Abstract

G-CSF mobilized peripheral blood CD34+ cells are now the preferred and major source of hematopoietic stem and progenitor cells harvested for both autologous and allogeneic transplantation. Several mechanisms, like SDF-1/CXCR4 interactions or degradation of adhesion molecules by proteolytic environnement, are involved in the mobilization process. However this phenomenon is still partially understood. Gene expression analysis has identified an overexpression of the caspase-3 gene in CD34+ mobilized cells, compared to CD34+ from normal bone marrow. Caspase-3 is the main effector of the terminal phase of apoptosis. However recent studies have provided evidence of its implication in non apoptotic cellular processes, such as differentiation, migration and cytoskeleton modelling. We evaluated by multicolour flow cytometry the expression of activated caspase-3 in G-CSF mobilized CD34+/CD45+ cells from blood (n=16), and from apheresis products (n=10). CD34+/CD45+ cells from normal bone marrow (n=4) served as control. Caspase-3 activity on fluorescent substrate (PhiPhiLux method) and apoptosis (Annexin V assay) were also evaluated. Finally we analysed the expression of anti apoptotic proteins Bcl-2, Bcl-Xl, and of Heat Shock Proteins HSP27, HSP70 and HSP90 in the same cell population. There was no significant difference for apoptosis between mobilized and bone marrow CD34+ cells (26% versus 33% apoptotic cells). Activated caspase-3 levels were significantly higher in mobilized CD34+ cells (mean fluorescence intensity 3.64 fold higher). This was consistent with cleavage of caspase-3 substrate observed in mobilized cells, but not in bone marrow CD34+ cells. An increased expression of HSP90 (of which caspase-3 is a client protein) was observed in peripheral CD34+ cells, but there was no variation of BCl-2 and Bcl-Xl expression. Our results show an activation of caspase-3 in the mobilized peripheral blood CD34+ cells, which appears to be independent of apoptosis induction. The role of this activation and possible control by HSPs warrants further analysis to establish its relationship with mobilization mechanisms.

Disclosures: No relevant conflicts of interest to declare.

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