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Response of U937 human acute myeloid leukemia cell line to differentiation treatment in co-culture with murine stromal cell line MS-5

Introduction Mesenchymal stromal cells (MSCs) form bone marrow niche that supports hematopoiesis. For investigation of hematopoietic stem cell (HSC) disorders such as acute myeloid leukemia (AML) it is important to understand interaction of HSCs and MSCs. Stromal cells influence the proliferation and apoptosis of leukemia cells and thus may contribute to the resistance of residual AML blasts to chemotherapy. Our previous study demonstrated that two drugs that inhibit pyrimidine synthesis, AICAr and brequinar, inhibit proliferation and induce differentiation of AML cells by activating DNA damage signaling pathway. AICAr, a precursor in purine biosynthesis and a wellestablished activator of AMP- kinase inhibited uridine monophosphate synthesis in an AMPK- independent manner, and brequinar inhibited the activity dihydroorotate dehydrogenase. Our recent data suggest that low doses cytarabine used the similar mechanism to induce differentiation of AML cells by activating Chk1. The murine bone marrow stromal cell line MS-5 is known to improve AML cell survival in vitro and attenuate cytarabine- induced cell killing, but the role of stromal cells on leukemia cell differentiation is unknown. The aim of this study was to test for the effects of marrow stromal cells on differentiation and growth arrest of AML cells in response to pyrimidine synthesis inhibitor and low dose cytarabine Materials and Methods: MS-5 cell line (ACC 441) and human AML cell line U937 (85011440) were obtained from DSMZ (Germany) and ECACC (UK), respectively. MS-5 cells were seeded in 6-well plates 24 hours before U937 cells, and treated with AICAr (0.2 mM), brequinar (0.5 µM) and cytarabine (10, 100, 1000 nM) for 72 hours. The number of viable cells was determinated by hemocytometer and trypan blue exclusion. The expression of differentiation markers CD11b and CD64, and cell cycle analysis of propidium iodidelabeled cells were determined by Attune acoustic focusing cytometer (Life Technologies, USA) and the data were analyzed by FlowJo v.10 platform (FlowJo LCC, USA). Morphology of MS-5 cells stained with May-Grünwald- Giemsa was examined using AxioVert 200 microscope and photographed using Axiocam MRc 5 camera (Carl Zeiss AG, Germany). Results: Our preliminary results suggest that co-culture of U937 cells with MS-5 increases number of viable U937 cells treated with 100 nM and 1000 nM cytarabine, and flow cytometry data confirmed that cytarabine mediated killing is attenuated by the presence of stromal cells. However, the presence of MS-5 cells did not inhibit the expression of differentiation markers in response to AICAR, brequinar and low doses of cytarabine. In addition, AICAr induced appearance of fibrocyte-like morphology in MS-5 cells grown in the presence or absence of U937 cells, and no such effects were observed in the presence of brequinar or cytarabine. The addition of nucleotides had no effects on the induction of fibrocyte-like cells suggesting that the effects of AICAr on MS-5 cells does not depend on inhibition of pyrimidine synthesis. However, the similar effects were observed in MS-5 cells treated with metformin, another AMPK agonist, suggesting that AICAr-mediated effects may be AMPK-dependent. Conclusions: Our preliminary data suggest that the presence of stromal cells attenuates killing by cytrabine but did not prevent differentiation induced by pyrimidine synthesis inhibitors and cytarabine. In addition, AICAr induces differentiation of MS-5 cells into fibrocyte-like cells, probably by activation of AMPK.

AML ; differentiation ; U937 ; stromal cells ; MS-5

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