Krill oil ameliorates benign prostatic hyperplasia by regulating G1-phase cell cycle arrest and altering signaling pathways and benign prostatic hyperplasia-associated markers

Krill oil(KO)exhibits various biological activities,such as anti-inflammatory and antitumor effects.However,the inhibitory effects of benign prostatic hyperplasia(BPH)in vitro and in vivo have not yet been studied.This study investigated the anti-BPH effects of KO extracted by an enzymatic hydrolysis method.KO treatment inhibited the proliferation of WMPY-1 and BPH-1 cells by induction of G0/G1 phase arrest through the modulation of positive and negative regulators in both prostate cell types.KO treatment stimulated phosphorylation of c-Jun N-terminal kinase(JNK)and p38 signaling.In addition,KO changed the expression of BPH-related markers(5α-reductase,androgen receptor,FGF,Bcl-2,and Bax)and the activity of the proliferation-mediated NF-κB binding motif.KO-induced levels of proliferation-mediated molecules of prostate cells were attenuated in the presence of siRNA-specific p-38(si-p38)and JNK(si-JNK).Furthermore,the administration of KO alleviated prostate size and weight and the cell layer thickness of prostate glands in a testosterone enanthate-induced BPH rat model.KO treatment altered the level of dihydrotestosterone in serum and the expression levels of BPH-related markers in prostate tissues.Finally,KO-mediated inhibition of prostatic growth was validated by histological analysis.These results suggest that KO has an inhibitory effect on BPH in prostate cells in vitro and in vivo.Thus,KO might be a potential prophylactic or therapeutic agent for patients with BPH.

Effect of cis-9,trans-11-conjugated linoleic acid on cell cycle of gastric adenocarcinoma cell line(SGC-7901)

AIM: To determine the effect of cis -9, trans -11-conjugated linoleic acid (c9, t11-CLA) on the cell cycle of gastric cancer cells (SGC-7901) and its possible mechanism in inhibition cancer growth. METHODS: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B(1), D(1), p16(ink4a) and p21(cip/waf1) of SGC-7901 cells which were treated with various c9, t11-CLA concentrations (25, 50, 100 and 200 micromol.L(-1))of c 9, t 11-CLA for 24 and 48h, with a negative control (0.1% ethane). RESULTS: The cell growth and DNA synthesis of SGC-7901 cells were inhibited by c9, t11-CLA.SGC-7901 cells. Eight day after treatment with various concentrations of c9, t11-CLA mentioned above, the inhibition rates were 5.92%, 20.15%, 75.61% and 82.44%, respectively and inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 micromol.L, 24h) showed significantly less (3)H-TdR incorporation than that in the negative controls (P【0.05 and P【0.01). Immunocytochemical staining demonstrated that SGC-7901 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA (the expression rates were 7.2-3.0%, 24h and 9.1-0.9% at 48h, respectively), Cyclin A (11.0-2.3%, 24h and 8.5-0.5%,48h), B(1) (4.8-1.8% at 24h and 5.5-0.6% at 48h)and D(1) (3.6-1.4% at 24h and 3.7%-0 at 48h) as compared with those in the negative controls(the expressions of PCNA, Cyclin A, B(1) and D(1) were 6.5% at 24h and 9.0% at 48h, 4.2% at 24h and 5.1% at 48h, 9.5% at 24h and 6.0% at 48h,respectively)(P【0.01), whereas the expressions of P16(ink4a) and P21(cip/waf1), cyclin-dependent kinases inhibitors(CDKI), were increased. CONCLUSION: The cell growth and proliferation of SGC-7901 cell is inhibited by c9, t11-CLA via blocking the cell cycle, with reduced expressions of cyclin A,B(1) and D(1) and enhanced expressions of CDKI(P16(ink4a) and p21(cip/waf1)).

