Bone Marrow Mesenchymal Stromal Cells Regulate the Metabolism of H₂O₂ In Human Leukemic Cells.

Abstract 1058

Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic cells and it has been recently established by the Guy Sauvageau's group (Institute for Research in Immunology and Cancer, Montréal, QC) that glutathione peroxydase 3 (GPx-3) promotes competitiveness of Hoxa9-Meis1 induced leukemic stem cells in which Gpx3 overexpression is concomitant of a decrease in H₂O₂ level and inactivation of p38 MAPK (Herault O et al, ASH annual meeting, 2010 - submitted). Leukemic cells located in the bone marrow (BM) are interacting with a microenvironment which plays a crucial role in the development and progression of leukemia. Mesenchymal stromal cells (MSCs) constitute a population of multipotential cells giving rise to the different hematopoietic microenvironmental cells (adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells). The aim of our study was to evaluate the effects of MSC-contact on the GPx-3/H₂O₂/p38 MAPK axis in human leukemic cells and to assess the cell cycle changes associated with the modification of H₂O₂ metabolism.

BM MSCs were obtained from informed and consenting patients undergoing orthopedic surgery, following a procedure approved by the local ethical committee. Nucleated cells harvested from the iliac crest were seeded (5.10⁴ cells per cm²) in α-MEM supplemented with 10% fetal calf serum (FCS), 20 mmol/l L-glutamine, and 100 units/mL penicillin G. Cells were incubated at 37°C in 95% humidified air and 5% CO2. When fully confluent, the layer of adherent cells was trypsinized (0.25% trypsin-EDTA), and the cells were resuspended in culture medium and seeded at 10³ cells per cm² (passage 1 - P1). BM MSCs were used at P2 in all experiments. The three-lineage mesenchymal differentiation of the BM MSCs used was systematically checked by culturing cells in adipogenic, chondrogenic, and osteogenic induction media as previously described (Delorme et al, Blood 2008, 111:2631). The human KG1a leukemic cell line (FAB M0/M1 CD34+ leukemic cells) was cultured in RPMI 1640 with 20 mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100 mg/mL streptomycin. KG1a cells were seeded at 1.5 10⁵ cells/cm² and cocultured with MSCs for 72 h. We have defined two distinct localizations of leukemic cells relative to MSC layer: those in supernatant (non-adherent cells) and cells adhering on the surface of MSCs. The expression of antioxydative enzymes, H₂O₂ level, p38 MAPK activation (T180/Y182), cell cycle, proliferation and immunophenotype of these two cell fractions were evaluated at day 0 and day 3. The expression of SOD1, SOD2, SOD3, CAT, TXN, TXN2, GLRX, GLRX2, GLRX3, GLRX5, PRDX, PRDX2, PDRX3, PRDX4, PRDX5, PRDX6, GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7 and GSR antioxydative genes and CDKN1A (p21CIP1) gene was quantified by qRT-PCR (Universal ProbeLibrary, Roche). SDS-PAGE and western-blot experiments were realized to quantify GPx-3 expression and p38 MAPK activation. Flow cytometry studies were performed: (a) to quantify H₂O₂ level by dichlorodihydrofluorescein diacetate (DCF-DA) staining; (b) to analyze the cell cycle by staining with 7-aminoactinomycin D (7AAD), Alexa Fluor®488-conjugated anti-human Ki67 and Alexa Fluor®488-conjugated anti-phospho-histone H3 (ser10) antibodies; (c) to track the cell divisions with carboxyfluorescein diacetate N-succinimidyl ester (CFSE).

Supernatant of MSCs did not modify the GPx-3/H₂O₂/p38 MAPK axis in KG1a cells. Conversely, when compared with cells in the supernatant of MSCs, adhering KG1a cells were characterized by the exclusive overexpression of GPX3 antioxydative gene, the induction of GPx-3 production, a major decrease in H₂O₂ concentration and the inactivation of p38 MAPK. These effects were concomitant of cell cycle inhibition: increase in G0 phase, decrease in S and M phase, overexpression of CDKN1A and reduced mitotic activity (CFSE).

Altogether these findings suggest that the bone marrow microenvironment plays a key role in the regulation of the oxidative metabolism of leukemic cells by promoting the inhibition of the H₂O₂/p38 MAPK axis via the induction of GPx-3. Modulation of the GPx-3/H₂O₂/p38 MAPK pathway by targeting of microenvironmental interactions in leukemia may have clinical relevance and it will be important to verify if these results can be extended in vivo to other models of human leukemias.