Identification of new hypoxia-regulated epithelial-mesenchymal transition marker genes labeled by H3K4 acetylation.

Hypoxia-induced epithelial-mesenchymal transition (EMT) involves the interplay between chromatin modifiers histone deacetylase 3 (HDAC3) and WDR5. The histone mark histone 3 lysine 4 acetylation (H3K4Ac) is observed in the promoter regions of various EMT marker genes (eg, CDH1 and VIM). To further define the genome-wide location of H3K4Ac, a chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) analysis was performed using a head and neck squamous cell carcinoma (HNSCC) FaDu cell line under normoxia and hypoxia. H3K4Ac was found to be located mainly around the transcription start site. Coupled with analysis of gene expression by RNA sequencing and using a HDAC3 knockdown cell line, 10 new genes (BMI1, GLI1, SMO, FOXF1, SIRT2, etc) that were labeled by H3K4Ac and regulated by HDAC3 were identified. Overexpression or knockdown of GLI1/SMO increased or repressed the in vitro migration and invasion activity in OECM-1/FaDu cells, respectively. In HNSCC patients, coexpression of GLI1 and SMO in primary tumors correlated with metastasis. Our results identify new EMT marker genes that may play a significant role in hypoxia-induced EMT and metastasis and further provide diagnostic and prognostic implications.

HSP60 overexpression increases the protein levels of the p110α subunit of phosphoinositide 3-kinase and c-Myc.

Heat shock protein 60 (HSP60) is a chaperone protein which plays an essential role in facilitating the folding of many newly synthesized proteins to reach their native forms. Increased HSP60 expression is observed in various types of human cancers. However, proteins induced by HSP60 to mediate transformation remain largely unknown. Here we show that HSP60 overexpression increases the protein levels of the p110α subunit of phosphoinositide 3-kinase (PI3K). The amino acid domain 288-383 of HSP60 is used to increase the protein levels. Overexpression of HSP60 also induces the levels of phosphorylated Akt. In addition, the amino acid domain 288-383 of HSP60 is used to induce c-Myc expression. Finally, a mono-ubiquitinated form of ß-catenin has a higher activity to activate ß-catenin downstream targets compared to wild-type ß-catenin. These results indicate that HSP60 overexpression induces the levels or activity of multiple oncogenic proteins to mediate transformation.

Positive therapy of andrographolide in vocal fold leukoplakia.

PURPOSE: Vocal fold leukoplakia is a premalignant precursor of squamous cell carcinoma. Although many efforts have been contributed to therapy of this disease, none exhibits a satisfactory result. The aims of this study were to investigate the effectiveness and feasibility of andrographolide therapy in vocal fold leukoplakia and to explore the preliminary mechanism underlying. MATERIALS AND METHODS: Forty-one eligible patients were enrolled in the study. The patients were treated for 10-minute exposures of 5 ml (25mg/ml) andrographolide injection aerosols twice a day, and 2 weeks was considered as one treatment course. Electronic laryngoscope was used to observe the condition of vocal fold leukoplakia during the treatment. Every patient received one or two treatment courses, and the follow-up was carried out for 12 months. Toxic reactions of treatments were evaluated on the basis of the standards of the United States MD Anderson Cancer Center. Moreover, laryngeal carcinoma cell line Hep2 was applied to explore the mechanism of effect of andrographolide. Anti-proliferative effect on Hep2, cell nuclear morphology, express of mitogen-activated protein kinases (MAPK) and pro-apoptotic protein were detected after andrographolide treatment. RESULTS: We found that andrographolide exhibited significant curative effects on treatments, which were accompanied by thinning of the lesion of leukoplakia, reduction in the whitish surface area, and return of pink or red epithelium. A complete response up to 85% was observed, and no toxic side effect events occurred during the study. No patient with a complete response had a recurrence in the follow-up. Moreover, cellular experiments in Hep2 indicated that andrographolide activated MAPK pathway and caspase cascade, and finally induced apoptosis in laryngeal carcinoma cell. CONCLUSIONS: The advantages of andrographolide are connected with minimally invasive and localized character of the treatment and no damage of collagenous tissue structures, which are more convenient and less painful for patients. These results suggest that andrographolide treatment is a viable strategy for curing vocal fold leukoplakia.

Analysis of Epigenetic Regulation of Hypoxia-Induced Epithelial-Mesenchymal Transition in Cancer Cells by Quantitative Chromatin Immunoprecipitation of Histone Deacetylase 3 (HDAC3).

Epigenetics plays a key role in gene expression control. Histone modifications including acetylation/deacetylation or methylation/demethylation are major epigenetic mechanisms known to regulate epithelial-mesenchymal transition (EMT)-associated gene expression during hypoxia-induced cancer metastasis. Chromatin immunoprecipitation (ChIP) assay is a powerful tool for investigation of histone modification patterns of genes of interest. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the epigenetic regulation of EMT marker genes by deacetylation of acetylated Histone 3 Lys 4 (H3K4Ac) under hypoxia in a head and neck cancer cell line FaDu cells. Not only a method of ChIP coupled by real-time quantitative PCR but also the detailed conditions are provided based on our previously published studies.

Epigenetic regulation of epithelial-mesenchymal transition by hypoxia in cancer: targets and therapy.

