BACKGROUND Mesenchymal stem cells (MSCs) have immense self-renewal capability. They can be differentiated into many cell types and therefore hold great potential in the field of regenerative medicine. MSCs can be converted into beating cardiomyocytes by treating them with DNA-demethylating agents. Some of these compounds are nucleoside analogs that are widely used for studying the role of DNA methylation in biological processes as well as for the clinical treatment of leukemia and other carcinomas. AIMS To achieve a better therapeutic option for cardiovascular regeneration, this study was carried out using MSCs treated with two synthetic compounds, zebularine and 5-azacytidine. It can be expected that treated MSCs prior to transplantation may increase the likelihood of successful regeneration of damaged myocardium. METHODS The optimized concentrations of these compounds were added separately into the culture medium and the treated cells were analyzed for the expression of cardiac-specific genes by RT-PCR and cardiac-specific proteins by immunocytochemistry and flow cytometry. Treated MSCs were cocultured with cardiomyocytes to see the fusion capability of these cells. RESULTS mRNA and protein expressions of GATA4, Nkx2.5, and cardiac troponin T were observed in the treated MSCs. Coculture studies of MSCs and cardiomyocytes have shown improved fusion with zebularine-treated MSCs as compared to untreated and 5-azacytidine-treated MSCs. CONCLUSION The study is expected to put forth another valuable aspect of certain compounds, that is, induction of transdifferentiation of MSCs into cardiomyocytes. This would serve as a tool for modified cellular therapy and may increase the probability of better myocardial regeneration.

UNLABELLED Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. CONCLUSION Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs.

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First, we observe an increase in the DNA binding when zebularine is incorporated into the DNA, compared to deoxycytidine and 5-fluorodeoxycytidine, together with a strong decrease in the dissociation rate. Second, we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible, differing thus from 5-fluorodeoxycytidine. Third, no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA, hindering the methylation and decreasing the dissociation, thereby trapping the enzyme and preventing turnover even at other sites.