Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways

International audienceBackground: Several studies show that bone marrow (BM) microenvironment and hypoxia condition can promote the survival of leukemic cells and induce resistance to anti-leukemic drugs. However, the molecular mechanism for chemoresistance by hypoxia is not fully understood.Methods: In the present study, we investigated the effect of hypoxia on resistance to two therapies, methotrexate (MTX) and prednisolone (PRD), in two cell models for acute lymphoblastic leukemia (ALL). To look for an implication of hypoxia in chemoresistance, cell viability, total cell density and cell proliferation were analyzed. Survival and death signaling pathways were also screened by "reverse phase protein array" (RPPA) and western blotting experiments conducted on selected proteins to confirm the results.Results: We found that hypoxia promotes chemoresistance in both ALL cell lines. The induction of drug-resistance by hypoxia was not associated with an increase in total cell density nor an increase in cell proliferation. Using RPPA, we show that chemoresistance induced by hypoxia was mediated through an alteration of cell death signaling pathways. This protective effect of hypoxia seems to occur via a decrease in pro-apoptotic proteins and an increase in anti-apoptotic proteins. The results were confirmed by immunoblotting. Indeed, hypoxia is able to modulate the expression of anti-apoptotic proteins independently of chemotherapy while a pro-apoptotic signal induced by a chemotherapy is not modulated by hypoxia.Conclusions: Hypoxia is a factor in leukemia cell resistance and for two conventional chemotherapies modulates cell death signaling pathways without affecting total cell density or cell proliferation

Additional file 3: Figure S2. of Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways

RPPA analysis of Nalm-6 cell lines treated with MTX. Signal intensities were normalized by Z-score and signals were used for a hierarchical cluster analysis. A black color indicates that protein from Nalm-6 cells treated with several concentrations of MTX in normoxia versus hypoxia, matches the medium expression level calculated for a specific protein in a particular experimental condition. Higher level expression than mean is shown as a red color, and a green color refers to a lower level expression than mean. Protein names are listed below and experimental conditions are mentioned on the right-hand side. All antibodies used for RPPA were validated by Western blot. All antibodies are listed in Additional file 7: Table S1. (DOCX 145 kb

Additional file 1: Protocol S1. of Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways

RPPA full protocol. (DOCX 15 kb

Additional file 6: Figure S5. of Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways

RPPA analysis of Reh cell lines treated with PRD. Signal intensities were normalized by Z-score and signals were used for a hierarchical cluster analysis. A black color indicates that protein from Reh cells treated with several concentrations of PRD in normoxia versus hypoxia, matches the medium expression level calculated for a specific protein in a particular experimental condition. Higher level expression than mean is shown as a red color, and a green color refers to a lower level expression than mean. Protein names are listed below and experimental conditions are mentioned on the right-hand side. All antibodies used for RPPA were validated by Western blot. All antibodies are listed in Additional file 7: Table S1. (DOCX 148 kb

Additional file 2: Figure S1. of Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways

Differential effect of MTX and PRD on leukemic cell proliferation independently of hypoxia. (A) Effect of hypoxia (5 % O2) on cell survival in leukemic cell (day 0, day 2 and day 6). (B) Effect of chemotherapies and hypoxia (5 % O2) on cell survival in leukemic cells (day 6). Nalm-6 and Reh cells were loaded with CFDA SE at day 0 and cultured either in normoxia or in hypoxia and in the presence of MTX or PRD. The relative decrease of CFDA SE staining was monitored by flow cytometry. (DOCX 111 kb

Nature and Decay Pathways of Photoexcited States in CdSe and CdSe/CdS Nanoplatelets

The nature and decay dynamics of photoexcited states in CdSe core-only and CdSe/CdS core/shell nanoplatelets was studied. The photophysical species produced after ultrafast photoexcitation are studied using a combination of time-resolved photoluminescence (PL), transient absorption (TA), and terahertz (THz) conductivity measurements. The PL, TA, and THz exhibit very different decay kinetics, which leads to the immediate conclusion that photoexcitation produces different photophysical species. It is inferred from the data that photoexcitation initially leads to formation of bound electron–hole pairs in the form of neutral excitons. The decay dynamics of these excitons can be understood by distinguishing nanoplatelets with and without exciton quenching site, which are present in the sample with close to equal amounts. In absence of a quenching site, the excitons undergo PL decay to the ground state. In nanoplatelets with a quenching site, part of the initially produced excitons decays by hole trapping at a defect site. The electron that remains in the nanoplatelet moves in the Coulomb potential provided by the trapped hole

Bimolecular Auger Recombination of Electron–Hole Pairs in Two-Dimensional CdSe and CdSe/CdZnS Core/Shell Nanoplatelets

We have determined the Auger recombination kinetics of electrons and holes in colloidal CdSe-only and CdSe/CdS/ZnS core/shell nanoplatelets by time-resolved photoluminescence measurements. Excitation densities as high as an average of 18 electron–hole pairs per nanoplatelet were reached. Auger recombination can be described by second-order kinetics. From this we infer that the majority of electrons and holes are bound in the form of neutral excitons, while the fraction of free charges is much smaller. The biexciton Auger recombination rate in nanoplatelets is more than 1 order of magnitude smaller than for quantum dots and nanorods of equal volume. The latter is of advantage for application in lasers, light-emitting diodes, and photovoltaics