Anna-Maria Barciszewska

Detection of MGMT, RASSF1A, p15INK4B, and p14ARF promoter methylation in circulating tumor-derived DNA of central nervous system cancer patients

Despite the growing understanding of the mechanisms of carcinogenesis, cancers of the central nervous system are usually associated with unfavorable prognosis. The use of an appropriate molecular marker may improve the treatment outcome by allowing early diagnosis and treatment susceptibility monitoring. Since methylation of tumor-derived DNA can be detected in the serum of cancer patients, this makes DNA methylation-based biomarkers one of the most promising diagnostic strategies. In this study, the methylation profiles of MGMT, RASSF1A, p15INK4B, and p14ARF genes were evaluated in serum free-circulating DNA and the corresponding tumor tissue in a group of 33 primary or metastatic central nervous system cancer patients. Gene promoter methylation was assessed using methylation-specific polymerase chain reaction (PCR). All the tested genes were found to be methylated to a different extent in both serum and tumor samples. In comparison to metastatic brain tumor patients, the patients with glial tumors were characterized by a higher frequency of gene hypermethylation. The hypermethylation of RASSF1A differentiated primary from metastatic brain cancers. Moreover, the gene methylation profiles observed in serum, in most cases, matched the methylation profiles detected in paired tumor samples.

Analysis of 5‐Methylcytosine in DNA of Breast and Colon Cancer Tissues

5‐methylcytosine (m5C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m5C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5‐methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C‐5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m5C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m5C in DNA as a good marker for diagnosis of neoplasia in clinical practice.

The methylation of a panel of genes differentiates low-grade from high-grade gliomas

Epigenetic changes play an important role in the pathogenesis of gliomas and have the potential to become clinically useful biomarkers. The aim of this study was the evaluation of the profile of promoter methylation of 13 genes selected based on their anticipated diagnostic and/or prognostic value. Methylation-specific PCR (MSP) was used to assess the methylation status of MGMT, ERCC1, hMLH1, ATM, CDKN2B (p15INK4B), p14ARF, CDKN2A (p16INK4A), RASSF1A, RUNX3, GATA6, NDRG2, PTEN, and RARβ in a subset of 95 gliomas of different grades. Additionally, the methylation status of MGMT and NDRG2 was analyzed using pyrosequencing (PSQ). The results revealed that the methylation index of individual glioma patients correlates with World Health Organization (WHO) tumor grade and patient’s age. RASSF1A, RUNX3, GATA6, and MGMT were most frequently methylated, whereas the INK4B-ARF-INK4A locus, PTEN, RARβ, and ATM were methylated to a lesser extent. ERCC1, hMLH1, and NDRG2 were unmethylated. RUNX3 methylation correlated with WHO tumor grade and patient’s age. PSQ confirmed significantly higher methylation levels of MGMT and NDRG2 as compared with normal, non-cancerous brain tissue. To conclude, DNA methylation of a whole panel of selected genes can serve as a tool for glioma aggressiveness prediction.

A New Epigenetic Mechanism of Temozolomide Action in Glioma Cells.

Temozolomide (TMZ) is an oral alkylating chemotherapeutic agent that prolongs the survival of patients with glioblastoma (GBM). Despite that high TMZ potential, progression of disease and recurrence are still observed. Therefore a better understanding of the mechanism of action of this drug is necessary and may allow more durable benefit from its anti-glioma properties. Using nucleotide post-labelling method and separation on thin-layer chromatography we measured of global changes of 5-methylcytosine (m5C) in DNA of glioma cells treated with TMZ. Although m5C is not a product of TMZ methylation reaction of DNA, we analysed the effects of the drug action on different glioma cell lines through global changes at the level of the DNA main epigenetic mark. The first effect of TMZ action we observed is DNA hypermethylation followed by global demethylation. Therefore an increase of DNA methylation and down regulation of some genes expression can be ascribed to activation of DNA methyltransferases (DNMTs). On the other hand hypomethylation is induced by oxidative stress and causes uncontrolled expression of pathologic protein genes. The results of brain tumours treatment with TMZ suggest the new mechanism of modulation epigenetic marker in cancer cells. A high TMZ concentration induced a significant increase of m5C content in DNA in the short time, but a low TMZ concentration at longer time hypomethylation is observed for whole range of TMZ concentrations. Therefore TMZ administration with low doses of the drug and short time should be considered as optimal therapy.

The degree of global DNA hypomethylation in peripheral blood correlates with that in matched tumor tissues in several neoplasia.

