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Nucleotide metabolism and activation of Chk1 in monocytic differentiation

INTRODUCTION: Inhibitors of pyrimidine synthesis are known to induce monocytic differentiation in leukemia cells. Our previous studies show that 5-aminoimidazol-4- carboxamide ribonucleoside (AICAr) inhibits UMP synthesis and induces S-phase arrest as well as cellular differentiation. Brequinar, inhibitor of dihydroorotate dehydrogenase, one of the key enzymes in pyrimidine de novo synthesis, exerts similar effects. AIM: The aim of this study is to further test for the role of Chk1 DNA-damage pathway in differentiation by comparing response to cytarabine, an antimetabolite analogue of cytidine, to the effects of pyrimidine synthesis inhibitors. MATERIALS AND METHODS: U937 cells were incubated in the presence of AICAr, brequinar and cytarabine. The expression of differentiation markers and DNA content for cell cycle analysis were determined by flow cytometry. Total cell lysates were analyzed for the level of specific proteins using western blot. Protein level was downregulated using RNA-interference. RESULTS: Results of our study show that the effects of cytarabine on cell viability and the expression of differentiation markers in monocytic leukemia cells are similar to the effects of AICAr and brequinar. Furthermore, western blot analysis confirmed the activation of Chk1 and inhibition of Cdk1 corelated with cell cycle arrest. Use of Torin2 and VE-821, pharmacological inhibitors of ATR/Chk1 DNAdamage signaling pathway, as well as transfection of U937 cells with siRNA targeting Chk1, reduced differentiative effects of all agents tested. CONCLUSION: Nucleotide metabolism perturbation induces monocytic differentiation through activation of ATR/Chk1 signaling pathway

nucleotides ; Chk1 ; differentiation

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