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Supplementary Data from Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

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 has 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.

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.

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.

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.

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.

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.

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.

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]. Keywords: Separase, Inhibitor, Sepin-1, Toxicity, Hematology, Clinal chemistry, Rats

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.

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.

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, 2009, Clin Cancer Res 15(8): 2703-2710] [1]. 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.

Fluorescence in situ hybridization (FISH) is the most widely used molecular technique to visualize chromosomal abnormalities. Here, we describe a novel 3D modeling approach to allow precise shape estimation and localization of FISH signals in the nucleus of human embryonic stem cells (hES) undergoing progressive but defined aneuploidy. The hES cell line WA09 acquires an extra copy of chromosome 12 in culture with increasing passages. Both diploid and aneuploid nuclei were analyzed to quantitate the differences in the localization of centromeric FISH signals for chromosome 12 as it transitions from euploidy to aneuploidy. We employed superquadric modeling primitives coupled with principal component analysis to determine the 3D position of FISH signals within the nucleus. A novel aspect of our modeling approach is that it allows comparison of FISH signals across multiple cells by normalizing the position of the centromeric signals relative to a reference landmark in oriented nuclei. Using this model we present evidence of changes in the relative positioning of centromeres in trisomy-12 cells when compared with diploid cells from the same population. Our analysis also suggests a significant change in the spatial distribution of at least one of the FISH signals in the aneuploid chromosome complements implicating that an overall change in centromere position may occur in trisomy-12 due to the addition of an extra chromosome. These studies underscore the unique utility of our modeling algorithms in quantifying FISH signals in three dimensions. © 2013 International Society for Advancement of Cytometry

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.

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G 2 phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin's emerging role in cancer and the potential issues that need be addressed in the future.

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.

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC50 alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3–7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2–5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ. [Mol Cancer Ther 2009;8(8):2232–42]

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.

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.

Purpose: A phase 1 study 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/m2 dose followed by a 1-week rest. Bortezomib pharmacokinetics and nuclear factor κB (NF-κB) binding activity were evaluated during the first treatment cycle. Results: Twelve patients (nine with acute lymphoblastic leukemia, three with acute myelogenous leukemia), median age 11 years (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/m2 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/m2 revealed a clearance of 11 mL/h/m2, a central volume of distribution of 6.7 L/m2, and a terminal half-life of 12.6 h. NF-κB 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/m2/dose. Although bortezomib treatment inhibited NF-κB activity, bortezomib had little activity as a single agent in this population.

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.

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.

Epithelial tumor metastasis is preceded by an accumulation of collagen cross-links that heighten stromal stiffness and stimulate the invasive properties of tumor cells. However, the biochemical nature of collagen cross-links in cancer is still unclear. Here, we postulated that epithelial tumorigenesis is accompanied by changes in the biochemical type of collagen cross-links. Utilizing resected human lung cancer tissues and a p21CIP1/WAF1-deficient, K-rasG12D-expressing murine metastatic lung cancer model, we showed that, relative to normal lung tissues, tumor stroma contains higher levels of hydroxylysine aldehyde-derived collagen cross-links (HLCCs) and lower levels of lysine aldehyde-derived cross-links (LCCs), which are the predominant types of collagen cross-links in skeletal tissues and soft tissues, respectively. Gain- and loss-of-function studies in tumor cells showed that lysyl hydroxylase 2 (LH2), which hydroxylates telopeptidyl lysine residues on collagen, shifted the tumor stroma toward a high-HLCC, low-LCC state, increased tumor stiffness, and enhanced tumor cell invasion and metastasis. Together, our data indicate that LH2 enhances the metastatic properties of tumor cells and functions as a regulatory switch that controls the relative abundance of biochemically distinct types of collagen cross-links in the tumor stroma.

