mRNA expression was measured using qRT-PCR. Flow cytometric analysis of cell cycle progression and apoptotic markers was used to determine cytotoxicity of the compounds. Overall changes in DNA methylation level were quantified by direct estimation of 5-methyl-2'-deoxycytidine- 5'-monophosphate by HPLC using digested genomic DNA. Methylation-specific PCR was employed to detect the efficiency of individual agents on cyclin-dependent kinase inhibitor 2B and thrombospondin-1 hypermethylated gene loci.
#KID3 CARDIOMYOCYTE SERIES#
A comparative study of hypomethylating activities of a series of 5- azacytidine nucleosides: 5- azacytidine (AC), 2'-deoxy- 5- azacytidine (DAC) and its α-anomer (α-DAC), 5,6-dihydro- 5- azacytidine (DHAC), 2'-deoxy- 5,6-dihydro- 5- azacytidine (DHDAC, KP-1212) and its α-anomer (α-DHDAC), and of a 2-pyrimidone ribonucleoside (zebularine) was conducted. Restoration of transcriptionally silenced genes by means of methyltransferases inhibitors plays a crucial role in the current therapy of myelodysplastic syndromes and certain types of leukemias. MatouÅ¡ová, Marika Votruba, Ivan Otmar, Miroslav TlouÅ¡Å¥ová, Eva Günterová, Jana MertlÃková-Kaiserová, Helena 5- Azacytidine also could be a potential therapeutic candidate for CMGTs.Ģ´-deoxy- 5,6-dihydro- 5- azacytidine - a less toxic alternative of 2´-deoxy- 5- azacytidine: a comparative study of hypomethylating potential. These results suggest that DAPK1 and MGMT methylation can serve as sensitive diagnostic biomarkers and therapeutic targets for CMGTs. Meanwhile, 5- azacytidine increased the expression of DAPK1 and MGMT mRNA.
However, the DAPK1 and MGMT genes were re-inducible in CHMm and CHMp cells treated with 5 μM 5- azacytidine. Furthermore, the DAPK1 and MGMT genes were hypermethylated in CHMm/CHMp cells and clinical malignant tumor samples, but not in normal female dogs' blood and tissue. 5- Azacytidine may suppress the proliferation of CHMm and CHMp cells. Effect of 5- azacytidine on the methylation of DAPK1 and MGMT gene, and DAPK1 and MGMT mRNA expression in CHMm and CHMp cells were analyzed by MSP assay and qRT-PCR assay, respectively. The DAPK1 and MGMT gene methylation patterns in CHMm and CHMp cells and CMGTs blood/tissue samples were analyzed by MSP assay. The effects of 5- azacytidine on CHMm and CHMp cell proliferation were evaluated by MTT assay. Therefore, we sought to demonstrate the effects of 5- azacytidine on the proliferation of CMGTs cell, and elucidate the potential molecular mechanisms of action in these cancerous cells. However, the methylation of the DAPK1 and MGMT is unknown with respect to CMGTs. 5- Azacytidine is a known specific demethylation drug that covalently binds to DNA methyltransferase. Aberrant DAPK1 and MGMT methylation associated with tumor formation and development in various cancers.
Ren, Xiaoli Li, Huatao Song, Xianyi Wu, Yuhong Liu, YunĬanine mammary gland tumors (CMGTs) are the most common, spontaneous types of neoplasias in female dogs. 5- Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells.
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