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.

By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3␤ activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3␤ and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/ Akt signaling axis by phosphorylation and stabilization of PTEN.Plk3, a member of the Polo-like kinase family, plays an important role in regulating cell cycle checkpoint control in response to genotoxic stresses (1, 2), as well as in cellular responses to hypoxia (3, 4). Plk3 is also strongly implicated in tumorigenesis. Aberrant expression of Plk3 is found in multiple tumors (5, 6). The human PLK3 gene is localized to the short arm of the chromosome 1 (1p32), a region that displays loss of heterozygosity or homozygous deletions in many types of cancer and has been proposed to harbor tumor susceptibility genes (7,8). A recent mouse genetic study showed that PLK3 Ϫ/Ϫ mice develop tumors in multiple organs at an enhanced rate (4). Many of the tumors developed in the PLK3 null mice are large in size and are highly vascularized (4), suggesting that this kinase may be involved in regulating the angiogenesis pathway.The PTEN 3 tumor suppressor is frequently mutated in cancer cells, and inherited PTEN mutations cause cancer-susceptibility conditions, including Cowden syndrome (9 -13). The PTEN level, as well as its activity, profoundly influences tumor susceptibility because haplo-insufficiency of PTEN results in tumor development in many organs in animal models (14, 15). Biochemically, PTEN dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate to generate phosphatidylinositol 3,4-bisphosphate and, by doing so, antagonizes the PI3K/Akt signaling pathway. Therefore, the PTEN tumor suppressor is a central negative regulator of the PI3K/PDK1/Akt signaling axis that controls multiple cellular functions, including cell growth, survival, proliferation, and angiogenesis (16). PTEN is also involved in regulating hypoxic responses and HIF-1␣ stability (17, 18). Loss of PTEN function and increased activities of PI3K/Akt are associated ...

Genomic instability is a characteristic of most cancer cells. It is an increased tendency of genome alteration during cell division. Cancer frequently results from damage to multiple genes controlling cell division and tumor suppressors. It is known that genomic integrity is closely monitored by several surveillance mechanisms, DNA damage checkpoint, DNA repair machinery and mitotic checkpoint. A defect in the regulation of any of these mechanisms often results in genomic instability, which predisposes the cell to malignant transformation. Posttranslational modifications of the histone tails are closely associated with regulation of the cell cycle as well as chromatin structure. Nevertheless, DNA methylation status is also related to genomic integrity. We attempt to summarize recent developments in this field and discuss the debate of driving force of tumor initiation and progression.

Chromosomal instability during cell division frequently causes cell death or malignant transformation. Orderly chromosome congression at the metaphase plate, a paramount process to vertebrate mitosis and meiosis, is controlled by a number of molecular regulators, including kinesins. Kinesin-8 (Kif18A) functions to control mitotic chromosome alignment at the mid-zone by negative regulation of kinetochore oscillation. Here the authors report that disrupting Kif18a function results in complete sterility in male but not in female mice. Histological examination reveals that Kif18a−/− testes exhibit severe developmental impairment of seminiferous tubules. Testis atrophy in Kif18a−/− mice is caused by perturbation of microtubule dynamics and spindle pole integrity, leading to chromosome congression defects during mitosis and meiosis. Depletion of KIF18A via RNAi causes mitotic arrest accompanied by unaligned chromosomes and increased microtubule nucleating centers in both GC-1 and HeLa cells. Prolonged depletion of KIF18A causes apoptosis due to perturbed microtubule dynamics. Further studies reveal that KIF18A silencing results in degradation of CENP-E and BubR1, which is accompanied by premature sister chromatid separation. KIF18A physically interacts with BubR1 and CENP-E, and this interaction is modulated during mitosis. Combined, the studies indicate that KIF18A is essential for normal chromosome congression during cell division and that the absence of KIF18A function causes severe defects in microtubule dynamics, spindle integrity, and checkpoint activation, leading to germinal cell aplasia in mice.

KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.