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.