Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-07-28
2002-08-27
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C536S023500
Reexamination Certificate
active
06440696
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions and methods of use for a novel putative GTPase activating protein (“GAP”) that interacts with papilloma virus E6 oncoprotein and is thereby targeted for degradation. Said novel protein, herein designated “E6TP1” for E6 targeted protein, its nucleic acid and polypeptide sequences, and derivatives, fragments, and analogues thereof, are described. The invention further relates to cells containing recombinant E6TP1 nucleic acid and protein sequences; animal models of the same, including transgenic and “knock-out” animal models; anti-E6TP1 specific antibodies, and derivatives, fragments, and analogues thereof; oligonucleotides hybridizable to sense or antisense E6TP1 nucleic acids; oligonucleotide pairs capable of PCR amplifying E6TP1; and use of any of the above compositions in diagnostics, therapeutics, and treatments of tumors. Specific embodiments include methods of use of E6TP1 in tumor suppression. In addition, the present invention relates to compositions and methods of use of E6TP1 in high-risk human papilloma virus (“HPV”) associated carcinomas of cervix and other anogenital tumors.
BACKGROUND OF THE INVENTION
Carcinomas, the tumors originating from epithelial cells, constitute nearly 80% of all human cancers. In the U.S. alone, a predicted 650,000 new cases of the carcinomas of lung, colon, breast, prostate and cervix will be diagnosed in 1998, and nearly 300,000 of these will be fatal. See, e.g., American Cancer Society Cancer Facts and Figures,
American Cancer Society, Inc.
(1998). Despite these astonishing statistics, much of our knowledge into cell biology of the oncogenic process stems from studies of fibroblasts, reflecting the available experimental models.
A critical event in carcinogenesis is the conversion of normal epithelial cells, with a finite proliferative potential, into cells that are endowed with an ability to multiply continuously, a trait that allows accumulation of further genetic alterations enroute to full malignancy. In vitro, this behavior manifests as continuous proliferation of cells beyond their limited life span. This process is referred to as immortalization. Understanding the biochemical basis of immortalization is therefore likely to identify crucial cellular pathways involved in cellular control. The recently identified ability of viral oncogenes to immortalize normally senescent epithelial cells has provided practical approaches to delineate the regulatory cascades involved in these critical paths.
For example, normal human mammoplasty-derived mammary epithelial cells reproducibly proliferate in vitro for about 20 passages, followed by their senescence. See, e.g., Band and Sager,
Proc Natl Acad Sci USA.,
86:1249-1253 (1989); Band,
Sem Cancer Biol,
6: 185-192 (1995). A similar finite life span is exhibited in vitro by other epithelial cells. A number of viral oncogenes, including SV40 large T and adenovirus E1A and E1B, were inefficient at immortalizing the epithelial cells, in sharp contrast to rodent fibroblasts which were efficiently transformed by these oncoprotein. See, e.g., Sager,
Cancer Cells,
2: 487-494 (1984). Strikingly, however, we found that transfection of the genome of high risk human papilloma viruses (“HPVs”)-16 or -18, which are naturally associated with epithelial oncogenesis in the genitourinary tract, led to reproducible and highly efficient immortalization of MECs. See, e.g., Band et al.,
Proc Natl Acad Sci USA,
87: 463-467 (1990). The HPV genome transfection also efficiently immortalized keratinocytes (a natural host epithelial cell type for these viruses) and other epithelial cell types. See, e.g., Kaur and McDougall, J Virol, 62: 1917-1924 (1988); Yeager et al.,
Cancer Res,
55: 493-497 (1995). In all of these cases, the HPV genome-transformed cells were immortal but not fully tumorigenic, as shown by their inability to grow in soft agar or form tumors when implanted in nude mice. However, when these immortal cells are transfected with additional oncogenes (e.g., activated H-ras or mutant erbB-2), they attained a tumorigenic phenotype. See, e.g., Woodworth, In: Neoplastic Transformation in Human Cell Culture: Mechanisms of Carcinogenesis, Eds. Rhim and Dritschilo, Humana Press, New Jersey, pp 153-161 (1991). Thus, introduction of the HPV genome into primary human epithelial cells induces a preneoplastic transformation.
