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Aurora kinases are known to play a key role in maintaining mitotic fidelity, and overexpression of aurora kinases has been noted in various tumors. Overexpression of aurora kinase activity is thought to promote cancer development through a loss of centrosome or chromosome number integrity. Here we observed augmentation of G12V-mutated HRAS-induced neoplastic transformation in BALB/c 3T3 A31-1-1 cells transfected with Aurora-A. Aurora-A-short hairpin RNA (shRNA) experiments showed that the expression level of Aurora-A determines susceptibility to transformation. Aurora-A gene amplification was noted in human patients with tongue or gingival squamous carcinoma (4/11). Amplification was observed even in pathologically normal epithelial tissue taken at sites distant from the tumors in two patients with tongue cancer. However, overexpression of Aurora-A mRNA was observed only within the tumors of all patients examined (11/11). Our data indicate that Aurora-A gene amplification and overexpression play a role in human carcinogenesis, largely due to the effect of Aurora-A on oncogenic cell growth, rather than a loss of maintenance of centrosomal or chromosomal integrity.

LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells. Here we report truncation of LyGDI after irradiation in mouse 3SB thymus cells. A 21-kDa fragment of LyGDI, resulting from activated caspase 3-induced cleavage at an N-terminal consensus site following the Asp(18) residue, accumulated at peak quantities between 5 and 12 h after irradiation. Cleavage of LyGDI was inhibited by the caspase inhibitor benzoyloxycarbonyl-Val-Asp-fluoromethylketone. Subcellular fractionation and immunofluorescence revealed the truncated 21-kDa fragment of LyGDI within the nuclear fraction of irradiated 3SB cells, whereas full-length LyGDI was found only in the cytoplasmic fraction. Truncated LyGDI within the nucleus had no association with the Rho family proteins RhoA and Rac1, since these proteins were observed only in the cytoplasmic fractions. These data demonstrate that regulation of Rho family GTPases by LyGDI is disrupted during apoptosis, suggesting that fragmentation of LyGDI implicates the transmission of a signal from the cytoplasm to the nucleus during Trp53-dependent apoptosis of thymus cells after irradiation.

Impaired regulation of Aurora-B/AIM-1 expression in human cells causes chromosomal abnormality and instability, and recent observations of high expression but not mutation of Aurora-B/AIM-1 in human cancers imply that Aurora-B/AIM-1 might be a candidate molecule for cancer progression. We analyzed the effects of modification of Aurora-B/AIM-1 expression on the growth of a human glioma cell line and the expression of Aurora-B/AIM-1 in astrocytomas.A glioma cell line, U251MG was transfected with wild type (WT) of Aurora-B/AIM-1 or kinase-inactive mutant of Aurora-B/AIM-1 in order to test the effects of overexpression of WT or kinase-inactive Aurora-B/AIM-1 on cell morphology and cell growth. Brain tissue samples were obtained during surgery and processed for reverse transcription-polymerase chain reaction, immunofluorescence in order to analyze the expression of Aurora-B/AIM-1 mRNA and protein.Exogenous overexpression of WT of Aurora-B/AIM-1 in cultured cells of U251MG produced multinuclearity and increased ploidy, and inhibited the growth of tumor cells. Exogenous overexpression of kinase-inactive Aurora-B/AIM-1 in a human glioma cell line also suppressed the tumor cell growth without affecting ploidy. Aurora-B/AIM-1 was highly expressed in astrocytomas and U251MG, and mRNA and protein levels of Aurora-B/AIM-1 in tumor tissues well correlated with their histological malignancy (World Health Organization grading). Survival time also negatively correlated with the levels of Aurora-B/AIM-1 mRNA in tumor samples.Aurora-B/AIM-1 was highly expressed in high-grade gliomas and its expression was well correlated with histological malignancy and clinical outcomes. The modification of the level of Aurora-B/AIM-1 expression might be a new target for glioma therapy.

Rho family GTPases play an important role in a number of processes related to metastasis, and RhoGDP dissociation, inhibitors (RhoGDIs) regulate Rho family proteins. We cloned genomic DNA from colon carcinoma SW480 cells capable of transforming nonmetastatic ras-transformed 1-1ras1000 cells into metastatic cells. This DNA contained a truncated human ras homolog gene family GDP dissociation inhibitor beta (ARHGDIB) gene, resulting in a C-terminal truncated form of LyGDI (Delta C-LyGDI, 166-201 deletion), a member of the RhoGDIs. The stable expression of Delta C-LyGDI induced pulmonary metastasis in 1-1ras1000 cells, whereas expression of full-length LyGDI did not induce metastasis. Delta C-LyGDI was preferentially localized in the membrane, detected in a NP-40-insoluble fraction, and co-purified with radixin, moesin, Rac1, Cdc42, and RhoA. In Delta C-LyGDI transfectant, an activation state of Rac1 was elevated and Delta C-LyGDI was associated with Rac1-GTP. In keeping with the observed localization of Rac1 to the cell membrane and the elevated level of Rac1-GTP, Delta C-LyGDI transfectants were found to be more invasive than mock transfectant. These results suggest that LyGDI functions in the cell membrane to afford spatial regulation of Rho family GTPase signaling through ezrin radixin moesin (ERM) proteins during metastasis.

