Fig 1.
Site specific DNA methylation with temozolomide.
The main product of DNA reaction with TMZ is 7- methylguanosine (m7G). Methylation of O6 guanosine is supposed to be the main mechanism of TMZ action, but takes place only in 5%.
Fig 2.
A. Flow chart of the analysis of m5C in genomic DNA. Isolated DNA was hydrolyzed to 3’mononucleotides (A—adenosine, G—guanosine, C—cytidine, T–thymidyne and X–other modifications). Furthermore they were labelled with [γ-32P], dephosphorylated of 3’ phosphate and separated with TLC in two dimensions. The chromatogram was evaluated with phosphoimager. B. Two dimensional cellulose thin layer chromatography (TLC) analysis of [5’-32P] labelled deoxynucleotides obtained by enzymatic hydrolysis of DNA from different types of cells. Pure DNA was isolated from non treated HeLa cells (left) and treated with TMZ (right) for 48 h.
Fig 3.
Time effect of TMZ on the DNA methylation status.
U118 cell line treated with 250 μM (empty circle) and 500 μM (gray circle) of TMZ at 3, 6, 9, 12, 24 and 48 h. DNA not treated with temozolomide was used as a control (black circle). The contents of m5C is indicated on the Y axis. The highest content of m5C in DNA was observed after incubation of the cells 24 h with 500 μM of TMZ.
Fig 4.
The effect of TMZ on 5 methylcytosine (m5C) contents in DNA of glioma.
(U138, T98G, C6) and HeLa cells at 3, 12, 24 and 48 h. HaCaT cell line was used as the reference. C stands for control cells treated with DMSO only.
Fig 5.
The effect of TMZ on the cell cycle.
Cell cycle phases distribution in human glioblastoma (T98G) versus non cancer (HaCaT) cell lines after 12 and 48h temozolomide (TMZ) treatment was carried out using FACS calibur flow cytometer by DNA staining with propidium iode. TMZ treatment resulted in G2M phase arrest in both tested cell lines in a dose dependent manner.
Table 1.
The effect of TMZ on the HaCaT and T98G cell cycle lines.
The percentage of cells in each phase of the cell cycle in cancer (T98G) versus non cancer (HaCaT) cell line after the drug treatment. The highest concentrations of TMZ (500 μM and 1000 μM) affected the most cell cycle phase distribution. The results are expressed as a mean ± SD. All experiments were done in triplicate.
Fig 6.
The effect on cell viability after temozolomide treatment estimated by MTT assay.
Normal (HaCaT) and tumor cells (HeLa, T98G and U118)were treated with different TMZ concentrations (1–2000 μM) for 3-48h. C- control cells cultured without TMZ; DMSO- cells treated with the highest DMSO concentration used in the experiments. The results are expressed as a mean ± SD.