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Perturbation of pyrimidine biosynthesis and activation of DNA damage response network by 5- aminoimidazole-4-carboxamide ribonucleoside (AICAr) in myeloid leukemia cells

INTRODUCTION: We have previously reported that 5- aminoimidazole-4-carboxamide ribonucleoside (AICAr), a widely used activator of AMP-activated kinase (AMPK), as well as a precursor in purine biosynthesis, not only inhibits proliferation, but also promotes differentiation of myeloid leukemia cells. AICAr was demonstrated to inhibit pyrimidine synthesis at a step downstream of the dihydroorotate dehydrogenase (DHODH), leading to cell differentiation and S-phase arrest. Nucleotide pool depletion is known to initiate a DNA damage response network via activation of the ataxia telangiectasia and RAD3-related (ATR)/checkpoint kinase 1 (Chk1) signaling pathway. AIM: The present study was undertaken in order to test for the possible role of ATR/Chk1 in AICAr mediated effects. MATERIALS AND METHODS: Flow cytometry analyses, immunoblotting, administration of pharmacological inhibitors and siRNA-mediated gene silencing were used in commercially available acute myeloid leukemia (AML) cell lines – HL60, MOLM-14, NB4, THP-1 and U937. RESULTS: Western blot analysis revealed that AICAr increased the level of Ser-345-phosphorylated Chk1, and the effect was abolished by the addition of either nucleosides or uridine. Furthermore, AICAr and the DHODH inhibitor brequinar had similar effects on the expression of the differentiation markers and cell cycle arrest, while pharmacological ATR/Chk1 pathway inhibition by caffeine and Torin2 abrogated both of their effects. Moreover, these effects were significantly reduced by siRNA-mediated Chk1 knockdown. Our preliminary data suggest that both AICAr and brequinar increase Tyr-15- phosphorylated Cdk1. In THP-1 cells, AICAr induces accumulation of PU.1 transcription factor which favors macrophage differentiation. CONCLUSION: AICAr induces cell cycle arrest and differentiation in monocytic cell lines via activation of ATR/Chk1 DNA-damage signaling pathway and these effects are abolished by the addition uridine.

AICAr ; leukemia cells ; DNA damage ; pyrimidines

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