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G_1 phase

Objective:To investigate the effects of an ethanol extract of Kalopanax septemlobus(Thunb.)Koidz.leaf(EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.Methods:Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis.Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting,and activation of eyclin-associaled kinases studied using kinase assays.Results:The EEKS suppressed cell proliferation in both HepG2 and Hep3 B cells,but showed a more sensitive anli-proliferative activity in HepG2 cells.Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G_1 phase cell cycle arrest in HepG2 cells,along with the dephosphorylation of retinoblastoma protein(pRB) and enhanced binding of pRB with the E2 F transcription factor family proteins.Treatment with EEKS also increased the expression of cyclin-dependent kinase(CDK) inhibitors,such as p21WAF1/CIP1 and p27KIP1.without any noticeable changes in G_1 cyclins and CDKs(except for a slight decrease in CDK4).Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6.which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities.Conclusions:Overall,our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G_1,cell cycle arrest Further studies arc required to identify the active compounds in EEKS.

6-OHDA Induces Cycle Reentry and Apoptosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway

This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine （6-OHDA） in PC12 cells. By using neural differentiated PC12 cells treated with 6-OHDA, the apoptosis model of dopaminergic neurons was established. Cell viability was measured by MTT. Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry. Western blot was used to detect the activation of extracellular regulator kinasel/2 （ERK1/2） pathway and the phosphorylation of retinoblastoma protein （RB）. Our results showed that after PC12 cells were treated wtih 6-OHDA, the viability of PC12 cells was declined in a concentration-dependent manner. Flow cytornetry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner. The percentage of ceils in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased. Simultaneously, ERK1/2 pathway was activated and phosphorylated RB increased. It was concluded that 6-OHDA could induce cell cycle reentry of dopaminergic neurons through the activation of ERK1/2 pathway and RB phosphorylation. The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

Anoctamin 5 regulates the cell cycle and affects prognosis in gastric cancer

BACKGROUND Anoctamin 5(ANO5)/transmembrane protein 16E belongs to the ANO/transmembrane protein 16 anion channel family.ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid scrambling and regulate other membrane proteins in numerous cell types.Previous studies have elucidated the roles and mechanisms of ANO5 activation in various cancer types.However,it remains unclear whether ANO5 acts as a plasma membrane chloride channel,and its expression and functions in gastric cancer(GC)have not been investigated.AIM To examine the role of ANO5 in the regulation of tumor progression and clinicopathological significance of its expression in GC.METHODS Knockdown experiments using ANO5 small interfering RNA were conducted in human GC cell lines,and changes in cell proliferation,cell cycle progression,apoptosis,and cellular movement were assessed.The gene expression profiles of GC cells were investigated following ANO5 silencing by microarray analysis.Immunohistochemical staining of ANO5 was performed on 195 primary tumor samples obtained from patients with GC who underwent curative gastrectomy between 2011 and 2013 at our department.RESULTS Reverse transcription-quantitative polymerase chain reaction(PCR)and western blotting demonstrated high ANO5 mRNA and protein expression,respectively,in NUGC4 and MKN45 cells.In these cells,ANO5 silencing inhibited cell proliferation and induced apoptosis.In addition,the knockdown of ANO5 inhibited G1-S phase progression,invasion,and migration.The results of the microarray analysis revealed changes in the expression levels of several cyclin-associated genes,such as CDKN1A,CDK2/4/6,CCNE2,and E2F1,in ANO5-depleted NUGC4 cells.The expression of these genes was verified using reverse transcription-quantitative PCR.Immunohistochemical staining revealed that high ANO5 expression levels were associated with a poor prognosis.Multivariate analysis identified high ANO5 expression as an independent prognostic factor for 5-year survival in patients with GC(P=0.0457).CONCLUSION ANO5 regulates the cell cycle progression by regulating the expression of cyclin-associated genes and affects the prognosis of patients with GC.These results may provide insights into the role of ANO5 as a key mediator in tumor progression and/or promising prognostic biomarker for GC.

