engleski

Cell cycle arrest and monocytic differentiation by activating checkpoint kinase 1

Our previous study demonstrated that 5- aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a widely used activator of AMP-activated kinase (AMPK), promotes differentiation and inhibits proliferation of monocytic U937 cells. Furthermore, AICAr was demonstrated to inhibit pyrimidine synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH), and AICAr-mediated effects on differentiation were prevented by the addition of uridine. Depletion of nucleotide pools is known to activate the DNA damage signaling pathway through activation of the ataxia teleangiectasia and RAD3- related (ATR)/checkpoint kinase 1 (Chk1)- mediated checkpoint in S-phase of the cell cycle. Present studies are aimed to test for the role of ATR/Chk1 in AICAr-mediated effects. Western blot analysis revealed that AICAr increased the level of Ser-345-phosphorylated Chk1, and the effect was abolished by addition of either nucleosides or uridine. The activation of Chk1 was observed in the presence of DHODH inhibitor brequinar, and the increase in the level of Ser-345-phosphorylated Chk1 in response to AICAr, brequinar and their combinations followed the same pattern as the expression of differentiation markers and S-phase arrest. Pharmacological inhibition of ATR/Chk1 pathway by caffeine, Torin2 and VE-821 prevented differentiation and cell cycle arrest in response to AICAr and brequinar. Transfection of U937 cells with siRNA targeting Chk1 decreased the level of Chk1 and significantly reduced the effects of AICAr and brequinar on the expression of differentiation markers and cell cycle arrest. These results demonstrate that AICAr-mediated differentiation of U937 cells is mediated by activation of DNA damage checkpoint kinase Chk1 induced by pyrimidine depletion.

monocytic differentiation ; Chk1 ; AICAr ; cell cycle

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