (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
FIG. 1 shows a flow chart depicting steps involved in the isolation
of total PCNA and csPCNA.
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.
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.
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.
FIG. 5A shows the results of a validation study on the viability
of the streptavidin surface of streptavidin-coated plates.
FIG. 5B shows the results of a study for determining the maximal
binding capability of biotinylated XPG-GST protein to streptavidin-coated
plates.
FIG. 6 shows denisitometric analyses of total PCNA in P4 fractions
from MCF7 and MCF10A cells.
FIGS. 7A-7C show the results of ELISAs using MCF7 P4 and MCF10A
P4 proteins.
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
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: (A) obtaining a tissue or body fluid
sample comprising csPCNA; (B) contacting said sample with a peptide
comprising the amino acid sequence LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArg
IleAspSerPhePheArgLeuAlaGlnGlnGluLys GluAspAlaLysArg (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 (C) isolating
csPCNA from said peptide-csPCNA complex so as to purify said csCNA.
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: (1) homogenizing cells constituting said tissue or body
fluid to obtain a homogenate (H); (2) separating said H into a nuclear
pellet fraction (NP) and a cytosolic fraction (S1); (3) extracting
nuclei from said NP to obtain a nuclear extract (NE); (4) subjecting
said S1 to centrifugation to obtain a post-mitochondrial cytosolic
supernatant (S2); (5) subjecting said S2 to centrifugation to obtain
a post-mitochondrial/post-microsomal cytosolic supernatant (S3);
(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);
(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); (8) dialyzing out
ethylene glycol present in the PSE to obtain a dialyzate; and (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.
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).
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.
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.
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.
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.
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.
The resulting preferred 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.
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.
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.
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).
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.
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.
Accordingly, in another embodiment, the above-described objects
of the present invention have been met by an immunoassay for detecting
csPCNA comprising: (1) contacting a test sample with a peptide comprising
the amino acid sequence LeuLysGlnLeuAspAlaGlnGlnThrGlnLeuArgIle
AspSerPhePheArgLeuAlaGlnGlnGluLysGluAsp 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 (2) contacting
said peptide-csPCNA complex with an anti-PCNA antibody and detecting
binding of said antibody to said complex.
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.
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.
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.
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.
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.)histadine.
The particular mode of immobilization of the fusion protein on
the solid support is not critical to the present invention.
It is preferable that the fusion protein is immobilized on the
solid support via biotin-streptavidin conjugation.
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
Total PCNA was isolated using a series of centrifugation and chromatographic
steps as shown in FIG. 1, and discussed in detail below.
A. Nuclear Extract (NE)
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 dounce 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.
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.
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.
The NE was then combined with the S3 to obtain an NE/S3 fraction.
B. Phosphocellulose Column
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.
C. Phenylsepharose Column
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).
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.
D. Q-Sepharose Column
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.
E. Western Blot Analysis
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).
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).
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
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).
A. Isolation of GST-XPG Fusion Protein
A peptide fragment of the protein XPG was cloned into E. coli as
a GST fusion protein, as described by Gary et al, supra.
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.
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.
B. Purification of PCNA
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 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.
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.
C. 2D PAGE
The XPGE was further analyzed by 2D PAGE as described below, to
determine which form of PCNA was present.
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.
As shown in FIG. 4, csPNCA is the only form of PCNA present in
the XPGE fraction.
D. Polymerase Assays
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 .delta., 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.).
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
A. Isolation of GST-XPG Fusion Protein
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).
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.
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.
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) Tween20). 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.
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.
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.
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.
B. ELISA
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.
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.
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.
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.
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) Tween20. 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.
As shown in FIG. 7A, XPG peptide is capable of distinguishing two
forms of PCNA in the ELISA assay.
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.
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.
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).
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) Tween20. 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.
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.
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.
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