Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry.
Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is overexpressed during G(2)/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34(+) hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34(+) cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34(+) cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G(0)/G(1). Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67(negative) and Cyclin D(negative) CD34(+) cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G(0) CD34(+) cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G(0) CD34(+) cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSE(bright) G(0) CD34(+) cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34(+) cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.
Fukuda
Seiji
S
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Pelus
Louis M
LM
D018952:Antigens, CD34 / Q000032:analysis*; D017209:Apoptosis; D002453:Cell Cycle; D002478:Cells, Cultured; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D019394:Ki-67 Antigen / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis*; D009363:Neoplasm Proteins; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D000077022:Survivin
D018952:Antigens, CD34; C107489:BIRC5 protein, human; D056741:Cyclin D; D016213:Cyclins; D016298:Hematopoietic Cell Growth Factors; D051034:Inhibitor of Apoptosis Proteins; D019394:Ki-67 Antigen; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin