**6. Consequence of nuclear residence**

To determine the consequences of expressing survivin in the nucleus we fused survivin cDNA to an NLS and transfected it into cultured human cells (Colnaghi et al., 2006; Connell et al., 2008a). Upon expression we noted the abundance of NLS-survivin was significantly reduced compared with wild type survivin, present in the cytoplasm. Moreover the levels of survivin-NLS in the nuclear fraction could be restored by treatment with the proteasome inhibitor, MG132, or siRNA mediated depletion of the APC/C activator cdh1. From our data we concluded that coercing survivin into the nucleus facilitated its removal from the cell in a 26S-proteasome/cdh1 dependent manner (Connell et al., 2008a). Hence, in addition to import-export dynamics, stability is another key determinant of intranuclear localisation of survivin.

But why is it so important to keep survivin out of the nucleus? From our data on the accelerated clearance of NLS-survivin, and the presence of two NES sequences, it is clear that the cell has put in place multiple measures to eliminate survivin from the nucleus, so what are the consequences of its presence should it become resident there? In an early study by Suzuki and co-workers (2000) it was noted that upon release from serum starvation ectopically expressed survivin accumulated in the nucleus of human hepatoma cells. Interestingly they found that translocation of survivin to the nucleus coincided with an Sphase shift. At the molecular level they explained this precocious entry into S-phase as a downstream consequence of a direct interaction between survivin and cdk4. In essence, upon binding to cdk4 survivin competitively inhibits the interaction between cdk4 and p16INKa, cdk4's natural inhibitor, allowing cdk4 to activate cdk2/cyclin E, which in turn hyperphosphorylates pRB derepressing transcription, and promotes passage through the G1 restriction point (Suzuki et al., 2000). Survivin-induced S-phase promotion has also been

of this tiny protein. As mentioned above redistribution of survivin into the nucleus is promoted by activation of GSK3B, and although it is not yet known whether survivin is a substrate of this kinase, GSK3B can bind survivin directly and has a bipartite NLS (Li et al.,

In addition to "wild type" survivin, alias survivin-alpha/BIRC5, several splice variants exist. The two most extensively studied are alternatively spliced at the exon 2-intron border, which generates the variants, survivin 2B, with an additional 23 aa after exon 2, and delta exon 3 (DeX3) which incurs a frame shift that results in a completely distinct C-terminal sequence (Mahotka et al., 2002; Mahotka et al., 1999). We, and others, have shown that consistent with the central positioning of the NES, 2B retains this sequence and is predominantly cytoplasmic when ectopically expressed in cultured cells, while DeX3 is nuclear, not only because it lacks the two NES sequences, but because it has its own, functional NLS in its C-terminus (Mahotka et al., 2002; Noton et al., 2006; Rodriguez et al., 2002). As for the nuclear-cytoplasmic debate, the prognostic importance of the expression of these forms is also a matter of controversy, and there are many reports correlating the expression of transcripts of these variants and localisation of their gene products with disease severity, most recently (Antonacopoulou et al., 2011) and (Takeno et al., 2010).

To determine the consequences of expressing survivin in the nucleus we fused survivin cDNA to an NLS and transfected it into cultured human cells (Colnaghi et al., 2006; Connell et al., 2008a). Upon expression we noted the abundance of NLS-survivin was significantly reduced compared with wild type survivin, present in the cytoplasm. Moreover the levels of survivin-NLS in the nuclear fraction could be restored by treatment with the proteasome inhibitor, MG132, or siRNA mediated depletion of the APC/C activator cdh1. From our data we concluded that coercing survivin into the nucleus facilitated its removal from the cell in a 26S-proteasome/cdh1 dependent manner (Connell et al., 2008a). Hence, in addition to import-export dynamics, stability is another key determinant of intranuclear localisation of

