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Immune cell infiltration in the tumor microenvironment is associated with cancer prognosis. Tumor-associated macrophages (TAMs) play an essential role in tumor initiation, progression, and metastasis. Follistatin-like protein 1 (Fstl1), a glycoprotein widely expressed in human and mouse tissues, is a tumor suppressor in various cancers and a regulator in macrophage polarization. However, how Fstl1 affects the crosstalk between breast cancer cells and macrophages remains unclear. Analyzing the public data, we found that Fstl1 expression significantly decreases in breast cancer tissues compared with normal breast tissues, and high expression of Fstl1 in patients means prolonged survival. Using flow cytometry, we found that total macrophages and M2-like macrophages increased dramatically in the metastatic lung tissues during breast cancer lung metastasis of Fstl1+/- mice. Cellular experiments in vitro showed that Fstl1 inhibited macrophage migration towards 4T1 cells via decreasing colony stimulating factor 1 (CSF-1), transforming growth factor-β1 (TGFB1) and vascular endothelial growth factor-α (VEGF-α) expression of 4T1 cells. We demonstrated that Fstl1 inhibited M2-like tumor-associated macrophages recruitment towards the lung and suppressed TGFB1/Smad2/3 pathway and relevant cytokines expression of 4T1 cells. Thereby we find a potential therapeutic strategy contributing to breast cancer.

This paper deals with the investigation of the corrosion situation of oil wells on site. On the basis of water quality analysis, the corrosion status of the site was studied on J55 steel used in the field. The most severe water sample was selected as the medium for electrochemical evaluation and on-site. The screening of four corrosion inhibitors was conducted in our study. Results showed that the salinity of the produced water is relatively high (average value of 29819 mg/L), the chloride ion content is high (average value of 17762.8 mg/L), and the corrosion rate is up to 0.1345 mm/a. Scanning electron microscopy (SEM) was used to analyze the corrosion products and morphology of the studied samples. In the electrochemical evaluation test, the TS-001 corrosion inhibitor has a good corrosion inhibition rate, which can reach up to 97.75% when the dosage is 100 mg/L.

Flame-retardant cycloaliphatic epoxy systems have long been studied; however, the research suffers from slow and unsatisfactory advances. In this work, we synthesized a kind of phosphorus-containing difunctional cycloaliphatic epoxide (called BCEP). Then, triglycidyl isocyanurate (TGIC) was mixed with BCEP to achieve epoxy systems that are rich in phosphorus and nitrogen elements, which were cured with 4-methylhexahydrobenzene anhydride (MeHHPA) to obtain a series of flame-retardant epoxy resins. Curing behaviors, flame retardancy, thermal behaviors, dielectric performance, and the chemical degradation behaviors of the cured epoxy system were investigated. BCEP-TGIC systems showed a high curing activity, and they can be efficiently cured, in which the incorporation of TGIC decreased the curing activity of the resin. As the ratio of BCEP and TGIC was 1:3, the cured resin (BCEP1-TGIC3) showed a relatively good flame retardancy with a limiting oxygen index value of 25.2%. In the cone calorimeter test, they presented a longer time to ignition and a lower heat release than the commercially available cycloaliphatic epoxy resins (ERL-4221). BCEP-TGIC systems presented good thermal stability, as the addition of TGIC delayed the thermal weight loss of the resin. BCEP1-TGIC3 had high dielectric performance and outperformed ERL-4221 over a frequency range of 1 HZ to 1 MHz. BCEP1-TGIC3 could achieve degradation under mild conditions in an alkali methanol/water solution. Benefiting from the advances, BCEP-TGIC systems have potential applications as electronic packaging materials in electrical and electronic fields.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies due to its high frequency of metastasis via the epithelial-mesenchymal transition (EMT) pathway. Hepatic stimulator substance (HSS) can protect hepatocytes from injury and promote liver growth. Recent studies indicated that HSS expression is increased in HCC tissues; however, whether HSS expression is potentially associated with HCC metastasis, particularly through the EMT pathway, remains largely unknown. In this study, the relationship between HSS expression and HCC metastasis was investigated in clinical samples of HCC. Meanwhile, the regulation of HCC metastasis and EMT progression by HSS were also analyzed in both in vitro and in vivo models. The results showed that the expression of 23 kDa HSS was significantly decreased among HCC tissues with angioinvasion. A decrease in HSS predicted poor prognosis with a lower survival rate. Furthermore, the growth of xenograft tumors after inoculating MHCC97H-HSS-shRNA (HCC) cells into nude mice was notably accelerated compared to those inoculated with HSS-expressing cells. Further analysis revealed that knockdown of HSS expression in both MHCC97H and HepG2 cells could enhance the migration of these HCC cells. Concurrently, interference of HSS expression by shRNA promoted conversion of morphologically epithelial-like HCC cells into mesenchymal-like cells, together with downregulations of epithelial markers (such as E-cadherin and zonula occludens-1) and upregulation of mesenchymal-like makers (such as α-SMA, β-catenin, and fibronectin). Furthermore, it was demonstrated that, as well as promoting EMT, HSS-shRNA induced the phosphorylation of extracellular signal-regulated kinase (ERK) and elevated the expression of the EMT-related transcription factor Snail. Specific inhibition of HSS-shRNA-induced ERK phosphorylation by PD98059 attenuated HCC cell migration in a dose-dependent manner. In conclusion, we demonstrated that downregulation of HSS expression contributes to HCC metastasis partially through the ERK-activated EMT pathway.

