DNA 5-methylcytosine Levels Research Articles

Revealing the epigenetic effect of temozolomide on glioblastoma cell lines in therapeutic conditions.

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20–30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10–50 μM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients’ survival.

Natural Larval Diet Differently Influences the Pattern of Developmental Changes in DNA 5-Methylcytosine Levels in Apis mellifera Queens as Compared with Workers and Drones.

The principal mechanism of gene activation/silencing is DNA 5-methylcytosine methylation. This study was aimed at determining global DNA methylation levels in larvae, prepupae, pupae, and 1-day-old adults of Apis mellifera queens, workers and drones. The Imprint Methylated DNA Quantification Kit MDQ1 was used. Percentages of DNA 5-methylcytosine were low and relatively similar in the larvae of all the castes until 4th day of larval development (3-5%). However, they were higher in the drone and worker larvae than in the queen larvae. Generally, the developmental patterns of changes in the DNA methylation levels were different in the queens in comparison with the drones and workers. While methylation increased in the queens, it decreased in the drones and workers. Methylated DNA methylcytosine percentages and weights in the queen prepupae (15%, 9.18ng) and pupae (21%, 10.74ng) were, respectively, three and four times higher than in the worker/drone brood of the same age (2.5-4%, 0.03-0.07ng). Only in the queens, after a substantial increase, did DNA methylation decrease almost twice between the pupal stage and queen emergence (from 21% and 10.74ng to 12% and 6.78ng). This finding seems very interesting, particularly for experimental gerontology.

Antitumour imidazotetrazines—XVIII: Modification of the level of 5-methylcytosine in DNA by 3-substituted imidazotetrazinones

The effect of 3-methyl(temozolomide) and 3-ethyl (CCRG 82019) substituted imidazotetrazinones on cytosine methylation has been studied in the human lymphoblastoid cell line GM892. There was a decrease in the 5-methylcytosine content of newly synthesized DNA in cells treated with the 3-methyl and a small increase in cells treated with the 3-ethyl analogue, which was maximal 4 days after drug treatment. There was a progressive decrease in nuclear DNA methyltransferase after treatment with temozolomide with complete inhibition at 11–12 hr after drug addition, followed by a re-establishment of enzyme levels towards control values. While the free drugs had no effect on DNA methyltransferase activity in vitro, DNA isolated from GM892 cells previously treated with temozolomide inhibited the transfer of methyl groups from S-adenosyl- l-methionine to M. lysodeiktious DNA. The maximum effect was observed at 6 hr after drug addition and was proportional to the concentration of temozolomide to which the cells had previously been exposed. These results suggest that temozolomide may induce a block in cellular replication as a result of an indirect inhibition of DNA methylation and cells which escape this block progress with hypomethylated DNA.

Antitumour imidazotetrazines and gene expression.

The effect of 3-alkyl substituted imidazotetrazinones on methylation of DNA has been studied in drug sensitive and resistant cell lines. The 3-methyl analogue (Temozolomide) has been shown to cause a decrease in the level of 5-methylcytosine in newly synthesized DNA in both cell lines, although the effect occurred at lower drug concentrations in the drug sensitive cell line. In order to investigate the mechanism of hypomethylation of DNA, calf thymus DNA was alkylated in vitro by both Temozolomide and the 3-ethyl analogue, CCRG 82019, and the alkylated DNA was shown to inhibit the transfer of methyl groups from S-adenosyl-L-methionine to M. lysodeikticus DNA by purified eukaryotic DNA methylase. Neither free drug alone or unmodified DNA affected the methylase reaction. Calf thymus DNA modified with CCRG 82019 was more effective as a methylase inhibitor than DNA modified with Temozolomide, which was a reverse of the order of potencies of the free drugs against tumour cells in culture. CCRG 82019 modified DNA also formed a more stable complex with nuclear proteins. Alterations in the level of 5-methylcytosine in DNA may be important in the alteration of gene expression by these agents.

Chemical carcinogen-mediated decreases in DNA 5-methylcytosine content of BALB/3T3 cells.

The genomic level of DNA cytosine methylation was significantly diminished in dividing BALB/3T3 A31 CL1-13 cells treated with several aromatic hydrocarbon carcinogens. Benzo[a]pyrene-induced decreases in DNA 5-methylcytosine levels were concentration dependent over the range of 0.1 to 1.0 microgram/ml when determined at the end of the 16 h treatment period. The enzymatic methylation of DNA cytosine residues was the most sensitive to inhibition 48 h after treatment as concentrations of benzo[a]pyrene as low as 0.033 micrograms/ml initiated significant reductions in 5-methylcytosine levels. This inhibition of DNA cytosine methylation may be mediated by alkylation of the DNA. Treatment of hemimethylated DNAs with (+/-)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro benzo[a]pyrene (anti-BPDE), (+/-)-r,7-t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE), and (+/-)-benzo[a]pyrene-4,5-epoxide inhibited the transfer of methyl moieties from [3H]S-adenosylmethionine to cytosine residues in the undermethylated strand in the presence of mouse spleen methyltransferase activity. The syn isomer of BPDE was the most potent in this action while the parent compound, benzo[a]pyrene, did not significantly decrease the methyl accepting abilities of treated DNAs. All chemical carcinogens that were tested and are known to transform BALB/3T3 cells initiated significant reductions in 5-methylcytosine formation by 48 h post-treatment. However, concentrations of some hydrocarbons which do not transform these cells 5- to 50-fold above effective concentrations of the transforming carcinogens also provided significant reductions in DNA cytosine methylation. Thus the inhibition of DNA methylation may be an important step in the initiation of oncogenic transformation of BALB/3T3 cells, but decreases in DNA 5-methylcytosine levels alone cannot account for the onset of this multi-step process.

DNA methylationin normal and tumour virus-transformed cells in tissue culture I. The level of DNA methylation in BHK21 cells and in BHK21 cells transformed by polyoma virus (PyY cells)

The proportion of the cytosine moieties in DNA which are methylated to 5-methylcytosine at the macromolecular level has been determined in BHK21 cells and compared with the proportion of this minor base in the DNA of polyoma virustransformed BHK21 cells under identical conditions. Almost twice as much 5-methylcytosine was detected in the DNA of the PyY cells as was present in the BHK21 cells, both quantities being calculated relative to the total cytosine in the DNA of the relevant cell line. The possibility that 5-methylcytosine could also arise from the amination of thymine within the DNA was investigated. No evidence for the operation of this pathway was detected within the experimental limits, which would have detected 0.2 % amination of the thymine of either cell line. When the cells were grown at lower densities the proportion of 5-methylcytosine in the DNA of both cell lines was increased, but the difference between the levels in each cell line was maintained. The possibility that the level of 5-methylcytosine in DNA in mammalian cells could alter with their rate of DNA synthesis, or their degree of contact inhibition, was investigated by synchronising the cells with deoxyadenosine. Different levels of methylation were observed in both cell lines at different rates of DNA synthesis.