Overexpression of protein kinase C-beta1 isoenzyme suppresses indomethacin-induced apoptosis in gastric epithelial cells

BACKGROUND & AIMS: We have previously reported that nonsteroidal anti-inflammatory drugs (NSAIDs) could induce apoptosis of gastric epithelial cells both in vivo and in vitro. This study investigated the role of protein kinase C (PKC) isoforms in the regulation of NSAID-induced apoptosis. METHODS: Protein levels of 12 PKC isoforms in AGS cells, in the presence or absence of indomethacin, were determined by Western blot. The effect of PKC-beta1 overexpression by transfection with its complementary DNA (cDNA) on indomethacin-induced apoptosis and apoptosis-related genes, including p53, p21(waf1/cip1), and c-myc, was further investigated. RESULTS: Treatment with indomethacin decreased the abundance of PKC-beta1 and increased that of PKC-beta2, eta, and epsilon, but did not alter the expression of PKC alpha, gamma, zeta, delta, iota, and micro. Overexpression of PKC-beta1 attenuated the apoptotic response of AGS cells to indomethacin, associated with overexpression of p21(waf1/cip1) in both messenger RNA and protein levels. Inhibition of PKC-beta1-mediated overexpression of p21(waf1/cip1) by its antisense cDNA partially reduced the antiapoptotic effect of PKC-beta1. CONCLUSIONS: Indomethacin-induced apoptosis in gastric cancer cells is partly mediated by differential regulation of PKC isoform expression. Enhanced expression of exogenous PKC-beta1 protects against indomethacin-induced apoptosis through up-regulation of p21(waf1/cip1).     

着色性干皮病基因B对白介素-6介导的血管平滑肌细胞增殖及凋亡的影响

目的 探讨着色性干皮病基因B(xeroderma pigmentosum B,XPB)对白介素-6(IL-6)介导人血管平滑肌细胞( VSMC)增殖及凋亡的影响.方法 用脂质体转染法将重组质粒pcDNA3.1-XPB和空载质粒pcDNA3.1稳定转染VSMC,然后给予100 U/ml的IL-6孵育48 h.实验分为6组:(1)空白对照组;(2) pcDNA3.1组;(3) pcDNA3.1-XPB组;(4)IL-6组;(5) IL-6+ pcDNA3.1组;(6)IL-6+ pcDNA3.1-XPB组.用反转录聚合酶链反应(RT-PCR)和蛋白质免疫印迹法(Western blotting)检测XPB、Bcl-2、Bax和野生型p53(wt-p53)表达量的变化;用比色法(MTT)观察细胞增殖活力;用流式细胞仪检测细胞周期和凋亡率.结果 重组质粒pcDNA3.1-XPB转染使细胞XPB表达增高(P＜0.05或P＜0.01),同时Bcl-2表达降低、Bax和wt-p53表达增高(P＜0.05或P＜0.01),抑制IL-6促进VSMC的Bcl-2高表达、Bax和wt-p53降表达(P＜0.05或P＜0.01);XPB高表达抑制了细胞增殖活力(q=2.95,P＜0.05),并抑制IL-6促进VSMC增殖,IL-6+ pcDNA3.1-XPB组和IL-6 +pcDNA3.1组VSMC存活率分别为(102.6±6.2)％和(124.5+7.9)％(q=3.49,P＜0.05);流式细胞仪检测结果显示,XPB高表达引起细胞G0/G1期增加、S期减少、凋亡率增加(q值分别为2.99、5.39、3.05,P＜0.05或P＜0.01),并抑制IL-6促进VSMC G0/G1期减少、S期增加、凋亡率降低的作用,IL-6+ pcDNA3.1-XPB组和IL-6 +pcDNA3.1组分别为(70.9±6.7)％与(54.8±2.9)％、(20.2±3.6)％与(36.4±7.2)％、(5.9±2.1)％与(0.3±0.1)％(q值分别为6.91、8.54、7.53,均P＜0.01).结论 XPB基因能抑制VSMC增殖并促进其凋亡,并能抑制IL-6促进VSMC增殖和降低其凋亡的作用,有望成为治疗动脉粥样硬化的靶点.     