Hepatocellular carcinoma (HCC) is highly chemoresistant and therefore challenges both physicians and patients. Augmenter of liver regeneration (ALR), previously also known as 'hepatic stimulator substance', is reported to inhibit the epithelial-mesenchymal transition (EMT) in HCC, one of the frequent events that occur in cancer metastasis, suggesting that ALR is involved in HCC. In this study, we report for the first time that the transfection of ALR enhances the antitumor effect of chemotherapy with doxorubicin, a typical anticancer drug, on HCC in vitro and in vivo. The efflux of doxorubicin from ALR-transfected HCC cells is efficiently suppressed. This implies the intracellular retention of doxorubicin in tumor cells, which is at least partly attributable to the effective inhibition of ABCB1 and ABCG2 transporter expression in ALR-expressing cells. The downregulation of ALR expression by short hairpin RNA diminishes the antitumor effect of ALR. We further demonstrate that ALR inhibits the AKT/Snail signaling pathway, resulting in the downregulation of ABCB1 and ABCG2 expression. In conclusion, our results suggest that ALR is a potential chemotherapeutic agent against HCC.

// Wei Huang 1, * , Juan Song 2, * , Xiao-Wei Jia 3, * , Yin-Xue Chen 1 , Jia Shi 1 , Xun Jiang 3 1 Department of Stomatology, Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Zhuhai 519099, China 2 Department of Clinical Laboratory, Taihe Hospital, Hubei University of Medicine, Shiyan 442000, China 3 Department of Stomatology, Guangzhou Hospital of Integrated Traditional and West Medicine, Guangzhou 510800, China * Co-first authors Correspondence to: Wei Huang, email: hwdentist@163.com Keywords: interleukin-10, polymorphism, head and neck neoplasms, mouth neoplasms, oropharyngeal neoplasms Received: February 13, 2017 Accepted: March 21, 2017 Published: March 29, 2017 ABSTRACT Background: The association of interleukin-10 rs1800896 polymorphism with head and neck cancer risk and its clinical stages has been investigated by many published studies, but the results remain inconsistent. Therefore, we conducted this meta-analysis for further investigation. Results: Six case-control studies involving 1,781 head and neck cancer patients and 1,978 controls were yielded. The results indicated an association between rs1800896 polymorphism and increased head and neck risk [odds ratio (95%confidence interval) for G vs. A, GA vs. AA, GG vs. AA, GA+GG vs. AA, and GG vs. AA + GA were 1.63 (1.30–2.04), 3.17 (2.11–4.76), 1.63 (1.17–2.26), 1.73 (1.25–2.39), and 2.73 (1.82–4.09), respectively]. The subgroup analyses all obtained similar results with overall populations. The results of clinical stages yielded a non-significant association. No publication bias was detected. Materials and Methods: The PubMed and Chinese National Knowledge Infrastructure databases were searched up to December 27, 2016. Two authors independently selected studies, extracted and analyzed the data using the RevMan 5 software. Either a fixed effect or a random effect model was used to estimate pooled odds ratio and its 95% confidence intervals. Conclusions: We concluded that interleukin-10 rs1800896 polymorphism was significantly associated with head and neck cancer risk but not with the clinical stages thereof.