Viral infections and cell cycle G2/M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast (Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well- characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins, which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest. Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

Cell Cycle Arrest Mediates Global DNA Methylation Patterns in Normal Human Keratinocytes, Epidermoid Carcinoma Cells and Murine Embryonic Fibroblasts

The 5-methylationcytosine (5-MC) DNA content of murine embryonic fibroblasts arrested in G1 by four growth conditions (Gc, Gn, Gd, and Gs) were hypermethylated relative to rapidly growing (RG) fibroblasts. Normal human keratinocytes (NHK) arrested in G1 by suspension were hypermethylated relative to RG cultures. Four RG cultures of epidermoid carcinoma cells (ECC) were hypomethylated relative to RG NHK cultures, and two cultures (SCC25 and A431) were further hypomethylated by SUS-induced arrest. Linear regression analyses established a positive linear correlation between growth rate and 5-MC content for three murine fibroblasts lines, and a negative correlation for both NHK and ECC lines.

Correlation of S100A13 and FOXA1 expression with cell cycle and cell invasion in fine needle aspiration thyroid carcinoma tissue

Objective: To study the correlation of S100A13 and FOXA1 expression with cell cycle and cell invasion in fine needle aspiration thyroid carcinoma tissue. Methods: Patients who received ultrasound-guided thyroid nodule fine needle aspiration in Haiyang People's Hospital between April 2015 and February 2017 were selected, and the tissues were divided into malignant thyroid tissue and benign thyroid nodules according to the pathological results after biopsy. The expression of S100A13, FOXA1, cell cycle molecules and cell invasion molecules were measured. Results: S100A13, FOXA1, CDK2, CyclinD1, MCM2, MCM7, SKP2, CLOCK, STAT3, STAT5, N-cadherin, MT1-MMP and ADAM17 mRNA expression in thyroid carcinoma tissue were significantly higher than those in benign thyroid nodule;CDK2, CyclinD1, MCM2, MCM7, SKP2 and CLOCK mRNA expression in thyroid carcinoma tissue with high FOXA1 expression were significantly higher than those in thyroid carcinoma tissue with low FOXA1 expression;STAT3, STAT5, N-cadherin, MT1-MMP and ADAM17 mRNA expression in thyroid carcinoma tissue with high S100A13 expression were significantly higher than those in thyroid cancer tissue with low S100A13 expression. Conclusions: High expression of S100A13 and FOXA1 in thyroid carcinoma can promote cell invasion and cell cycle progression.

Regulation of Survivin and CDK4 by Epstein-Barr virus encoded latent mem-brane protein 1 in nasopharyngeal carcinoma cell lines

Latent membrane protein 1 （LMP1）, an important protein encoded by Epstein Barr virus （EBV）, has been implied to link with the pathogenesis of nasopharyngeal carcinoma （NPC）. Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line- CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/ M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMPI. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.

Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells

AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines,HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological observation,flow cytometry analysis,Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells,as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner,which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition,G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53,pRb,p27,cyclins D1,D2,E,cyclin-dependent kinase (Cdk) 2,Cdk4,and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option,as a potential anticancer agent,in the treatment of hepatoma.

The involvement of p38 MAPK in transforming growth factor β1-induced apoptosis in murine hepatocytes

We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.

