Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Comparison of Biochemical Substances in the Fiber Development of Different Cotton Varieties

Zhongmian 42 and Xinluzao 36 were used as raw materials to determine the contents of soluble sugar and protein, as well as dynamic changes of enzyme activities after flowering during cotton fiber growth. The results showed that contents of soluble protein in the two species sharply declined 7 to 21 days after flowering, as the soluble sugar in Zhongmian 42 leveling off after 21 days of flowering while the soluble sugar in Xinluzao 36 dropped notably after 21 days of flowering before remaining stable after seven days later. The soluble sugar decreased 7 to 14 days after flowering before sharply rising to the maximum seven days later, and then began to decline quickly. The soluble sugar was the minimum after 35 days of flowering and then remaining stable. Peroxidase activity generally increased. Indole-3- acetic acid oxidase activities were low at 7 days after flowering. IAAO activity reached to the peaks on the 14th and 28th day after flowering. IAAO activity of two varieties decreased with the same trend 35 days after flowering.

Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor

The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFbB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber,Apostichopus japonicus(Selenka),during aestivation

The sea cucumber, Apostichopusjaponicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling （DNA （cytosine-5）-methyltransferase l, Methyl-CpG-binding domain protein 2）, and Chromodomain-helicase-DNA-binding protein 5） was significantly higher during aestivation （Days 20 and 40）. Similarly, we observed an increase in the expression of genes involved in histone acetylation （Histone deacetylase 3） and Histone-binding protein RBBP4） during the early （Days 5 and 10） and late phases （Days 20 and 40） of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers （Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5） was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in A. japonicus.

Targeting histone deacetylases： perspectives for epigenetic-based therapy in cardio-cerebrovascular disease

Although the pathogenesis of cardio-cerebrovascular disease （CCVD） is multifactorial, an increasing number of experimental and clinical studies have highlighted the importance of histone deacetylase （HDAC）-mediated epigenetic processes in the development of cardio-cerebrovascular injury. HDACs are a family of enzymes to balance the acetylation activities of histone acetyltransferases on chromatin remodeling and play essential roles in regulating gene transcription. To date, 18 mammalian HDACs are identified and grouped into four classes based on similarity to yeast orthologs. The zinc-dependent HDAC family currently consists of 11 members divided into three classes （class I, II, and IV） on the basis of structure, sequence homology, and domain organization. In comparison, class III HDACs （also known as the sirtuins） are composed of a family of NAD＋-dependent protein-modifying enzymes related to the Sir2 gene. HDAC inhibitors are a group of compounds that block HDAC activities typically by binding to the zinc-containing catalytic domain of HDACs and have displayed an- ti-inflammatory and antifibrotic effects in the cardio-cerebrovascular system. In this review, we summarize the current knowledge about classifications, functions of HDACs and their roles and regulatory mechanisms in the cardio-cerebrovascular system. Pharmacological tar- geting of HDAC-mediated epigenetic processes may open new therapeutic avenues for the treatment of CCVD.

Structural insights into selective histone H3 recognition by the human Polybromo bromodomain 2

The Polybromo （PB） protein functions as a key component of the human PBAF chromatin remodeling complex in regulation of gene transcription. PB is made up of modular domains including six bromodomains that are known as acetyl-lysine binding domains. However, histone-binding specificity of the bromodomains of PB has remained elusive. In this study, we report biochemical characterization of all six PB bromodomains＇ binding to a suite of lysine-acetylated peptides derived from known acetylation sites on human core histones. We demonstrate that bromodomain 2 of PB preferentially recognizes acetylated lysine 14 of histone H3 （H3K14ac）, a post-translational mark known for gene transcriptional activation. We further describe the molecular basis of the selective H3K14ac recognition of bromodomain 2 by solving the protein structures in both the free and bound forms using X-ray crystallography and NMR, respectively.

Nuclear effects of ethanol-induced proteasome inhibition in liver cells

Alcohol ingestion causes alteration in several cellular mechanisms, and leads to inflammation, apoptosis, immunological response defects, and fibrosis. These phenomena are associated with significant changes in the epigenetic mechanisms, and subsequently, to liver cell memory. The ubiquitin-proteasome pathway is one of the vital pathways in the cell that becomes dysfunctionial as a result of chronic ethanol consumption. Inhibition of the proteasome activity in the nucleus causes changes in the turnover of transcriptional factors, histone modifying enzymes, and therefore, affects epigenetic mechanisms. Alcohol consumption has been associated with an increase in histone acetylation and a decrease in histone methylation, which leads to gene expression changes. DNA and histone modifications that result from ethanol-induced proteasome inhibition are key players in regulating gene expression, especially genes involved in the cell cycle, immunological responses, and metabolism of ethanol. The present review highlights the consequences of ethanol-induced proteasome inhibition in the nucleus of liver cells that are chronically exposed to ethanol.