// Wei Huang 1, * , Bing-Yang He 2, * , Jun Shao 3, * , Xiao-Wei Jia 3 and Ya-Di Yuan 3 1 Department of Stomatology, Zhuhai People’s Hospital, Zhuhai Hospital Affiliated With Jinan University, Zhuhai 519099, Guangdong Province, China 2 Department of Stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China 3 Department of Stomatology, Guangzhou Hospital of Integrated Traditional and West Medicine, Guangzhou 510800, Guangdong Province, China * Wei Huang, Bing-Yang He and Jun Shao are the co-first authors Correspondence to: Wei Huang, email: hwdentist@163.com Keywords: interleukin-1, periodontitis, periodontal disease, meta-analysis, polymorphism Received: December 12, 2016 Accepted: January 17, 2017 Published: February 22, 2017 ABSTRACT Association between interleukin-1 beta ( IL-1β ) rs1143627 polymorphism and periodontal disease susceptibility was inconsistent; hence we performed this meta-analysis to explore the precise correlation between them. The degree of association was appraised through calculating pooled odds ratio (OR) and its 95% confidence interval (CI). The databases known as PubMed, Embase, and Chinese National Knowledge Infrastructure were searched up to October 26, 2016. A total of 8 eligible case-control studies were finally included, which involved 229 aggressive periodontitis patients, 382 chronic periodontitis patients, and 555 healthy controls. All the five genetic models revealed a non-significant association between IL-1β rs1143627 polymorphism and periodontal disease susceptibility (TT vs. CC: OR = 1.22, 95% CI = 0.80-1.87; CT+TT vs. CC: OR = 0.66, 95% CI = 0.44-1.01; TT vs. CT + CC: OR = 1.19, 95% CI = 0.81-1.74; T vs. C: OR = 0.92, 95% CI = 0.81-1.12; CT vs. CC: OR = 0.92, 95% CI = 0.69-1.23). Sensitivity analyses indicated that the results were robust and the subgroup analyses reached similar conclusions. IL-1β rs1143627 polymorphism is not related to periodontal disease susceptibility in the overall population based on the current evidence, but further studies are required in more large scale sample size with risk factor adjusted.

Background. Previous studies have revealed that gene polymorphisms of inflammatory factors may influence the development or progression of periodontitis, a main cause of tooth loss in adults; however, due to limitations of individual studies, inconsistent findings were reported.Objective. To meta-analytically investigate the relationship between periodontitis and the Interleukin-4 (IL-4) and Interleukin-4 receptor (IL-4R) gene polymorphisms.Methods. Databases were searched for relevant case-control studies. After study selection based on the predefined selection criteria, methodological quality assessment and data extraction were conducted independently by two reviewers, before subsequent statistical analyses.Results. 37 studies involving 4,385 patients and 5,168 controls were included. All the studied IL-4 polymorphisms were not significantly associated with periodontitis, except the -33C/T (CT versus CC: OR = 0.50, 95% CI = 0.28–0.88) associated with reduced AgP susceptibility. Positive association was found between IL-4R Q551 polymorphism and periodontitis susceptibility in three genetic models (R versus Q: OR = 1.59, 95% CI = 1.14–2.22; QR versus QQ: OR = 1.84, 95% CI = 1.21–2.80; RR + QR versus QQ: OR = 1.82, 95% CI = 1.22–2.72).Conclusions. A positive association exists between the IL-4R Q551R polymorphism and occurrence of CP. The IL-4 -33 CT genotype is negatively associated with the occurrence of AgP.

Objective: To investigate the influence of rosuvastatin on the blood lipids, inflammatory factors, oxidative stress and matrix metalloproteinases (MMPs) in patients with chronic heart failure (CHF).Methods: Totally 114 patients with CHF admitted to Yanan Hospital of TCM were collected from December, 2014 to December 2015, and randomly assigned into observation group (n=57) and control group (n=57). The patients in control group were given conventional treatment for CHF, while those in observation group orally took Rosuvastatin Calcium, 20 mg per time, qd.. The course of treatment was 3 months. The changes of cardiac function parameters, blood lipids-related indexes, inflammatory factors, oxidative stress and matrix metalloproteinases (MMPs) were all observed in two groups before and after treatment.Results: After treatment, left ventricular ejection fraction (LVEF) and level of high-density lipoprotein cholesterol (HDL-C) increased notably, while left ventricular end systolic diameter (LVESD), left ventricular end-diastolic dimension (LVEDD) and levels of triglyceride (TG), total cholesterol (TC) as well as low-density lipoprotein cholesterol (LDL-C) decreased obviously in two groups, which changed more significantly in observation group than those in control group (P0.05). The levels of MDA, MMP-2 and MMP-9 were lower, while the level of SOD was evidently higher in observation group than those in control group, and the differences were shown statistically significant (P