The involvement of protein kinase C (PKC) and arachidonic acid (AA) pathways were investigated in the GnRH regulation of oocyte meiosis and follicular testosterone production in the goldfish ovary. The results clearly demonstrate differences in the postreceptor mechanisms involving the stimulatory and inhibitory actions of GnRH peptides on basal and gonadotropin (GtH)-induced reinitiation of oocyte meiosis and steroidogenesis. In isolated goldfish follicles in vitro, the observed stimulatory effects of both salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) on germinal vesicle breakdown were completely blocked by addition of PKC inhibitors, suggesting the involvement of PKC, presumably through activation of phospholipase C/diacylglycerol pathways in the GnRH-induced reinitiation of oocyte meiosis. Administration of an AA metabolism inhibitor, however, only blocked the stimulatory effect of sGnRH without affecting cGnRH-II-induced meiosis. As observed previously, in the presence of GtH, sGnRH was found to inhibit GtH-induced resumption of meiosis and testosterone production, whereas cGnRH-II was without effect. The inhibitory effect of sGnRH on GtH-induced meiosis and steroidogenesis was completely reversed by addition an AA metabolism inhibitor, whereas PKC inhibitors had no effect. These findings provide functional evidence in support of the novel hypothesis that goldfish ovarian follicles contain GnRH-receptor subtypes with different ligand selectivity mediating stimulatory and inhibitory actions of sGnRH and cGnRH in the goldfish ovary.

This study investigated the effects of gonadotropin-releasing hormones (GnRH) present in the goldfish brain (sGnRH and cGnRH-II) as well as a number of other GnRH variants on the reinitiation of meiosis and testosterone production in the follicle-enclosed goldfish oocytes, in vitro. All GnRH peptides tested individually stimulated oocyte meiosis in vitro, as determined by germinal vesicle breakdown (GVBD) as well as histone H1 kinase activity, which is an indicator of maturation promoting factor (MPF) production. The GnRH peptides tested had no detectable effect on basal follicular testosterone production with the exception of lGnRH-III, which had a relatively small stimulatory effect. In the presence of gonadotropin hormone (GTH), however, both sGnRH and lGnRH-III inhibited GTH-induced meiosis and steroidogenesis, whereas other GnRH peptides had no effect on GTH-induced responses. Addition of a GnRH antagonist effectively blocked the stimulatory effect of all GnRH peptides on oocyte meiosis, but was without effect on the inhibitory actions of sGnRH and lGnRH-III on GTH-induced meiosis, suggesting the involvement of different pathways mediating the stimulatory and inhibitory actions of sGnRH and lGnRH-III. These GnRH peptides were found to bind to the GnRH receptors in the goldfish ovary with different affinities (equilibrium association constant, K(a)). The findings provide novel information on the activity of GnRH variants in the goldfish ovary and provide a strong support for the hypothesis that GnRH plays a paracrine/autocrine role in the regulation of ovarian function in goldfish.

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

The presence of ovarian GnRH and/or compounds with GnRH-like activity was investigated in the goldfish ovary. Goldfish ovary was extracted using an acetone/HCl/ether mixture and was purified by Waters C-18 Sep-Pak columns (ovarian extract). The goldfish ovarian extract was found to 1) stimulate gonadotropin release and subunit messenger RNA production in the goldfish pituitary that was inhibited by a GnRH antagonist; 2) stimulate germinal vesicle breakdown in the prophase-I arrested follicle-enclosed goldfish oocytes in vitro, which was inhibited by a GnRH antagonist; 3) immunoreact with various GnRH antisera; and 4) bind to GnRH receptors in the goldfish pituitary and ovary as well as rat pituitary. Further purification with HPLC revealed the presence of two compounds with GnRH-like activity. One with identical chromatographic characteristics, amino acid composition, and primary structure to that of the salmon GnRH (sGnRH), and the other a novel compound with GnRH-like activity.

In the present study, both lamprey GnRH-I and -III stimulated steroidogenesis and induced ovulation in adult female sea lampreys during their final reproductive stage. One injection of lamprey GnRH-III at 0.1 or 0.2 microg/g lamprey stimulated plasma estradiol levels in lampreys held at each of three water temperatures, 13 degrees , 17 degrees , and 19 degrees , corresponding to increasing stages of maturation. Four successive injections, 3 to 4 days apart, of lamprey GnRH-III at 0.1 or 0.2 microg/g body weight induced ovulation in 100 or 88% of lampreys, respectively, compared to 21% in controls by Day 31. Lamprey GnRH-III also had a direct stimulatory effect on estradiol production in the sea lamprey gonads in vitro. Lamprey GnRH-III at 100 or 1000 ng/ml stimulated estradiol levels in media incubated with either lamprey ovaries or testes. In contrast to a previous finding in which lamprey GnRH-III was more potent than lamprey GnRH-I in inducing spermiation in adult male sea lampreys (Deragon and Sower, 1994), the results from the present study indicate that lamprey GnRH-I and -III are equally potent in inducing ovulation and stimulating steroidogenesis in female sea lampreys. In addition, GnRH binding sites have been demonstrated for the first time in both the testis and the ovary of the adult sea lamprey using an analog of mammalian GnRH ([D-Lys6] mammalian GnRH) as a labeled ligand. Scatchard analysis suggested the presence of a high affinity binding site in both the testis and the ovary. In summary, lamprey GnRH-III is biologically active in stimulating the pituitary-gonadal axis in adult female sea lampreys. This is the first report demonstrating the presence of a GnRH binding site in the gonads of an Agnathan. The evidence for a direct stimulatory effect of lamprey GnRH in the gonads, the presence of GnRH binding site, and the absence of GnRH in the plasma suggest that, like other vertebrates including rat, rabbit, teleost fish, and human, there may be a GnRH-like factor produced in the gonads of the lamprey and it may act as a paracrine/autocrine modulator of gonadal function. This study further strengthens the paracrine regulatory role of GnRH peptides in the gonads of vertebrates, which appear to be evolutionarily conserved.

