Death receptor-independent cytochrome c release and caspase activation mediate thymidine kinase plus ganciclovir-mediated cytotoxicity in LN-18 and LN-229 human malignant glioma cells

Suicide gene therapy using viral transfer of herpes simplex virus type 1 (H SV-1) thymidine kinase (TK) and subsequent ganciclovir (GCV) chemotherapy w as the first approach used in clinical trials of somatic gene therapy for g lioblastoma. The molecular pathways mediating TK/GCV-induced cell death rem ain to be elucidated. Here, we report that adenoviral (Ad)-TK/GCV-induced d eath is p53-independent and does not involve altered CD95 or CD95L expressi on. Ectopic expression of the preferential caspase 8 inhibitor, crm-A, inhi bits Ad-CD95L-induced cell death but has no effect on TK/GCV cytotoxicity. LN-18 glioma cells selected for resistance to death receptor-mediated cell death do not acquire cross-resistance to TK/GCV. TK/GCV triggers mitochondr ial cytochrome c release and activation of caspases 3, 7, 8 and 9 in a deat h receptor-independent manner. These events are associated with the loss of BCL-X-L. Forced expression of a BCL-X-L transgene, or co-exposure to a pse udosubstrate caspase inhibitor, zVAD-fmk, inhibit TK/GCV cytotoxicity. Doub le-transfected cell lines expressing crm-A and enhanced green fluorescent p rotein (eGFP) show that the bystander effect in vitro is also death recepto r- and caspase 8-independent. TK/GCV therapy does not kill glioma cells in synergy with cancer chemotherapy drugs, including lomustine, temozolomide a nd topotecan. In contrast, there is strong synergy of TK/GCV and CD95L. Thu s, TK/GCV-induced cell death involves a mitochondria-dependent loop of casp ase acvtivation that can be synergistically enhanced by death receptor agon ists such as CD95L. TK/GCV-mediated sensitization of glioma cells to CD95L expressed on immune effector cells or parenchymal brain cells might account for the immune system's and bystander effects of TK/GCV therapy observed i n rodent glioma models in vivo.