Metaphase

About: Metaphase is a research topic. Over the lifetime, 6925 publications have been published within this topic receiving 291590 citations. The topic is also known as: GO:0007091 & mitotic metaphase/anaphase transition.

Overexpression and Mislocalization of the Chromosomal Segregation Protein Separase in Multiple Human Cancers

Abstract: 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.

Calcium—cell cycle regulator, differetiator, killer, chemopreventor, and maybe, tumor promoter

Abstract: Ca2+ and Ca(2+)-binding proteins are involved in running the cell cycle. Ca2+ spikes and signals from integrin-activated focal adhesion complexes and Ca2+ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca2+ spike trigger mitotic prophase. Another Ca2+ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca2+ finally stimulates cytoplasmic division (cytokinesis). However, Ca2+ does more than this in epithelial cells, such as those lining the colon, and skin keratinocytes. These cells also need Ca2+, integrin signals, and only a small amount (e.g., 0.05-0.1 mM) of external Ca2+ to start DNA replication. Signals from their surface Ca2+ receptors trigger a combination of differentiation and apoptosis ("diffpoptosis") when external Ca2+ concentration reaches their setpoints. The skin's steep, upwardly directed, Ca2+ gradient has a low concentration in the basal layer to allow stem and precursor keratinocytes to proliferate, and higher concentrations in the suprabasal layers to trigger the differentiation-apoptosis ("diffpoptosis") mechanism that converts granular cells into protective, hard-shelled, dead corneocytes. A similar Ca2+ gradient may exist in the colon crypt allowing the stem cell and its amplifying transit or precursor offspring to cycle in the lower parts of the crypt, while stopping proliferation and stimulating terminal differentiation in the upper crypt and flat mucosa. Raising the amount of Ca2+ in fecal water above a critical level reduces proliferation and thus colorectal carcinogenesis in normal rats and some high-risk humans. But during carcinogenesis the Ca2+ sensors malfunction or their signals become ineffective: high Ca2+ does not stop, and may even stimulate, the proliferation of initiated mutants. Therefore, Ca2+ may either not affect, or even promote, the growth of epithelial cells in carcinogen-initiated rat colon and human adenoma patients. Clearly, a much greater understanding of how Ca2+ controls the proliferation and differentiation of epithelial cells and why initiated cells lose their responsiveness to Ca2+ are needed to assess the drawbacks and advantages of using Ca2+ as a chemopreventor.

High-resolution FISH on super-stretched flow-sorted plant chromosomes.

Abstract: A novel high-resolution fluorescence in situ hybridisation (FISH) strategy, using super-stretched flow-sorted plant chromosomes as targets, is described. The technique that allows longitudinal extension of chromosomes of more than 100 times their original metaphase size is especially attractive for plant species with large chromosomes, whose pachytene chromosomes are generally too long and heterochromatin patterns too complex for FISH analysis. The protocol involves flow cytometric sorting of metaphase chromosomes, mild proteinase-K digestion of air-dried chromosomes on microscopic slides, followed by stretching with ethanol:acetic acid (3 : 1). Stretching ratios were assessed in a number of FISH experiments with super-stretched chromosomes from barley, wheat, rye and chickpea, hybridised with 45S and 5S ribosomal DNAs and the [GAA](n) microsatellite, the [TTTAGGG](n) telomeric repeat and a bacterial artificial chromosome (BAC) clone as probes. FISH signals on stretched chromosomes were brighter than those on the untreated control, resulting from better accessibility of the stretched chromatin and maximum observed sensitivity of 1 kbp. Spatial resolution of neighbouring loci was improved down to 70 kbp as compared to 5-10 Mbp after FISH on mitotic chromosomes, revealing details of adjacent DNA sequences hitherto not obtained with any other method. Stretched chromosomes are advantageous over extended DNA fibres from interphase nuclei as targets for FISH studies because they still retain chromosomal integrity. Although the method is confined to species for which chromosome flow sorting has been developed, it provides a unique system for controlling stretching degree of mitotic chromosomes and high-resolution bar-code FISH.

Highly effective cell synchronization in plant roots by hydroxyurea and amiprophos-methyl or colchicine.

Abstract: Effective somatic cell synchronization in root-tip meristems and improved chromosome spreading were achieved in white campion, wheat, rye, and barley by application of hydroxyurea and amiprophos-methyl or colchicine, combined with a pretreatment of ice water and modified fixative, as well as enzymatic digestion of the meristems. The protocol provides metaphase indices of approximately 50%. The chromosomes and chromosomal DNA were with minimum distortion, providing useful material for chromosome banding studies, in situ DNA-DNA hybridization, microdissection, and microcloning.