Cohesin Subunit RAD21 Regulates the Differentiation and Self-Renewal of Hematopoietic Stem and Progenitor Cells.

Recent studies suggest that chromosomal cohesin complex proteins are important in regulating hematopoiesis and may contribute to myeloid malignancies. To investigate the effects of perturbing the cohesin subunit protein RAD21 on normal hematopoiesis, we used conditional knockout (cKO) mouse models. While cohesin is vital for hematopoietic stem cell (HSC) function, Rad21 haploinsufficiency (Rad21Δ/+) led to distinct hematopoietic phenotypes. Our findings revealed that Rad21Δ/+ cells exhibited decreased hematopoietic reconstitution in competitive bone marrow transplantation assays. This reduction in peripheral blood chimerism was specifically observed in the lymphoid compartment, while the chimerism in the myeloid compartment remained unaffected. Rad21 haploinsufficiency also resulted in changes in the hematopoietic stem and progenitor cells (HSPC) and myeloid progenitor compartments, with a significant accumulation of granulocyte-macrophage progenitors in the bone marrow. We observed differential gene expression in Rad21Δ/+ LSK (Lin- Sca1-Kit+) cells, including genes required for HSPC function and differentiation, such as Setdb1, Hmga2, Ncor1, and Myb. In addition, we observed a notable decrease in the expression of genes related to the interferon response and a significant reduction in the expression of genes involved in the IL2-STAT5 signaling pathways. Our studies suggest that RAD21 protein and level of its post-translational modifications in the bone marrow cells may play a potential role in hematopoiesis. Overall, Rad21 haploinsufficiency impairs hematopoietic differentiation and increases HSC self-renewal.

Haploinsufficiency of cohesin protease, Separase, promotes regeneration of hematopoietic stem cells in mice.

Cohesin recently emerged as a new regulator of hematopoiesis and leukemia. In addition to cohesin, whether proteins that regulate cohesin's function have any direct role in hematopoiesis and hematologic diseases have not been fully examined. Separase, encoded by the ESPL1 gene, is an important regulator of cohesin's function. Canonically, protease activity of Separase resolves sister chromatid cohesion by cleaving cohesin subunit-Rad21 at the onset of anaphase. Using a Separase haploinsufficient mouse model, we have uncovered a novel role of Separase in hematopoiesis. We report that partial disruption of Separase distinctly alters the functional characteristics of hematopoietic stem/progenitor cells (HSPCs). Although analyses of peripheral blood and bone marrow of Espl1+/Hyp mice broadly displayed unperturbed hematopoietic parameters during normal hematopoiesis, further probing of the composition of early hematopoietic cells in Espl1+/Hyp bone marrow revealed a mild reduction in the frequencies of the Lin- Sca1+ Kit- (LSK) or LSK CD48+ CD150- multipotent hematopoietic progenitors population without a significant change in either long-term or short-term hematopoietic stem cells (HSCs) subsets at steady state. Surprisingly, however, we found that Separase haploinsufficiency promotes regeneration activity of HSCs in serial in vivo repopulation assays. In vitro colony formation assays also revealed an enhanced serial replating capacity of hematopoietic progenitors isolated from Espl1+/Hyp mice. Microarray analysis of differentially expressed genes showed that Separase haploinsufficiency in HSCs (SP-KSL) leads to enrichment of gene signatures that are upregulated in HSCs compared to committed progenitors and mature cells. Taken together, our findings demonstrate a key role of Separase in promoting hematopoietic regeneration of HSCs.

PDS5A and PDS5B in Cohesin Function and Human Disease.

Precocious dissociation of sisters 5 (PDS5) is an associate protein of cohesin that is conserved from yeast to humans. It acts as a regulator of the cohesin complex and plays important roles in various cellular processes, such as sister chromatid cohesion, DNA damage repair, gene transcription, and DNA replication. Vertebrates have two paralogs of PDS5, PDS5A and PDS5B, which have redundant and unique roles in regulating cohesin functions. Herein, we discuss the molecular characteristics and functions of PDS5, as well as the effects of its mutations in the development of diseases and their relevance for novel therapeutic strategies.

