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.

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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 ...

Purpose: A phase1study to determine the maximum-tolerated dose, dose-limiting toxicity, pharmacokinetics, and biological effects of bortezomib in children with recurrent/refractory leukemia. Experimental Design: Bortezomib was administered twice weekly for 2 consecutive weeks at either 1.3 or 1.7 mg/m 2 dose followed by a 1-week rest. Bortezomib pharmacokinetics and nuclear factor nB (NF-nB) binding activity were evaluated during the first treatment cycle. Results:Twelve patients (nine with acute lymphoblastic leukemia, three with acute myelogenous leukemia), median age 11years (range, 1-18 years), were enrolled between May 2004 and November 2005, of whom seven were not fully evaluable for toxicity due to rapidly progressive disease or uncontrolled infection. Dose-limiting toxicities occurred in two patients at the 1.7 mg/m 2 dose level. One patient experienced grade 3 confusion and the other patient had grade 4 febrile neutropenia associated with grade 4 hypotension and grade 3 creatinine. Pharmacokinetic analysis at 1.3 mg/m 2 revealed a clearance of 11 mL/h/m 2 , a central volume of distribution of 6.7 L/m 2 , and a terminal half-life of 12.6 h. NF-nB activity was examined in five patients and was noted to transiently increase and then decrease 4-to 6-fold by 24 h following bortezomib in two patients. There were no objective clinical responses. Conclusions: For children with leukemia, the recommended phase 2 dose of bortezomib, administered twice weekly for 2 weeks followed by a 1-week rest, is 1.3 mg/m 2

RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, we have generated a hypomorphic allele of Rad51c in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, we propose that this late function may be associated with HJ resolution.

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G 2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G 2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.Eukaryotes have a complex yet conserved response to DNA damage including changes in cell cycle kinetics (23) and transcriptional induction of multiple genes (17). Some of the genes responsible for these DNA damage-inducible cell cycle arrests or checkpoints have been identified (11). In at least one instance, the human checkpoint gene mutated in the disease ataxia telangiectasia, ATM (47), has sequence and functional homologs in many eukaryotes, including Drosophila melanogaster (MEI41 [22]), Saccharomyces cerevisiae (TEL1 and MEC1/ ESR1 [20,29,38]), and Schizosaccharomyces pombe (rad3, [50]). In contrast, other genes such as mammalian p53, a regulator of the damage-inducible G 1 checkpoint (31), do not appear to be conserved in lower eukaryotes. Germ line mutations in human checkpoint genes ATM and p53 result in a predisposition to malignancy, perhaps due to the unstable nature of the genome which results when checkpoint function is absent (reviewed by Weinert and Lydall [61]).Multiple checkpoint genes in both budding yeast (RAD17, RAD24, RAD9, RAD53/MEC2/SAD1, MEC3, and POL2 [4,23,60,62]) and fission yeast (hus1, hus3, rad1, rad9, rad17, rad24, rad25, rad26, cds1, and chk1 [2, 3, 13, 16, 28, 37, 45, 52]) are required for normal checkpoint function. Examination of these mutant phenotypes reveals that certain gene products, e.g., Mec1 (60) and Rad3 (28), are required for response to both incomplete replication and DNA damage. Other mutant strains have specific defects in the response to DNA damage, e.g., rad9, rad24 (60) of S. cerevisiae and chk1 (56) and rad27 (3) of S. pombe. The number of proteins required for establishment of cell cycle arrest after DNA damage suggest that a complex signal transduction pathway ...

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 ...