Sally P. Wheatley

Survivin is required for stable checkpoint activation in taxol-treated HeLa cells

Survivin is an essential chromosomal passenger protein whose function remains unclear. Here, we have used RNA interference to specifically repress Survivin in cultured HeLa cells. Immunoblot analysis showed that Survivin was no longer detectable in cultures 60 hours after transfection with Survivin-specific siRNA. Live cell analysis showed that many Survivin-depleted cells were delayed in mitosis, and immunofluorescence analysis of fixed specimens revealed that Survivin-depleted cells accumulated in prometaphase with misaligned chromosomes. The chromosomal passenger proteins, INCENP and Aurora-B, which can interact directly with Survivin, were absent from the centromeres of Survivin-depleted cells. These data contribute to the emerging picture that Survivin operates together with INCENP and Aurora-B to perform its mitotic duties. Some Survivin-depleted cells eventually exited mitosis without completing cytokinesis. This resulted in a gradual increase in the percentage of multinucleated cells in the culture. Time-lapse imaging of synchronized cultures revealed that control and Survivin-depleted cells arrested in mitosis in the presence of nocodazole; however, the latter failed to arrest in mitosis when treated with taxol. Immunofluorescence studies revealed that Survivin-depleted cells were unable to stably maintain BubR1 at the kinetochores in the presence of either taxol or nocodazole. Our data reveal that Survivin is not required for the spindle assembly checkpoint when it is activated by the loss of microtubules. However, Survivin is required for the maintenance of the checkpoint when it is activated by taxol, which is generally thought to cause a loss of spindle tension.

INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. First, interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis [1-7] (see [8] for review). Second, INCENP and Aurora-B exist in a complex in Xenopus eggs [9] and in mammalian cultured cells [7]. Third, interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates [5, 7, 9]. Here, we provide evidence that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. Our data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex.

Survivin: A Protein with Dual Roles in Mitosis and Apoptosis

Survivin is a fascinating little protein that acts as a component of the chromosomal passenger complex, which is essential for cell division, and as an inhibitor of apoptosis. With dual roles in promoting cell proliferation and preventing apoptosis, it is considered a protein that interfaces life and death. Interest in survivin has been fueled by its abundance in human cancers, where it has potential as a prognostic marker for cancer, and as a target for chemotherapy. Accordingly, since its discovery in 1997, publications on survivin have risen exponentially in basic and clinical fields alike. This review highlights the key advances in our understanding of the cellular function of this protein.

Aurora-B Phosphorylation in Vitro Identifies a Residue of Survivin That Is Essential for Its Localization and Binding to Inner Centromere Protein (INCENP) in Vivo

The chromosomal passengers, aurora-B kinase, inner centromere protein (INCENP), and survivin, are essential proteins that have been implicated in the regulation of metaphase chromosome alignment, spindle checkpoint function, and cytokinesis. All three share a common pattern of localization, and it was recently demonstrated that aurora-B, INCENP, and survivin are present in a complex in Xenopus eggs and Saccharomyces cerevisiae. The presence of aurora-B kinase in the complex and its ability to bind the other components directly suggest that INCENP and survivin could potentially be aurora-B substrates. This hypothesis was recently proven for INCENP in vitro. Here we report that human survivin is specifically phosphorylated in vitro by aurora-B kinase at threonine 117 in its carboxyl α-helical coil. Mutation of threonine 117 to alanine prevents survivin phosphorylation by aurora-B in vitro but does not alter its localization in HeLa cells. By contrast, a phospho-mimic, in which threonine 117 was mutated to glutamic acid, was unable to localize correctly at any stage in mitosis. Mutation at threonine 117 also prevented immunoprecipitation of INCENP with survivin in vivo. These data suggest that phosphorylation of survivin at threonine 117 by aurora-B may regulate targeting of survivin, and possibly the entire passenger complex, in mammals.

Separating the Anti-apoptotic and Mitotic Roles of Survivin

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and has an essential role in mitosis. To date whether these two functions can be divorced has not been addressed. Here we show that the linker region between the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin and COOH-terminal α helix may be the key to separating its roles. When overexpressed survivin is present in interphase cells and shuttles between the cytoplasm and nucleus. Here we identify a rev-like nuclear exportation signal (NES) in the central domain of survivin and demonstrate that point mutations within this region cause accumulation of survivin in the nucleus. Interestingly cells expressing NES mutants exhibit reduced survival after X-irradiation. Moreover, cells expressing survivinL98A-green fluorescent protein (GFP) showed increased poly(ADP-ribose) polymerase-cleavage and caspase-3 activity after tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment compared with cells expressing full-length survivin-green fluorescent protein. These data suggest a direct link between the interphase localization of survivin and cellular responsiveness to apoptotic stimuli. Using a cell proliferation assay, we also found that ectopic expression of NES mutants can complement for depletion of endogenous survivin, indicating that they can execute the mitotic duties of survivin. Thus we demonstrate for the first time that 1) survivin has a functional NES; 2) nuclear accumulation of overexpressed survivin correlates with increased sensitivity of cells to ionising radiation; and 3) the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its NES. Separating these two functions of survivin could open up new possibilities for therapeutic strategies aimed at eliminating cancer cells yet preserving normal cell viability.

