It has been our goal to determine how ovarian cancer cells become resistant to cisplatin therapy. To ascertain the reasons, we have studied in vitro models of cisplatin resistance and cell lines from ovarian cancer patients refractory to high dose platinum therapy. Although an understanding of this process is not complete, we can say that resistance can occur by several pathways and often involves the changes in expression of many genes. Furthermore, our data damage tolerance is repair of cisplatin DNA damage. Hence, DNA repair not only defines the innate sensitivity of a cell to cisplatin but also if the function of this pathway is enhanced produces resistance to cisplatin. These data suggest that perturbation of the DNA repair process may be one way to increase the cytotoxicity of cisplatin and in part reverse resistance to the drug. Therefore, we propose to test the hypothesis that genetic inactivation of ERCC1, an indispensable DNA repair [protein, will disruption the repair of cisplatin DNA damage, and make cisplatin more cytotoxic and that we can add specificity to this effect using an ovarian specific promote and cisplatin damage inducible promoter. We propose to use optimal circumstances to test our ideas and improve upon them by work on the following Specific Aims.
SPECIFIC AIM #1 : Determine the impact of down-regulation of ERCC1 n cisplatin sensitivity. We will eliminate functional ERCC1 from ovarian cancer cells with plasmids which produce antisense ERCC1, dominant negative ERCC1, or intrabodies against ERCC1. In this optimal test, transcription will be driven by the CMV promoter. These gene inactivation strategies will be tested for effects on ERCC1 amounts, function, and cisplatin cytotoxicity.
SPECIFIC AIM #2 : Modify an Ovarian Specific Transcript promoter to enhance its ability to drive gene expression in ovarian cancer cells but maintain and/or improve specificity. The promoter we have identified and will improve is a approximately 500bp portion of an endogenous rat retroviral- like element that encompasses the U3 region of the LTR of one of these genomic units. These units are specifically transcriptionally active in the rat ovary and the U3 region will regulate reporter gene expression in human ovarian cancer cells but not diverse other cell types.
SPECIFIC AIM #3 : Test the ability of an optimized tissue specific promoter and cisplatin damage inducible promoter to confer specificity tot the best method for ERCCl inactivation. The best method for ERCCl inactivation, as determined in Specific Aim #1, will be utilized in the construction of plasmids where the inactivation product will be expressed under the control of an optimized ovarian tissue specific promoter and by the promoter for P21 which showed marked inducibility when ovarian cancer cells are exposed to platinum.
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| Cvetkovic, Dusica; Pisarcik, Debra; Lee, Chan et al. (2004) Altered expression and loss of heterozygosity of the LOT1 gene in ovarian cancer. Gynecol Oncol 95:449-55 |
| Nikitin, Alexander Y; Connolly, Denise C; Hamilton, Thomas C (2004) Pathology of Ovarian Neoplasms in Genetically Modified Mice. Comp Med 54:26-8 |
| Connolly, Denise C; Bao, Rudi; Nikitin, Alexander Yu et al. (2003) Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer. Cancer Res 63:1389-97 |
| Abdollahi, Abbas; Gruver, Briana N; Patriotis, Christos et al. (2003) Identification of epidermal growth factor-responsive genes in normal rat ovarian surface epithelial cells. Biochem Biophys Res Commun 307:188-97 |
| Hamilton, T C; Connolly, D C; Nikitin, A Y et al. (2003) Translational research in ovarian cancer: a must. Int J Gynecol Cancer 13 Suppl 2:220-30 |
| Abdollahi, Abbas; Pisarcik, Debra; Roberts, David et al. (2003) LOT1 (PLAGL1/ZAC1), the candidate tumor suppressor gene at chromosome 6q24-25, is epigenetically regulated in cancer. J Biol Chem 278:6041-9 |
| Selvakumaran, Muthu; Pisarcik, Debra A; Bao, Rudi et al. (2003) Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines. Cancer Res 63:1311-6 |
| Cvetkovic, Dusica; Williams, Stephen J; Hamilton, Thomas C (2003) Loss of cellular retinol-binding protein 1 gene expression in microdissected human ovarian cancer. Clin Cancer Res 9:1013-20 |
| Bao, Rudi; Selvakumaran, Muthu; Hamilton, Thomas C (2002) Targeted gene therapy of ovarian cancer using an ovarian-specific promoter. Gynecol Oncol 84:228-34 |
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