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Uncovering the action of Temozolomide in brain tumor cells

Anna-Maria Barciszewska, Department of Neurosurgery and Neurotraumatology, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, 60-355 Poznan, Poland, Pawel Glodowicz, Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12, 61-704 Poznan, Poland, Monika Piwecka, Institute of Bioorganic Chemistry of the Polish Academy of Sciences, Noskowskiego 12, 61-704 Poznan, Poland, Stanislaw Nowak, Department of Neurosurgery and Neurotraumatology, Karol Marcinkowski University of Medical Sciences, Przybyszewskiego 49, 60-355 Poznan, Poland


Introduction
Malignant gliomas are most aggressive brain tumors with a dismal prognosis despite optimal treatment. 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 chmoresistance. 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. We’ve showed that in addition to O6 methylation of guanines, TMZ induces methylation of cytosine at short times and then causes demethylation of DNA through reactive oxygen species (ROS) induced damage of 5-methylcytosine (m5C). The observed global hypomethylation of the genome contributes to regulation of gene expression on epigenetic level.
Aim of study
The aim of our study was to evaluate the molecular mechanism of temozolomide action, the drug of choice in glioblastoma treatment.
Material and Methods
We have treated the C6, glioblastoma and HeLa (as a control) cell lines with TMZ dissolved in DMSO with different time. 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 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
We’ve showed that TMZ treatment affects m5C formation in DNA. m5C amount in gliomas’ DNA increased significantly after short treatment with TMZ, whereas longer treatment caused 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.

Format: Oral communication

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