The cohesin complex is responsible for the accurate separation of sister chromatids into two daughter cells. Several models for the cohesin complex have been proposed, but the one-ring embrace model currently predominates the field. However, the static configuration of the embrace model is not flexible enough for cohesins to perform their functions during DNA replication, transcription, and DNA repair. We used coimmunoprecipitation, a protein fragment complement assay, and a yeast two-hybrid assay to analyze the protein–protein interactions among cohesin subunits. The results show that three of the four human cohesin core subunits (Smc1, Smc3, and Rad21) interact with themselves in an Scc3 (SA1/SA2)-dependent manner. These data support a two-ring handcuff model for the cohesin complex, which is flexible enough to establish and maintain sister chromatid cohesion as well as ensure the fidelity of chromosome segregation in higher eukaryotes.

A neuploidy (aberrant chromosome number) is a hallmark feature of human malignancies (1, 2) and has also been proposed as a necessary event for tumorigenesis (2). Although there have been many proposed hypotheses, there is no general agreement as to why aneuploidy is so highly prevalent in cancer cells, and how it contributes to tumor progression (3, 4). Importantly, if aneuploidy forms an underlying cause of human cancer, it has not been fully substantiated. The mechanisms of aneuploidy also remain a fundamental unresolved problem in cancer biology.To understand how aneuploidy might originate in mammalian tissues, we have focused on the elements that regulate chromosomal segregation, particularly those involved in sister chromatid cohesion and separation, because chromosome missegregation, for example during mitosis, can lead to aneuploidy. A key gene in our analysis is ESPL1, which encodes an endopeptidase called Separase that separates sister chromatids by cleaving cohesin Rad21/Mcd1/Scc1 during the metaphase to anaphase transition. The hypothesis we tested is that hormonal stimulation of the p53-null mouse mammary gland results in misexpression of the ESPL1 gene, thus promoting aneuploidy and breast cancer formation. Dysregulation of the mitotic machinery that helps maintain chromosomal stability in mammary cells can result in aneuploidy and subsequently, cancer formation. We focused on Separase for the following reasons that have important implications for breast cancer: (i) Separase plays a central role in promoting faithful chromosome segregation; (ii) our previous studies strongly indicated that hormonal stimulation of p53-null mice mammary gland results in overexpression of the ESPL1 and Separase protein, which may be a direct cause of aneuploidy (5); and (iii) siRNA-mediated knockdown of Separase and Separase deficient mouse embryonic fibroblasts results in genomic instability (6-8).An evolutionarily conserved protein complex called cohesin and an endopeptidase named Separase play pivotal roles in the accurate segregation of sister chromatids into two daughter cells. Cohesion along the length of the sister chromatids is formed during DNA replication in S phase. Cohesion along the chromosomal arms is removed during prophase and from centromeric regions at the metaphase-to-anaphase transition when Separase is activated after its inhibitory chaperone securin is degraded (9, 10).To understand how aberration in sister chromatid separation may contribute to chromosomal missegregation, we investigated the role of Separase overexpression in mouse mammary cells by using a mammary epithelial transplant model (11) as well as various biochemical and functional assays. Our results indicate that conditional overexpression of Separase alone in mammary epithelial cells with a p53 mutant background is sufficient to induce aneuploidy and tumorigenesis in vitro and in vivo. Results Conditional Expression of Mouse Separase (mSeparase) Results inAneuploidy in Mouse Mammary Epithelial Cells. To examine the direct effect of ...

Rad21 is one of the major cohesin subunits that holds sister chromatids together until anaphase, when proteolytic cleavage by separase, a caspase-like enzyme, allows chromosomal separation. We show that cleavage of human Rad21 (hRad21) also occurs during apoptosis induced by diverse stimuli. Induction of apoptosis in multiple human cell lines results in the early (4 h after insult) generation of 64-and 60-kDa carboxyterminal hRad21 cleavage products. We biochemically mapped an apoptotic cleavage site at residue Asp-279 (D 279 ) of hRad21. This apoptotic cleavage site is distinct from previously described mitotic cleavage sites. hRad21 is a nuclear protein; however, the cleaved 64-kDa carboxy-terminal product is translocated to the cytoplasm early in apoptosis before chromatin condensation and nuclear fragmentation. Overexpression of the 64-kDa cleavage product results in apoptosis in Molt4, MCF-7, and 293T cells, as determined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and Annexin V staining, assaying of caspase-3 activity, and examination of nuclear morphology. Given the role of hRad21 in chromosome cohesion, the cleaved C-terminal product and its translocation to the cytoplasm may act as a nuclear signal for apoptosis. In summary, we show that cleavage of a cohesion protein and translocation of the C-terminal cleavage product to the cytoplasm are early events in the apoptotic pathway and cause amplification of the cell death signal in a positive-feedback manner.Normal development and homeostasis require the orderly regulation of both cell proliferation and cell survival. Cell cycle progression and control of apoptosis are thought to be intimately linked processes. Activation of the cell cycle plays a significant role in the regulation of apoptosis (16); in some cell types and under certain conditions, apoptosis has been shown to occur only at specific stages of the cell cycle (24). Mitosis and apoptosis are also closely interrelated (25), and the mitotic index is the most important determinant of the apoptotic index (25). Although proteins that regulate apoptosis have been implicated in the restraint of cell cycle entry (14) and the control of ploidy (29), the effector molecules at the interface between cell proliferation and cell survival have remained elusive.Studies with yeast and higher eukaryotes, including humans, have indicated that an evolutionarily conserved protein complex, called cohesin, and its subunit, Mcd1/Scc1/hRad21, are required for appropriate arrangement of chromosomes during normal cell division (11, 28; for a review, see references 20, 30, 31, and 36). Analyses of Rad21 function in fission yeast, Schizosaccharomyces pombe, and of Scc1/Mcd1 function in budding yeast, Saccharomyces cerevisiae, have demonstrated that the nuclear phosphoprotein is required for appropriate chromosomal cohesion during the mitotic cell cycle and double-strandbreak repair after DNA damage (1, 30). Biochemical analysis of cohesin indicates that it acts as a molecular glu...

