Identification and Characterization of Sur3γ and Sur3γV, Two Novel Splice Variants of Survivin in HSPC

Survivin is a member of the inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. We have previously shown that Survivin is expressed in primitive hematopoietic stem and progenitor (HSPC) cells and required for HSC maintenance and for entry into cell cycle. Several alternative Survivin splice variants have been described that are associated with differential prognosis in patients with cancer and leukemia, however, the function of alternative splice variants in normal and cancer cells is not fully understood. Using rapid amplification of cDNA ends (RACE) with mRNA from HL-60 leukemia cells we have identified two novel alternative splice variants, Survivin-3γ (Sur3γ) and Survivin-3γ-Variant (Sur3γV) that differ by only 4 bases, however due to changes in the reading frame, they result in proteins with very different C-termini, with Sur3γV being the predominant variant. We have characterized the expression of Sur3γV and Sur3γ in normal HSPC, leukemia and solid cancer cells and evaluated their anti-apoptotic function in cancer cell lines and primary hematopoietic cells. In contrast to cell lines and most cancers, where SurWT is the predominant transcript, Sur3γV is expressed at higher levels than SurWT in a number of normal human tissues including bone marrow, pericardium, placenta, seminal vesicles, testis and uterus. In placenta and bone marrow, Sur3γV mRNA is expressed at 25-fold and 65-fold higher levels respectively, than wild-type Survivin (SurWT). Sur3γV is expressed at ~16-fold higher levels in CD34+ HSPC and at ~80-fold higher level in primary CD34+ CD38− HSPC. In CD34+ cells, stimulation with SCF, TPO, FLT3L was associated with 30 – 55 fold increase in expression of SurWT, however expression of Sur3γ and Sur3γV were not altered, suggesting that these variants may be associated with HSPC maintenance/quiescence. Sur3γV and SurWT are expressed at equivalent levels in AML however, in blast crisis CML, Sur3γV is expressed at 5-fold higher level than SurWT. Over expression of Sur3γ and Sur3γV in Yac1, a mouse lymphoma cell line, confers ~ 2-fold increased resistance to Taxol similar to that observed using Survivin-WT. Similarly, Yac-1 cells over expressing Sur3γV or Sur3γ were equally resistant to Etoposide as cells over expressing SurWT. Over-expression of Sur3γ and Sur3γV using the bi-cistronic MIEG vector in primary murine bone marrow mononuclear cells results in 1.7-fold increase in total CFU-GM, similar to SurWT. In addition, over-expression of Sur3γV and Sur3γ protected CFU-GM from apoptosis induced by delayed growth factor addition resulting in a 1.6 fold increase in the number of CFU-GM colonies detected. These studies clearly indicate that both Sur3γ and Sur3γV possess anti-apoptotic function at least equivalent to SurWT. In summary, we have identified 2 novel splice variants of Survivin that differ from one another by 4 bases. Unlike other Survivin splice variants that are expressed at low levels, Sur3γV is expressed at a higher level than SurWT in primitive HSPC and in some primary leukemia samples. Expression of Sur3γ and Sur3γV is not altered by hematopoietic growth factors that stimulate HSC proliferation. Over-expression of Sur3γ and Sur3γV protects cells from chemotherapeutic drugs and ectopic expression of Sur3γ and 3γV stimulates HPC proliferation and protects primary mouse HSPC from apoptosis. These studies also suggest that differential expression of Survivin splice variants may play a critical role in HSC function and differentiation.