Objectives: TP53 is an important tumor suppressor gene to maintain genomic integrity, and its mutations increase the susceptibility to oral carcinoma. Previous published studies have reported the relation of TP53 codon 72 polymorphism with the risk of oral carcinoma, but the results remain controversial and inconclusive. Methods: We therefore utilized meta-analysis based on a comprehensive search in PubMed, EMBASE, and Google of Scholar databases up to August 19, 2017. Results: Total 3,525 cases and 3,712 controls from 21 case-control studies were selected. We found no significant association between TP53 codon 72 polymorphism and oral carcinoma susceptibility in all genetic contrast models, including subgroup analysis based on control source and ethnicity. Furthermore, TP53 codon 72 polymorphism was not significant associated with oral carcinoma susceptibility in tobacco or alcohol use, and HPV infection status. Our results were confirmed by sensitivity analysis and no publication bias was found. Conclusions: Taken together, our data indicate that TP53 codon 72 polymorphism is not associated with the susceptibility to oral carcinoma.

Quality problem or issue Chinese medical institutions need clinical guidelines to improve healthcare quality. Unfamiliarity with clinical methodology and procedures leads to poor quality. Initial assessment This study examined 327 clinical guidelines made in China during the period of 2006–10 and found these clinical guidelines have many problems in terms of guideline making procedures-compliant process, conflicts of interest disclosure. Choice of solution Chinese Medical Association organized a working group in 2014 to make a national [Guideline for Clinical Guidelines Constitution/Amendment] and invited multidiscipline experts to prove its possibility. Implementation Experts investigated and reviewed numerous domestic and foreign published literature within the past 2 years, concluded that a clinical guideline should have following seven components: I. Objective; II. General Principle; III. Procedure and Methodology; IV. Confirmation, Publication and Dissemination; V. Update and Amendment; VI. Implementation and Outcome Validation; VII. Reference. Evaluation The [Guideline for Clinical Guidelines Constitution/Amendment] will improve the quality of Chinese clinical guidelines and regulate applications, as well as outcome evaluations of clinical guidelines in China. Lessons learned Standardized methodology and procedures are important for constituting high-quality clinical guidelines.

To avoid the defects of physical detection methods, such as road spoiling, complex algorithms and equipment maintenance, SVM (Support Vector Machine) and PHOW (Pyramid Histogram of Words) are explored to detect the traffic and road condition. Experimental results show that the average accuracies go beyond 82.5% in the case of different weather. The proposed method can effectively classify 4 types of traffic and road condition images.

In order to avoid the drawbacks of physical detection methods, such as spoiling the road, having complex algorithms and affected by weather factors, the detection methods of traffic and road condition are explored using the Adaboost algorithm and its three variants based on PHOG (pyramid histogram of edge orientation gradients) image feature. Experimental results show that this method can effectively classify 4 types of traffic and road condition images, and Gentle Adaboost algorithm has the best performance for the noisy samples.

Through analyzing and comparing existing wireless communication protocols, this paper selects Bluetooth protocol suitable for CNC manufacturing and constructs communication system base on Bluetooth protocol.this system can solve some shortcomings of the wired network process, and make CNC machining faster, more flexible, more efficient.

