Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Reexamination Certificate
1999-10-15
2003-09-09
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
C530S300000, C530S350000, C530S387100, C530S388800, C435S007100
Reexamination Certificate
active
06617432
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to nuclear matrix proteins, called “NMPs” here, and more specifically to novel nuclear matrix proteins of the prostate which are associated with cell-proliferative disorders.
The early diagnosis of prostate cancer is central to the effective treatment of the disease. In addition, the ability to differentiate disease with metastatic ability from prostate cancer that is not able to metastasize is important. Nuclear structural alterations are so prevalent in cancer cells that they are commonly used as a pathological marker of transformation for many types of cancer. Nuclear shape is determined in part by the nuclear matrix, the dynamic skeleton of the nucleus.
The nuclear matrix is the structural component of the nucleus that determines nuclear morphology, organizes the DNA in a three-dimensional fashion which is tissue specific, and has a central role in the regulation of a number of nuclear processes including the regulation of gene expression. The nuclear matrix has been demonstrated to play a central role in the regulation of important cellular processes such as DNA replication and transcription. Getzenberg,
J. Cell Biochem
. 55: 22-31 (1994). The nuclear matrix is the framework or scaffolding of the nucleus and consists of the peripheral laminas and pore complexes, an internal ribonucleic protein network, and residual nucleoli. Berezney et al.,
Biochem. Biophys. Res. Comm
. 60: 141017 (1974). The nuclear matrix consists of approximately 10% of the nuclear proteins and is virtually devoid of lipids, DNA and histones. Fey et al.,
Critical Reviews in Eukaryotic Gene Expression
1: 127-44 (1991).
A majority of the known NMPs are common to all cell types and physiologic states. A number of laboratories have identified NMPs which may be unique to certain cell types or states. Mitogenic stimulation and the induction of differentiation have been demonstrated to alter the composition of nuclear matrix proteins and structure. The nuclear matrix contains a number of associated proteins that have been demonstrated to be involved in transformation. Berezney first showed that the nuclear matrix is altered in transformation, examining hepatoma nuclear matrix proteins. Berezney et al.,
Cancer Res
. 39: 3031-39 (1979). Fey and Penman demonstrated that tumor promoters induce a specific morphologic signature in the nuclear matrix-intermediate filament scaffold of kidney cells. Fey et al.,
Proc. Nat'l Acad. Sci
. USA 81: 859-66 (1984). Fey and Penman went on to demonstrate that the pattern of NMPs differed between normal and tumorigenic cell lines. Fey et al., loc. cit. 85: 121-25 (1989). An antibody to a nuclear matrix protein, termed NM-200.4, was raised from the breast carcinoma cell line T47D. Weidner et al.,
Am. J. Path
. 138: 1293-98 (1991). This antibody reacts strongly with human breast carcinoma specimens as well as specimens from lung, thyroid, and ovarian cancers, but does not react with normal epithelial cells of similar origin, raising the possibility of the use of certain anti-NMP antibodies as diagnostic tools.
U.S. Pat. No. 5,824,490 discloses certain nuclear matrix proteins associated with prostate tissue, including one denoted “PC-1” used to identify prostate cancer. When human prostate samples were examined, nuclear matrix proteins were identified that (1) were present only in the normal prostate and were missing in both prostate cancer and benign prostatic hyperplasia (BPR) (normal pattern), (2) were found only in the prostate cancer cells and missing in the normal prostate and BPH (prostate cancer pattern), and (3) were found in both normal and BPH samples but were absent from prostate cancers. See also Getzenberg et al.,
Cancer Res
., 1991(51):6514-20.
Additional nuclear matrix proteins associated with prostate tissue are disclosed in Partin et al., Cancer Res. 1993(53): 744-746.
SUMMARY OF THE INVENTION
The present invention relates to novel nuclear matrix proteins that are able to differentiate cancerous cells from normal cells and metastatic cancer cells from non-metastatic disease, polynucleotide sequences encoding them, polynucleotide sequences hybridizing to the sequences encoding them, antibodies directed against them, and their methods of use. In particular, these proteins are useful for diagnosing and producing treatments for cell proliferative disorders of the prostate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to one aspect, the present invention comprehends a purified nuclear matrix protein (NMP) or a fragment thereof, which is absent in normal prostate cells but present in cancerous prostate cells. Preferably, the protein of this embodiment is selected from the those, as described below, that are designated “D-1,” “D-2,” and “D-3.” The proteins D-1, D-2, and D-3 are present in both metastatic and non-metastatic cancerous prostate cells.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is present in normal prostate cells but absent in cancerous prostate cells. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated NDP-1, NDP-2, NDP-3, NDP-4, NDP-5, NDP6, NDP-7, NDP-8, NDP-9, and NDP-10.
According to another aspect, the present invention is directed generally to a method for differentiating a metastatic cell (preferably, prostate) from a non-metastatic cell (preferably prostate) comprising determining the presence or absence of a nuclear matrix protein unique to a metastatic cell or a non-metastatic cell. In particular, nuclear matrix proteins of the prostate are disclosed herein which are capable of differentiating between a metastatic cancer cell and non-metastatic cell of the prostate.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is present in metastatic but absent in non-metastatic prostate tumors and absent in normal prostate cells. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated as AM-1 and AM-2.
According to another aspect, the present invention is directed to a purified NMP or a fragment thereof, which is absent in metastatic but present in non-metastatic prostate tumors. Preferably, the protein of this embodiment is selected from the proteins described below which have been designated as G-1 and G-2. These two proteins, G-1 and G-2, are also not present in normal prostate cells.
Preferably, the fragments of the NMPs in the above embodiments are immunogenic fragments. It also is preferable that, in the embodiments discussed above, the NMPs are derived from humans.
Unless otherwise specified, the terms “a”, “an” or “the” mean one or more.
The phrase “purified nuclear matrix protein” means a protein of the nuclear matrix which has been separated from at least one cellular component. The phrase covers both purified nuclear matrix proteins produced recombinantly and those produced by extraction from a natural source.
Another embodiment of the present invention is a purified polynucleotide sequence encoding the above-identified NMPs or NMP fragments of the preceding embodiments. Another embodiment is a purified polynucleotide sequence which hybridizes to the polynucleotide sequence encoding the above-mentioned NMPs or NMP fragments.
Another embodiment is a host cell transformed with a polynucleotide sequence encoding the above-mentioned NMPs or NMP fragments. Transformation of a host cell with recombinant DNA may be carried out by conventional techniques known in the art. Where the host is prokaryotic, such as
E. coli
, competent cells which are capable of DNA uptake can be prepared from cells harvested after the exponential growth phase and subsequently treated by the CaC
1
2
method by procedures well known in the art. Alternatively, MgC
1
2
or RbC
1
can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods
Davis Minh Tam
Ungar Susan
University of Pittsburgh
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