Intratumoral hypoxia followed by stabilization/activation of hypoxia-inducible factor 1 (HIF-1) and its downstream transcriptional factors, is one of the most important mechanisms inducing epithelial-mesenchymal transition (EMT), which has been widely accepted as a crucial step to generate early stage of tumor metastasis. Accumulating evidence suggests that epigenetic mechanisms play important roles in hypoxia-induced EMT and metastasis. These epigenetic regulations are mediated by various players including chromatin modifiers, transcriptional co-regulators, microRNAs, etc. In this review, we discuss how his tone-modifying enzymes and transcriptional co-regulators regulate EMT under hypoxic conditions. Developed or potential anticancer agents targeting epigenetic molecules regulating hypoxia-induced EMT are also discussed.

Interaction between NBS1 and the mTOR/Rictor/SIN1 complex through specific domains.

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome. The NBS gene product, NBS1 (p95 or nibrin), is a part of the Mre11-Rad50-NBS1 complex. SIN1 is a component of the mTOR/Rictor/SIN1 complex mediating the activation of Akt. Here we show that NBS1 interacted with mTOR, Rictor, and SIN1. The specific domains of mTOR, Rictor, or SIN1 interacted with the internal domain (a.a. 221-402) of NBS1. Sucrose density gradient showed that NBS1 was located in the same fractions as the mTOR/Rictor/SIN1 complex. Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets. Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity. These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.

Quantitative detection of Streptococcus mutans and bacteria of dental caries and no caries groups in permanent teeth from a north China population.

BACKGROUND: Streptococcus mutans (S. mutans) is the prime pathogen of dental caries. There are few reports that studied the relationship between S. mutans, bacteria and dental caries in permanent teeth when compared to those in primary teeth. This study aimed to detect S. mutans and bacteria of dental caries and non-caries groups in permanent teeth from a north China population by real-time polymerase chain reaction (PCR) and compare the relationship between the number of these bacteria and the prevalence of dental caries in permanent teeth. METHODS: Human saliva samples were collected from 142 subjects with permanent teeth. According to their dental tooth (DT), 142 subjects were divided into a dental caries group (DT ≥ 1) and a non-caries group (DT = 0). With specific primers for S. mutans and 16S rRNA, the total number of S. mutans and total bacteria of 142 saliva samples were detected by real-time PCR and statistically analyzed. RESULTS: There was no significant difference between the detection rates of S. mutans (P = 0.118) and medians of S. mutans (P = 0.115). The ratio of S. mutans to total bacteria in people with dental caries was significantly higher than in those without caries (P < 0.001), but the total number of bacteria in people with dental caries was significantly lower than in those without caries (P < 0.001). CONCLUSIONS: S. mutans had different effects on caries in the permanent teeth of several individuals from a north China population. The ratios of S. mutans to total bacteria in saliva detected by real-time PCR with Sm479F/R and 16S RNA primers were closely associated with the prevalence of dental caries in the same population. These assays may be useful for the assessment of an individual's risk of dental caries.

Molecular regulation of telomerase activity in aging.

The process of aging is mitigated by the maintenance and repair of chromosome ends (telomeres), resulting in extended lifespan. This review examines the molecular mechanisms underlying the actions and regulation of the enzyme telomerase reverse transcriptase (TERT), which functions as the primary mechanism of telomere maintenance and regulates cellular life expectancy. Underpinning increased cell proliferation, telomerase is also a key factor in facilitating cancer cell immortalization. The review focuses on aspects of hormonal regulations of telomerase, and the intracellular pathways that converge to regulate telomerase activity with an emphasis on molecular interactions at protein and gene levels. In addition, the basic structure and function of two key telomerase enzyme components-the catalytic subunit TERT and the template RNA (TERC) are discussed briefly.

[Quantitative detection of Streptococcus mutans in different people with dental caries by real-time polymerase chain reaction].

OBJECTIVE: To establish a quantity detection method of Streptococcus mutans (Sm) and bacteria and compare the relationship between the number of these bacteria and the prevalence of dental caries in different people. METHODS: With specific primers for a unique sequence in a 14 kb HaeIII restriction fragment consistently presenting during detecting Sm by chromosomal DNA fingerprints, the total number of Sm and bacteria of 99 saliva samples were detected by real-time polymerase chain reaction (PCR) and statistically analyzed. RESULTS: The primers were specific for Sm and the minimum detectable level by real-time PCR was 0.1 microg/L. The total number of bacteria in the dental caries and people without caries was 51.4 x 10(8) cell copies/L and 221.6 x 10(8) cell copies/L respectively, in which the ratio of Sm to bacteria was 0.0193 and 0.0059 respectively. The differences were significantly different between the people with dental caries and those without caries in the total number of bacteria and the ratio of Sm to bacteria. CONCLUSIONS: The primers can be used to detect the Sm by real-time PCR. The ratio of Sm to bacteria was closely associated with the prevalence of dental caries.

[Effect of carbon and nitrogen sources on the accumulation of poly-beta-hydroxybutyrate by purple non-sulfur photosynthetic bacteria].

The effect of carbon and nitrogen sources on the accumulation of Poly-beta-Hydroxybutyrate (PHB) by purple non-sulfur photosynthetic bacteria (PNSB)was investigated. The results showed that ammonia nitrogen and organic nitrogen could benefit the PNSB accumulating PHB; under certain ratio of carbon and nitrogen low carbon concentration can accumulate more PHB, but high carbon concentration is better for PNSB accumulating PHB with the growth of PNSB. As to different substrates, butyrate is the best for accumulating of PHB, but acetate is better and propionate is the poorest. Mixed substrate with butyrate as the main content are better than single substrate; carbon dioxide have certain effect on the PNSB accumulating PHB, and high carbon dioxide concentration can improve PHB content when using acetate or butyrate as substrate.