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 (O6MeG), 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 O6MeG adducts are required to elicit cytotoxic responses. Adduct quantification revealed that O6MeG 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 mmol/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 mmol/L, which is achievable in a therapeutic setting, we determined that 14,000 adducts give rise to 32 DSBs (gH2AX foci) in A172 cells. This leads to 12% cell death and 35% of cells entering senescence. In LN229 cells, 20 mmol/L TMZ induced 20,600 O6MeG adducts, 66 DSBs (gH2AX 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 O6MeG adduct level, DSBs, and p53 activation, the low curative effect of TMZ results presumably from the low rate of apoptosis compared to senescence.