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 G2 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 G2 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.

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.

Separase is an endopeptidase that cleaves cohesin subunit Rad21, facilitating the repair of DNA damage during interphase and the resolution of sister chromatid cohesion at anaphase. Separase activity is negatively regulated by securin and Cdk1-cyclin B in vivo. Separase overexpression is reported in a broad range of human tumors, and its overexpression in mouse models results in tumorigenesis. To elucidate further the mechanism of separase function and to test if inhibition of overexpressed separase can be used as a strategy to inhibit tumor-cell proliferation, small-molecule inhibitors of separase enzyme are essential. Here, we report a high-throughput screening for separase inhibitors (Sepins). We developed a fluorogenic separase assay using rhodamine 110-conjugated Rad21 peptide as substrate and screened a small-molecule compound library. We identified a noncompetitive inhibitor of separase called Sepin-1 that inhibits separase enzymatic activity with a half maximal inhibitory concentration (IC50) of 14.8 µM. Sepin-1 can inhibit the growth of human cancer cell lines and breast cancer xenograft tumors in mice by inhibiting cell proliferation and inducing apoptosis. The sensitivity to Sepin-1 in most cases is positively correlated to the level of separase in both cancer cell lines and tumors.

Fluorescence in situ hybridization (FISH) is the most widely used molecular technique to visualize chromosomal abnormalities. Here, we describe a novel 3D modeling approach to allow precise shape estimation and localization of FISH signals in the nucleus of human embryonic stem cells (hES) undergoing progressive but defined aneuploidy. The hES cell line WA09 acquires an extra copy of chromosome 12 in culture with increasing passages. Both diploid and aneuploid nuclei were analyzed to quantitate the differences in the localization of centromeric FISH signals for chromosome 12 as it transitions from euploidy to aneuploidy. We employed superquadric modeling primitives coupled with principal component analysis to determine the 3D position of FISH signals within the nucleus. A novel aspect of our modeling approach is that it allows comparison of FISH signals across multiple cells by normalizing the position of the centromeric signals relative to a reference landmark in oriented nuclei. Using this model we present evidence of changes in the relative positioning of centromeres in trisomy-12 cells when compared with diploid cells from the same population. Our analysis also suggests a significant change in the spatial distribution of at least one of the FISH signals in the aneuploid chromosome complements implicating that an overall change in centromere position may occur in trisomy-12 due to the addition of an extra chromosome. These studies underscore the unique utility of our modeling algorithms in quantifying FISH signals in three dimensions.

Recently we have demonstrated that the human hepatocellular carcinoma-derived cell lines, HepG2 and HuH7, contain gonadotropin-releasing hormone (gonadoliberin) receptors and respond to various molecular forms of gonadoliberin in terms of suppressed proliferation in vitro. This study provides the first demonstration that gonadoliberin inhibits the zinc-induced production of metallothionein mRNA in HepG2 and HuH7 cells. Administration of gonadoliberin agonist (gonadoliberin-A) inhibited the Zn-induced metallothionein mRNA level in a time-related and dose-related manner. The effect of gonadoliberin-A was found to be specific, because concomitant treatment with a gonadoliberin antagonist (gonadoliberin-ANT) blocked gonadoliberin-A inhibition of metallothionein mRNA accumulation. Furthermore, the gonadoliberin-A-induced inhibition of Zn-mediated metallothionein accumulation was found to correlate closely with suppresion of cell proliferation and [3H] thymidine uptake in these cells. It is known that the metal-binding protein metallothionein plays an important role in tumor cell pathobiology and resistance to chemotherapeutic drugs. The present findings may have important implications in the development of an effective chemotherapy for treatment of human liver cancer, in part, by improving the sensitivity of tumor cells through suppression of metallothionein production by gonadoliberin peptides.