大肠癌细胞周期G_1/S期检查点调控的研究进展

大肠癌细胞周期G_1/S期检查点具有重要作用,是目前肿瘤研究的热点之一。全文就大肠癌细胞周期的研究进展作一综述。

蛋白激酶Cα对人正常肝和肝癌细胞周期及G_1期相关调控因子cyclinD1、cyclinE的影响

目的 探讨蛋白激酶Cα(PKCα)对人正常肝和肝癌细胞周期的作用和对G1 期相关调控因子的影响。 方法 通过细胞转染技术将PKCαcDNA正向插入的真核表达质粒PXJ4 1 PKCα导入正常肝细胞 (L 0 2 ) ,并利用本室已构建的表达反义PKCα的BEL 74 0 2细胞 (HT6 )检测细胞的生长曲线 ,细胞周期以及细胞对G1 期相关调控因子cylinD1和cyclinE的影响。 结果 构建了稳定过表达PKCα的人正常肝细胞模型 (LT3) ,过表达PKCα可促进L 0 2细胞增殖 ,促进细胞由G1 期向S期的过渡 ;cyclinD1和cyclinE的蛋白水平上升 ,反之表达反义PKCα的BEL 74 0 2细胞 (HT6 )增殖被抑制 ,阻抑细胞由G1 期向S期的过渡 ,cyclinD1和cyclinE的蛋白水平下降。 结论 从正反两个方面表明 ,PKCα可通过作用于G1 期相关周期蛋白的水平影响G1 S期的进程。

宫颈癌组织中miR-183与GSPT1的表达及临床意义

目的探讨宫颈癌组织中微小RNA(miR)-183与细胞周期G1到S期的转换1(GSPT1)的表达及与预后的关系。方法收集2014年1月至2016年12月接受根治性手术的91例宫颈癌患者。采用实时荧光定量PCR检测91例宫颈癌和癌旁组织中miR-183和GSPT1的表达。分析宫颈癌组织中miR-183与GSPT1表达的相关性,并采用生物信息学方法预测两者之间的结合位点。进一步分析miR-183和GSPT1表达与宫颈癌临床病理特征及预后的关系。结果宫颈癌组织中miR-183的表达(0.521±0.065)低于癌旁组织(1.241±0.286),差异有统计学意义(P=0.000)。宫颈癌组织中GSPT1的表达(2.034±0.374)高于癌旁组织(0.708±0.157),差异有统计学意义(P=0.000)。宫颈癌组织中miR-183与GSPT1的表达呈负相关(r=-0.621,P=0.001)。生物信息学预测结果显示,GSPT1 mRNA第981至987碱基存在与miR-183相互作用的位点。宫颈癌组织中miR-183和GSPT1表达与FIGO分期、分化程度、肌层浸润和淋巴结转移有关(P<0.05),与年龄和病理类型无关(P>0.05)。全组1、3、5年无病生存率分别为86.8%、73.6%和61.5%。miR-183高表达组1、3、5年无病生存率分别为91.1%、86.6%和80.0%,优于miR-183低表达组的82.6%、60.9%和43.5%,差异有统计学意义(P=0.012)。GSPT1高表达组1、3、5年无病生存率分别为86.0%、60.4%和39.5%,低于GSPT1低表达组的87.5%、85.4%和81.2%,差异有统计学意义(P=0.007)。结论宫颈癌组织中miR-183表达降低,GSPT1表达升高,两者共同促进宫颈癌进展,有望成为宫颈癌预后评估的标志物。

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.

下调垂体瘤转化基因1对胶质瘤细胞SHG44血管生成拟态及细胞周期的影响

目的研究下调垂体瘤转化基因1(PTTG1)对胶质瘤细胞SHG44血管生成拟态及细胞周期的影响,探索PTTG1影响人恶性胶质瘤细胞SHG44细胞周期的可能机制。方法用PTTG1 siRNA干扰胶质瘤细胞SHG44基因表达,通过定量聚合酶链式反应和蛋白质印迹分析在mRNA和蛋白质水平上评估PTTG1表达下降对AKT、p-AKT、C-myc、CyclinD1表达的影响,并进一步探究PTTG1对SHG44血管生成拟态及细胞周期的影响。结果 PTTG1 siRNA可以显著抑制PTTG1基因和蛋白的表达。降低PTTG1表达导致细胞不能形成网格状结构,即血管生成拟态形成能力受到抑制。抑制PTTG1的表达导致S期和G2期细胞百分比显著降低,即细胞增殖受到明显抑制。抑制PTTG1表达导致C-myc和CyclinD1 mRNA水平明显降低;p-AKT、C-myc和CyclinD1蛋白表达水平明显降低;说明PTTG1通过AKT/C-myc/CyclinD1信号通路影响SHG44细胞周期。结论下调PTTG1可以抑制胶质瘤的发生发展进程,为其治疗提供新思路。

