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

Abstract

Temozolomide 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 is the minor lesion O6-methylguanine (O6MeG), which is responsible for nearly all genotoxic, cytotoxic and cytostatic effects of TMZ 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 (DSBs) and p53ser15. Regarding apoptosis, hockeystick modeling indicated a possible threshold for A172 cells at 2.5 µM TMZ, while 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 µM, 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 µM TMZ induced 20,600 O6MeG 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 makes 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.

Targeting c-IAP1, c-IAP2, and Bcl-2 Eliminates Senescent Glioblastoma Cells Following Temozolomide Treatment

“…It is important to note that even low concentrations of TMZ (1–25 µM) induce a linear increase in the activation of the DNA damage response, cell death, and senescence [ 43 , 44 ]. However, the frequency of senescent cells was only 25% at 10 µM and 35% at 25 µM TMZ ( Figure A1 A,B).…”

“…LN-229 showed a higher frequency of cell death at 25 and 50 µM, whereas concentrations of 5 and 10 µM were not toxic but strongly affected clonogenic survival in all cell lines (present publication, [ 8 ]). It is important to note that in the low concentration ranges between 1 and 25 µM TMZ, a linear increase with dose in the activation of the DNA damage response, cell death, and senescence was observed without a no-effect threshold [ 43 , 44 ]. For in vitro experiments, higher concentrations are often necessary, since established cell lines are more robust and not as responsive as cells in vivo.…”

Accumulation of Temozolomide-Induced Apoptosis, Senescence and DNA Damage by Metronomic Dose Schedule: A Proof-of-Principle Study with Glioblastoma Cells

“…The fraction of apoptotic and late apoptotic/necrotic cells was determined by flow cytometry 5 d after the last treatment using annexin V/FITC and propidium iodide (A/PI) staining of cells [ 23 ]. In brief, cells in the supernatant and trypsinized cells were collected, washed with PBS, and stored on ice.…”

“…Data was analyzed using the Flowing Software 2 program (Perttu Terho, Turku Center for Biotechnology, University of Turku, Turku, Finland). Apoptotic cells were defined as annexin V+/PI−, whereas late apoptotic/necrotic cells were defined as annexin V+/PI+ cells (for representative plots see [ 23 ]).…”

“…Untreated, proliferating cells were used as the control. Cells with a fluorescence intensity higher than the control were defined as senescent (for representative plots see [ 23 ]).…”