gms | German Medical Science

Objective: The gold standard treatment of glioblastoma, the most lethal glioma subtype, includes surgery followed by the combination of radiotherapy and chemotherapy with temozolomide (TMZ). The active metabolite of TMZ methylates DNA bases in several positions, from which methylation of the O6 position of guanine is regarded as the lethal lesion. However, the effect of TMZ appears to be limited by the occurrence of chemoresistance. Therefore there’s an urgent need to adequately elucidate the mechanisms of TMZ action and the pathways by which glioma cells escape from death. We present the other possible point of TMZ action at the different, epigenetic level of the genome. The aim of our study was to evaluate the cellular effects of temozolomide action, the drug of choice in glioblastoma treatment.

Methods: We have treated the C6, U118, U138, T98G, HeLa, and HaCaT (control) cell lines with TMZ dissolved in DMSO in different time periods and drug concentration, adjusted to drug concentrations in target tissue and standard treatment scheme. DNA from cultured cells was isolated with commercially available DNA isolation kit, hydrolysed into nucleotides and separated after labelling with 32P-ATP and T4-polynucleotide kinase. Separation of 32P-labelled nucleotides was done on cellulose thin layer chromatography (TLC) plates in two dimensions. Chromatograms were then evaluated using phosphoimager and the amounts of 5-methylcytosine (m5C) calculated as a ratio (R) of spot intensities of m5C to m5C+C+T. Thymine and cytosine were included in the formula because they are also products of damage of m5C.

Results: Our experiments showed that TMZ treatment affects m5C contents in DNA. m5C amount in DNA increases after short treatment with TMZ in a nonlinear manner. However, treatment with any drug concentration longer than 24h causes demethylation.

Conclusion: The results of the study put a new light on the mechanism of action of TMZ situating it as an epigenetic modificator that acts not only as an directly destroying agent, but also indirectly by influencing the gene expression regulation. TMZ modifies epigenetic pattern through changing the 5-methylcytosine level in DNA. We showed that TMZ induces methylation of cytosine at short times and then causes demethylation of DNA through reactive oxygen species induced damage of 5-methylcytosine. The observed global hypomethylation of the genome contributes to regulation of gene expression on epigenetic level.