Adenovirus-mediated Thymidine Kinase Gene Therapy Induces Apoptosis in Human Epithelial Ovarian Cancer Cells and Damages PARP-1

Adenoviral (ADV) gene therapy with the thymidine kinase gene (TK) under control of the Rous sarcoma virus (RSV) promotor followed by the administration of acyclovir leads to replication errors in transcription and to cell death. This concept of ADV-RSV-TK has been established for the treatment of ovarian cancer cells. 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, whether 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 was examined using the epithelial cell lines OVCAR-3 and MDAH-2774, 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. 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. ADV-RSV-TK gene therapy in combination with acyclovir administration may in the future play a role in the treatment of BRCA1- and 2-related familial ovarian cancer. Adenoviral (ADV) gene therapy with the thymidine kinase gene (TK) under control of the Rous sarcoma virus (RSV) promotor followed by the administration of acyclovir leads to replication errors in transcription and to cell death. This concept of ADV-RSV-TK has been established for the treatment of ovarian cancer cells (1). To date, it has not been clearly established whether with this type of gene therapy induced cell death is secondary to necrosis or apoptosis. Diverse groups of molecules are involved in the apoptosis pathway. One set of mediators implicated in apoptosis belong to the aspartate-specific cysteinyl proteases or caspases. A member of this family, caspase-3 (CPP32, apopain, YAMA) has been identified as being a key mediator of apoptosis of mammalian cells. Kothakota et al. (2) screened the translation products of a murine protein library to find the substrates that are susceptible to cleavage by caspase-3. They found that in cells exposed to Fas, gelsolin was cleaved in vivo in a caspase-dependent manner. The cleaved fragments of gelsolin led to the cleavage of the actin filaments in a Ca 2+ -independent manner. The expression of the gelsolin fragments also led to the apoptosis of cells.