Gastric cancer cell lines induced by trichostatin A

AIM： To explore the effect of trichostatin A （TSA） on apoptosis and acetylated histone H3 levels in gastric cancer cell lines BGC-823 and SGC-7901. METHODS： The effect of TSA on growth inhibition and apoptosis was examined by MTT, fluorescence microscopy and PI single-labeled flow cytometry. The acetylated histone H3 level was detected by Western blot. RESULTS： TSA induced apoptosis in gastric cancer cell lines BGC-823 and SGC-7901 was in a dose and time-dependent manner. Apoptotic cells varied significantly between TSA treated groups （37.5 ng/mL 72 h for BGC-823 cell line and 75 ng/mL 72 h for SGC-7901 cell line） and control group （0.85 ± 0.14 vs 1.14 ± 0.07, P = 0.02; 0.94 ± 0.07 vs 1.15 ± 0.06, P = 0.02）. Morphologic changes of apoptosis, including nuclear chromatin condensation and fluorescence strength, were observed under fluorescence microscopy. TSA treatment in BGC-823 and SGC-7901 cell lines obviously induced cell apoptosis, which was demonstrated by the increased percentage of sub-G1 phase cells, the reduction of Gl-phase cells and the increase of apoptosis rates in flow cytometric analysis. The result of Western blot showed that the expression of acetylated histone H3 increased in BGC-823 and SGC-7901 TSA treatment groups as compared with the control group.CONCLUSION： TSA can induce cell apoptosis in BGC-823 and SGC-7901 cell lines. The expression of acetylated histone H3 might be correlated with apoptosis.

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

Expression and purification of the complete PreS region of hepatitis B Virus

AIM: To express the complete PreS region of HBV in E.coli with good solubility and stability, and to establish an effective method for purification of the recombinant PreS protein. METHODS: The complete PreS region (PreS1 and PreS2) was fused into a series of tags including glutathione S-transferase (GST), dihydrofolate reductase (DHFR), maltose binding protein (MBP), 6× histidine, chitin binding domain (CBD), and thioredoxin, respectively. Expression of recombinant PreS fusion proteins was examined by SDS-PAGE analysis and confirmed by Western blot. Two fusion proteins, thio-PreS, and PreS-CBD, with desirable solubility and stability, were subjected to affinity purification and further characterization. RESULTS: Recombinant PreS fusion proteins could be synthesized with good yields in E.coli. However, most of these proteins except for thio-PreS and PreS-CBD were vulnerable to degradation or insoluble as revealed by SDS-PAGE and Western blot. Thio-PreS could be purified by affinity chromatography with nickel-chelating sepharose as the matrix. However, some impurities were also co-purified. A simple freeze-thaw treatment yielded most of the thio-PreS proteins in solution while the impurities were in the precipitate. Purified thio-PreS protein was capable of inhibiting the binding of HBV virion to a specific monoclonal antibody against an epitope within the PreS1 domain. CONCLUSION: Increased solubility and stability of the complete PreS region synthesized in E.coli can be achieved by fusion with the thioredoxin or the CBD tag. A simple yet highly effective method has been established for the purification of the thio-PreS protein. Purified thio-PreS protein likely assumes a native conformation, which makes it an ideal candidate for studying the structure of the PreS region as well as for screening antivirals.

From Bench to Bedside： Targeting Epigenetics for Cancer Therapy

The initiation and progression of cancer not only involves genetic abnormalities, but also epigenetic alterations, such as DNA methylation and histone modifications. Epigenetics refers to the heritable changes that do not involve any structural changes in the target gene, i.e., DNA sequence and protein sequence. Thus, these epigenetic aberrations are potentially reversible, allowing the malignant cells to revert to a state with more normal characteristics. The use of epigenetics is emerging as an effective and promising approach to treat cancer. Epigenetic drugs, which target two well- known epigenetic pathways, namely, DNA methyltransferases and histone deacetylases, are already being applied for the cancer treatment. In the current study, an overview regarding the under-standing of epigenetic alterations in the development of cancer and the current state of epigenetic drug discovery is provided.

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.

Globulin-platelet model predicts minimal fibrosis and cirrhosis in chronic hepatitis B virus infected patients

AIM： To establish a simple model consisting of the rou- tine laboratory variables to predict both minimal fibrosis and cirrhosis in chronic hepatitis B virus （HBV）-infected patients. METHODS： We retrospectively investigated 114 chron- ic HBV-infected patients who underwent liver biopsy in two different hospitals. Thirteen parameters were analyzed by step-wise regression analysis and correla- tion analysis. A new fibrosis index [globulin/platelet （GP） model] was developed, including globulin （GLOB） and platelet count （PLT）. GP model = GLOB （g/mL） x 100/PLT （x 109/L）. We evaluated the receiver operating characteristics analysis used to predict minimal fibrosis and compared six other available models. RESULTS： Thirteen clinical biochemical and hemato- logical variables [sex, age, PLT, alanine aminotransfer- ase, aspartate aminotransferase （AST）, albumin, GLOB, total bilirubin （T.bil）, direct bilirubin （D.bil）, glutamyl-transferase, alkaline phosphatase, HBV DNA and pro- thrombin time （PT）] were analyzed according to three stages of liver fibrosis （F0-F1, F2-F3 and F4）. Bivariate Spearman＇s rank correlation analysis showed that six variables, including age, PLT, T.bil, D.bil, GLOB and PT, were correlated with the three fibrosis stages （FS）. Cor- relation coefficients were 0.23, -0.412, 0.208, 0.220, 0.314 and 0.212; and P value was 0.014, 〈 0.001, 0.026, 0.018, 0.001 and 0.024, respectively. Univariate analysis revealed that only PLT and GLOB were signifi- cantly different in the three FS （PLT： F = 11.772, P 〈 0.001; GLOB： F = 6.612, P = 0.002）. Step-wise multiple regression analysis showed that PLT and GLOB were also independently correlated with FS （R2 = 0.237）. By Spearman＇s rank correlation analysis, GP model was significantly correlated with the three FS （r = 0.466, P 〈 0.001）. The median values in F0-F1, F2-F3 and F4 were 1.461, 1.720 and 2.634. Compared with the six available models （fibrosis index, AST-platelet ratio, FIB-4, fibrosis-cirrhosis index and age-AST model and age-PLT ratio）, GP model showed a highest correlation coefficient. The sensitivity and positive predictive value at a cutoff value 〈 1.68 for predicting minimal fibrosis F0-F1 were 72.4% and 71.2%, respectively. The speci- ficity and negative predictive value at a cutoff value 〈 2.53 for the prediction of cirrhosis were 84.5% and 96.7%. The area under the curve （AUC） of GP model for predicting minimal fibrosis and cirrhosis was 0.762 [95% confidence interval （CI）： 0.676-0.848] and 0.781 （95% CI： 0.638-0.924）. Although the differences were not statistically significant between GP model and the other models （P all 〉 0.05）, the AUC of GP model was the largest among the seven models. CONCLUSION： By establishing a simple model using available laboratory variables, chronic HBV-infected patients with minimal fibrosis and cirrhosis can be di- agnosed accurately, and the clinical application of this model may reduce the need for liver biopsy in HBV- infected patients.

The proliferation inhibition of Hela cells by histone deacetylases- 1 siRNA

Objective： To investigate the anti-proliferative effect of histone deacetylases-1 （HDAC-1） knockdown in Hela cells. Methods： The HDAC-1 protein was knockdowned using siRNA. The expression of HDAC-1 was detected by Western blotting. Apoptosis was assessed by flow cytometry. The inhibition of cell growth was assesses by MTT assay. Results： HDAC-1 siRNA knockdowned the expression of HDAC-1 protein. HDAC siRNA inhibited the proliferation of Hela cells. HDAC- 1 siRNA induced apoptosis. Conclusion： HDAC-1 siRNA may inhibit the growth of Hela cells by inducing apoptosis.

Expression of p21^(WAF1) is related to acetylation of histone H3 in total chromatin in human coiorectai cancer

AIM： To explore the relationship between acetylation of histone in total chromatin and p21^WAF1 expression regulation in human colorectal carcinoma. METHODS： We analyzed the expression of tumor suppressor gene p21^WAF1 mRNA by RT-PCR or realtime PCR in 33 samples of colorectal cancerous tissue, corresponding para-cancerous tissue and normal colorectal mucosa, and also examined the level of acetylated histone H3 in total chromatin using Western blotting. RESULTS： The expression level of p21^WAF1 mRNA was significantly lower in colorectal cancerous tissue from 33 patients than in para-cancerous tissue and normal colorectal mucosa （2377.95 ± 865.80 vs 3216.58 ± 1149.42 and 3541.61 ± 1433.17 respectively, P 〈 0.01）. In addition, when p21^WAF1 mRNA expression was undectectable or at very low level （50% less than that in adjacent tissue and normal colorectal mucosa） in all tissues, the level of acetylated histone H3 in colorectal cancerous tissue was significantly lower than that in corresponding para-cancerous tissue and normal colorectal mucosa in five of seven （71.43%） cases. The transcriptional level of p21^WAF1 in colorectal carcinoma might not be associated with its biological behaviors.CONCLUSION： The down-regulation of p21^WAF1 transcription is involved in the tumorigenesis and development of colorectal carcinoma. The down-expression of p21^WAF1 mRNA in colorectal carcinoma might be associated with histone hypoacetylation in chromatin but not with biological behaviors.

Progress in clinical trial of histone deacetylase(HDAC) inhibitors for non-small cell lung cancers

Histone deacetylase(HDAC) inhibitors, which represent a structurally diverse group of molecules, have emerged as a novel therapeutic class of molecules with significant anticancer potential. Vorinostat and romidepsin, known as the first generation of HDAC inhibitors, were approved in the United States for the treatment of T-cell lymphomas. Preliminary activity of HDAC inhibitors has also been observed in non-small cell lung cancer(NSCLC) in combination with the existing treatment regimens, of which is the focus of the current review.

Increased exchange rate of histone H1 on chromatin by exogenous myo-genin expression

To explore the molecular mechanism of chromatin remodeling involved in the regulation of transcriptional activation of specific genes by a myogenic regulatory factor Myogenin, we used NIH3T3 fibroblasts with a stably integrated Hl.l-GFP fusion protein to monitor histone HI movement directly by fluorescence recovery after photobleaching (FRAP) in living cells. The observation from FRAP experiments with myogenin transfected fibroblasts showed that the exchange rate of histone HI in chromatin was obviously increased, indicating that forced expression of exogenous Myogenin can induce chromatin remodeling. The hyper-acetylation of histones H3 and H4 from myogenin transfected fibroblasts was detected by triton-acid-urea (TAU)/SDS (2-D) electrophoresis and Western blot with specific antibodies against acetylated N-termini of histones H3 and H4. RT-PCR analysis indicated that the nAChR β-subunit gene was expressed in the transfected fibroblasts. These results suggest that the expression of exogenous Myogenin can induce chromatin remodeling and activate the transcription of Myogenin-targeted gene in non-muscle cells.

Expression Grade Sirtuin-1 （SIRT-1） in Tumor Tissue in Women with Breast Cancer： A New Biomarker Prognosis？

Breast cancer is a complex and heterogeneous disease with different clinical outcomes. The investigations of new biomolecular markers are essential to know the prognosis and improve the clinical management of patients. The SIRT-1 （sirtuin- 1） is a histone deacetylase implicated in various epigenetic critical functions for the cells and the maintenance of genomic stability. The objective of this study is to investigate the grade of expression of the SIRT-I （sirtuin-1） and the prognostic value in overall survival of women with breast cancer. Retrospective cohort of 457 women with breast cancer has been researched, undergoing treatment in Erechim-RS from 2003 to 2013 and followed until July 2015. The degree of SIRT-1 expression was investigated by immunohistochemistry in 123 patients （26.9%） of the cohort. The OS （overall survival） from specific disease and risk of death from breast cancer were estimated by Kaplan-Meier and Cox＇s proportional risks. The median age of 57.4 years cohort with OS of 79.6% in 5 years and 69.1% at 10 years, with follow-up time of 61.9 months are revealed in this work. The SIRT-1 overexpression was found in 6.5% of cases and characterized a subgroup of women with shorter survival and increased risk of death from breast cancer （HR = 2.66; 95% CI 1.03 to 6.86; p = 0.043） and adjusted by age （HR = 2.86; 95% CI 1.11 to 7.38; p = 0.030）, histology （HR = 2.79; 95% CI 1.07 to 7.28; p = 0.036）, lymph nodes （HR = 2.73; 95% CI 1.06 to 7.04; p = 0.037）, Her-2 （HR = 2.82; 95% CI 1.07 to 7.44; p = 0.036）; chemotherapy （HR = 2.90; 95% CI 1.11 to 7.60; p = 0.030） and radiotherapy （HR = 2.71; 95% CI 1.05 to 7.01; p = 0.040）. In regressive multivariate models adjusted for age, status of axillary lymph nodes, Her-2 expression and proliferation index （Ki-67）, the grade of expression of the SIRT-1 maintained association with poor prognosis. From the study, it can be concluded that the assessment of the SIRT-1 expression is an independent prognostic biomarker in breast cancer.