| Background & Objective Cellular growth was regulated by many cell cycle-regulatory factors. The key checkpoints of cell cycle is located at G1/S. The p16 protein may act as a cyclin-dependent kinase inhibitor by binding competitively to Cdk4 and thereby preventing the interaction of Cdk4 with cyclin Dl. The inactivation of either Rb or p16 gene would, therefore, abrogate an important pathway for inhibiting cell growth and thus promote tumorigenesis. In normal cell, progression through the G1 phase of the cell cycle requires function inactivation of Rb through its phosphorylation by a complex of cyclin Dl and Cdk4 or cyclin-dependent kinase 6. p16 is one of the most relevant genetically inactivated tumor suppressor genes. The inactivation of p1 6 in cell cycle may cause cell lost the ability to activate pRB and grow out of control. Alteration of the p16 gene for the carcinogenesis in the liver involves different molecular mechanisms, including deletion, mutation or methylation.More recently, it has become clear that epigenetic alterations, particularly tumor suppressor gene silencing via DNA methylation, also plays a critical role in the initiation and progression of cancer. Aberrant methylation conformed through the convertion cytosine of S-adenosylmethionine (SAM)into 5-methylcytosine (5mC) by the action of methionine adenosyltransferase (MAT).Studies using DNA methyltransferase inhibitors like 5-azacytidine (5-aza-CR) and 5-aza-2'- deoxycytidine (decita-bine) in order to arrest DNA methylation, show that demethylation of 5' gene sequences can activate gene expression. In order to analyse the correlation between DNA methylation alternation and hepatocellular carcinoma cell line, the hepatoma cell line HuH-7 were exposed to the agent 5-Aza-CR to study the effects of 5-Aza-CR on the growth of the cell line.Methods Cell morphology, growth speed, cell cycle distribution, apoptotic rate, the level of methylation and unmethylation status of pi 6, expression of pi 6 mRNA, and tumorigenicity in nude mice of HuH-7 cell after treatment with 5-aza- CR were observed under phase contrast microscope, MTT assay, flow cytometer, methylation specific PCR and RT-PCR.Results Afer treatment with 5-aza-CR displayed that supermicro structure inclined to normal and a slowed growth, and that the cell growth rate decreased in a dose- and time-dependent manner. When 5-aza-CR increased to 5/imol/L, most of the cells were dead. The growth inhibiting rate moved up as the concentration increased. The cell cycle was influenced under the well-chosen concentration of 5-aza-CR (5/imol/L). In the cell, Orl increased by 41.1%, S and G2+M decreased by 39.9% and 2.2%, and apoptotic rate increased by 30.9%. In xenografted of nude mice, Gl increased by 27.4%, S and G2+M decreased by 25.8% and 1.6%, and apoptotic rate increased by 2.9%. DNA was abstracted before and after the drug application, Only methylating PCR product appeared before treatment with drugs, Conversely, Only demethylating PCR amplification product showed after treatment with drugs. For the xenografted of nude mice, methylating PCR product appearedin control group, however, methylating and demethylating PCR amplification product showed in test group. Both cell and xenografted of nude mice showed the expression of pl6 mRNA in test group, conversely, showed no expression of pl6mRNA in control group. Tumorigenicities of nude mice of test group were lower than control group.Conclusion Hepatocellular carcinoma is associated with alternation of DNA methylation. 5-aza-CR, as a well-established inhibitor of DNA methylation, may slow the growth of tumor cells by demethylation and reactivating growth-regulatory genes silenced by de novo methylation and genes silenced by promoter hypermathylation (such as tumor suppressor pi6 gene) at cell line and xenografted of nude mice. The study may prompt pi6 express after demethylation by the drug, then restrained cell growth by inhibition of pRb phosphorylation and Gi arrest.
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