Cell division and cell survival in the absence of survivin Dun Yang*, Alana Welm, and J. Michael Bishop G. W. Hooper Research Foundation and Department of Microbiology and Immunology, University of California, 513 Parnassus Avenue, San Francisco, CA 94143 Contributed by J. Michael Bishop, September 9, 2004 The survivin protein contains structural features of the inhibitor of uncertain which of these effects are primary, and which are apoptosis protein family. Previous studies have suggested that secondary. Because cancer cell lines have been widely used to survivin is essential for cell survival because it counteracts an study the function of survivin in the past, interpretation of the otherwise constitutive propensity to apoptosis during mitosis. In results is complicated by the abnormal ploidy and multiple addition, survivin appears to be a component of the chromosomal genetic defects characteristic of these cells, which may modify the passenger protein complex that participates in multiple facets of outcome of survivin depletion. cell division. Here we report that euploid human cells do not die in We used RNA interference (RNAi) to specifically reduce the absence of survivin. Instead, depletion of survivin caused survivin expression in the IMR-90 strain of human primary lung defects in cell division, followed by an arrest of DNA synthesis due fibroblasts and in a human retinal pigment epithelial (RPE) cell to activation of a checkpoint involving the tumor suppressor line that has normal ploidy and functioning checkpoints for the protein p53. During anaphase mitosis in survivin-deficient cells, cell cycle and mitotic spindle (9). In contrast to studies with sister chromatids disjoined normally, but one or more of the sister cancer cells (3, 7, 10, 11), we report that RPE and IMR-90 cells chromatids frequently lagged behind the main mass of segregating can survive in the absence of survivin but suffer frequent chromosomes, probably because of merotelic kinetochore attach- missegregation of sister chromatids and lack the spindle midzone ments. Survivin-deficient cells initiated but failed to complete and midbody microtubules during late mitosis. These abnormal- cytokinesis, apparently because the spindle midzone and midbody ities result in a previously described failure of cytokinesis and a microtublues were absent during late mitosis. The abnormalities of previously unrecognized p53-dependent arrest of DNA synthe- both chromosome segregation and cytokinesis could be attributed sis. A deficiency of p53 allows DNA endoreduplication with- to a defect in the chromosomal passenger protein complex, with a out completion of cytokinesis in survivin-depleted RPE cells, consequent mislocalization of the kinesin-like motor protein resulting in cell-division defects typically found in survivin- MKLP-1 playing a more immediate role in the microtubule abnor- depleted p53-deficient cancer cells, such as abnormalities of cen- malities. Depletion of another chromosomal passenger protein, trosome number and multipolar spindles (3). We conclude that aurora-B, recapitulated the survivin RNA interference phenotypes. survivin may not be essential for cell survival, but the protein is We conclude that survivin can be essential for the proliferation of required for cellular proliferation, because as a CPP, it has an normal human cells by virtue of its contributions to accurate sister ͞ essential role in both the segregation of sister chromatids and the chromatid segregation and assembly stabilization of microtubules ͞ in late mitosis. However, the protein is not inevitably required for assembly stabilization of microtubules late in mitosis. the survival of normal cells. Materials and Methods Preparation of Small Hairpin RNA (shRNA). apoptosis ͉ cytokinesis ͉ chromosome segregation ͉ chromosomal shRNAs were prepared passenger proteins ͉ mitosis as described in ref. 12. Each RNA had a 24-bp stem that is complementary with a unique sequence within the target gene. All RNAs had an extra GGG at the 5Ј end,aUUatthe3Ј end, he protein survivin has attracted attention because of its and a UUGAGAG loop. The complete sequences of shRNAs Tabundant expression in various human cancers and its po- are available upon request. suv1 and suv2 are complementary to tential as a target in cancer therapy (1). Because survivin sequences 575–598 and 586–609, respectively, within the sur- contains a BIR domain (baculoviral inhibitor of apoptosis vivin mRNA (National Center for Biotechnology Information protein repeats), it was assigned to the inhibitor of apoptosis mRNA record no. NM࿝001168). protein family (2). Numerous studies have shown that survivin overexpression confers cytoprotection against a variety of apo- Western Blot Analysis of Cell