The receptor binding and biological activity of epidermal growth factor-urogastrone (EGF) was characterized in the follicle-enclosed goldfish oocyte. The binding of 125I-labeled mouse EGF (mEGF) to goldfish ovarian membrane preparation was peptide specific, saturable, reversible, and dependent on time and tissue concentration. Binding data analysis indicated the presence of a single class of high-affinity binding sites with an estimated equilibrium dissociation constant of 4.4 +/- 1.8 x 10(-10) M. The 125I-mEGF binding was displaced by unlabeled mEGF and by human recombinant transforming growth factor-alpha (hTGF-alpha). Both mEGF and hTGF-alpha were found to stimulate reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown (GVBD). Treatment with mEGF and hTGF-alpha stimulated GVBD from a basal level of 8.5 to approximately 30% with an estimated half-maximal effective dose for EGF of 5.80 +/- 0.82 + 10(-10) and for hTGF-alpha, 1.9 +/- 1.0 x 10(-10) M. Furthermore, treatment with mEGF marginally increased 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP)-induced GVBD without significantly influencing the gonadotropin-induced response. Treatment with either mEGF or hTGF-alpha significantly reduced human chorionic gonadotropin-stimulated testosterone production in a concentration-related manner. These data suggest that members of the EGF family may play a role in the regulation of ovarian function in goldfish.

This study provides the first demonstration that human hepatocarcinoma-derived cell line HepG2 contains GnRH receptors and responds to various molecular forms of GnRH in terms of inhibition of proliferation in a time- and dose-related manner. In addition, GnRH peptides also significantly inhibited the uptake of [3H]thymidine by these cells. The effect of GnRH was specific because GnRH antagonists reversed the GnRH-mediated inhibition, and non-GnRH peptides had no effect on HepG2 cell proliferation. An important finding is that certain fish GnRH molecules such as lamprey GnRH, which has little gonadotropin (LH) release activity in mammals, suppress hepatocarcinoma cell proliferation with similar potency to a superactive mammalian GnRH analog, [D-Lys6]GnRH. These findings may have profound implications in the development of an effective chemotherapy for treatment of human liver cancer. An added advantage is that fish GnRH forms will likely exert little side effect in terms of human pituitary gonadotropin release, gonadal steroidogenesis, and reproductive functions in general.

Hematopoiesis—the process that generates distinct lineage-committed blood cells from a single multipotent hematopoietic stem cell—is a complex process of cellular differentiation regulated by a set of dynamic transcriptional programs. Cytokines and growth factors, transcription factors, chromatin remodeling, and modifying enzymes have been suggested to enact critical roles during hematopoiesis, leading to the development of myeloid, lymphoid, erythroid and platelet precursors. How is such a complex process orchestrated? Is there a higher order of hematopoiesis regulation? These are some of the unresolved questions in the field of hematopoiesis. Here, we suggest that cohesin, which is known to mediate chromosomal cohesion between sister chromatids, may have a central role in the orchestration of hematopoiesis and serve as a master transcriptional regulator.

GnRH binding was characterized in the African catfish ovary by use of an analog of salmon GnRH (sGnRH- ; [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH) as a labeled ligand. Binding of sGnRH-A to catfish ovarian membrane preparation was found to be saturable, displaceable, reversible, and dependent on time, temperature, and tissue concentration. Optimal binding was achieved after 70 min of incubation at room temperature (approximately 22 degrees C) at pH 7.6. Addition of unlabeled sGnRH-A displaced the bound 125I-sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of one class of high-affinity binding sites with a equilibrium dissociation constant (Kd) of 0.27 +/- 0.036 nM. Bound 125I-sGnRH-A was also found to be displaceable by catfish GnRH (cfGnRH; [His5, Leu7, Asn8]-GnRH), chicken GnRH-II (cGnRH-II; [His5, Trp7, Tyr8]-GnRH), and salmon GnRH (sGnRH; [Trp7, Leu8]-GnRH); all the peptides were found to bind with lower affinities than sGnRH-A to the catfish ovarian GnRH binding sites. Further experiments using ovarian extracts indicated the presence of compounds with GnRH-like activity in the ovary of African catfish. The crude ovarian extract was found to stimulate pituitary gonadotropin release from goldfish pituitary, as well as displacing 125I-sGnRH-A binding in the catfish ovary. HPLC analysis of the catfish ovarian extract revealed the presence of two fractions that bind specifically to the catfish ovary and release gonadotropin from cultured goldfish pituitary. These fractions include an early eluting peak that does not correspond with the retention time of known GnRH forms in addition to a fraction that co-elutes with the mammalian GnRH. Overall, the study provided characterization of GnRH binding sites in the catfish ovary, and evidence for the presence of compounds with GnRH-like activity in the catfish ovary.

