The list of brain tumor types identified in 183 patients for whom DNA from brain tumor tissue and peripheral blood samples was isolated and analyzed for the content of m⁵C in DNA.

<p>Specific R coefficient was calculated as (m5dC/m5dC+dC+dT)×100 on the basis of analysis TLC plate exposed to Phosphoimager. Histopathological analysis revealed the WHO grade. Sex is also mentioned.</p

Comparison of m⁵C content in DNA in tissue and blood with malignancy of brain tumors.

<p><b>A</b>. Pearson r correlation (0.9; p<0.0001) of genomic m⁵C contents of DNA from peripheral blood and from brain tumor tissues of the same subjects. <b>B</b>. Amounts of m⁵C in DNA (R) from blood and in tumor tissues of the same subjects with brain tumors (astrocytoma, anaplastic astrocytoma and glioblastoma) of different malignancy. Data on human brain metastasis and meningioma were also analyzed. Data were evaluated with ANOVA test.</p

Comparison of content of m⁵C in DNA from tissue and blood.

<p>5-methylcytosine content (R) in DNA from 183 patients with brain tumors: cancer tissues (upper panel) and blood (bottoms panel). The figure clearly shows that m⁵C content, expressed as R [ = (m5dC/m5dC+dC+dT)×100] coefficient, in DNA from tumor tissue matches the results from blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092599#pone.0092599-Barciszewska2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092599#pone.0092599-Barciszewska3" target="_blank">[26]</a>.</p

Effect of brain tumor tissue handling on content of m⁵C in DNA.

<p>The level of m⁵C content (R) in DNA isolated from resected meningioma tissue (WHO grade I) stored at −80°C (grey bar), formalin-fixed paraffin-embedded (FFPE) (empty bar) and exposed to room temperature for 3 h (black bar). Analysis was done for 5 samples in each conditions. Standard deviations for R is shown.</p

The diagram showing the relation of m⁵C content (R values with deviations errors) in DNA isolated from peripheral blood of patients with different brain tumors, breast and colon cancers and arterial hypertension.

<p>As one can see there is strict relation of R with different diseases. R decreases as malignancy increases. The amount of m⁵C suggests a possibility of a disease occurrence. As a control samples from healthy patients of age group 19–50 were used.</p

Patients with brain tumors analyzed in this study classified according to age.

<p>The 183 patients were divided into 6 groups of different age. The largest group consisted of patients within the age range of 51–60 years.</p

DataSheet_1_Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.pdf

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 μM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.</p

DataSheet_2_Cross-reactivity between histone demethylase inhibitor valproic acid and DNA methylation in glioblastoma cell lines.pdf

Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 μM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.</p

The effect on cell viability after temozolomide treatment estimated by MTT assay.

<p>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.</p

Analysis of m⁵C in DNA.

<p>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.</p