The JmjC-domain-containing histone demethylases (JHDMs) can remove histone lysine-methylation and thereby regulate gene expression. The JmjC-domain uses iron Fe (II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl-lysine. We hypothesize that reactive oxygen species will oxidize Fe (II) to Fe (III), thereby attenuating the activity of JmjC-domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3 hours) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H2O2) for 3 hours, exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; pre-incubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′, 7′-dichlorofluorescein (DCF), GSH/GSSG ratio and protein carbonyl content. A cell free system indicated H2O2 inhibited histone demethylase activity where increased Fe (II) rescued this inhibition. TET protein also showed a decreased activity under oxidative stress. Cells exposed to a low dose and long term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3 ؊/؊ murine embryonic fibroblasts contained an enhanced level of HIF-1␣ under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1␣ under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1␣ in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer halflife than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1␣ stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.

Since its discovery several years ago, Shugoshin 1 (SGO1) has emerged as a crucial regulator of the cell cycle. [1][2][3][4][5][6] At cellular and molecular levels, SGO1 functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. [5][6][7] Depletion of SGO1 by small interfering RNA (siRNA) leads to premature sister chromatid separation. [5][6][7][8] During mitosis, SGO1 localizes to centromeres in a manner that appears to be dependent on Bub1, Aurora B and survivin.7-13 SGO1 works in concert with protein phosphatase 2A (PP2A) to protect centromeric cohesion during mitosis and meiosis. 14,15 It is implicated in microtubule dynamics and required for tension generation at the kinetochore. 2,6 In addition to the function of SGO1 in centromeres, sSGO1, a major splice variant of SGO1, has an important function in centrosome dynamics through mediating centriole cohesion. 16 A recent study supports the centrosomal function of Sgo1 in further detail. 17Importantly, both cohesin and Sgo1 are shown to be involved in engagement of centrioles and thus in centrosomal integrity. 17Given the importance of centromeric cohesion and centrosome Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1 +/-mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AoM), a colon carcinogen. together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation. Key words: SGO1, mouse genetics, chromosomal instability, centrosome, colon cancer Abbreviations: SGO1, shugoshin 1; CIN, chromosome instability; AOM, azoxymethane; siRNA, small interfering RNA; MEFs, mouse embryonic fibroblasts; FACS, fluorescence activated cell sorter; ACF, aberrant crypt foci; WT, wild type; COX2, cyclooxygenase-2 dynamics in the maintenance of chromosomal stability during cell division, it is conceivable that deregulated function of SGO1 would lead to major chromosomal instability. Chromosomal instability has long been appreciated as a drivi...

Aneuploidy is defined as numerical abnormalities of chromosomes and is frequently (>90%) present in solid tumors. In general, tumor cells become increasingly aneuploid with tumor progression. It has been proposed that enhanced genomic instability at least contributes significantly to, if not requires, tumor progression. Two major modes for genomic instability are microsatellite instability (MIN) and chromosome instability (CIN). MIN is associated with DNA-level defects (e.g. mismatch repair defects), and CIN is associated with mitotic errors such as chromosome mis-segregation. The mitotic spindle assembly checkpoint (SAC) ensures that cells with defective mitotic spindles or defective interaction between the spindles and kinetochores do not initiate chromosomal segregation during mitosis. Thus, the SAC functions to protect the cell from chromosome mis-segregation and anueploidy during cell division. A loss of the SAC function results in gross aneuploidy, a condition from which cells with an advantage for proliferation will be selected. During the past several years, a flurry of genetic studies in mice and humans strongly support the notion that an impaired SAC causes enhanced genomic instabilities and tumor development. This review article summarizes the roles of key spindle checkpoint proteins {i.e. Mad1/Mad1L1, Mad2/Mad2L1, BubR1/Bub1B, Bub3/Bub3 [conventional protein name (yeast or human)/mouse protein name]} and the modulators (i.e. Chfr/Chfr, Rae1/Rae1, Nup98/Nup98, Cenp-E/CenpE, Apc/Apc) in genomic stability and suppression of tumor development, with a focus on information from genetically engineered mouse model systems. Further elucidation of molecular mechanisms of the SAC signaling has the potential for identifying new targets for rational anticancer drug design.