Apoptosis induction with
5-fluorocytosine/cytosine deaminase gene therapy for human malignant glioma
cells mediated by adenovirus
Kazuhiko Kurozumi, Takashi
Tamiya, Yasuhiro Ono, Shinji Otsuka, Hirokazu Kambara, Yoshiaki Adachi,
Tomotsugu Ichikawa, Hirofumi Hamada and Takashi Ohmoto
of Neurological Surgery, Okayama University Graduate School of Medicine and
Dentistry, Okayama (K.K., T.T., Y.O., S.O., H.K., Y.A., T.I., T.O.); Department
of Molecular Medicine, Sapporo Medical University, Sapporo, Japan (H.H.)
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 [Gli36Δ5 (mutated p53)
and U87MG (wild p53)].
The treatment with 5-FC/CD gene-therapy-induced apoptosis both in Gli36Δ5
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
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
Key words: adenovirus
vector, apoptosis, caspase, cytosine deaminase, 5-fluorocytosine, gene therapy,
malignant gliomas, mitochondria
© 2004 Kluwer Academic
Publishers. Printed in the Netherlands.