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Introduction Interaction between acute myeloid leukemia (AML) cells and bone marrow stromal (BMS) cells is known to contribute to development of resistant AML clones. Our recent study demonstrated that two drugs that inhibit pyrimidine synthesis, 5-aminoimidazole-4- carboxamide ribonucleoside (AICAr) and brequinar, induced differentiation of AML cells by activating checkpoint kinase-1 (Chk1). In addition, Chk1 was necessary for AML differentiation in response to low doses of cytarabine. The murine BMS cell line MS-5 is known to attenuate cytarabine-mediated cytotoxicity, but the role of stromal cells on AML differentiation is unknown. The aim of this study was to investigate the effects of drugs that activate Chk1 in a co-culture system. Material and Methods MS-5 cells (ACC 441, DSMZ) were seeded 24 hours prior to human AML cell lines U937 (85011440, ECACC) and THP-1 (a kind gift from Paolo Gallipoli). The cells were incubated with AICAr (0.2 mM), brequinar (0.5 µM) or cytarabine (10, 100, 1000 nM) for 72 hours. In some experiments, nucleosides were added 15 minutes after the addition of agents. Viable cells were counted using hemocytometer and trypan blue exclusion. Cells were stained with May-Grünwald- Giemsa and morphology was analyzed by AxioVert 200 microscope and Axiocam MRc 5 camera. Cell cycle analysis of propidium iodide-labeled cells and the expression of differentiation markers were analyzed by flow cytometry. Results and Discussions In AML cell lines, the presence of MS- 5 reduced cytarabine-mediated toxicity and S-phase arrest caused by pyrimidine synthesis inhibitors, but inhibited the expression of differentiation markers only in U937 cells treated with low-dose cytarabine. However, AICAr and high-dose cytarabine induced significant changes in morphology of stromal cells. AICAr inhibited the growth and induced fibrocyte-like appearance in MS-5 cells. The addition of nucleosides completely prevented cell cycle arrest and differentiation of AML cells induced by AICAr and brequinar, but had no effects on AICAr-induced changes in stroma. Preliminary data suggest that AICAr activates AMPK and inhibits mTOR in MS-5 cells. Conclusion The presence of stromal cells decreases differentiation of leukemia cells only in response to low-dose cytarabine. AICAr inhibits proliferation and induces fibrocyte-like changes in stromal cells that are independent of pyrimidine synthesis inhibition. These results suggest that mechanisms responsible for phenotypic changes in response to AICAr are different in AML and stromal cells. |
