Molecular Dosimetry of Temozolomide: Quantification of Critical Lesions, Correlation to Cell Death Responses, and Threshold Doses.

Abstract

Temozolomide (TMZ) is a DNA-methylating agent used in cancer chemotherapy, notably for glioblastoma multiforme (GBM), where it is applied as a front-line drug. One of the DNA alkylation products of TMZ is the minor lesion O6 -methylguanine (O6 MeG), which is responsible for nearly all genotoxic, cytotoxic, and cytostatic effects induced in the low-dose range relevant for cancer therapy. Here, we addressed the question of how many O6 MeG adducts are required to elicit cytotoxic responses. Adduct quantification revealed that O6 MeG increases linearly with dose. The same was observed for DNA double-strand breaks (DSB) and p53ser15. Regarding apoptosis, hockeystick modeling indicated a possible threshold for A172 cells at 2.5 μmol/L TMZ, whereas for LN229 cells no threshold was detected. Cellular senescence, which is the main cellular response, also increased linearly, without a threshold. Using a dose of 20 μmol/L, which is achievable in a therapeutic setting, we determined that 14,000 adducts give rise to 32 DSBs (γH2AX foci) in A172 cells. This leads to 12% cell death and 35% of cells entering senescence. In LN229 cells, 20 μmol/L TMZ induced 20,600 O6 MeG adducts, 66 DSBs (γH2AX foci), 24% apoptosis, and 52% senescence. The linear dose response and the genotoxic and cytotoxic effects observed at therapeutically relevant dose levels make it very likely that the TMZ target concentration triggers a significant cytotoxic and cytostatic effect in vivo Despite a linear increase in the O6 MeG adduct level, DSBs, and p53 activation, the low curative effect of TMZ results presumably from the low rate of apoptosis compared to senescence.