Extracts. Immunoblotting was per- ptotic stimuli, whereas loss of survivin expression or function formed as described in ref. 12. Mouse monoclonal antibodies causes spontaneous apoptosis or sensitizes cancer cells to apo- were used to detect p53 and actin, and rabbit polyclonal anti- ptotic stimuli (1). It has been proposed that survivin functions at the interface between cell division and cell survival by counter- bodies were used for all other analysis: p21Cip1, cyclin D1, cyclin acting a constitutive pathway that induces apoptosis during E, cyclin B1, cyclin A, pRB (Ser-780 phosphorylation-specific), mitosis (3, 4). But the existence of that pathway remains and proliferating cell nuclear antigen (PCNA). The antiserum hypothetical, and little is known of how survivin might be for phosphorylated pRB was from Cell Signaling Technology involved in the cellular machinery that mediates apoptosis and (Beverly, MA) and the others were from Santa Cruz Biotech- cell division. nology. Horseradish peroxidase-conjugated anti-mouse and an- Survivin has been defined as one of the chromosomal pas- ti-rabbit immunoglobulins were from Amersham Pharmacia. senger proteins (CPPs), which also include the aurora-B kinase, the inner centromere protein, and the telophase disk antigen Abbreviations: CPP, chromosomal passenger protein; CREST, calcinosis, Raynaud’s phenom- (TD-60) (5). These proteins appear to function as part of a enon, esophageal dysfunction, sclerodactyly, and telangiectasia; DAPI, 4Ј,6-diamidino-2- multiprotein complex, referred to as the CPP complex, which phenylindole; RNAi, RNA interference; RPE, retinal pigment epithelial; shRNA, small hair- plays multiple roles during cell division (5). Depletion of survivin pin RNA. in human cells has been reported to cause apoptosis and *To whom correspondence should be addressed. E-mail: [email protected]. pleiotropic defects in cell division (3, 6–8). However, it remains © 2004 by The National Academy of Sciences of the USA 15100–15105 ͉ PNAS ͉ October 19, 2004 ͉ vol. 101 ͉ no. 42 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406665101 Downloaded by guest on September 29, 2021 Western blots were developed with the ECL detection kit (Amersham Pharmacia). Fluorescence Microscopy. Immunofluorescence staining was per- formed as described in ref. 13. Mouse monoclonal antibodies were used to detect p53, survivin, AIM-1, ␤-tubulin, and ␥- tubulin. Rabbit polyclonal antibodies were used for MKLP-1, survivin, and inner centromere protein. Human autoimmune serum from a patient with calcinosis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (CREST) was used for staining kinetochores. Antibodies to p53, MKLP-1, and survivin (rabbit) were from Santa Cruz Biotech- nology. Antibodies to ␤- and ␥-tubulin were from Sigma, and monoclonal antibody for survivin was from Cell Signaling Tech- nology. Antibodies to AIM-1 were from BD Transduction Laboratories (Lexington, KY). CREST antibody was a gift from B. R. Brinkley (Baylor College of Medicine, Houston). Anti- bodies to inner centromere protein were from T. Stukenberg (University of Virginia, Charlottesville) and W. C. Earnshaw (University of Edinburgh, Edinburgh). Primary antibodies were detected with Texas red-conjugated or fluorescein isothiocya- nate-conjugated secondary antibodies, purchased from Jackson ImmunoResearch. After immunostaining, we mounted cells on Fig. 1. Depletion of survivin elicits arrest of cellular proliferation. (A) Selec- microscope slides with 4Ј,6-diamidino-2-phenylindole (DAPI)- tive silencing of survivin expression by RNAi. Immunoblotting of whole-cell containing Vectashield mounting solution from Vector Labo- extracts from mock-treated cells (lane 1), and cells treated with shRNAs against firefly luciferase (Fluc) (lane 2) or survivin (suv1, suv2, and suv1m) ratories. For fluorescence detection, we used an Axioskop 50 (lanes 3–5) was performed 48 h after RNAi treatment. Actin was used as a immunofluorescence microscope (Zeiss). loading control. (B) Proliferation of RPE cells. Cells were treated with either suv1 or suv1m for 16 h at 70–90% confluence and then passaged into fresh BrdUrd Cellular Proliferation Assay. Cells were incubated for 16–24 medium at 5% confluence. Cells were harvested at daily intervals and counted h in the presence of 10 ␮M BrdUrd and fixed in 75% ethanol. with a hemocytometer. Each data point represents the average of three Nuclear incorporation of BrdUrd was visualized by immuno- independent experiments, and each experiment was done in triplicate. Error staining with a cell proliferation kit (Oncogene Research Prod- bars represent SD. ucts, San Diego). DNA synthesis determined by BrdUrd incorporation assays Histochemistry Staining. Cells were fixed and stained for nuclei and cytoplasm