Abstract
The inhibitor of apoptosis protein Survivin is barely detectable in most normal adult tissues but is over-expressed in almost all cancers. Survivin regulates apoptosis, cell division and cell cycle, making anti-Survivin therapy an attractive cancer treatment strategy. We reported that Survivin is expressed and regulated by hematopoietic growth factors in normal human CD34+ cells and that over-expression of wild-type Survivin in bone marrow cells enhances in vitro proliferation and survival of normal hematopoietic progenitor cells, whereas disrupting Survivin reduced their proliferation and survival. These results suggest that Survivin regulates normal hematopoietic progenitor cell function. Although targeted anti-Survivin therapies for cancers demonstrate efficacy without overt toxicity in animal models, the consequences of in vivo Survivin disruption in normal hematopoietic stem and progenitor cells (HSPC) has not been determined. In order to understand the physiological roles of Survivin in normal HSPC function in vivo, we created Cre-ER™/Survivin flox/flox mice, in which the Survivin gene can be excised by Tamoxifen treatment and characterized HSPC growth following Survivin gene deletion. RT-PCR analysis showed that Survivin mRNA is expressed in freshly isolated normal mouse marrow Sca-1+, c-kit+, lin− (SKL) cells and more primitive CD34−SKL cells, which contain long term repopulating hematopoietic stem cells (HSC). Administration of 5mg of Tamoxifen for 6 days (3 days injection, 3 days off, 3 additional days and analyzed 14 days after final injection) in Cre-ER™/Survivin flox/flox mice induced Survivin gene deletion in marrow cells, but had little effect on peripheral blood cell count, marrow cellularity (3.5+/−7.1%, NS) or the proportion or total number of lineage committed cells (Gr-1+, Mac-1+, B220+, CD4+ and/or CD8+) in marrow and in peripheral blood. In contrast, short term Survivin deletion significantly decreased the frequency and the absolute number of undifferentiated linneg cells (37+/−6% reduction), c-kit+, lin− cells (35.2+/−8.4% reduction,), CFU-GM (31+/−9 % reduction), Lin−, IL7Ra−, Sca-1−, c-kit+, CD34+, Fcglow common myeloid progenitor cells (52+/−13% reduction), SKL cells (56.8+/−5.4% reduction) and CD34−SKL cells (60.6+/−5.5% reduction) in bone marrow compared to control mice. The effect of Survivin gene deletion was more dramatic on primitive hematopoietic populations compared to mature cells, which is consistent with down-regulation of Survivin in hematopoietic cells with terminal differentiation. Similarly, treatment of bone marrow cells from Cre-ER™/Survivin flox/flox mice with 1uM of Tamoxifen in vitro significantly reduced the number of CFU-GM, (c-kit+, lin−) KL, SKL and CD34−SKL cells cultured with hematopoietic cytokines and increased apoptosis measured by Annexin-V staining. These results suggest that Survivin is required and regulates normal hematopoietic stem and progenitor function in vivo and that Survivin function may be selectively essential for growth and differentiation of primitive hematopoietic cells. In addition, acute ablation of Survivin may cause adverse toxicity on HSPC that provide long term hematopoiesis in the patients receiving anti-Survivin target therapies.
Platelets secrete stromal cell–derived factor 1α and recruit bone marrow–derived progenitor cells to arterial thrombi in vivo