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.

Cancer gene therapy is now being developing to provide new strategies for the treatment of human tumors. Cationic lipids represent one of the powerful mediators for DNA delivery. The liposome-plasmid DNA complex itself is known to inhibit tumor cell growth, but other effects on cancer cell behaviors have not been reported so far.Six commercially available cationic liposomes complexed with plasmid DNA were applied to cancer cells and their metastatic potentials were measured.The liposome-plasmid DNA complexes affected metastatic capability in three different ways: TM-TPS:DOPE and DOTAP:DOPE had no effect on metastatic capability; a suppressive effect was observed in DOSPA:DOPE and DMRIE:cholesterol; while an augmentative effect was observed in DOTMA:DOPE and Effectene. These effects are likely to be DNA sequence independent, because different plasmids have the same effects.Liposome-plasmid DNA complexes influence cancer metastasis capability, dependent upon the cationic liposome formulations.

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.

Phosphorylation of histone H3 at Ser-10 is required for maintenance of properchromosome dynamics during mitosis. AIM-1, a mammalian Ipl1/aurora kinase involved in H3 phosphorylation, is transcriptionally overexpressed in many tumor cell lines. Increased expression of the AIM-1 gene has been observed in human colorectal tumors of advanced grade and stage. Here we report that forced exogenous overexpression of AIM-1 in Chinese hamster embryo cells causes increased mitotic Ser-10 phosphorylation with concomitant induction of lagging chromosomes during mitosis. Lagging chromosomes could also be induced by transfection with mutated histone H3 (S10E), which is thought to maintain Ser-10 in the phosphorylated state. In the present study, chromosome number instability and increased tumor invasiveness were noted in constitutively AIM-1-overexpressing cells in vivo. Increased mitotic Ser-10 phosphorylation was also observed in various colorectal tumor cells with high AIM-1 expression levels. These data suggest that increased H3 histone phosphorylation as a result of AIM-1 overexpression is a major precipitating factor of chromosome instability and, thus, may play a role in carcinogenesis.

Phosphorylation of myosin II regulatory light chain (MRLC) is important for cell motility and cytokinesis in nonmuscle cells. Although the regulation of monophosphorylated MRLC at serine 19 throughout the cell cycle was examined in detail, MRLC diphosphorylation at both threonine 18 and serine 19 is still unclear. Here we found that Rho-kinase has an activity for MRLC diphosphorylation in nonmuscle cells using sequential column chromatographies. Transfection of Rho-kinase-EGFP induced the excess diphosphorylated MRLC and the bundling of the actin filaments. Conversely, the treatment of cells with a specific inhibitor of Rho-kinase, Y-27632, resulted in the decrease of endogenous diphosphorylated MRLC and actin stress fibers. Immunolocalization studies showed that both diphosphorylated MRLC and Rho-kinase accumulated and colocalized at the contractile ring and the midbody in dividing cells. Taken together, it is suggested that Rho-kinase contributes to MRLC diphosphorylation and reorganization of actin filaments in nonmuscle cells.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

To analyze specific genes related to radiation-induced apoptosis, 12 apoptosis-resistant clones were isolated from cells of the radiosensitive mouse thymic lymphoma 3SB line after treatment with ethyl methanesulfonate. Five of 12 clonal cell lines were recloned and were examined for their susceptibility to X-ray-induced apoptosis. A cell survival assay showed that all five secondary cell lines were two to three times more resistant to X rays than 3SB cells. When 3SB cells were exposed to 5 Gy of X rays, the fraction of cells stained with erythrosin B increased quickly within 8 h of incubation after irradiation. However, no apoptosis occurred in these secondary mutant cells. In particular, the percentage of cells undergoing apoptosis in one clone, 1B1C4, was low even after incubation for 48 h. In contrast to X rays, after exposure to 20 J/m2 UV radiation, the proportion of apoptotic cells in these mutant cells increased and reached about 60 to 100% at 24 h, indicating a difference in the ability of X rays and UV radiation to induce apoptosis. A similar radioresistance was observed using agarose gel electrophoresis of DNA from cells of all X-irradiated secondary lines. Western blot analysis and a sequence-specific DNA-binding assay demonstrated that 1B1C4 cells had a functional defect in p53 protein, but the other four cell lines displayed wild-type p53 after X irradiation. Our results suggest the existence of separate radiation-specific p53-dependent and independent apoptosis in thymic lymphoma cells. Thus these apoptosis-resistant cell lines provide a useful tool to identify the genes involved in the signaling pathways leading to X-ray-specific apoptosis.