Pharmacological Inhibition of Protein Kinase C Activity Could Induce Apoptosis in Gastric Cancer Cells by Differential Regulation of Apoptosis-Related Genes

The protein kinase C (PKC) signaling pathwayplays a key role in tumor cell proliferation,differentiation, and apoptosis. Gastric cancer usuallypossesses a higher level of PKC activity than normaltissue. We evaluated inhibition of PKC activity inapoptosis induction of gastric cancer cells and theexpression profile of apoptosis-related genes. Gastriccancer cells (AGS) were incubated with two highlyspecific PKC inhibitors (RO-31-8220 and chelerythrine).Cell viability and cell cycle were determined bymethyl-tetrazolium (MTT) assay and flow cytometry,respectively. Apoptosis was characterized by acridineorange staining, DNA gel electrophoresis, and flowcytometry. The expression of p53,p21^waf/cip1, c-myc, bcl-2, and bax wasdetermined by western blot. The results showed that bothPKC inhibitors hindered cell growth, arrested cells atG₀/G₁ phase and induced apoptosis.The protein level of p53, p21^waf/cip1, c-myc,and bax was elevated while bcl-2 kept unchangedfollowing drug exposure. In conclusion, PKC inhibitorssuppress growth of gastric cancer cells throughapoptosis induction and cell cycle quiescence, which maybe regulated by differential expression ofapoptosis-related genes.     

p53 functional impairment and high p21waf1/cip1 expression in human T- cell lymphotropic/leukemia virus type I-transformed T cells

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53- regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I- infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I- transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T- cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.     

Inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma

AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC). METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized P33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%). CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wildtype p53.     

乙型肝炎病毒X基因下调miR-192对人肝癌细胞株HepG2凋亡的影响

目的 探讨乙型肝炎病毒X基因(HBx)通过调节人肝癌细胞株HepG2中miR-192的表达而抑制其凋亡的机制.方法 设立3个细胞组:稳定转染HBx基因的HepG2细胞(HepG2/HBx),稳定转染空载体pcDNA3.1的HepG2细胞(HepG2/pcDNA3.1)以及未作转染的HepG2细胞.用流式细胞术分析3个细胞组的凋亡率差异,用Taqman探针荧光定量PCR检测3组细胞中miR-192的表达水平.转染miR-192后,用流式细胞术检测HepG2细胞凋亡率的变化,同时用SYBR Green荧光定量PCR和Western blot检测细胞中p53、PUMA表达的变化.计量资料均数的比较用单因素方差分析.结果 HepG2/HBx细胞的凋亡率为2.37％±0.35％,较HepG2/pcDNA3.1、HepG2细胞(11.46％±0.69％、12.50％±0.66％)明显降低(F=171.722,P＜0.01).miR-192表达在HepG2/HBx细胞中为49.1％±5.9％,较HepG2/pcDNA3.1、HepG2细胞(98.0％±8.9％,100％)也明显下调(F=14.319,P＜ 0.05).转染miR-192后HepG2细胞的凋亡率(15.74％±1.17％)较转染相应阴性对照的HepG2细胞的凋亡率(10.74％±1.15％)显著升高(F=18.415,P＜0.05),同时,p53、PUMA基因在mRNA (953:1.68±0.12比0.90±0.09,F=43.115,P＜0.05 ; PUMA:1.66±0.10比0.98±0.06,F=22.541,P＜0.05)和蛋白质水平(p53:3.07比1,PUMA:2.13比1)的表达均显著上升.结论 miR-192促进HepG2细胞凋亡,HBx通过下调miR-192抑制HepG2细胞凋亡.     

The histone-deacetylase inhibitor Trichostatin A blocks proliferation and triggers apoptotic programs in hepatoma cells

BACKGROUND/AIMS: Effective treatment for hepatocellular carcinoma is urgently needed. The histone-deacetylase inhibitor Trichostatin A (TSA) was shown to induce apoptosis in non-hepatic cells at submicromolar concentrations. However, the effect of TSA on hepatoma cells is unknown. METHODS: The hepatoma cells HepG2, MH1C1, Hepa1-6 and Hep1B as well as human fibroblasts (control cells) were exposed to TSA (10(-6) to 10(-9)M). Cell proliferation was assessed by measuring DNA-synthesis and cell numbers. Apoptosis was quantified by flow cytometry and by the TdT-mediated dUTP nick-end labeling method. Expression patterns of cell cycle- and/or apoptosis-associated p27, p21(cip/waf), bax, bcl-2, cyclin A and (pro)-caspase 3 were studied using quantitative Western blotting. Activation of caspase 3 was analyzed via a colorimetric assay. RESULTS: 10(-6)M TSA inhibited DNA-synthesis by 46% (HepG2) to 64% (MH1C1) after 24h, inducing a G(2)/M-phase arrest and apoptosis. TSA increased activation of caspase 3 and expression of cyclin A, p2l(cip/waf), bax and (pro)-caspase 3, while bcl-2 was downregulated. Human fibroblasts remained unaffected. CONCLUSIONS: TSA inhibits hepatoma cell growth in vitro, which are otherwise particularly resistant to chemotherapy. Its anti-proliferative activity is paralleled by a comparable rate of apoptosis. TSA may be a promising agent for treatment of hepatocellular carcinoma in vivo.     

