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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

Gene Therapy volume 8, pages 469–476 (2001)Cite this article

Abstract

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-XL. Forced expression of a BCL-XL 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.

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Acknowledgements

This work was supported by a grant from the State of Baden-Württemberg to MW. TG was supported by a grant from the Federal Ministry of Education, Science, Research and Technology (Fö. 01KS9602) and the Interdisciplinary Center of Clinical Research Tübingen (IZKF). MGC and PRL acknowledge the support of European Union Biomed Program grants BHH4-CT98–3277 and QLK3-CT-1999–00364, and Cancer Research Campaign (UK) grant SP2332–01. PRL is a Research Fellow of the Lister Institute of Preventive Medicine.

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  1. Department of Neurology, Laboratory of Molecular Neuro-Oncology, University of Tübingen, Tübingen, Germany

    T Glaser & M Weller

  2. Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, Manchester, UK

    M G Castro & P R Löwenstein

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  1. T Glaser
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  2. M G Castro
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  3. P R Löwenstein
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  4. M Weller
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Glaser, T., Castro, M., Löwenstein, P. et al. 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. Gene Ther 8, 469–476 (2001). https://doi.org/10.1038/sj.gt.3301415

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