Different Responses of Two Highly Permissive Cell Lines Upon HCV Infection

The construction of the first infectious clone JFH-1 speeds up the research on hepatitis C virus (HCV). However, Huh7 cell line was the only highly permissive cell line for HCV infection and only a few clones were fully permissive. In this study, two different fully permissive clones of Huh7 cells, Huh7.5.1 and Huh7-Lunet-CD81 (Lunet-CD81) cells were compared for their responses upon HCV infection. The virus replication level was found slightly higher in Huh7.5.1 cells than that in Lunet-CD81 cells. Viability of Huh7.5.1 cells but not of Lunet-CD81 cells was reduced significantly after HCV infection. Further analysis showed that the cell cycle of infected Huh7.5.1 cells was arrested at G1 phase. The G1/S transition was blocked by HCV infection in Huh7.5.1 cells as shown by the cell cycle synchronization analysis. Genes related to cell cycle regulation was modified by HCV infection and gene interaction analysis in GeneSpring GX in Direct Interactions mode highlighted 31 genes. In conclusion, the responses of those two cell lines were different upon HCV infection. HCV infection blocked G1/S transition and cell cycle progress, thus reduced the cell viability in Huh7.5.1 cells but not in Lunet-CD81 cells. Lunet-CD81 cells might be suitable for long term infection studies of HCV.

GSTO1抑制TGFβ诱导的大鼠心脏成纤维细胞增殖和活化

目的:研究谷胱甘肽S-转移酶ω1(GSTO1)在转化生长因子β(TGFβ)诱导心脏成纤维细胞增殖与活化中的作用。方法:分离乳大鼠心脏成纤维细胞并进行体外培养。采用含绿色荧光蛋白(GFP)的重组腺病毒质粒构建对照(Ad-GFP)或GSTO1过表达(Ad-GSTO1)质粒并转染心脏成纤维细胞,再用大鼠TGFβ(rTGFβ)刺激细胞24 h,实验分为4组:Ad-GFP+PBS、Ad-GFP+rTGFβ、Ad-GSTO1+PBS和Ad-GSTO1+rTGFβ。运用qPCR和免疫荧光染色确定转染的有效性;Western blot检测GSTO1蛋白表达;划痕实验和EdU掺入实验评估细胞迁移和增殖;CCK-8实验评估细胞活力;Western blot和细胞免疫荧光染色检测心脏成纤维细胞活化标志物α-平滑肌肌动蛋白(α-SMA)、I型胶原(Col I)和纤连蛋白(FN)的表达水平,并检测心脏成纤维细胞内P38 MAPK和Smad3信号通路相关蛋白水平。结果:与Ad-GFP+rTGFβ组相比,GSTO1过表达抑制TGFβ诱导的心脏成纤维细胞增殖(P<0.05),降低TGFβ刺激后α-SMA、Col I和FN的表达水平(P<0.05),抑制P38 MAPK/Smad3信号通路激活(P<0.05)。结论:GSTO1过表达通过抑制P38 MAPK/Smad3信号通路而阻遏TGFβ诱导的大鼠心脏成纤维细胞增殖和活化。

Mec1-Dependent Phosphorylation of the Scc3 Subunit of Cohesin during Mitosis in Budding Yeast

Cohesin is an evolutionary conserved complex that controls chromosome segregation during mitosis. Here we show that, in response to DNA damage, Saccharomyces cerevisiae Scc3, one of the major regulatory subunits of the Smc1-Smc3-Scc1 cohesin ring, is phosphorylated on S/T-Q residues. This event depended on the Mec1 checkpoint kinase as well as on cell cycle arrest triggered by the DNA damage checkpoint network. This phosphorylation event also took place during mitosis of an unperturbed cell cycle. The present finding that S. cerevisiae Scc3 is phosphorylated during mitosis represents a potentially important new regulatory step in cohesin’s mitotic functions.