The Flame pyrolysis method used to synthesize carbon nanotubes was studied in this work. In order to improve the quality of synthesized carbon nanotubes, it is important to change the corresponding natures of the catalyst. Two catalyst preparation methods, namely, the sol-gel method and the impregnation method, were compared in this experiment. The properties of the catalyst are analyzed in depth by energy dispersive spectrometer (EDS), x-ray diffraction (XRD), temperature program reduction (TPR). The generation of carbon nanotubes was systematically analysed through scanning electron microscope (SEM), molecule dynamics (MD), raman spectroscopy and transmission electron microscope (TEM). The results show that the catalysts prepared by the impregnation method are stickier, dispersed and easier to dip onto the probe or substrate, which is beneficial for the large-scale production of carbon tubes. The specific surface area of alumina is larger and the iron and molybdenum oxide are more evenly dispersed on the surface of alumina. The carbon nanotubes produced by the catalysts prepared by impregnation method are flatter and have less impurities. The ratio of ID/IG+ is 29.7% lower than that of the sol-gel method in the Raman spectra. The TEM statistics show that the average diameter of the carbon tubes decreases by 23.3%. Therefore, the impregnation method can improve the quality of carbon nanotubes in the case of a similar degree of difficulty in the preparation of the catalyst.

Population fluctuation is a risk factor for TB in Beijing., To determine the role of the migrant population in the transmission of tuberculosis (TB), we investigated the distribution and magnitude of TB in permanent residents and migrant populations of Beijing, People’s Republic of China, from 2000 through 2006. An exploratory spatial data analysis was applied to detect the “hot spots” of TB among the 2 populations. Results, using the data obtained from 2004–2006, showed that people who migrated from the western, middle, and eastern zones of China had a significantly higher risk of having TB than did permanent residents. These findings indicate that population fluctuations have affected the rate of TB prevalence in Beijing, and interventions to control TB should include the migrant population.

Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.kod-ssb may in future be used to enhance DNA and cDNA amplification.

To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica.HELFs were divided into 4 groups: control group, curcumin (20 µmol/L for 1 h) group, silica (200 µg/ml for 24 h) group and curcumin plus silica group, i.e. after exposure to 20 µmol/L curcumin for 1h, the HELFs were treated with 200 µg/ml silica for 24 h. Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1, CDK4 and E2F1/4. Flow cytometry was used to detect the cell cycle progression, the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes.The expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously, but the expression level of E2F-1 did not significantly change in silica group. The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group, as compared with control group (P0.05). When suppressing the expression of cyclin D1 or CDK4, the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change, as compared with control group. When suppressing AP-1 activity, the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group, as compared with silica group.The results of present suggested that 200 µg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4, resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.

To investigate the roles of mitogen-activated protein kinases (MAPK) on silica-induced cell cycle changes.After cells were treated with 200 microg/ml silica, Western blot and Immunofluorescence assays were utilized to detect the expression of cyclin D1, CDK4 and E2F-4, Flow cytometry was used to detect cell cycle progression, the dominant negative mutants techniques were used to investigate silica-induced signal pathway and the effects of which in silica-induced cell cycle changes.After cells were exposed to 200 microg/ml silica 24 h, the results of present study showed the proportion of cells in G1 phases was decreased. Silica-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutants of ERK or JNK, but not p38. It was found that ERK and JNK were involved in silica-induced cyclin D1 and CDK4 overexpression and the decreased expression of E2F-4.ERK and JNK, but not p38, mediated silica-induced cell cycle changes in human embryo lung fibroblasts.

To study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF).Ku80 siRNA expression vectors were transfected into HELF by lipofectamine. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression level of Ku80, p53 and p21 proteins or the phosphorylation levels of p53-ser15 after cells were exposed to silica.The expression levels of Ku80 protein increased in concentration-dependent and time-dependent manners after cells were exposed to silica. The proportion of G1 phases in H-NC cells (controls) decreased from 89.28% +/- 2.19% to 68.93% +/- 3.79% after exposure to silica, and the proportion of G1 phases in HELF cells (H-Ku80) decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% after exposure to silica (P0.05). The expression levels of Ku80, p53 proteins or p21 proteins or phosphorylation level of p53-ser15 were obviously suppressed in H-Ku80, as compared with H-NC.Ku80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.

To study the roles of DNA dependent protein kinase (DNA-PK)in silica-induced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).The expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 microg/ml silica for 0, 3, 6, 12, 24 h.The proportion of G1 phases in negative control cells decreased from 83.53% +/- 2.24% to 69.11% +/- 3.12% after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% and from 75.06% +/- 2.23% to 58.32% +/- 1.35%, respectively (P0.05). The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.DNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.