Synthesis and activity of benzimidazole-1,3-dioxide inhibitors of separase.

Due to the oncogenic activity of cohesin protease, separase in human cancer cells, modulation of separase enzymatic activity could constitute a new therapeutic strategy for targeting resistant, separase-overexpressing aneuploid tumors. Herein, we report the synthesis, structural information, and structure-activity relationship (SAR) of separase inhibitors based on modification of the lead molecule 2,2-dimethyl-5-nitro-2H-benzimidazole-1,3-dioxide, named Sepin-1, (1) identified from a high-throughput-screen. Replacement of -NO2 at C5 with other functional groups reduce the inhibitory activity in separase enzymatic assay. Substitution of the two methyl groups with other alkyl chains at the C2 moderately improves the effects on the inhibitory activity of those compounds. Modifications on 2H-benzimidazole-1,3-dioxide or the skeleton have variable effect on inhibition of separase enzymatic activity. Density-functional theory (DFT) calculations suggest there may be a correlation between the charges on the oxide moieties on these compounds and their activity in inhibiting separase enzyme.

Overexpression and constitutive nuclear localization of cohesin protease Separase protein correlates with high incidence of relapse and reduced overall survival in glioblastoma multiforme.

Separase, an enzyme that cleaves the chromosomal cohesin during mitosis, is overexpressed in a wide range of human epithelial cancers of breast, bone and prostate (Meyer et al., Clin Cancer Res 15(8):2703-2710, 2009). Overexpression of Separase in animal models results in aneuploidy and tumorigenesis. We have examined the expression and localization of Separase protein in adult and pediatric glioblastoma and normal brain specimens. Immunofluorescence microscopy and Western blot analysis showed significant overexpression of Separase in all adult and a subset of pediatric glioblastoma cells. Tumor status and patient survival strongly correlate with the mislocalization of Separase into the nucleus throughout all stages of the cell cycle. Unlike exclusively nuclear localization in mitotic control cells, glioblastoma samples have a significantly higher number of resting (interphase) cells with strong nuclear Separase staining. Additionally, patient survival analysis demonstrated a strong correlation between overexpression of Separase protein in adult glioblastoma and a high incidence of relapse and reduced overall survival. These results further strengthen our hypothesis that Separase is an oncogene whose overexpression induces tumorigenesis, and indicate that Separase overexpression and aberrant nuclear localization are common in many tumor types and may predict outcome in some human malignancies.

A cohesin-RAD21 interactome.

The cohesin complex holds the sister chromatids together from S-phase until the metaphase-to-anaphase transition, and ensures both their proper cohesion and timely separation. In addition to its canonical function in chromosomal segregation, cohesin has been suggested by several lines of investigation in recent years to play additional roles in apoptosis, DNA-damage response, transcriptional regulation and haematopoiesis. To better understand the basis of the disparate cellular functions of cohesin in these various processes, we have characterized a comprehensive protein interactome of cohesin-RAD21 by using three independent approaches: Y2H (yeast two-hybrid) screening, immunoprecipitation-coupled-MS of cytoplasmic and nuclear extracts from MOLT-4 T-lymphocytes in the presence and absence of etoposide-induced apoptosis, and affinity pull-down assays of chromatographically purified nuclear extracts from pro-apoptotic MOLT-4 cells. Our analyses revealed 112 novel protein interactors of cohesin-RAD21 that function in different cellular processes, including mitosis, regulation of apoptosis, chromosome dynamics, replication, transcription regulation, RNA processing, DNA-damage response, protein modification and degradation, and cytoskeleton and cell motility. Identification of cohesin interactors provides a framework for explaining the various non-canonical functions of the cohesin complex.

Interaction of Sororin protein with polo-like kinase 1 mediates resolution of chromosomal arm cohesion.

