Promoter Methylation Status of Survival-Related Genes in MOLT- 4 Cells Co-Cultured with Bone Marrow Mesenchymal Stem Cells under Hypoxic Conditions.

OBJECTIVES

DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes.

MATERIALS AND METHODS

In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes' promoter regions.

RESULTS

The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes.

CONCLUSIONS

Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies.