ATM modulates transcription in response to histone deacetylase inhibition as part of its DNA damage response

Chromatin structure has a crucial role in a diversity of physiological processes, including development, differentiation and stress responses, via regulation of transcription, DNA replication and DNA damage repair. Histone deacetylase (HDAC) inhibitors regulate chromatin structure and activate the DNA damage checkpoint pathway involving Ataxia-telangiectasia mutated (ATM). Herein, we investigated the impact of histone acetylation/deacetylation modification on the ATM-mediated transcriptional modulation to provide a better understanding of the transcriptional function of ATM. The prototype HDAC inhibitor trichostain A (TSA) reprograms expression of the myeloid cell leukemia-1 (MCL1) and Gadd45alpha genes via the ATM-mediated signal pathway. Transcription of MCL1 and Gadd45alpha is enhanced following TSA treatment in ATM+ cells, but not in isogenic ATM- or kinase-dead ATM expressing cells, in the ATM-activated E2F1 or BRCA1-dependent manner, respectively. These findings suggest that ATM and its kinase activity are essential for the TSA-induced regulation of gene expression. In summary, ATM controls the transcriptional upregulation of MCL1 and Gadd45alpha through the activation of the ATM-mediated signal pathway in response to HDAC inhibition. These findings are important in helping to design combinatory treatment schedules for anticancer radio- or chemo-therapy with HDAC inhibitors.

Co-treatment with hepatocyte growth factor and TGF-beta1 enhances migration of HaCaT cells through NADPH oxidase-dependent ROS generation

Wound healing requires re-epithelialization from the wound margin through keratinocyte proliferation and migration, and some growth factors are known to influence this process. In the present study, we found that the co-treatment with hapatocyte growth factor (HGF) and TGF-beta1 resulted in enhanced migration of HaCaT cells compared with either growth factor alone, and that N-acetylcysteine, an antioxidant agent, was the most effective among several inhibitors tested, suggesting the involvement of reactive oxygen species (ROS). Fluorescence-activated cell sorter analysis using 2',7'-dichlorofluorescein diacetate (DCF-DA) dye showed an early (30 min) as well as a late (24 h) increase of ROS after scratch, and the increase was more prominent with the growth factor treatment. Diphenyliodonium (DPI), a potent inhibitor of NADPH oxidase, abolished the increase of ROS at 30 min, followed by the inhibition of migration, but not the late time event. More precisely, gene knockdown by shRNA for either Nox-1 or Nox-4 isozyme of gp91phox subunit of NADPH oxidase abolished both the early time ROS production and migration. However, HaCaT cell migration was not enhanced by treatment with H2O2. Collectively, co-treatment with HGF and TGF-beta1 enhances keratinocyte migration, accompanied with ROS generation through NADPH oxidase, involving Nox-1 and Nox-4 isozymes.

Presence of autocrine hepatocyte growth factor-Met signaling and its role in proliferation and migration of SNU-484 gastric cancer cell line

Autocrine stimulation via coexpression of hepatocyte growth factor (HGF) and its receptor (Met) has been reported in many human sarcomas, but few in carcinomas. In this report, we found that one gastric cancer cell line, SNU-484, among 11 gastric cell lines tested has an autocrine HGF- Met stimulation. RT-PCR, ELISA and scattering assay using MDCK cells revealed that SNU-484 cells secreted a significant amount of active HGF (about 1.25 +/- 0.41 ng/24 h/106 cells) into conditioned medium. Resultantly, Met in this cell line was constitutively phosphorylated. Neutralizing antibodies against HGF reduced the tyrosine phosphorylation of Met, resulting in the inhibition of cell proliferation and migration (P <0.005). To the best of our knowledge, this is the first report on autocrine HGF-Met signaling in a gastric cancer cell line. Our observations with SNU-484 cells suggest that HGF is involved in the development and/or progression of some gastric carcinoma through an autocrine mechanism.

Centrosome amplification and multinuclear phenotypes are Induced by hydrogen peroxide

Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (> or =3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.

Dimethylnitrosamine-Induced Liver Cirrhosis and Expression of Hepatocyte Growth Factor (HGF), its Receptor c-Met, and the Transforming Growth Factor (TGF)-beta1 in Sprague-Dawley Rats

BACKGROUND: Liver fibrosis and cirrhosis are the ultimate histologic consequences of chronic liver damage. Efforts have been made to study the mechanisms of cirrhosis and to discover effective therapeutic strategies. However, to date, no animal model reproduces the disease in man. The purpose of this work is to establish a model of DMN-induced liver cirrhosis for treatment of liver cirrhosis, to understand the basic characteristics of DMN-induced liver cirrhosis, and to confirm the expression of HGF, its receptor c-Met, and TGF-beta1 in Sprague-Dawley rats. METHODS: Five-week-old male Sprague-Dawley rats (n=56) were used for this study. Liver cirrhosis was induced in the rats by using DMN (1 ml/kg body weight, i.p.) given 3 consecutive days a week for 6 weeks. Changes in the portal vein pressure were measured by a venous catheter during the duration of the DMN-treatment. The levels of serum albumin, bilirubin, and ammonia were determined in a clinical laboratory by routive methods. Pieces of the median lobe were cut and fixed in 10% buffered neutral formalin, embedded in paraffin, and stained by hematoxylin-eosin (H&E) & masson-trichrome (M&T). Changes in the extracellular matrix were measured by image analysis and hydroxyproline content. Immunohistochemical staining of alpa-smooth muscle actin was performed to confirm the activation ofhepatic stellate cells. Northern blot analyses were performed to confirm the expression of HGF and TGF-beta1 and western blotting was performed c-Met, HGF receptor. RESULTS: Pressures in the portal vein were significantly increased during the DMN-treatment time (p<0.05). Biochemical parameters were significantly correlated with the progression of liver cirrhosis. H&E staining of 4-week DMN-treated rats demonstrated fibrous tissue bridging between the periportal and the pericentral areas with gradual widening of fibrous bands. Both the extracellular matrix measured by image analysis of the M&T staining and the hydroxyproline content rose continuously throughout the 6 weeks of DMN treatment. alpa-smooth muscle actin was observed in the stellate cells of DMN-treated rats. The northern blot analyses showed that the expression of HGF mRNA decreased with the progression of DMN-induced liver cirrhosis but that of TGF-beta1 mRNA did not. The western blot analyses showed that the expression of the c-Met receptor protein increased continuously, but the expression of HGF mRNA a decreased. CONCLUSION:The model of cirrhosis induced by chronic, discontinuous treatment with a low dose of DMN in rats was simple and predictable and displayed many of the features of human cirrhosis. The decrease in the expression of HGF mRNA may be responsible for the reduced hepatocyte regeneration in liver cirrhosis. The expression of the c-Met protein was related with the decreased expression of HGF. The exact significance of TGF-beta1 was not determined in this study.