Unlike in budding yeast, sister chromatid cohesion in vertebrate cells is resolved in two steps: cohesin complexes are removed from sister chromatid arms during prophase via phosphorylation, whereas centromeric cohesins are removed at anaphase by Separase. Phosphorylation of cohesin subunit SA2 by polo-like kinase 1 (Plk1) is required for the removal of cohesins at prophase, but how Plk1 is recruited to phosphorylate SA2 during prophase is currently not known. Here we report that Sororin, a cohesin-interacting protein essential for sister chromatid cohesion, plays a novel role in the resolution of sister chromatid arms by direct interaction with Plk1. We identified an evolutionarily conserved motif (ST(159)P) on Sororin, which was phosphorylated by Cdk1/cyclin B and bound to the polo box domain of Plk1. Mutating Thr(159) into alanine prevented the interaction of Plk1 and Sororin and inhibited the resolution of chromosomal arm cohesion. We propose that Sororin is phosphorylated by Cdk1/cyclin B at prophase and acts as a docking protein to bring Plk1 into proximity with SA2, resulting in the phosphorylation of SA2 and the removal of cohesin complexes from chromosomal arms.

Synthesis and Evaluation of a Class of Compounds Inhibiting the Growth of Stromal Antigen 2 (STAG2)-Mutant Ewing Sarcoma Cells.

STAG2 (SA2) is a critical component of the cohesin complex that regulates gene expression and the separation of sister chromatids in cells. Mutations in STAG2 have been identified in over thirty different types of cancers including myeloid leukaemia, non-small cell lung, bladder and Ewing sarcoma. Selectively inhibiting cancer cells lacking of STAG2 is an attractive approach for the cancer therapy. Here we report that a small molecule, StagX1, identified through a high-throughput screening, inhibits the growth of Ewing sarcoma cells possessing mutant STAG2. A new synthetic route to the StagX1 scaffold and new versions of the molecule along with their activity in a cell viability assay are reported.

Overexpression of Separase induces aneuploidy and mammary tumorigenesis.

Separase is an endopeptidase that separates sister chromatids by cleaving cohesin Rad21 during the metaphase-to-anaphase transition. Conditional expression of Separase in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells with both p53 WT and mutant (Ser-233-234) alleles of unknown physiological significance develops aneuploidy within 5 days of Separase induction in vitro. Overexpression of Separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In an in vivo mouse mammary transplant model, induction of Separase expression in the transplanted FSK3 cells for 3-4 weeks results in the formation of aneuploid tumors in the mammary gland. Xenograft studies combined with histological and cytogenetic analysis reveal that Separase-induced tumors are clonal in their genomic complements and have a mesenchymal phenotype suggestive of an epithelial-mesenchymal transition. Induction of Separase resulted in trisomies for chromosomes 8, 15, and 17; monosomy for chromosome 10; and amplification of the distal region of chromosomes 8 and 11. Separase protein is found to be significantly overexpressed in human breast tumors compared with matched normal tissue. These results collectively suggest that Separase is an oncogene, whose overexpression alone in mammary epithelial cells is sufficient to induce aneuploidy and tumorigenesis in a p53 mutant background.

Overexpression and mislocalization of the chromosomal segregation protein separase in multiple human cancers.

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.

Cohesin subunit RAD21: From biology to disease.

