Method of identifying inhibitors of EBNA-1

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid

Reexamination Certificate

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C435S007100, C435S091330, C435S069100

Reexamination Certificate

active

06811983

ABSTRACT:

BACKGROUND OF THE INVENTION
Epstein-Barr virus (EBV) contributes causally to Burkitt's lymphoma, nasopharyngeal carcinoma, and B-cell lymphomas in immunocompromised individuals (de-The, et al., 1978; Hanto, et al., 1981; Zeng, et al., 1983; Zeng, 1985). EBV may also contribute causally to a portion of T-cell lymphomas, Hodgkin's disease, gastric carcinomas, and, possibly even, breast carcinoma (Bonnet, et al., 1999; Fina, et al., 2001; Glaser, et al., 1997;Imai, et al., 1994; Siebert, et al., 1995; Shibata and Weiss, 1992; Tokunaga, et al., 1993). The growing number of malignancies potentially associated with EBV drives our need to understand EBV's biology in order to develop specific therapeutic tools to treat its associated cancers.
EBV establishes a latent infection in human cells, in which its DNA is maintained extrachromosomally with only a limited number of its genes being expressed (reviewed in Rowe, 1999). Among them, EBV nuclear antigen-1 (EBNA-1) is the only viral gene known to be expressed as a protein in all EBV-associated tumor cells. EBNA-1 dimerizes and binds specifically to two clusters of its cognate sites within the origin of DNA synthesis of EBV's replicon, oriP. EBNA-1's binding to oriP has been shown to promote both the initiation of DNA synthesis and the extrachromosomal maintenance of small replicons derived from EBV in proliferating cells (Rawlins, et al., 1985; Yates, et al., 1985). EBNA-1 contributes to the initiation of DNA synthesis at oriP by associating directly or indirectly with ORC and the MCM complex (Chaudhuri, et al., 2001; Schepers, et al., 2001)). EBNA-1 also enhances transcription from promoters near oriP that drive expression of the viral genes EBNA-2, EBNA-3a, EBNA-3c, and latent membrane protein-1 (LMP-1), all of which are known to be required for the initiation and/or maintenance of proliferation of EBV-immortalized B cells (Cohen, et al., 1989; Gahn and Sugden, 1995; Hammerschmidt and Sugden, 1989; Kaye, et al., 1993; Kempkes, et al., 1995; Kilger, et al., 1998; Sugden and Warren, 1989; Tomkinson, et al., 1993; Zimber-Strobl, et al., 1996). These observations make it likely that EBNA-1's functions are required for EBV-infected cells to proliferate. However, what EBNA-1 contributes to those tumor cells in which it is expressed alone is unknown.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method assaying potential inhibitors of EBNA-1 comprising the steps of (a) obtaining an EBV-positive cell line and an EBV-negative cell line; (b) exposing the cell lines to a test compound, and (c) observing the effect of the compound on induction of apoptosis in the cell lines, wherein significant apoptosis in the EBV-positive cell line and lack of significant apoptosis in the EBV-negative cell line indicates a compound that specifically inhibits EBNA-1.
In a particularly advantageous form of the present invention, the EBV-positive cell line comprises 721 cells and the EBV-negative cell comprise BJAB cells.
It is an object of the present invention to assay potential inhibitors of EBNA-1.
Other objects, features and advantages of the present invention will become apparent to one of skill in the art after examination of the specification, claims and drawings.


REFERENCES:
Cohen, J. I., Wang, F., Mannick, J., and Kieff, E. (1989). Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation, Proc. Natl. Acad. Sci. USA 86, 9558-62.
Gahn, T. A., and Sugden, B. (1995). An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene, J. Virol. 69, 2633-6.
Hammerschmidt, W., and Sugden, B. (1989). Genetic analysis of immortalizing functions of Epstein-Barr virus in human B lymphocytes, Nature 340, 393-7.
Kang, M. S., Hung, S. C., and Kieff, E. (2001). Epstein-Barr virus nuclear antigen 1 activates transcription from episomal but not integrated DNA and does not alter lymphocyte growth, Proc. Natl. Acad. Sci. USA 98, 15233-8.
Kaye, K. M., Izumi, K. M., and Kieff, E. (1993). Epstein-Barr virus latent membrane protein 1 is essential for B- lymphocyte growth transfromation, Proc. Natl. Acad. Sci. USA 90, 9150-4.
Kempkes, B., Spitkovsky, D., Jansen-Durr, P., Ellwart, J. W., Kremmer, E., Delecluse, H. J., Rottenberger, C., Bornkamm, G. W., and Hammerschmidt, W. (1995). B-cell proliferation and induction of early G1-regulating proteins by Epstein-Barr virus mutants conditional for EBNA2, EMBO J. 14, 88-96.
Kilger, E., Kieser, A., Baumann, M., and Hammerschmidt, W. (1998). Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor, EMBO J. 17, 1700-9.
Kirchmaier, A. L., and Sugden, B. (1997). Dominant-negative inhibitors of EBNA-1 of Epstein-Barr virus, J. Virol. 71, 1766-75.
Mackey, D., and Sugden, B. (1997). Studies on the mechanism of DNA linking by Epstein-Barr virus nuclear antigen 1, J. Biol. Chem. 272, 29873-29879.
Mackey, D., and Sugden, B. (1999). The linking regions of EBNA1 are essential for its support of replication and transcription, Mol. Cell Biol. 19, 3349-59.
Sugden, B., and Warren, N. (1989). A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection, J. Virol. 63, 2644-9.
Tomkinson, B., Robertson, E., and Kieff, E. (1993). Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation, J. Virol. 67, 2014-25.
Zimber-Strobl. U., Kempkes, B., Marschall, G., Zeidler, R., Van Kooten, C. Banchereau, J., Bornkamm, G. W., and Hammerschmidt, W. (1996). Epstein-Barr virus latent membrane protein (LMP1) is not sufficient to maintain proliferation of B cells but both it and activated CD40 can prolong their survival, EMBO J. 15, 7070-8.

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