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Chk1-mediated DNA damage response and monocytic differentiation

Introduction: Depletion of nucleotide pools is known to induce monocytic differentiation in leukemia cells. Our recent study demonstrated that 5- aminoimidazol-4- carboxamide ribonucleoside (AICAr) inhibited UMP synthase and induced S-phase arrest, as well as cellular differentiation. Its effects mimicked those of brequinar, a dihydroorotate dehydrogenase inhibitor. Both agents inhibited key enzymes in de novo pyrimidine synthesis leading to nucleotide depletion and activation of the ataxia telangiectasia and RAD3-related (ATR)/checkpoint kinase 1 (Chk1) DNA-damage signaling pathway. Cytarabine, an antimetabolite analogue of cytidine and a well-known chemotherapeutic, interferes with DNA synthesis exerting not only cytotoxic effects, but also monocytic differentiation. However, the exact mechanism responsible for cellular differentiation in response to cytarabine remains to be elucidated. Aim: This study is aimed to test for the role of Chk1 activation in monocytic differentiation by comparing response to cytarabine to the effects of pyrimidine synthesis inhibitors. Methods: Flow cytometry, western blotting, administration of pharmacological inhibitors Torin2 and VE-821, and siRNA transfection were used in commercially available monocytic leukemia cell lines and/or non-adherent mononuclear cells from bone marrow samples of five acute myeloid leukemia (AML) patients. Results: Effects of cytarabine on cell viability, expression of differentiation markers, and the level of Ser-345-phosphorylated Chk1, a marker of Chk1 activation, were dose-dependent and similar to the effects of AICAr and brequinar. In addition, administration of pharmacological inhibitors of ATR/Chk1 pathway, as well as transfection of U937 cells with siRNA targeting Chk1, significantly reduced cellular differentiation in response to cytarabine, AICAr and brequinar. Furthermore, cytarabine dose- dependently induced differentiation in two primary AML samples that were responsive to pyrimidine synthesis inhibitors. Conclusion: Cytarabine-mediated monocytic differentiation occurs through the activation of DNA-damage checkpoint kinase Chk1.

nucleotides ; cytarabine ; AICAr ; leukemia

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