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 I (HSV-1) thymidine kinase (TK) and subsequent ganciclovir (GCV) chemotherapy was the first approach used in clinical trials of somatic gene therapy for glioblastoma. The molecular pathways mediating TK/GCV-induced cell death remain to be elucidated. Here, we report that adenoviral (Ad)-TK/GCV-induced death is p53-independent and does not involve altered CD95 or CD95L expression. Ectopic expression of the preferential caspase 8 inhibitor, crm-A, inhibits 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 mitochondrial cytochrome c release and activation of caspases 3, 7, 8 and 9 in a death 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 pseudosubstrate caspase inhibitor, zVAD-fmk, inhibit TK/GCV cytotoxicity. Double-transfected cell lines expressing crm-A and enhanced green fluorescent protein (eGFP) show that the bystander effect in vitro is also death receptor- and caspase 8-independent. TK/GCV therapy does not kill glioma cells in synergy with cancer chemotherapy drugs, including lomustine, temozolomide and topotecan. In contrast, there is strong synergy of TK/GCV and CD95L. Thus, TK/GCV-induced cell death involves a mitochondria-dependent loop of caspase acvtivation that can be synergistically enhanced by death receptor agonists 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 in rodent glioma models in vivo.
Glaser
T
T
Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, Tübingen, Germany.
Castro
M G
MG
Löwenstein
P R
PR
Weller
M
M
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D001932:Brain Neoplasms / Q000628:therapy*; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D015774:Ganciclovir / Q000627:therapeutic use; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D005910:Glioma / Q000628:therapy*; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D051381:Rats; D013937:Thymidine Kinase / Q000235:genetics; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism
D000998:Antiviral Agents; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D019014:fas Receptor; D013937:Thymidine Kinase; D020169:Caspases; D015774:Ganciclovir