In vitro and In vivo Radiosensitization Induced by the DNA Methylating Agent Temozolomide

Abstract

Purpose:Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. Experimental Design: The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (gH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. Results: Exposure of each cell line to temozolomide for 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G 2 cell cycle checkpoint. As a measure of DNA double strand breaks, gH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gH2AX foci per cell was significantly greater at 24 h after the combined modality compared with the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. Conclusions: These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase in mitotic catastrophe.Temozolomide has known anticancer effects against a broad range of tumor histologies including gliomas (1, 2). Temozolomide is a lipophilic molecule that can be given p.o. and crosses the blood-brain barrier. At physiologic pH, temozolomide is converted to the active metabolite methyltriazenoimidazole-carboxamide, which forms methyl adducts at O 6 -position of guanine in DNA. The formation of O 6 -methylguanine then results in mismatch pairing with thymine during subsequent cycles of DNA replication, followed by DNA strand-break formation and eventually cell death (2). Critical to its effectiveness as an antitumor agent is the cellular expression of O 6 -methlyguanine-DNA-methyltransferase (MGMT; refs. 3,4), which removes the O 6 -methyl adducts and, thus, acts to repair temozolomide-induced DNA damage (2, 3). In a recently published study by Stupp et al. (5), patients with primary glioblastoma multiforme (GBM) treated...

A mathematical model of brain tumour response to radiotherapy and chemotherapy considering radiobiological aspects

“…Therefore the adjuvant schedule may have only a marginal benefit compared to the concomitant phase. This result is consistent with published data of in vitro and in vivo radiosensitization of TMZ (Chakravarti et al, 2006;Kil et al, 2008). The model suggests that TMZ increases More specifically, the survival fraction distribution after one dose of radiation is less skewed with the addition of TMZ (Fig.…”

“…Our modelling suggests that the addition of TMZ produces a considerable extension of by Kil et al (2008) concerning in vivo tumour growth delay in mice bearing xenografts treated with radiotherapy and TMZ.…”

“…Alternatively, the concomitant administration of TMZ may inhibit the repair of doublestrand DNA breaks (Chakravarti et al, 2006;Kil et al, 2008).…”

Radiation-induced autophagy in normal and cancer cells: Towards novel cytoprotection and radio-sensitization policies?

“…72,73 The reluctance of glioma cells to undergo apoptosis together with the clear evidence of autophagy induction by radiotherapy and by temozolomide suggest that radio-sensitization policies aiming to autophagy stimulation may be of clinical importance. Of particular interest is the study on the induction of autophagy by the chemotherapeutic agent temzolomide, which has established in vitro radio-sensitizing properties 74 and its combination with radiotherapy has been recently approved as the standard regimen for the therapy of patients with gliomas. 66,74,75 Human breast cancer cell lines.…”

“…Of particular interest is the study on the induction of autophagy by the chemotherapeutic agent temzolomide, which has established in vitro radio-sensitizing properties 74 and its combination with radiotherapy has been recently approved as the standard regimen for the therapy of patients with gliomas. 66,74,75 Human breast cancer cell lines. Autophagy protects breast cancer cells from mitochondrial-mediated apoptosis following treatment with the antiestrogen tamoxifen.…”