There are no good blood and serum biomarkers for detection, follow up, or prognosis of brain tumors. However, they are needed for more detailed tumor classification, better prognosis estimation and selection of an efficient therapeutic strategy. The aim of this study was to use the epigenetic changes in DNA of peripheral blood samples as a molecular marker to diagnose brain tumors as well as other diseases. We have applied a very precise thin-layer chromatography (TLC) analysis of the global amount of 5-methylcytosine (m5C) in DNA from brain tumors, colon and breast cancer tissues and peripheral blood samples of the same patients. The m5C level in tissue DNA from different brain tumor types, expressed as R coefficient, changes within the range of 0.2–1.6 and overlaps with R of that of blood samples. It negatively correlates with the WHO malignancy grade. The global DNA hypomethylation quantitative measure in blood, demonstrates a big potential for development of non-invasive applications for detection of a low and a high grade brain tumors. We have also used this approach to analyze patients with breast and colon cancers. In all these cases the m5C amount in DNA cancer tissue match with data of blood. This study is the first to demonstrate the potential role of global m5C content in blood DNA for early detection of brain tumors and others diseases. So, genomic DNA hypomethylation is a promising marker for prognosis of various neoplasms as well as other pathologies.

Diagnosis of Brain Tumors Through Global Specific DNA Methylation Analysis

We describe a new, simple and reliable method for diagnosis of a neoplastic disease. It is based on the cellulose thin layer chromatography (TLC) quantitative determination of 5-methylcytosine (m5C) in DNA from tumor tissue. Currently, there is a wealth of data showing that oxidative stress effecting through reactive oxygen species (ROS) plays a critical role in the etiology and progression of a number of human diseases. Oxidative damage to DNA, lipids, and proteins is deleterious for the cell. 5-methylcytosine, along with other DNA constituents, is a target for ROS, which results in the appearance of a variety of modified nucleic acid bases. Therefore, m5C moiety constitutes the mutational hotspot; it occurs either within a structural gene or its regulatory regions. Here, we show the results of the analysis of DNA extracted from brain tumor and other tissues. DNA was isolated and sheared by enzymatic digestion into nucleotides which, after labeling with [γ-32p]ATP, were separated on cellulose TLC. A chromatogram was evaluated using phosphoimager and an amount of m5dC was calculated as a ratio (R) of m5dC to m5dC+dC+dT spots intensities. The R values decrease as the malignancy of brain tumors increase. It is the important factor for differentiating low and high grade gliomas. It seems that DNA methylation pattern might be a useful tool for the diagnosis of brain tumors or as a marker for the early detection of the relapse of the disease.

Molecular Diagnostics of Brain Tumours by Measuring the 5-Methylcytosine Level in Their DNA

Brain tumours form a group of neoplasms with distinct histological characteristics and different malignancies [Maher 2001]. Various molecular alterations occurring in brain tumors may have diagnostic and predictive values as they are connected with histologically determined tumour types and malignancy grades [Martinez et al. 2009; Martinez and Esteller 2010; Sciume et al. 2010]. Methylation of DNA cytosine residue at the carbon 5 position (m5C) is a common epigenetic marker in many eukaryotes and is often found in the sequence context of CpG. It is assumed that ca 5% of all cytosine residues, i.e. 1% of the nucleic bases, in mammalian genomes are methylated. Although DNA methylation has been viewed as a stable epigenetic mark, studies in the past decade have revealed that this modification is not as static [Wu and Zhang 2010]. In fact, loss of DNA methylation (DNA hypomethylation), has been observed in the specific context and can occur through active, passive or random modification mechanisms. Although the genome in each cell within the body is identical, celland tissue-specific profiles of gene transcription, posttranscriptional modification, modifications and translation are specifically regulated by epigenetic mechanisms that include DNA methylation, histone modification and noncoding RNAs [Robertson 2005]. In the central nervous system epigenetic mechanisms serve as main regulators of homeostasis and plasticity development, which are sensitive to local and global environmental, vascular and systemic factors [Martinez and Esteller 2010]. It is generally accepted that cancer initiation and progression are linked to the disruption of red-ox balance of the cell [Grek and Tew 2010]. Current evidences support an idea that cancer cells are generated by enhanced reactive oxygen species (ROS) generation, their accumulation, and down regulation of antioxidant enzymes [Essick and Sam 2010]. The oxidative damage to the cell caused by ROS plays a critical role in the etiology and progression of different neoplasms in humans [Johnstone and Baylin 2010; Jomova and Valko 2011]. Oxygen radicals cause damage to DNA and chromosomes, induce epigenetic alterations, interact with oncogenes or tumour suppressor genes, and finally change the immunological mechanisms [Robertson 2005; Pelizzola and Ecker 2010].