RAD21 (also known as KIAA0078, NXP1, HR21, Mcd1, Scc1, and hereafter called RAD21), an essential gene, encodes a DNA double-strand break (DSB) repair protein that is evolutionarily conserved in all eukaryotes from budding yeast to humans. RAD21 protein is a structural component of the highly conserved cohesin complex consisting of RAD21, SMC1a, SMC3, and SCC3 [STAG1 (SA1) and STAG2 (SA2) in metazoans] proteins, involved in sister chromatid cohesion. This function is essential for proper chromosome segregation, post-replicative DNA repair, and prevention of inappropriate recombination between repetitive regions. In interphase, cohesin also functions in the control of gene expression by binding to numerous sites within the genome. In addition to playing roles in the normal cell cycle and DNA DSB repair, RAD21 is also linked to the apoptotic pathways. Germline heterozygous or homozygous missense mutations in RAD21 have been associated with human genetic disorders, including developmental diseases such as Cornelia de Lange syndrome (CdLS) and chronic intestinal pseudo-obstruction (CIPO) called Mungan syndrome, respectively, and collectively termed as cohesinopathies. Somatic mutations and amplification of the RAD21 have also been widely reported in both human solid and hematopoietic tumors. Considering the role of RAD21 in a broad range of cellular processes that are hot spots in neoplasm, it is not surprising that the deregulation of RAD21 has been increasingly evident in human cancers. Herein, we review the biology of RAD21 and the cellular processes that this important protein regulates and discuss the significance of RAD21 deregulation in cancer and cohesinopathies.

Data set on Separase Inhibitor-Sepin-1 toxicity on organ weights, hematology and clinical parameters in Sprague-Dawley rats.

Separase Inhibitor-Sepin-1 has shown great promise as a developmental chemotherapeutic agent to treat Separase-overexpressing tumors, however, very little is known about its toxicity profile. Here we present the data set of organ weights, hematology, and clinical chemistry parameters in Sepin-1-treated Sprague-Dawley rats. The data set was generated from two study groups-Main Study and Recovery Study, with in-life duration of 29 and 57 days, respectively. Rats in both groups were dosed with 0, 5, 10 and 20 mg/kg of Sepin-1 once daily for 28 consecutive days. Blood samples from rats in Main Study were collected and their organs were weighed on day 29. The animals in Recovery Study were on a dose-off period of 28 days after dosed with Sepin-1 for 28 days, and their blood samples and organ weight data were collected on day 57. Body weights of rats in both Main and Recovery Study were collected twice a week. Hematology parameters of whole blood samples, such as hemoglobin concentration, counts of platelet and blood cells etc., were determined. Clinical chemistry parameters of serum, such as concentrations of albumin, glucose, cholesterol, triglyceride, alanine/aspartate aminotransferase, ect., were measured. Further analysis may yield useful information regarding the toxicity of Sepin-1 in Sprague-Dawley Rats. Data presented here are related to a research article entitled "Toxicity study of separase inhibitor-Sepin-1 in Sprague-Dowley rats", available in Pathology - Research and Practice Journal [1].

Toxicity study of separase inhibitor-Sepin-1 in Sprague-Dawley rats.

Sepin-1 is a small compound that inhibits enzymatic activity of Separase and growth of cancer cells. As part of the IND-enabling studies to develop Sepin-1 as a chemotherapeutic agent, herein we have profiled the toxicity of Sepin-1 in Sprague-Dawley rats in a good laboratory practice (GLP) setting. The maximum tolerated dose (MTD) of Sepin-1 in rats is 40 mg/kg in single dose study and 20 mg/kg in the study dosed for 7 consecutive days. The toxicity study consists of two parts-Main Study and Recovery Study. Sepin-1 with 0 (control), 5 (low dose), 10 (median dose), and 20 (high dose) mg/kg was administered by bolus intravenous injection to rats once daily for 28 consecutive days. The animals in the Main Study were euthanized on Day 29, whereas animals in the Recovery Study were allowed to recover for 28 days following the 28-day Sepin-1 dose before they were euthanized on Day 29 of the off-dose period. Although the effects of Sepin-1 at low and median doses are minimal, hematological analysis shows that high-dose Sepin-1 is associated with decrease of red blood cells and hemoglobin, and increase in the number of reticulocytes and platelets as well as mean corpuscular volume. Clinical chemistry indicates that Sepin-1 causes increase of total bilirubin and decrease of creatine kinase. Histopathology analysis indicates Sepin-1 results in minimal bone marrow erythroid hyperplasia, minimal to moderate splenic extramedullary hematopoiesis, minimal splenic lymphoid depletion, minimal to mild thymic lymphoid depletion, and minimal to mild mandibular lymph node lymphoid hyperplasia in male and female rats in the Main Study. Those abnormal changes are Sepin-1 dose-dependent and mostly reversible after a 28-day recovery period in animals from the Recovery Study. Based on our results, we conclude that Sepin-1 at pharmacologic doses (5-10 mg/kg) is well tolerable, with no significant rates of mortality or morbidity, and can further be developed as a potential new drug to treat Separase-overexpressed tumors.