Molecular analysis of survivin isoforms : Evidence that alternatively spliced variants do not play a role in mitosis

Survivin is a protein with proposed roles in cell division and apoptosis. Transcripts encoding splice variants of human survivin have been described and their expression correlated with cancer progression. As survivin forms homodimers in vitro, it has been suggested that these isoforms could interfere with wild type function by forming heterodimers. Here we show that survivin-2β and survivin-δEx3 can interact with wild type survivin but have reduced affinity for the partner protein of survivin, borealin, and thus do not localize with the chromosomal passenger complex in vivo. Furthermore, we demonstrate that overexpression of survivin-2β-green fluorescent protein (GFP) or survivin-δEx3-GFP does not impede cell cycle progression. We also report that wild type survivin, but not survivin-2β-GFP or survivin-δEx3-GFP, can rescue cell proliferation inhibited by small interfering RNA-mediated survivin depletion. These data suggest that, despite their ability to interact with wild type survivin, neither of these isoforms acts as its competitor during mitosis nor has an essential function.

Nuclear survivin has reduced stability and is not cytoprotective

Survivin is an essential mitotic protein that is overexpressed in many cancers, and its presence is correlated with increased resistance to radiation and chemotherapy. Here we demonstrate that sending survivin into the nucleus accelerates its degradation in a cdh1-dependent manner, abolishes the radio resistance normally conferred to cells by its overexpression, and prevents survivin from inhibiting apoptosis without affecting its mitotic localization. Our data suggest that targeting survivin to the nucleus provides an efficient means of eliminating it from the cell and may prove a novel strategy in cancer treatment, particularly in combination with radiotherapy.

Involvement of centrosome amplification in radiation-induced mitotic catastrophe.

Cells exposed to ionising radiation die via different mechanisms, including apoptosis and mitotic catastrophe. To determine the frequency of mitotic catastrophe in tumour cells after irradiation, we used timelapse imaging to track centrin-1 and histone H2B in U2OS osteosarcoma cells. We observed a dose-dependent increase in the frequency of mitotic catastrophe after irradiation, although a consistent 30% of cell death occurred through mitotic failure at doses from 2- 10 Gy. One potential cause of mitotic catastrophe is centrosome amplification, which is induced by irradiation, and which can result in the formation of multipolar mitotic spindles. Up to 60% of mitotic catastrophes occurred in cells with >2 centrosomes after irradiation. We observed multipolar mitoses in p53+ and p53- tumour cells after irradiation and found that the spindle assembly checkpoint is active in multipolar mitotic cells. However, we did not detect active caspase-3 in multipolar mitoses. These data demonstrate that a significant proportion of cell death induced by ionising irradiation is through an apoptosis-independent mechanism involving centrosome amplification and mitotic catastrophe.

Endopolyploidy in irradiated p53-deficient tumour cell lines: Persistence of cell division activity in giant cells expressing Aurora-B kinase

Recent findings including computerised live imaging suggest that polyploidy cells transiently emerging after severe genotoxic stress (and named ‘endopolyploid cells’) may have a role in tumour regrowth after anti‐cancer treatment. Until now, mostly the factors enabling metaphase were studied in them. Here we investigate the mitotic activities and the role of Aurora‐B, in view of potential depolyploidisation of these cells, because Aurora‐B kinase is responsible for coordination and completion of mitosis. We observed that endopolyploid giant cells are formed via different means in irradiated p53 tumours, by: (1) division/fusion of daughter cells creating early multi‐nucleated cells; (2) asynchronous division/fusion of sub‐nuclei of these multi‐nucleated cells; (3) a series of polyploidising mitoses reverting replicative interphase from aborted metaphase and forming giant cells with a single nucleus; (4) micronucleation of arrested metaphases enclosing genome fragments; or (5) incomplete division in the multi‐polar mitoses forming late multi‐nucleated giant cells. We also observed that these activities can release para‐diploid cells, although infrequently. While apoptosis typically occurs after a substantial delay in these cells, we also found that ∼2% of the endopolyploid cells evade apoptosis and senescence arrest and continue some form of mitotic activity. We describe here that catalytically active Aurora‐B kinase is expressed in the nuclei of many endopolyploid cells in interphase, as well as being present at the centromeres, mitotic spindle and cleavage furrow during their attempted mitotes. The totally micronucleated giant cells (containing sub‐genomic fragments in multiple micronuclei) represented only the minor fraction which failed to undergo mitosis, and Aurora‐B was absent from it. These observations suggest that most endopolyploid tumour cells are not reproductively inert and that Aurora‐B may contribute to the establishment of resistant tumours post‐irradiation.

Phosphorylation by Aurora-B Negatively Regulates Survivin Function During Mitosis

Survivin operates in a complex with aurora B kinase and is phosphorylated by it on threonine 117 in vitro. Here we ask whether phosphorylation of survivin by aurora B kinase regulates its function during mitosis in vivo. Using a phospho-specific antibody we first establish that survivin is phosphorylated at T117 during mitosis and is present at the midbody during cytokinesis. Next we use two independent RNAi complementation approaches to investigate threonine 117 mutants in survivin depleted cells. Our data suggest that while non-phosphorylatable survivin, survivinT117A, can substitute for the wild type protein, a phosphomimic, survivinT117E cannot restore viability, nor can it complement chromosome congression and spindle checkpoint defects that arise due to depletion of endogenous survivin. Fluorescence imaging and fluorescence recovery after photobleaching analysis suggest that the phosphomimic has reduced affinity for centromeres compared with the non-phosphorylatable form. We conclude that survivin is phosphorylated at T117 during mitosis, and once phosphorylated, dephosphorylation is crucial for chromosome congression and progression into anaphase.