Delayed repletion of O6-methylguanine-DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity.
OBJECTIVE
Temozolomide (TMZ)-induced O6-methylguanine (MG) DNA lesions, if not removed by MG-DNA methyltransferase (MGMT), mispair with thymine, trigger rounds of futile mismatch repair (MMR), and in glioma cells lead to prolonged G2-M arrest and ultimately cell death. Depletion of MGMT by O6-benzylguanine (BG) sensitizes tumor cells to TMZ, and this combination is currently used in clinical trials. The use of the TMZ+BG combination in gliomas, however, is complicated by the prolonged TMZ-induced G2-M arrest, which may delay activation of poorly defined cell death pathways and allow for MGMT repletion and reversal of toxicity.


METHODS
To address these issues, the actions of TMZ were monitored in DNA MMR-proficient SF767 glioma cells depleted of MGMT by BG, and in cells in which BG was removed at various times after TMZ exposure. In MGMT-depleted cells, TMZ exposure led to DNA single-strand breaks and phosphorylation of cdc2, followed by G2-M arrest, induction of p53/p21, and DNA double-strand breaks. Although DNA single-strand breaks, phosphorylation of cdc2, and G2-M arrest could be reversed by repletion of MGMT up to 5 days after TMZ exposure, TMZ-induced cytotoxicity could only be prevented if MGMT was replenished within 24 hours of the onset of G2-M arrest, and before the creation of DNA double-strand breaks.


CONCLUSIONS
These results indicate that although SF767 glioma cells undergo a prolonged G2-M arrest in response to TMZ, their ability to escape TMZ-induced cytotoxicity by MGMT repletion is limited to an approximately 24-hour period after the onset of G2-M arrest.
Hirose
Yuichi
Y
Brain Tumor Research Center, Department of Neurological Surgery, and the University of California at San Francisco Cancer Center, University of California at San Francisco, California 94115, USA.
Kreklau
Emiko L
EL
Erickson
Leonard C
LC
Berger
Mitchel S
MS
Pieper
Russell O
RO
D000970:Antineoplastic Agents / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003114:Colony-Forming Units Assay; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D000077204:Temozolomide; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D018797:Cell Cycle Proteins; C064976:O(6)-benzylguanine; D006147:Guanine; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D000077204:Temozolomide