Genome-wide DNA methylation patterns are frequently deregulated in cancer. There is considerable interest in targeting the methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5-aza-2-deoxycytidine (5-azadC). 5-azadC exerts its antitumor effects by reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. We sought to better characterize the DNA damage response of tumor cells to 5-azadC and the role of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively) in modulating this process. We demonstrate that 5-azadC treatment results in growth inhibition and G 2 arrest-hallmarks of a DNA damage response. 5-azadC treatment led to formation of DNA double-strand breaks, as monitored by formation of ␥-H2AX foci and comet assay, in an ATM (ataxiatelangiectasia mutated)-dependent manner, and this damage was repaired following drug removal. Further analysis revealed activation of key strand break repair proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 by Western blotting and immunofluorescence. Significantly, DNMT1-deficient cells demonstrated profound defects in these responses, including complete lack of ␥-H2AX induction and blunted p53 and CHK1 activation, while DNMT3B-deficient cells generally showed mild defects. We identified a novel interaction between DNMT1 and checkpoint kinase CHK1 and showed that the defective damage response in DNMT1-deficient cells is at least in part due to altered CHK1 subcellular localization. This study therefore greatly enhances our understanding of the mechanisms underlying 5-azadC cytotoxicity and reveals novel functions for DNMT1 as a component of the cellular response to DNA damage, which may help optimize patient responses to this agent in the future.DNA methylation is an essential epigenetic modification required for normal mammalian development, gene regulation, genomic imprinting, and chromatin structure (9). Methylation occurs at the C-5 position of cytosine within the CpG dinucleotide and is carried out by a family of DNA methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B (32). DNMT1 acts primarily as a maintenance methyltransferase by copying existing methylation patterns following DNA replication (53). DNMT3A and DNMT3B exhibit de novo methyltransferase activity and are required for establishing new methylation patterns during embryonic development (72). Deregulation of the DNA methylation machinery has been identified in several disease states, particularly cancer (78), and leads to global DNA hypomethylation of repetitive DNA, which has been linked to genomic instability (21) and, concomitantly, hypermethylation at the promoter regions of certain genes (tumor suppressors), leading to their aberrant silencing (43).The process of cytosine methylation is reversible and may be altered by biochemical and biological manipulation, making it an attractive target for therapeutic intervention. Demethylation and consequent reactivation of tumor ...

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9-and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G 2 /M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3␤. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G 2 /M arrest. HCT116 human colon cancer-derived Chk2 ؊/؊ cells were significantly more resistant to G 2 /M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G 2 /M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.Epidemiological studies have revealed an inverse correlation between the dietary intake of cruciferous vegetables and the risk for certain types of cancers, including prostate cancer (1-5). Laboratory studies indicate that the anticancer effect of cruciferous vegetables is caused by isothiocyanates that exist as thioglucoside conjugates (glucosinolates) in a variety of edible plants including broccoli, cabbage, watercress, and so forth (6 -9). Cruciferous vegetable-derived organic isothiocyanates are generated by hydrolytic cleavage of corresponding glucosinolates through catalytic mediation of myrosinases, which are released when the plant cells are damaged because of cutting or chewing (6 -9). Sulforaphane (SFN) 1 is one such isothiocyanate analog that has received a great deal of attention not only because it is present in high concentrations in certain varieties of broccoli but also because of its potent anticancer activity (10 -15). For example, oral administration of SFN (1-isothiocyanato-4-(methylsulfinyl)butane; CH 3 -SO-(CH 2 ) 4 -NϭCϭS)) caused a statistically significant reduction in 9,10-dimethyl-1,2-benzanthr...

Abstract. Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-I. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence microscopy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle--dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re-initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.

SUMMARY Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD+) exhibits axon-protective effects in cultured neurons, however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD+ levels in mice by administering nicotinamide riboside (NR), a recently described NAD+ precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD+-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD+ biosynthetic enzymes. These findings reveal that administration of NR activates a NAD+-SIRT3 pathway that reduces neurite degeneration caused by noise exposure.