But why is it so important to keep survivin out of the nucleus? From our data on the accelerated clearance of NLS-survivin, and the presence of two NES sequences, it is clear that the cell has put in place multiple measures to eliminate survivin from the nucleus, so what are the consequences of its presence should it become resident there? In an early study by Suzuki and co-workers (2000) it was noted that upon release from serum starvation ectopically expressed survivin accumulated in the nucleus of human hepatoma cells. Interestingly they found that translocation of survivin to the nucleus coincided with an Sphase shift. At the molecular level they explained this precocious entry into S-phase as a downstream consequence of a direct interaction between survivin and cdk4. In essence, upon binding to cdk4 survivin competitively inhibits the interaction between cdk4 and p16INKa, cdk4's natural inhibitor, allowing cdk4 to activate cdk2/cyclin E, which in turn hyperphosphorylates pRB derepressing transcription, and promotes passage through the G1 restriction point (Suzuki et al., 2000). Survivin-induced S-phase promotion has also been

2008).

**5. Isoforms** 

survivin.

**6. Consequence of nuclear residence** 

noted in breast cancer cells, T-lymphocytes, and haemopoietic cells, reviewed in (Fukuda and Pelus, 2006). Thus one manifestation of nuclear residence is accelerated entry into the cell cycle.

A second consequence of nuclear residence of survivin is the loss of its cytoprotective activity. Converging data from analysis of NES mutants (Colnaghi et al., 2006), NLS fusions (Connell et al., 2008a), LMB treatment (Knauer et al., 2007), GSK3β-activated cells (Li et al., 2008), and the response of cells in which nuclear survivin naturally occurs (Temme et al., 2007), have clearly demonstrated that nuclear survivin can no longer inhibit cell death, invoked by either the intrinsic or extrinsic apoptotic pathways. An apparent paradox here is that while cytoplasmic localisation of survivin is a requirement for it to protect cells from irradiation (IR), irradiation itself induces translocation of survivin to the nucleus (Chakravarti et al., 2004). In our hands, IR induces nuclear foci of survivin, which form as early as 60 minutes post-IR and can persist for many hours (Figure 1B), although a more dynamic response has been noted by others (Capalbo et al., 2010). This paradox, however, may be explained in part by data from Chan et al., (2010) who recently reported that during early stages of cell death, the CRM1/exportin pathway collapses. Although in their case retention of survivin in the nucleus as a result of compromised export, resulted in its rapid Ub-proteasome dependent rapid clearance (Chan et al., 2010), it is formally possible that failed export contributes to an initial sequestration of survivin in the nucleus, which may be followed by an altered rate of turnover, and eventually focus formation. Another plausible explanation for these data may be that the cells are arresting in G2 in response to DNA damage and that this is a time when survivin normally enters the nucleus in preparation for mitosis. Whatever the means by which these foci form have been shown to colocalise with the DNA damage marker, gamma-H2AX, and the DNA repair proteins, DNA-PK and Ku70 (Capalbo et al., 2010). Moreover, survivin translocation may be effected by the downstream chk1 and chk2 effector kinases, which respond to the PIK-family of kinases, ATR and ATM respectively, both pivotal upstream kinases in the DNA damage-repair pathways, and DNA-PK knock out cells accumulate nuclear survivin after UV irradiation (Asumen et al., 2010). Consistent with this, we identified DNA-PK and Ku70 as potential partners by Mass Spec analysis of in survivin co-immunoprecipitating proteins post-X-irradiation (Connell and Wheatley, unpublished), and similar data was published by (Capalbo et al., 2010). Whether survivin then aids in DNA repair per se remains to be determined, but early work from (Chakravarti et al., 2004) reported fewer comet tails post-IR in glioblastoma cell lines expressing survivin compared with control cells, indicating either less damage sustained or more efficient repair of DNA strand breaks.

