Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous clinical experience shows that the efficacy of suicide gene transfer in tumor therapy is limited, resulting from inefficient gene transfer or alternatively, from intrinsic resistance of the tumor in vivo. Herpes simplex virus thymidine kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy system, has been described to exert its cytotoxic effect, at least in part, by inducing apoptosis in target cells. Here, we report that mitochondria amplify TK/GCV-induced apoptosis by regulating p53 accumulation and the effector phase of apoptosis. Treatment with TK/GCV led to mitochondrial perturbations including loss of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol, inducing caspase activation and nuclear fragmentation. Inhibition of TK/GCV-induced mitochondrial perturbations by Bcl-2 overexpression or by the mitochondrion-specific inhibitor bongkrekic acid also strongly inhibited TK/GCV-induced activation of caspases and apoptosis. TK/GCV-induced mitochondrial perturbations depended on caspase activity possibly initiated by death receptor signaling. Perturbation of mitochondrial function mediated accumulation of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid, or inhibition of mitochondrial protein synthesis with chloramphenicol strongly reduced TK/GCV-induced accumulation of wild-type p53 protein. These findings suggest that TK/GCV therapy may be less efficient in tumors in which the mitochondrial amplification of TK/GCV-induced apoptosis is blocked, e.g., by Bcl-2 overexpression. Given the low efficacy of currently used gene therapy systems, our data on molecular mechanisms that regulate sensitivity or resistance toward TK/GCV-induced cytotoxicity might have important implications to improve the clinical application of suicide gene therapy.
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PMID:Mitochondrial amplification of death signals determines thymidine kinase/ganciclovir-triggered activation of apoptosis. 1086 13

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.
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PMID: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. 1131 26

Although ganciclovir (GCV) is most often used in suicide anticancer gene therapy, the mechanism of GCV-induced cell killing and apoptosis is not fully understood. We analysed the mechanism of apoptosis triggered by GCV using a model system of CHO cells stably transfected with HSV-1 thymidine kinase (HSVtk). GCV-induced apoptosis is due to incorporation of the drug into DNA resulting in replication-dependent formation of DNA double-strand breaks and, at later stages, S and G2/M arrest. GCV-provoked DNA instability was likely to be responsible for the observed initial decline in Bcl-2 level and caspase-9/-3 activation. Further decline in the Bcl-2 level was due to cleavage of the protein by caspase-9, as demonstrated by use of caspase inhibitors and transfection with trans-dominant negative caspase expression vectors. Bcl-2 cleavage resulted in the appearance of a pro-apoptotic 23 kDa Bcl-2 fragment and in excessive cytochrome c release, dephosphorylation of BAD, cleavage of PARP and finally DNA degradation. Since Fas/CD95 and caspase-8 were only slightly activated we conclude GCV-induced apoptosis to occur in this cell system mainly by activating the mitochondrial damage pathway. This process is independent of p53 for which the cells are mutated. Caspase-9 mediated cleavage of Bcl-2 accelerates the apoptotic process and may explain the high potential of GCV to induce apoptosis. Data are also discussed as to implications for HSVtk gene therapy utilizing GCV.
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PMID:Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing cells: critical role of DNA breaks, Bcl-2 decline and caspase-9 activation. 1194 97

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G(2)/M phases, immediately followed by the appearance of sub-G(0)/G(1) cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B(1) before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.
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PMID:Ectopic expression of herpes simplex virus-thymidine kinase gene in human non-small cell lung cancer cells conferred caspase-activated apoptosis sensitized by ganciclovir. 1240