Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-02-11
2001-01-23
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06177244
ABSTRACT:
BACKGROUND OF THE INVENTION
Prostate growth, from normal to neoplastic, can be viewed as a change from paracrine regulation of epithelial growth to autocrine regulation (Culig et al. (1996)
The Prostate
28:392-405)). Additionally, prostate cancers, especially advanced tumors, are frequently insensitive to the normal mitogenic action of growth factors, suggesting that prostate cancer expresses constitutively one or more of those genes upregulated by growth factors. The identification of genes expressed differentially in prostate cancers of dissimilar phenotypes could give clues to genes important in malignant transformation and progression. Moreover, these genes could serve as prognostic and diagnostic markers as well as new targets for therapy. However, the identification of growth regulatory genes in prostate cancer has been difficult and has lagged behind comparable studies in other cell types.
Methods to identify differentially expressed genes in other tissues have included differential screening of cDNA libraries with selective probes, subtractive hybridization utilizing DNA/DNA hybrids or DNA/RNA hybrids, RNA fingerprinting and differential display (Mather et al. (1981)
Cell
23:369-378; Hedrick et al. (1984)
Nature
308:149-153; Davis et al. (1992)
Cell
51:987-1000; Welsh et al. (1992)
Nucleic Acids Res.
20:4965-4970;
and Liang and Pardee (1992)
Science
257:967-971). Recently, PCR-coupled subtractive processes have also been reported (Straus and Ausubel (1990)
Proc. Natl. Sci. USA
87:1889-1893; Sive and John (1988)
Nucleic Acids Res.
16:10937; Wieland et al. (1990)
Proc. Natl. Acad. Sci. USA
87:2720-2724; Wang and Brown (1991)
Proc. Natl. Acad Sci. USA
30 88:11505-11509; Lisitsyn et al. (1993)
Science
259:946-951; Zeng et al. (1994)
Nucleic Acids Res.
22:4381-4385; Hubank and Schatz (1994)
Nucleic Acids Res.
22:5640-5648). Each of these methods has achieved some success but each has some inherent limitations. For example, problems associated with differential display include identification of “false positives,” redundancy, and under-representation of certain mRNA species. Liang and Pardee (1992)
Science
257:967-971. In addition, cDNA-RDA (Hubank and Schatz (1994)
Nucleic Acids Res.
22:5640-5648; Lisitsyn et al. (1993)
Science
259:946-951) is a labor-intensive process, and its efficiency remains to be evaluated. Some advances have been made in isolating genes expressed preferentially in prostate cancer versus normal tissue (Shen et al. (1995)
PNAS USA
92:6778-6782; Wang et al. (1996)
Cancer Res.
56:3634-3637; Liu et al. (1997)
The Prostate
30:145-153). Less data exist regarding differential expression of novel genes in human prostate cancers of different growth and metastatic potential (Blok et al. (1995)
The Prostate
26:213-224).
Thus a need still exists to develop a more efficient method for identification of differentially expressed genes and differences in genomic sequences.
Accordingly, an object of the invention is to provide a differentially expressed gene isolated by the methods described herein.
SUMMARY OF THE INVENTION
The transition from androgen-dependent to aggressive androgen-independent growth is an important development in prostate cancer, and the identification of genes expressed differentially between these two phenotypes can provide new therapeutic targets as well as prognostic markers.
The inventors have developed a novel method termed “Linker Capture Subtraction” (LCS), which overcomes many of the problems associated with present methods for identifying differentially expressed genes and differences in genomic sequences. Unlike other methods such as representational difference analysis (RDA) (Hubank and Schatz (1994)
Nucleic Acids Res.
22:5640-5648; Lisitsyn et al. (1993)
Science
259:946-951) LCS is a subtraction method coupled to PCR amplification which does not rely on a kinetic mechanism of enrichment of selected sequences. Rather, LCS achieves enrichment by specifically preserving PCR-priming sites of target sequences, using a nuclease which digests single-stranded nucleic acid as the mediator. Moreover, LCS is also a less labor-intensive process.
Using LCS, several genes expressed differentially between the human prostate carcinoma cell lines LNCaP and PC-3, which are androgen-dependent and androgen-independent, respectively, have now been isolated. One of these genes, designated NPG-1, was expressed by the majority of primary tumors examined. Moreover, the degree of NPG-1 expressed correlated with an aggressive histopathology.
Accordingly, the invention pertains to isolated nucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequence encoding an NPG-1 protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of NPG-1 -encoding nucleic acid (e.g., mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:1, or the coding region or a complement of this nucleotide sequence. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more homologous to the entire nucleotide sequence shown in SEQ ID NO:1, or a portion of this sequence. In other preferred embodiments, the isolated nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2. The preferred NPG-1 proteins of the present invention also preferably possess at least one of the NPG-1 activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2, e.g., sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains an NPG-1 activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to modulate tumor cell adhesion, e.g., malignant prostate cell adhesion. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more homologous to the amino acid sequence of SEQ ID NO:2.
In yet another embodiment, the isolated nucleic acid molecule is derived from a human and encodes a portion of a protein which includes a type I thrombospondin repeat domain. Preferably, the type I thrombospondin repeat domain encoded by the human nucleic acid molecule is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more homologous to the type I thrombospondin repeat domain (i.e., amino acid residues 73-109) of SEQ ID NO:2 which is shown as a separate sequence designated SEQ ID NO:3.
In another preferred embodiment, the isolated nucleic acid molecule is derived from a human and encodes a protein (e.g., an NPG-1 fusion protein) which includes a type I thrombospondin repeat domain which is at least about 50% or more homologous to SEQ ID NO:3 and has one or more of the following activities: 1) it can bind to the extracellular matrix, e.g., the basal lamina; 2) it can interact with a growth factor, e.g., VEGF; 3) it can interact with a cytokine; 4) it can modulate tumor cell adhesion; 5) it can modulate tumor cell invasion; 6) it can modulate tumor cell migration; 7) it can modulate tumor metastasis; 8) it can modulate tumor angiogenesis; 9) it can modulate extracellular matrix degradation, e.g., via tumor secreted proteases; 10) it can modulate cell proliferation; and 11) it can modulate tissue architecture and differentiation, e.g., cellular architecture and differentiation.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1. Preferably, the isolated nucleic acid molecu
Sytkowski Arthur J.
Yang Meiheng
Beth Israel Deaconess Medical Center Inc.
Campbell Eggerton A.
Fish & Richardson P.C.
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