Finally, could expression of survivin in the nucleus interfere with the cell's transcriptional programme? Although survivin has a zinc finger in its BIR domain, this fold is distinct from zinc rich domains found in many TFs, and accordingly survivin itself cannot bind directly to DNA(Klein et al., 2006). However, it has been suggested that survivin suppresses the cytokine activated transcription factor, STAT3, and that this association may be regulated acetylation of survivin at lysine, K129, in the carboxyl alpha helix (Wang et al., 2010). Although the consequences of this interaction are not clear at present, it is nevertheless intriguing that this could be one reason to eliminate survivin from the nucleus. In this regard it is important to note that significant new insight in the mitosis field has revealed that survivin interacts directly with the NH2 tail of histone H3, which protrudes from the

Nuclear Survivin: Cellular Consequences and Therapeutic Implications 339

Molecular structures were drawn using the UCSF Chimera package (www.cgl.

Ambrosini, G., C. Adida, G. Sirugo, and D.C. Altieri. 1998. Induction of apoptosis and

Antonacopoulou, A.G., K. Floratou, V. Bravou, A. Kottorou, F.-I. Dimitrakopoulos, S.

Asumen, M.G., T.V. Ifeacho, L. Cockerham, C. Pfandl, and N.R. Wall. 2010. Dynamic

Barrett, R.M.A., R. Colanghi, and S.P. Wheatley. 2011. Threonine 48 in the BIR domain of

Beardmore, V.A., L.J. Ahonen, G.J. Gorbsky, and M.J. Kallio. 2004. Survivin dynamics

Brennan, D.J., E. Rexhepaj, S.L. O'Brien, E.M. McSherry, D.P. O'Connor, A. Fagan, A.C.

Capalbo, G., K. Dittman, C. Weiss, S. Reichert, E. Hausmann, C. Rodel, and F. Rodel. 2010.

Chakravarti, A., G.G. Zhai, M. Zhang, R. Malhotra, D.E. Lathman, M.A. Delaney, P. Robe, U.

Chan, K.-S., C.-H. Wong, Y.-F. Huang, and H.-L. Li. 2010. Survivin withdrawal by nuclear

Colnaghi, R., C.M. Connell, R.M.A. Barrett, and S.P. Wheatley. 2006. Separating the

Connell, C.M., R. Colnaghi, and S.P. Wheatley. 2008a. Nuclear survivin has reduced stability

and is not cytoprotective. *J. Biological Chemistry.* 238:3289-96.

inhibition of cell proliferation by survivin gene targeting. *J. Biological Chemistry.*

Marousi, M. Stavropoulos, A.K. Koutras, C.D. Scopa, and H.P. Kalofonos. 2011. The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival. *Cell Oncology*. epub 3rd May

changes to survivin subcellular localization are initiated by DNA damage.

survivin is critical to its mitotic and anti-apoptotic activities and can be

increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity. *Journal Cell Sci.* 117:4033-

Culhane, D.G. Higgins, K. Jirstrom, R.C. Millikan, G. Landberg, M.J. Duffy, S.M. Hewitt, and W.M. Gallagher. 2008. Altered cytoplasmic-to-nuclear ratio of survivin is a prognostic indicator in breast cancer. . *Clinical Cancer Research* 

Radiation-induced survivin nuclear accumulation is linked to DNA damage repair.

Nestler, Q. Song, and J. Loeffler. 2004. Survivin enhances radiation resistance in primary human glioblastoma cells via caspase-independent mechanisms. *Oncogene*.

export failure as a physiological switch to commit cells to apoptosis. *Cell Death and* 

anti-apoptotic and mitotic roles of survivin. *J. Biological Chemistry.* 281:33450-

**9. Acknowledgements** 

**10. References** 

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nucleosome, and that this interaction is dependent upon phosphorylation of threonine 3 in histone H3's (Kelly et al., 2010; Wang et al., 2010). As this phosphorylation event is placed by the mitosis specific kinase, haspin, it is unclear whether such an interaction could occur in non-mitotic cells, however, indirect interactions in addition to direct binding between survivin and H2A also facilitates chromatin association (Kelly et al., 2010; Yamagishi et al., 2010), and borealin has been demonstrated to bind dsDNA directly in vitro (Klein et al., 2006). Hence, although not a transcription factor itself, it is indeed plausible that survivin could indirectly modulate transcription.