The human papilloma viruses (“HPVs”) are associated with epithelial tumors or benign lesions, especially those of anogenital origin. See, e.g., Zur Hausen and Schneider, “The Papillomaviruses” In: The Papillomaviruses, Howley and Salzman (ed.), Vol. 2 of The Papovaviridae, (Plenum, New York) pp 245-263 (1987). These viruses are grouped into low-risk HPVs, such as HPV6 and HPV11, which are usually associated with benign warts, and high-risk HPVs, such as HPV 16 and HPV18, which are associated with carcinomas of cervix and other genital tumors. See, e.g., Zur Hausen, ibid. Previous studies have identified two viral oncoproteins “E6” and “E7”, which are expressed in the majority of HPV-associated carcinomas, and which functionally inactivate cellular tumor suppressor proteins p53 and retinoblastoma (Rb), respectively. See, e.g., Dyson et al.,
Science
243: 934-937 (1989); Huibregtse et al.,
Mol Cell Biol
13: 4918-4927 (1993); Scheffner et al.,
Cell
75: 495-505 (1993). This is thought to provide the basis for the ability of high-risk, but not the low-risk, HPVs to promote oncogenesis.
In recent years, a distinct mechanism of viral oncoprotein-induced inactivation of tumor suppressor protein function has emerged, involving the targeting of tumor suppressor protein(s) to ubiquitin-proteasome mediated degradation machinery. See, e.g., Boyer et al., ibid.; Ciechanover,
Cell
79: 13-21 (1994); Huibregtse et al.,
Mol Cell Biol
13: 4918-4927 (1993). The ubiquitin-proteasome pathway participates in physiological regulation of the levels of cell-cycle related proteins such as cyclins, cdks, and tumor suppressor proteins such as p53 and Rb proteins. See, e.g., Boyer et al., ibid.; Ciechanover, ibid.; Maki et al.,
Cancer Res
56: 2649-2654 (1996). Viral oncoproteins target cellular tumor suppressor proteins to this pathway for enhanced degradation. See, e.g., Huibregtse et al.,
EMBO J
10: 4129-4135 (1991); Scheffner et al.,
Cell
63: 1129-1136 (1990). High-risk HPV E6 oncoproteins associate with E6-AP, a ubiquitin ligase, which in turn interacts with p53 and targets it for degradation. See, e.g., Huibregtse et al (1991), ibid.; Scheffner et al., ibid. We have since found that the high-risk HPV16 E7 oncoprotein also uses the ubiquitin proteasome pathway for enhanced degradation of bound Rb protein. See, e.g, Boyer et al.,
Cancer Res
56: 4620-4624 (1996).
Characterization of oncogenesis-related cellular targets of HPV oncoproteins has been facilitated by the ability of HPV to dominantly immortalize primary human cells in vitro. See, e.g., supra; Band et al.,
Proc Natl Acad Sci USA
87: 463-467 (1990); Woodworth et al.,
J Virol
63: 159-164 (1989). Both E7 and E6 proteins of high-risk HPVs are required for efficient immortalization of cervical keratinocytes, E6 alone is not enough. See, e.g., Hawley-Nelson et al.,
EMBO J
8: 3905-3910 (1989); Munger et al.,
J Virol
63: 4417-4421 (1989).
Surprisingly, we found that HPV E6 alone is sufficient to immortalize a subtype of normal human mammary epithelial cells (“MECs”). See, e.g., Band et al.,
J Virol
65: 6671-6676 (1991). Use of HPV16 DNA constructs with disruption of individual open reading frames of early (“E”) viral genes demonstrated that E1, E2, E4 and E7 genes were dispensable for MEC immortalization. In contrast, the E6 open reading frame was indispensable. See, e.g., Band et al., ibid. Introduction of the E6 gene alone demonstrated that this gene was sufficient to immortalize MECs. See, e.g., Band et al.,
EMBO J,
12: 1847-1852 (1993). Thus, immortalization of the human MECs by E6 provides a simple single-oncogene model wherein to define the cellular pathways who
Band Vimla
Gao Qingshen
Beattie Ingrid A.
Mintz Levin Cohn Ferris Glovsky & Popeo, P.C
New England Medical Center
Nguyen Dave T.
Shukla Ram R.
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