Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-05
2002-01-29
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C530S350000
Reexamination Certificate
active
06342594
ABSTRACT:
BACKGROUND OF THE INVENTION
In order for metastasis of cancer to occur, several hurdles must be overcome, such as degradation of the extracellular matrix and basal membrane, intra- and extravasation of vessels of the blood and of the lymphatic system, escape by the attack of the immune system, and homing and colonization of distant organs (Pardee, A. B., Advances in Cancer Res. 65 (1994) 213-227; Ponta, H., et al., Biochem. Biophys. Acta 1198 (1994) 1-10). A further level of complexity is achieved in that different types of cancers make use of different molecular mechanisms for metastasis and exhibit different tropism of metastasis.
Metastasizing and non-metastasizing human melanoma cell lines have been important tools in identifying differentially expressed genes and for investigation of their role in metastasis (Weterman, M .A .J., et al., Cancer Res. 52 (1992) 1291-1296; Weterman, M. A. J., et al., Int. J. Cancer 53 (1993) 278-284; Van Groningen, J. M., et al., Cancer Res. 55 (1995) 6237-6243; Weterman, M. A. J. , et al., Int. J. Cancer 60 (1995) 73-81; van Muijen, G. N. P., et al., Int. J. Cancer 48 (1991) 85-91; van Muijen, G. N. P., et al., Clin. Exp. Metastasis 9 (1991) 259-272). Cell adhesion molecules play an important role in the invasion, dissemination, extravasation and lodging of tumor cells. The interaction of disseminated tumor cells with endothelium and tissue stroma is supposed to be one of the critical steps in tumor progression and metastasis formation (Ebnet, K., et al., Annu. Rev. Immunol. 14 (1996) 155-177; Varner, J. A., and Cheresh, D. A., Curr. Opin. Cell Biol. 8 (1996) 724-730; Albelda, S. M., Lab. Invest. 68 (1993)4-17).
SUMMARY OF THE INVENTION
In accordance with the present invetion, a protein, termed URIM, is provided which is upregulated in metastatic cancer cells as compared to their non-metastatic counterparts. URIM may be involved in promotion of several steps of the metastatic cascade. The URIM gene codes for a polypeptide of SEQ ID NO:2.
The present invention provides an isolated nucleic acid which is upregulated in metastatic tumor cells and which codes for a polypeptide which induces tumor progression or metastasis, the nucleic acid being selected from the group consisting of:
(a) SEQ ID NO: 1;
(b) a nucleic acid sequence which hybridizes under stringent conditions with a nucleic acid probe selected from the group consisting of the complementary sequence of (a), SEQ ID NO:6 and SEQ ID NO:7;
(c) a nucleic acid sequence which, because of the degeneracy of the genetic code, is not a sequence of (a) or (b), but which codes for a polypeptide having exactly the same amino acid sequence as a polypeptide encoded by a sequence of (a) or (b); and
(d) a nucleic acid sequence which is a fragment of any of the sequences of (a), (b) or (c).
The present invention further provides a purified and isolated polypeptide having a sequence of SEQ ID NO:2.
The present invention further provies a process for detecting the presence or absence of at least one specific nucleic acid or mixture of nucleic acids, or distinguishing between two different sequences in said sample, wherein the sample is suspected of containing said sequence or sequences, which process comprises the following steps in order:
(a) incubating said sample under stringent hybridization conditions with a nucleic acid probe which is selected from the group consisting of;
(i) a nucleic acid sequence taken from the group consisting of SEQ ID NO: 1, SEQ ID NO:6 and SEQ ID NO:7;
(ii) a nucleic acid sequence which is exactly complementary to any nucleic acid sequence of (i);
(iii) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (i); and
(iv) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (ii); and
(b) determining whether said hybridization has occurred.
Moreover, the present invention provides a process for determining whether or not a cancer cell-containing test sample has potential for tumor progression or metastasis, wherein the test sample and a cancer cell-containing sample wherein is free from metastasis are obtained from the same individual or different individuals of the same species, which process comprises the following steps:
(a) incubating each respective sample under stringent hybridization conditions with a nucleic acid probe which is selected from the group consisting of:
(i) a nucleic acid sequence taken from the group consisting of SEQ ID NO: 1, SEQ ID NO:6 and SEQ ID NO:7;
(ii) a nucleic acid sequence which is exactly complementary to any nucleic acid sequence of (i);
(iii) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (i); and
(iv) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (ii); and
(b) determining the approximate amount of hybridization of each respective sample with said probe, and
(c) comparing the approximate amount of hybridization of the test sample to an approximate amount of hybridization of the sample which is free from metastasis, to identify whether or not the test sample contains a greater amount of the specific nucleic acid or mixture of nucleic acids than does the sample which is free from metastasis.
Furthermore, the present invention provides an isolated nucleic acid which inhibits a nucleic acid in inducing tumor progression and metastasis, said isolated nucleic acid having a sequence selected from the group consisting of:
(a) a nucleic acid sequence which is exactly complementary to SEQ ID NO: 1; and
(b) a nucleic acid sequence which hybridizes under stringent conditions with the sequence of (a).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides the new gene URIM (Up-Regulated In Metastasis), a protein coded thereby, and use of the URIM gene for diagnostics and therapeutics, especially in the field of cancer. In particular, the invention involves the identification of said gene URIM in mammalian, especially in malignant tumor cells. The invention also relates to diagnosis of the metastatic and progression potential of tumor cells and to gene therapy methods to inhibit URIM in its function in tumor cells.
The invention comprises a nucleic acid molecule (URIM) which has upregulated expression in metastatic tumor cells and which is capable of inducing tumor progression and/or metastasis, especially in malignant melanoma and mammary carcinoma cells. The nucleic acid (URIM) has the sequence SEQ ID NO:1 or it is a nucleic acid which, because of the degeneracy of the genetic code, differs from SEQ ID NO:1, but which encodes the amino acid sequence encoded by the nucleic acid of SEQ ID NO:1.
The invention further comprises a recombinant polypeptide which is coded by the nucleic acid sequences according to the invention, preferably by the DNA sequence shown in SEQ ID NO:1.
The isolated URIM polypeptide can occur in natural allelic variations which differ from individual to individual. Such variations of the amino acids are usually amino acid substitutions. However, they may also be deletions, insertions or additions of amino acids to the total sequence. The URIM protein according to the invention—depending, both in respect of the extent and type, on the cell and cell type in which it is expressed—can be in glycosylated or non-glycosylated form. Polypeptides with metastatic activity can be identified by transfection of URIM-negative non-metastasizing tumor cells with expression vectors for URIM, establishment of stable transfectants and evaluation of in vitro invasiveness in Matrigel invasion assays and their metastatic capacity after xenografting into nude mice.
“Polypeptide with URIM activity or URIM” means also proteins with minor amino acid variations but with substantially the same URIM activity. Substantially the same means that the activities are of the same biological properties and the polypeptides show at least 90% homology (identity) in amino acid sequence.
The term “nucleic acid molecule or nucleic acid” denotes a polynucleotide molecule which can be, for example, a DNA, RNA
Hildebrandt Tobias
Van Muijen Goosen
Weidle Ulrich
Ebel Eileen M.
Helms Larry R.
Huff Sheela
Johnston George W.
Rocha-Tramaloni Patricia S.
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