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Changes of cell cycle regulation in HepG2 cells through contribution of four independent pathways after targeted silencing of AHCY hydrolase

Most methylating enzymes use AdoMet as methyl group donor to methylate their substrate. AdoHyc is additional product of transmethylation reactions and is also proven to be one of the strongest competitive inhibitors of methyltransferases. Proper activity of AHCY hydrolase is therefore essential for fast removal of AdoHyc in order to avoid global alterations of cell methylation. Recently, it has been reported that level of AHCY hydrolase expression might have impact on changes in cell characteristics usually associated with so called cancer phenotype: cell cycle regulation, cell proliferation and migration. To investigate possible changes, targeted silencing of AHCY hydrolase in hepatocellular carcinoma cell line was performed using shRNA. Western blott was used to determine the amount of cell cycle regulating proteins involved in various independent signaling pathways. Metabolic cell activity was measured by MTT viability test and Neutral red uptake assay to analyze differences in cell proliferation, while transwell chamber assay was used to study changes in cell migration. Western blott analysis suggests G2/M cell cycle arrest induced through p53 independent GADD45a pathway. GADD45a could both act through strong Tyr-15 phosphorylation of Cdc2 or interaction with p21. High levels of p27 indicate G1/S arrest through inactivation of CyclinE/CDK2 complex. Further changes include Ras independent activation of MEK/ERK pathway possibly through regulation by p21/PAK/PcRaf(Ser338) cascade. Other than MEK/ERK activation, G1 passage promotion is also caused by degradation of p53 through Chk2 signaling. We found that both metabolic activity tests show decrease and indicate reduced cell proliferation. Also, slower cell migration is seen using transwell assay. Progression to cancer phenotype usually starts with alterations of cell cycle since its control is necessary for preventing unrestrained cellular growth. Our research indicates complex changes in regulation of cell cycle after AHCY hydrolase silencing with competing contribution of various pathways. Inhibitors of AHCY are currently being investigated as potential cancer treatments, thus understanding their effect on cell cycle is critical to predict possible treatment outcomes as well as for effective mixed drug therapies.

methylation, AHCY hydrolase, cell cycle, proliferation, cancer

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