Myelosuppression is a major and frequently dose-limiting side effect of anticancer

Myelosuppression is a major and frequently dose-limiting side effect of anticancer therapy and is responsible for most treatment-related morbidity and mortality. available.11,12 The aim of this study was to identify substances that protect human HSPCs from irradiation-induced apoptosis Sunitinib Malate reversible enzyme inhibition and to delineate their effects on the BCL-2 protein family. BCL-2 proteins are the master regulators of the intrinsic apoptosis pathway and have either pro- or anti-apoptotic function. Anti-apoptotic BCL-2 proteins (i.e. BCL-2, BCL-XL, MCL-1 and A1/BFL) protect cells from apoptotic stimuli by binding and inactivating their pro-apoptotic antagonists. The pro-apoptotic family members can be subdivided into the downstream effector proteins, BAK and BAX, and the BH3-only proteins (e.g. BIM, PUMA, BMF, BAD and others) that act upstream as cell stress sensors. Upon activation, BH3-only proteins activate BAX and BAK either directly or indirectly through inhibition of the anti-apoptotic BCL-2 proteins. BAX/BAK activation leads to outer mitochondrial membrane permeabilization, caspase activation and cell death.13 Radiotherapy as well as most conventional chemotherapeutic drugs converge at the level of BCL-2 proteins and engage the intrinsic apoptosis pathway.2 A particularly attractive candidate for our study was the epidermal growth factor (EGF) that was recently described to Sunitinib Malate reversible enzyme inhibition prevent irradiation-induced apoptosis of murine HSPCs expansion of human CD34+ cells. We have shown earlier that their pro-survival activity can be attributed to reduced transcription of and mRNA.18 None of these molecules have been tested yet for possible protective effects on human hematopoiesis and, in addition, developed a xenograft model to analyze stress resistance and regeneration of human hematopoiesis nor to promote hematopoietic regeneration following sublethal irradiation apoptotic susceptibility of human HSPCs to taxol and etoposide. This could, however, be ascribed to reduced proliferation rather than to a change in BCL-2 protein regulation. Accordingly, PGE2 did not accelerate regeneration of the human hematopoietic system mice were irradiated at five weeks of age with 3 Gy Sunitinib Malate reversible enzyme inhibition and 6C8 hours (h) later they were injected intravenously into the retrobulbar venous plexus with 3105 human CD34+ cells. Four weeks later, animals were irradiated again. Subsequently, xenograft mice were treated once daily intraperitoneally (i.p.) with human EGF (0.5 g/g body weight), murine EGF (0.5 g/g), human dmPGE2 (2 g/g), human FLT3L (40 ng/g), human TPO (40 ng/g), combinations thereof, or respective carrier solutions (Figure 1). At indicated time points, mice were sacrificed for analysis. Alternatively, mice were treated once daily for seven days with etoposide (20 mg/k, i.p.), and the anti-apoptotic substances were given simultaneously. Open in a separate window Figure 1. Xenograft model for evaluation of radioprotective substances. Cord blood-derived human CD34+ cells were transplanted into sublethally irradiated 5-week old mice. Four weeks later, xenograft Sunitinib Malate reversible enzyme inhibition mice were again irradiated with 3 Gy in order to subject human hematopoiesis to sublethal stress. Subsequently, mice were treated intraperitoneally (i.p.) once daily with the indicated molecules. Control mice were treated with the respective carrier solution (saline Mouse monoclonal to CK1 or ethanol). At day 8 after second irradiation, mice were sacrificed for analysis. Single cell suspensions were obtained from bone marrow and spleen. h: hours; hu EGF: human epidermal growth factor; mu EGF: murine epidermal growth factor; hu dmPGE2: human 16,16-dimethyl-PGE2; hu FLT3L: human FLT3L; TPO: human thrombopoietin. Proliferation, apoptosis and colony formation assays Cell cycle status and proliferation were determined by double staining for Ki-67 (BioLegend) and DAPI (Sigma-Aldrich) or incubation with CFSE (1 M; Sigma-Aldrich). Apoptosis was determined by combined staining with 7-AAD and Sunitinib Malate reversible enzyme inhibition Annexin-V. Specific apoptosis triggered by stress was calculated as follows: (induced apoptosis C spontaneous apoptosis)/(100 C spontaneous apoptosis). For colony forming assays, 150,000 human CD45+ cells isolated.