Concurrent treatment with the methylating agent
temozolomide during
radiotherapy has yielded the first significant improvement in the survival of adult
glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose
tumors display CpG methylation of the
O(6)-methylguanine (O(6)-meG)-DNA
methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that
temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O(6)-meG from
DNA. Here, we systematically examined the
temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O(6)-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of
temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O(6)-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to
temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing
alkyladenine-DNA glycosylase, the only activity in human cells that excises
3-methyladenine from
DNA, reduces the
temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by
3-methyladenine as well as by O(6)-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by
temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent
temozolomide radiotherapy.