Stability and pharmacokinetics of separase inhibitor-Sepin-1 in Sprague-Dawley rats.

Separase, a sister chromatid cohesion-resolving enzyme, is an oncogene and overexpressed in many human cancers. Sepin-1 (2,2-dimethyl-5-nitro-2H-benzimidazole-1,3-dioxide) is a potent separase inhibitor that impedes cancer cell growth, cell migration, and wound healing, suggesting that Sepin-1 possesses a great potential to target separase-overexpressing tumors. As a part of the IND-enabling studies to bring Sepin-1 to clinic, herein we report the results from a 28-day repeat-dose pharmacokinetic study of Sepin-1 in rats. Sepin-1 was intravenously administered to Sprague-Dawley rats once daily for 28 days at three different (5, 10, and 20 mg/kg) doses. Blood samples were collected after administration of doses on days 1 and 28. Sepin-1 is unstable and isomerizes in basic solutions, but it is stable in acidic buffer such as citrate-buffered saline (pH 4.0). UHPLC-MS analysis indicated Sepin-1 was rapidly metabolized in vivo. One of the major metabolites was an amine adduct of 2,2-dimethyl-5-nitro-2H-benzimidazole (named Sepin-1.55). The concentration of Sepin-1.55 in blood samples was Sepin-1 dose-dependent and used for pharmacokinetic analysis of Sepin-1. Tmax was approximately 5-15 min. The data suggest that no Sepin-1 accumulation occurred from daily repeat dosing and similar exposures on the first and final day of dosing. Data also suggest a gender difference, namely that female rats have more exposure and slower clearance than male rats. The data support that Sepin-1 is a potential drug candidate that can be further developed to treat Separase-overexpressing human tumors.

Development and validation of a fluorogenic assay to measure separase enzyme activity.

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.

Separase Inhibitor Sepin-1 Inhibits Foxm1 Expression and Breast Cancer Cell Growth.

Sepin-1, a potent non-competitive inhibitor of separase, inhibits cancer cell growth, but the mechanisms of Sepin-1-mediated growth inhibition are not fully understood. Here we report that Sepin-1 hinders growth of breast cancer cells, cell migration, and wound healing. Inhibition of cell growth induced by Sepin-1 in vitro doesn't appear to be through apoptosis but rather due to growth inhibition. Following Sepin-1 treatment caspases 3 and 7 are not activated and Poly (ADP-ribose) polymerase (Parp) is not cleaved. The expression of Forkhead box protein M1 (FoxM1), a transcription factor, and its target genes in the cell cycle, including Plk1, Cdk1, Aurora A, and Lamin B1, are reduced in a Sepin-1-dependent manner. Expressions of Raf kinase family members A-Raf, B-Raf, and C-Raf also are inhibited following treatment with Sepin-1. Raf is an intermediator in the Raf-Mek-Erk signaling pathway that phosphorylates FoxM1. Activated FoxM1 can promote its own transcription via a positive feedback loop. Sepin-1-induced downregulation of Raf and FoxM1 may inhibit expression of cell cycle-driving genes, resulting in inhibition of cell growth.

The Metabolism of Separase Inhibitor Sepin-1 in Human, Mouse, and Rat Liver Microsomes.

