AbstractsBiology & Animal Science

Survivin, the smallest and functionally unique member of the inhibitor of apoptosis protein (IAP) family, is frequently overexpressed in malignant cells and has been acknowledged as a predictive molecular marker for metastases and cancer patient survival following radiation therapy. The role of this protein in cellular radiation response, however, far exceeds a simple inhibition of apoptotic cell death involving non-caspase dependent mechanisms such as regulation of cell cycle and DNA damage repair. To investigate in more detail the role of Survivin in cellular radiation response and tumour cell motility, the present study aimed to establish and stably express several Survivin enhanced green fluorescent protein (EGFP)-tagged deletion or phosphorylation mutant constructs in SW480 colorectal cancer cells. To this end, Survivin wild-type (Surv. wt) and recombinant proteins lacking the binding sites for X-linked IAP (XIAP), Microtubules and heat shock protein 90 (Hsp90) (Survivin ΔXIAP/ΔMicTub/ΔHsp90) or the baculovirus IAP repeat (BIR) domain (Survivin ΔBIR) as well as phosphorylation mutants for the sites S20, T34 and T117 (Survivin S20A/S20D/T34A/T34D/T117A/T117D) were expressed in a pEGFP-N1 vector system. Subsequently, these mutant clones were subjected to more physiologic three-dimensional (3D) colony forming assays, immunofluorescence staining of DNA double strand break markers γH2AX and 53BP1, analysis of cell cycle distribution, induction of apoptosis, caspase 3/7 activity and transmigration assays following irradiation with doses ranging from 1 to 6 Gy. While knockdown of endogenous Survivin by RNA interference (siRNA) resulted in a significantly decreased 3D radiation survival in line with elevated numbers of residual γH2AX/53BP1 foci in pEGFP-N1 vector control, Survivin ΔXIAP, ΔBIR and T34A clones, both radiation clonogenic survival and the capacity to regulate DNA repair was rescued in clones stably overexpressing Survivin wt, ΔMicTub, ΔHsp90 and Survivin T34D mutant cells. Moreover, deletion of the BIR domain, XIAP, Microtubules and Hsp90 binding sites resulted in a G2/M cell cycle arrest, an elevated percentage of apoptotic SubG1 cells, an increased caspase 3/7 activity and significantly hampered transmigration activity of SW480 cells following depletion of endogenous Survivin. On a molecular level, constructs derived from Survivin ΔXIAP and T34A phospho-mutant overexpressing clones did not co-immunoprecipitate with the DNA repair protein DNA-dependent protein kinase catalytic subunit (DNA-PKcs), whereas Survivin wt and T34D constructs co-precipitated with the protein. These data confirm Survivin to act as a radiation resistance factor modulating cellular radiation response by multiple mechanisms including DNA DSB repair, induction of cell cycle arrest and apoptosis. It is for the first time indicated that Survivin’s XIAP binding and T34 phosphorylation sites, but not its Microtubules and Hsp90 binding sites, are essential for radiation clonogenic survival and modulation of DNA damage repair, at least in…