Gouqing Ge

Osteosarcoma is a primary malignant tumor of bone arising from primitive bone-forming mesenchymal cells and accounts for approximately 60% of malignant bone tumors. Our comparative genomic hybridization (CGH) studies have identified frequent amplification at 6p12-p21, 12q13-q15, and 17p11.2 in osteosarcoma. Of these amplified regions, 6p12-p21 is particularly interesting because of its association with progression and poor prognosis in patients with osteosarcoma. In an attempt to identify aberrantly expressed gene(s) mapping to the 6p12-p21 amplicon, a region-specific array was generated using 108 overlapping BAC and P1 clones covering a 28.8-Mb region at 0.26-Mb intervals. Based on array CGH analysis, the 6p amplicon was refined to 7.9 Mb between the clones RP11-91E11 and RP1-244F2 and 10 amplified clones, with possible target genes, were identified. To study the expression pattern of the target genes from the hotspot amplicon and known candidate genes from 6p12-21, we did quantitative reverse transcription-PCR analysis of MAPK14, MAPK13, CDKN1A, PIM1, MDGA1, BTB9, DNAH8, CCND3, PTK7, CDC5L, and RUNX2 on osteosarcoma patient samples and seven cell lines. The combined array CGH and quantitative reverse transcription-PCR analysis identified amplification and overexpression of CDC5L, CCND3, and RUNX2. We screened these three genes for protein expression by Western blotting and immunohistochemistry and detected overexpression of CDC5L. Furthermore, we used an in vivo assay to show that CDC5L possesses potential oncogenic activity. These results indicate that CDC5L, a cell cycle regulator important for the G2-M transition, is the most likely candidate oncogene for the 6p12-p21 amplicon found in osteosarcoma.

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.

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.

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(+/-)) also show a significantly reduced life span with an altered tumor spectrum of carcinomas and sarcomas compared to p53(+/-) mice alone. Furthermore, ESPL1(+/hyp), p53(-/-) 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(-/-) mice provide a new animal model for mechanistic study of aggressive lymphoma and also for preclinical evaluation of new agents for its therapy.