Jia and colleagues describe how a combination of increased domestic funding, supplemented by foreign loans and donations since 2002, have led to a dramatic increase in tuberculosis case finding in China.

To study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF).Control HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h.After treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%.Silica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.

To study the role of p53 in silica-induced cell cycle alternation and DNA double strand breaks repair in human embryo lung fibroblasts (HELF).Neutral comet assay was applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair competence was calculated. The expression levels and phosphorylation of protein in HELF were determined by Western blot. Cell cycle changes were identified by flow cytometry in HELF.After treatment with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h), the expression levels and phosphorylation of p53 increased in a time-dependent manner, reaching maximum at 12 h and then decreasing at 24 h. After treatment with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h, the expression levels and phosphorylation of p53 increased in concentration-dependent manner. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased (DRC = 87.68%), compared with the negative control cell induced by silica (DRC = 57.19%). Silica increased the percentage of S phase (31.8 +/- 1.1)% compared with the controls (24.3 +/- 3.8)% (P0.05). When p53 expression was inhibited, the number of S phase cells was significantly increased, (41.4 +/- 0.6)% compared with the controls (25.4 +/- 1.9)% (P0.05).The silica dramatically increases the expression levels and phosphorylation of p53. The increased expression of p53 mediates silica-induced cell cycle change and inhibits silica-induced DNA double strand breaks repair.

To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF).Two stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay.After treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P0.05).Silica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.

To investigate the roles of p53 in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo(a) pyrene[ B(a) P], and relationships between p53 and p21, E2F-1.Cells transfected with p53 siRNA plasmid (p53-H) and CMV vector (HELF/CMV) were cultured with serum-free R/MINI-1640 for 48 hours, then treated with 2 micromol/L B(a)P for 24 hours. Flow cytometry assay was used for detecting the cell cycle alteration after being exposed to B(a)P. Changes of p53 and p21 expressions were checked using Western blot assay, and the cytoplasmic and nuclear extraction was used to observe the subcellular localizations of p53 and p21. The immunofluorescence assay was used to check changes of E2F-1 expression and the distribution of E2F-1 in nuclear and cytoplasm after exposed to B (a)P. p53siRNA plasmid and the chemical inhibitor of p53 [pifithrin-alpha (PFT)] were used to observe effects of p53 in B(a)P induced cell cycle changes and the relationships of p53 and p21, E2F-1.After 2 micromol/L B(a)P exposure, the ratio of G1 phase cells (71 +/- 5)% was decreased to (39 +/- 4)% (P0.05). p53, p21 and E2F-1 expressions were increased significantly, and over expressed proteins were mostly located in nuclear after B(a)P treatment. When p53 expression was inhibited by p53 siRNA or PFT, the decreases of G1 phase in response to B(a)P treatment still existed, and over expression of p21 induced by B(a)P was attenuated, especially in nuclear, but E2F-1 over expression was not changed significantly.B(a)P could induce cell cycle changes through p53 independent pathways. And p53 could regulate p21 expression positively, but not E2F-1.

To investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.Cells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.After B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.AP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.

To investigate the alteration of activator protein-1 (AP-1) luciferase activity in human embryo lung fibroblasts (HELF) after exposed to silica, and the role of mitogen activated protein kinase (MAPK)/AP-1 pathway on silica-induced cell cycle changes.After HELF cells were treated with 200 microg/ml silica, immunofluorescence assays were employed to detect the translocation and the phosphorylation level of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK), flow cytometry was used to detect the distributions of cell cycle, the dominant negative mutant of ERK, JNK and p38 were applied to detect the upstream or downstream relationship of signaling pathways.After HELF-AP-1 cells were exposed to 200 microg/ml silica 6, 12, 24 h respectively, silica exposure lead to AP-1 activation in a time-dependent manner, inducing significant AP-1 activation at 6 h, reaching a maximum activation at 12 h, and having a little decrease at 24 h. After silica exposure 1 h, phosphorylation level of ERK and JNK increased mainly in cytoplasm, however, after exposure 2 h, they translocated to nucleus. The proportion of cells in G1 phases was decreased from (63.80 +/- 9.57)% to (32.23 +/- 7.22)%, and the proportion of cells in S phases was increased from (35.17 +/- 10.33)% to (66.00 +/- 8.07)% after exposed to silica 24 h. Curcumin, a chemical inhibitor of AP-1, impaired the decrease of cells in G1 phases. Furthermore we found expression of dominant-negative mutant of ERK and JNK impaired silica-induced AP-1 activation, whereas, dominant-negative mutant of p38 did not show the effect.These result suggested that 200 microg/ml silica exposure can induce AP-1 activation, induce cell cycle changes through ERK, JNK/AP-1-dependent pathway.

To investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).HELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.B(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.JNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.

To study the phosphorylation level of mitogen activated protein kinase (MAPK) in human embryonic lung fibroblasts (HELF), and the expression level of cyclin D1-CDK4 protein in S-HELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in S-HELF.Two kinds of treatment: (1) Cells were harvested after stimulation 2 h for the detection of cytokines. (2) Cells were stimulated by quartz for a long time (2 months) for transformation characters (S-HELF). The MAP kinase was detected by western blot. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle.Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in S-HELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S-HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduced the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on S-HELF.The phosphorylation levels of ERK1/2, JNK1/2, and p38 increased in HELF exposed to quartz. The phosphorylation levels of ERK1/2 and JNK1 increased, but the phosphorylation level of p38 decreased in S-HELF. The expression level of cyclin D1-CDK4 protein increased in S-HELF. Overexpression of cyclin D1-CDK4 is due to the activation of ERKs, JNKs/AP-1 signaling pathway in S-HELF.

To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.B[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.

To study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.B (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.

To study the role of mitogen activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in benzo(a)pyrene (B(a)P)-induced changes of cell cycle in human embryo lung fibroblasts (HELF).AP-1 luciferase activity was determined by the Luciferase reporter gene assay using a luminometer. The expression levels and activity of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. The dominant negative mutant of ERK2, JNK1 and p38 were applied to detect the upstream or downstream relationship of signaling pathways.B(a)P treatment resulted in a marked activation of AP-1 and its upstream MAPK, including ERK, JNK and p38 in human embryo lung fibroblasts (HELF). B(a)P exposure also led to increase the population of cells at S phase compared to control (P0.01) with a concomitant decline of cells at G(1) phase. B(a)P-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutant of ERK2 or JNK1, but not p38. B(a)P-induced AP-1 transactivation was inhibited by the overexpression of dominant-negative mutant of ERK2 or JNK1, but not p38. Inhibition of the activation of AP-1 by curcumin, a chemical inhibitor of AP-1, significantly inhibited the cell cycle changes in response to B(a)P treatment.ERK and JNK, but not p38, mediated benzo(a)pyrene-induced cell cycle changes by AP-1 transactivation in HELF.

To investigate the roles of cyclin D1/CDK4-E2F-1/4 pathway in cell cycle changes of human embryo lung fibroblasts (HELF) induced by two different benzo(a)pyrene [B(a)P] treatment models.Two B(a)P treatment models: HELF were treated by 2 micromol/L B(a)P for 24 hours; HELF were treated by 100 micromol/L B(a)P three times 24 hours each and provide with some characteristics of transformed cells (T-HELF). Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometry and Western bolt analysis.After 24 hours 2 microml/L B(a)P treatment, the HELFs in the G(1) phase was decreased. In HELF transfected with antisense cyclin Dl (A-Dl) and antisense CDK4 (A-K4), the expression of cyclin Dl and CDK4 blocked the cell cycle changes from the G(1) phase to the S phase induced by B(a)P. The overexpression of cyclin Dl and E2F-1 in HELF was induced by B(a)P. The E2F-1 overexpression in A-D1 induced B(a)P was inhibited. The E2F-4 expression was decreased and the CDK4 expression was increased significantly in A-K4 after B(a)P treatment. Most of T-HELF transfected with antisense cyclin Dl (T-A-Dl) and antisense CDK4 (T-A-K4) were retained in G(1) phase. The cyclin Dl expression in T-HELF was increased significantly compared with that in HELF. The E2F-4 expression in T-A-Dl and T-A-K4 was increased significantly compared with that in T-HELF.B(a)P induces the cell cycle changes through cyclin D1/CDK4-E2F-1/4 pathway in HELF treated by 2 micromol/L B(a)P while it induces cell cycle changes through cyclin D1/CDK4-E2F-4 pathway in T-HELF.

To investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).After HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.After treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.ATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.