P59 Involvement of CD9 in Erythroid Progenitor Cell Apoptosis

Abstract

Introduction The study of erythroid progenitor cell proliferation and differentiation in vitro is essential if we are to achieve our goal of producing therapeutic quantities of red cells by cell culture. The mechanisms of cell regulation need to be appreciated and a cell culture environment developed for optimal cell renewal and growth. Changes in cell surface molecules and understanding of their function form an important part of the study together with knowledge about the genes involved in the erythropoietic process, including those genes involved in apoptosis. CD9, a member of the tetraspanin super family, is reportedly involved in tumour progression and metastasis and has been implicated in apoptosis of cancer cells.Methods Apoptosis was induced in erythroid progenitor cells by depriving the cultures of erythropoietin (EPO). CD34+ cells were isolated from the peripheral blood of adults and cultured for 5 days in serum free medium with IL‐3 (1 ng mL−1), SCF (10 ng mL−1) and EPO (3 IU mL−1). On day 6 the culture was divided and half the cells deprived of EPO; both cultures were analysed on day 8. Total RNA was extracted and run on Affymetrix microarray chips to compare the mRNA profile of the apoptotic and control cells.Results One gene that was consistently upregulated in the apoptotic cells was CD9. This result was confirmed by real‐time PCR. Immunofluorescent staining and confocal microscopy revealed that CD9 protein expression changed during apoptosis from low level expression within the cell membrane to a much higher level, which was cytoplasmic as well as membranous. To determine whether this CD9 upregulation was an initiator of apoptosis or a consequence, CD34+ cells were cultured with anti‐CD9 (ALB6) antibody added to the culture medium. Ligation of CD9 by anti‐CD9 did not change the apototic profile of the cultures.Conclusion It can be concluded that although CD9, by itself, is not an initiator of apoptosis in erythroid progenitor cells it is obviously an important target in the subsequent apoptotic pathway. Continued research into CD9 and other mechanisms of apoptosis may allow manipulation of cells in culture with a possible extension of their life span and increase in the number of cells produced.