The promoter region of the human N-methylpurine-DNA glycosylase (MPG) gene was cloned and characterized. The cloned segment contains two first exons that were earlier identified and named exons 1a and 1b. These were found to be separated by approximately 800 bp. The minimal promoter region was identified upstream to the distal exon 1a, by transient transfection, and no promoter activity was found in the region in between exons 1a and 1b, suggesting that transcription starts at a single site which is then processed to generate mRNAs of the isoforms. The promoter sequence is G and C rich and contains neither TATA box, nor apparent CAAT sequences, although a partially matched CAAT sequence was identified just downstream to the minimal promoter.

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.

The induction of DNA synthesis in quiescent, density-arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so-called "competence factors" and "progression factors." Here we examined this induction pathway in Balb/c 3T3 A31-I variants, which showed differential susceptibilities to radiation- and chemical-induced neoplastic transformation despite their similar susceptibilities to radiation- or chemical-induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31-1-13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31-I-1). The competent state constitutively produced by an autologous mechanism in the highly transformation-susceptible A31-I-13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.

The motility of individual mammalian cells is crucial for many biological processes. This report describes a new technique to quantitate cell motility, momentary alterations of cell shape, based on trace images obtained by video-image analyses and computer techniques. By means of this system, quantitation of cell motility could be automatically done without human observation or subjective judgement. Quantitative data from transformed and nontransformed rodent fibroblasts revealed that the cell motility measured here was related to the expression of such transformed phenotypes as morphological changes and tumorigenicity.

An automated, video-driven system has been developed which can quantitate dynamic cell morphology in cultured mammalian cells. This system is based upon the Personal Image Analysis System and is assisted by a video-enhanced contrast microscopy with a computer-aided digital image processing unit and a time-lapse video technique. Various parameters for cell motility including locomotion (vectorial translation) and accompanying shape changes can be simultaneously analyzed. Here, we describe this system and demonstrate its application in Balb/c 3T3 cell culture. This system represents a new tool for exploring subtleties of mammalian cell behavior.

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.

Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiecstasia (AT) lymphoblastoid cells and normal human cells. Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in T cells than in normal cells as previously reported by other investigators. However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistunguishable from that in normal cells. Moreover, no significant difference in cell viability was notd between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar. These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to mor stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there. The possible reasons fro this greater accumulation are discussed.

Background Survivin, a member of the inhibitor of apoptosis (IAP) gene family, has a dual role in mitosis and in apoptosis. It is abundantly expressed in every human tumor, compared with normal tissues. During mitosis Survivin assembles with the chromosomal passenger complex and regulates chromosomal segregation. Here, we aim to explore whether interference with the mitotic function of Survivin is linked to p53-mediated G1 cell cycle arrest and affects chromosomal stability. Methods In this study, we used HCT116, SBC-2, and U87-MG and generated corresponding isogenic p53-deficient cells. Retroviral vectors were used to stably knockdown Survivin. The resulting phenotype, in particular the mechanisms of cell cycle arrest and of initiation of aneuploidy, were investigated by Western Blot analysis, confocal laser scan microscopy, proliferation assays, spectral karyotyping and RNAi. Results In all cell lines Survivin-RNAi did not induce instant apoptosis but caused polyplodization irrespective of p53 status. Strikingly, polyploidization after knockdown of Survivin resulted in merotelic kinetochore spindle assemblies, γH2AX-foci, and DNA damage response (DDR), which was accompanied by a transient p53-mediated G1-arrest. That p53 wild type cells specifically arrest due to DNA damage was shown by simultaneous inhibition of ATM and DNA-PK, which abolished induction of p21waf/cip. Cytogenetic analysis revealed chromosomal aberrations indicative for DNA double strand break repair by the mechanism of non-homologous end joining (NHEJ), only in Survivin-depleted cells. Conclusion Our findings suggest that Survivin plays an essential role in proper amphitelic kinetochore-spindle assembly and that constraining Survivin’s mitotic function results in polyploidy and aneuploidy which cannot be controlled by p53. Therefore, Survivin critically safeguards chromosomal stability independently from p53.