Separase, a known oncogene, is widely overexpressed in numerous human tumors of breast, bone, brain, blood, and prostate. Separase is an emerging target for cancer therapy, and separase enzymatic inhibitors such as sepin-1 are currently being developed to treat separase-overexpressed tumors. Drug metabolism plays a critical role in the efficacy and safety of drug development, as well as possible drug-drug interactions. In this study, we investigated the in vitro metabolism of sepin-1 in human, mouse, and rat liver microsomes (RLM) using metabolomic approaches. In human liver microsomes (HLM), we identified seven metabolites including one cysteine-sepin-1 adduct and one glutathione-sepin-1 adduct. All the sepin-1 metabolites in HLM were also found in both mouse and RLM. Using recombinant CYP450 isoenzymes, we demonstrated that multiple enzymes contributed to the metabolism of sepin-1, including CYP2D6 and CYP3A4 as the major metabolizing enzymes. Inhibitory effects of sepin-1 on seven major CYP450s were also evaluated using the corresponding substrates recommended by the US Food and Drug Administration. Our studies indicated that sepin-1 moderately inhibits CYP1A2, CYP2C19, and CYP3A4 with IC50 < 10 µM but weakly inhibits CYP2B6, CYP2C8/9, and CYP2D6 with IC50 > 10 µM. This information can be used to optimize the structures of sepin-1 for more suitable pharmacological properties and to predict the possible sepin-1 interactions with other chemotherapeutic drugs.

Linking sister chromatid cohesion and apoptosis: role of Rad21.

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 carboxy-terminal 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.

Biology and insights into the role of cohesin protease separase in human malignancies.

Separase, an enzyme that resolves sister chromatid cohesion during the metaphase-to-anaphase transition, plays a pivotal role in chromosomal segregation and cell division. Separase protein, encoded by the extra spindle pole bodies like 1 (ESPL1) gene, is overexpressed in numerous human cancers including breast, bone, brain, and prostate. Separase is oncogenic, and its overexpression is sufficient to induce mammary tumours in mice. Either acute or chronic overexpression of separase in mouse mammary glands leads to aneuploidy and tumorigenesis, and inhibition of separase enzymatic activity decreases the growth of human breast tumour xenografts in mice. This review focuses on the biology of and insights into the molecular mechanisms of separase as an oncogene, and its significance and implications for human cancers.

Hormone-induced chromosomal instability in p53-null mammary epithelium.

The absence of p53 function increases risk for spontaneous tumorigenesis in the mammary gland. Hormonal stimulation enhances tumor risk in p53-null mammary epithelial cells as well as the incidence of aneuploidy. Aneuploidy appears in normal p53-null mammary epithelial cells within 5 weeks of hormone stimulation. Experiments reported herein assessed a possible mechanism of hormone-induced aneuploidy. Hormones increased DNA synthesis equally between wild-type (WT) and p53-null mammary epithelial cells. There were two distinct responses in p53-null cells to hormone exposure. First, Western blot analysis demonstrated that the levels of two proteins involved in regulating sister chromatid separation and the spindle checkpoint, Mad2 and separase (ESPL1) were increased in null compared with WT cells. In contrast, the levels of securin and Rad21 proteins were not increased in hormone-stimulated p53-null compared with WT cells. ESPL1 RNA was also increased in p53-null mouse mammary cells in vivo by 18 h of hormone stimulation and in human breast MCF7 cells in monolayer culture by 8 h of hormone stimulation. Furthermore, both promoters contained p53 and steroid hormone response elements. Mad2 protein was increased as a consequence of the absence of p53 function. The increase in Mad2 protein was observed also at the cellular level by immunohistochemistry. Second, hormones increased gene amplication in the distal arm of chromosome 2, as shown by comparative genomic hybridization. These results support the hypothesis that hormone stimulation acts to increase aneuploidy by several mechanisms. First, by increasing mitogenesis in the absence of the p53 checkpoint in G2, hormones allow the accumulation of cells that have experienced chromosome missegregation. Second, the absolute rate of chromosome missegregation may be increased by alterations in the levels of two proteins, separase and Mad2, which are important for maintaining chromosomal segregation and the normal spindle checkpoint during mitosis.