Purpose: Separase, an endopeptidase, plays a pivotal role in chromosomal segregation by separating sister chromatids during the metaphase to anaphase transition. Using a mouse mammary tumor model we have recently shown that overexpression of Separase induces aneuploidy and tumorigenesis (Zhang et al., Proc Natl Acad Sci 2008;105:13033). In the present study, we have investigated the expression level of Separase across a wide range of human tumors. Experimental Design: To examine the expression levels and localization of Separase in human tumors, we have performed immunofluorescence microscopy using human Separase antibody and tumor tissue arrays from osteosarcoma, colorectal, breast, and prostate cancers with appropriate normal controls. Results: We show that Separase is significantly overexpressed in osteosarcoma, breast, and prostate tumor specimens. There is a strong correlation of tumor status with the localization of Separase into the nucleus throughout all stages of the cell cycle. Unlike the normal control tissues, where Separase localization is exclusively cytoplasmic in nondividing cells, human tumor samples show significantly higher number of resting cells with a strong nuclear Separase staining. Additionally, overexpression of Separase transcript strongly correlates with high incidence of relapse, metastasis, and lower 5-year overall survival rate in breast and prostate cancer patients. Conclusion: These results further strengthen our hypothesis that Separase might be an oncogene, whose overexpression induces tumorigenesis, and indicates that Separase overexpression and aberrant nuclear localization are common in many tumor types and may predict outcome in some human cancers.An evolutionarily conserved protein complex called cohesin holds sister chromatids together to allow accurate separation of sister chromatids into two daughter cells. At the onset of anaphase, Separase, an endopeptidase, is activated and cleaves the cohesin subunit Rad21 (also called SCC1 or MCD1), which releases sister chromatid cohesion. Separase activity is tightly regulated via several mechanisms (for details, see refs. 1 -3) to ensure accurate and precise activation of cohesin Rad21 cleavage during the metaphase to anaphase transition (2 -4).Separase is activated after its inhibitory chaperone securin is degraded by APC-mediated phosphorylation and ubiquitinmediated degradation (1, 5 -8). Additionally, phosphorylation of Separase on Ser 1126 and Thr 1326 residues is a second mechanism to inhibit Separase activity (9, 10). Therefore, Securin null cells are viable and appear to have a nearly normal cell cycle (11 -13). However, premature separation of sister chromatids, for example, by premature activation of Separase or by insufficient inhibition of overexpressed Separase, is thought to result in aneuploidy (14).Knockout of the Separase gene results in embryonic lethality in mice (13,15). Small interfering RNA-mediated knockdown of Separase results in genomic instability (8, 16), also seen in Separase-deficient mouse ...

Background: Sister chromatid arm cohesion is resolved at prophase/prometaphase, but its mechanism remains unclear. Results: The ST 159 P motif on Sororin is phosphorylated by Cdk1/cyclin B, and its subsequent interaction with the polo box domain of Plk1 facilitates the resolution of arm cohesion. Conclusion: Sororin mediates Plk1-mediated phosphorylation of SA2 to resolve sister chromatid arm cohesion. Significance: The mechanism of chromatid resolution is important to understand chromosomal missegregation often seen in cancer.

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Separase, a protease encoded by the ESPL1 gene, cleaves the chromosomal cohesin during mitosis. Separase protein and transcripts are overexpressed in a wide range of human cancers (Meyer et al., Clin Cancer Res 2009; 15: 2703-2710). To investigate the physiological consequence of Separase overexpression in animals, we have generated a transgenic MMTVEspl1 mouse model that overexpresses Separase protein in the mammary glands. MMTV-Espl1 mice in a C57BL/6 genetic background develop aggressive, highly aneuploid, and estrogen receptor alpha positive (ERα+) mammary adenocarcinomas with an 80% penetrance. The mammary tumors caused by overexpression of Separase, alone or combined with p53 heterozygosity, in mammary epithelium mimic several aspects of the most aggressive forms of human breast cancer, including high levels of genetic instability, cell cycle defects, poor differentiation, distant metastasis, and metaplasia. Histopathologically, MMTV-Espl1 tumors are highly heterogeneous showing features of both luminal as well as basal subtypes of breast cancers, with aggressive disease phenotype. In addition to aneuploidy, Separase overexpression results in chromosomal instability (CIN) including premature chromatid separation (PCS), lagging chromosomes, anaphase bridges, micronuclei, centrosome amplification, multi nucleated cells, gradual accumulation of DNA damage, and progressive loss of tumor suppressors p53 and cadherin gene loci. These results suggest that Separase overexpressing mammary cells are not only susceptible to chromosomal missegregation-induced aneuploidy but also other genetic instabilities including DNA damage and loss of key tumor suppressor gene loci, which in combination can initiate tumorigenesis and disease progression.