Apoptosis induction with 5-fluorocytosine/cytosine deaminase gene therapy for human malignant glioma cells mediated by adenovirus.
Previously, we evaluated the therapeutic efficacy of the adenovirus-mediated transduction of the cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) for malignant gliomas. However, the molecular pathways that mediate the 5-FC/CD gene therapy-induced cell death remains to be elucidated. In this study, we examined the induction of apoptosis and the role of caspases in 5-FC/CD gene therapy using human malignant glioma cells [Gli36delta5 (mutated p53) and U87MG (wild p53)]. The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36delta5 cells and in U87MG cells according to flow cytometric analysis. Immunoblot analysis revealed that caspases 3 and 9 were processed in response to 5-FC/CD in a concentration- and time-dependent manner, but caspase 8 was not. Each caspase 3 and 9 inhibitor significantly reduced apoptosis triggered by 5-FC/CD, but the caspase 8 inhibitor did not affect apoptosis induction. 5-FC/CD significantly promoted the release of cytochorme c from mitochondria in a concentration-dependent manner. These results indicate that 5-FC/CD gene therapy induces apoptosis in human malignant glioma cells and that the apoptotic cell death is mediated by the activation of mitochondrial caspase cascades involving caspases 3 and 9. This is the first report concerning the apoptotic mechanism of 5-FC/CD gene therapy, and these findings could be used to increase the efficacy of suicide gene therapy systems for the treatment of malignant glioma.
Kurozumi
Kazuhiko
K
Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Tamiya
Takashi
T
Ono
Yasuhiro
Y
Otsuka
Shinji
S
Kambara
Hirokazu
H
Adachi
Yoshiaki
Y
Ichikawa
Tomotsugu
T
Hamada
Hirofumi
H
Ohmoto
Takashi
T
D000256:Adenoviridae; D000963:Antimetabolites / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D043525:Cytosine Deaminase / Q000235:genetics*; D004354:Drug Screening Assays, Antitumor; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005437:Flucytosine / Q000627:therapeutic use*; D015316:Genetic Therapy; D005822:Genetic Vectors; D005910:Glioma / Q000201:enzymology / Q000378:metabolism / Q000628:therapy*; D006801:Humans
D000963:Antimetabolites; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D045304:Cytochromes c; D005437:Flucytosine; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D043525:Cytosine Deaminase