Defining the mechanisms of chromosomal cohesion and dissolution of the cohesin complex from chromatids is important for understanding the chromosomal missegregation seen in many tumor cells. Here we report the identification of a novel cohesin-resolving protease and describe its role in chromosomal segregation. Sister chromatids are held together by cohesin, a multiprotein ring-like complex comprised of Rad21, Smc1, Smc3, and SA2 (or SA1). Cohesin is known to be removed from vertebrate chromosomes by two distinct mechanisms, namely, the prophase and anaphase pathways. First, PLK1-mediated phosphorylation of SA2 in prophase leads to release of cohesin from chromosome arms, leaving behind centromeric cohesins that continue to hold the sisters together. Then, at the onset of anaphase, activated separase cleaves the centromeric cohesin Rad21, thereby opening the cohesin ring and allowing the sister chromatids to separate. We report here that the calcium-dependent cysteine endopeptidase calpain-1 is a Rad21 peptidase and normally localizes to the interphase nuclei and chromatin. Calpain-1 cleaves Rad21 at L192, in a calcium-dependent manner. We further show that Rad21 cleavage by calpain-1 promotes separation of chromosome arms, which coincides with a calcium-induced partial loss of cohesin at several chromosomal loci. Engineered cleavage of Rad21 at the calpain-cleavable site without activation of calpain-1 can lead to a loss of sister chromatid cohesion. Collectively, our work reveals a novel function of calpain-1 and describes an additional pathway for sister chromatid separation in humans.

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.

Background: Cohesin protease Separase plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Homozygous deletion of ESPL1 gene that encodes Separase protein results in embryonic lethality in mice and Separase overexpression lead to aneuploidy and tumorigenesis. However, the effect of Separase haploinsufficiency has not been thoroughly investigated. Methodology/Principal Findings: Here we examined the effect of ESPL1 heterozygosity using a hypomorphic mouse model that has reduced germline Separase activity. We report that while ESPL1 mutant (ESPL1 +/hyp ) mice have a normal phenotype, in the absence of p53, these mice develop spontaneous T- and B-cell lymphomas, and leukemia with a significantly shortened latency as compared to p53 null mice. The ESPL1 hypomorphic, p53 heterozygous transgenic mice (ESPL1 +/hyp , p53 +/2 ) also show a significantly reduced life span with an altered tumor spectrum of carcinomas and sarcomas compared to p53 +/2 mice alone. Furthermore, ESPL1 +/hyp , p53 2/2 mice display significantly higher levels of genetic instability and aneuploidy in normal cells, as indicated by the abnormal metaphase counts and SKY analysis of primary splenocytes. Conclusions/Significance: Our results indicate that reduced levels of Separase act synergistically with loss of p53 in the initiation and progression of B- and T- cell lymphomas, which is aided by increased chromosomal missegregation and accumulation of genomic instability. ESPL1 +/hyp , p53 2/2 mice provide a new animal model for mechanistic study of aggressive lymphoma and also for preclinical evaluation of new agents for its therapy.

Gonadotropin-releasing hormone (GnRH) binding sites were characterized in the goldfish testis using an analog of salmon GnRH ([D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to goldfish testicular membrane homogenate was found to be saturable, reversible, and dependent on time, temperature, and tissue concentration. Addition of unlabeled sGnRH-A displaced the bound 125 I-sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis indicated the presence of two classes of binding sites, a high affinity/low capacity site and a low affinity/high capacity site in the goldfish testis. Bound 125I-sGnRH-A was also found to be displaceable by sGnRH ([Trp7, Leu8]-GnRH) and cGnRH-II ([His5, Trp7, Tyr8,]-GnRH) which occur naturally in the goldfish brain. sGnRH-A and cGnRH-II were found to bind with greater affinity than sGnRH to the goldfish testicular GnRH binding sites. The findings provide the first characterization of testicular GnRH binding sites in a teleost species. © 1993 Wiley-Liss, Inc.