MECHANISM OF ACTION AND DURATION OF TRANSLATION OF THE THYMIDINE KINASE GENE IN ADENOVIRUS-MEDIATED GENE THERAPY OF OVARIAN CANCER

Adenovirus (ADV)-mediated gene therapy with the thymidine kinase (TK) Gene under control of the Rous sarcoma virus (RSV) promoter followed by the administration of acyclovir has been established in vitro for the treatment of ovarian cancer cells and has been used as the basis for intraperitoneal phase I clinical trials. The purpose of this investigation was to clarify whether cell death after ADV-RSV-TK gene therapy and acyclovir administration is indeed due to apoptosis induction. It was tested if the synergistic effect of ADV-RSV-TK gene therapy with chemotherapy was limited to the primary mechanism of action or whether the vector transduction itself exerted any pro-apoptotic effect. It is unclear how long a significant degree of transgene translation can be expected after adenovirus-mediated TK transduction, where the transcriptional complex is localized in the nucleus in an episomal fashion and thus without stable integration. The possible interaction of Acyclovir pretreatment with subsequent ADV-RSV-TK transduction also remains to be elucidated. The epithelial cell lines OVCAR-3 and MDAH-2774 were established from human poorly differentiated serous ovarian cancer Fluorimetric assay of caspase-3 activity was performed as well as ELISA of the CK 18 split product M30. PARP cleavage was analysed by Western blotting. Transgene expression and cell killing efficacy were analysed based on multiplicity of infection (MOI) and MTT assay. Anti-TK-antibody 1397 was used for immunocytochemistry and Western Blot analysis of TK expression. After transduction with ADV-RSV-TK at an MOI of 66, TK translation increased strongly in MDH 2774 and OVCAR-3 cell lines during the initial 48 hours. Apoptosis induction was established in this investigation as the mechanism of the ADV-RSV-TK gene therapy effect of acyclovir administration by caspase activity and subsequent CK 18 cleavage. Neither acyclovir nor vector administration alone showed any apoptotic activity. The synergistic effect of TK gene therapy and chemotherapeutic agents was shown to be TK induced. Significant anti-PARP 1 activity was found to be an ADV-RSV-TK treatment effect after acyclovir addition. CAR expression was observed on the membranes but also intracellular translocation of CAR takes place dependent on cellular growth patterns TK gene expression is dependent on multiplicity of infection (MOI) and thus on vector dose in a linear fashion. Neither TK expression nor ADV transduction influence CAR expression