Dihydrodiol dehydrogenase mediates cisplatin resistance

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DESCRIPTION (provided by applicant): Cisplatin is one of the most effective forms of chemotherapy for advanced stage ovarian cancer although it is generally used as a carboplatin taxol combination. However some patients relapse due to the development of a resistant cell population. In order to identify clinically relevant markers for cisplatin/carboplatin responsiveness, a stable cisplatin-resistant human ovarian carcinoma cell line (2008/C13*) derived from the parental human ovarian carcinoma cell line (2008) was analyzed by microarray analysis followed by semiquantitative RT-PCR. Four genes were found to be upregulated in the cisplatin-resistant ceils: tropomyosin, apolipoprotein J (clusterin), glucose dehydrogenase and dihydro dehydrogenase. All four were upregulated by cisplatin treatment of the 2008 cells. Transfection experiments showed that forced overexpression of only DDH1 induced cisplatin/carboplatin resistance in the parental cell lines. DDH mRNA expression posttransfection was monitored by RT-PCR, and protein expression by enzyme activity assays and immunohistochemistry. Transfection of DDH1 into two other human ovarian carcinoma cell lines, 2780 and SKOV3 resulted in cisplatin carboplatin resistance, but not to other anticancer drugs. Analysis of a human lung squamous cell carcinoma cell line (A549) which has a very high levels of endogenous DDH2 mRNA exhibited a degree of cisplatin resistance greater than that of the 2008/C13* cell line. We propose to continue this work by (a) transfection of non-ovarian cell lines, including Tera-2 (germ cell tumors), A431(cervical carcinoma cells), HTB56 (lung adenocarcinoma ceils) and H69 (small cell lung carcinoma cell), to show that DDH1/2 expression produces cisplatin carboplatin resistance in a variety of cell lines derived from cancers from different organs. (b) We will also attempt to determine the transcriptional control mechanisms responsible for DDH1 upregulation as well as the (c) mechanisms by which DDH1/2 produces resistance to cisplatin (e.g. possible involvement of reactive oxygen species and apoptotic pathways involved). (d) Finally, we will study human ovarian and lung carcinoma tissues prior to and post-chemotherapy (with platinum containing drugs) utilizing monoclonal antibodies to DDH1/2 to determine if DDH expression can be used as a surrogate marker to identify whether, or not a tumor will respond to platinum-based chemotherapy.