Pioglitazone inhibits growth of carcinoid cells and promotes TRAIL-induced apoptosis by induction of p21waf1/cip1.

BACKGROUND/AIMS: We investigated the effect of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone on growth and TRAIL-induced apoptosis in carcinoid cells. METHODS: Carcinoid cells were incubated without and with pioglitazone. Effects on growth were examined by cell count and cell cycle analysis. p21waf1/cip1 expression was determined by Western blotting. Cytotoxicity assay was performed by FACS analysis. RESULTS: Pioglitazone suppressed the growth and induced apoptosis of carcinoid cells. Additionally, pioglitazone significantly enhanced carcinoid cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The enhancement of TRAIL-induced apoptosis was associated with an upregulation of cyclin-dependent kinase inhibitor p21waf1/cip1 in pioglitazone-treated carcinoid cells. Importantly, overexpression of p21waf1/cip1 in carcinoid cells by adenoviral gene transfer of p21 sensitized them to TRAIL-induced apoptosis. CONCLUSIONS: These results suggest that pioglitazone inhibits cell growth and sensitizes cells to TRAIL-induced apoptosis by induction of p21waf1/cip1. Therefore, pioglitazone can be an effective therapeutic adjuvant for the treatment of carcinoid tumors.     

HCC和HepG2细胞表达HCV C蛋白、p14ARF、p21WAF1的意义探讨

目的检测HCVC蛋白、p14、p21在HCC和表达野生p53HepG2中的表达，初步探讨C蛋白在HCC和HepG2中对p14-p53-p21凋亡通路的作用。方法收集42例HCC石蜡组织，采用免疫组织化学EnVision法检测HCC组织中核心蛋白、p14和p21的表达，用统计学方法及临床联系分析它们之间的关系；用细胞化学EnVision法和免疫荧光法检测核心蛋白、p53、p14和p21在HepG2细胞中的表达。结果C蛋白、p14和p21的阳性表达主要定位于细胞核膜和细胞核中；HCC组织中C蛋白、p14和p21阳性率分别为40．5％、45．24％、19．05％；3组间的Kruskal-Wallis检验P=0．03，差异显著；C蛋白与p14、p21间及p14与D21间蛋白阳性强度相关性分析显示，P值分别为0．000、0．43、-0．34，相关系数rs分别为0．64、-0．29、-0．33。HepG2细胞有较高的C蛋白和p53表达及少量的p14、p21蛋白表达。结论在C蛋白阳性的HCC中p14的表达与C蛋白有关，HCC中D21表达缺陷是十分常见的；C蛋白在HCC中可能影响p53通路，下调p21的表达，阻止其凋亡作用；HepG2细胞永生化特性可能与HCV或HCVC蛋白有关。     

姜黄素对血管平滑肌细胞增殖及cyclinD1和p21waf1/cip1蛋白表达的影响

目的 观察姜黄素（curcumin）抑制大鼠血管平滑肌细胞(VSMC)增殖和诱导细胞周期停滞的作用,以及对细胞周期蛋白cyclinD1,p21waf1/cip1表达的影响。方法 采用组织贴块法体外培养大鼠VSMC,MTT法测定姜黄素对VSMC增殖的抑制作用,流式细胞仪分析细胞周期分布,Western印迹法检测cyclinD1,p21waf1/cip1蛋白的变化。结果 MTT示不同浓度姜黄素(7.5～120 μmol/L)在24～72 h范围内,浓度和时间依赖性抑制VSMC增殖;30 μmol/L以上浓度姜黄素显著抑制增殖的VSMC细胞周期进程,使S及G2/M期减少(P<0.05),G0/G1期增加(P<0.05);30 μmol/L的姜黄素可抑制cyclinD1表达,促进p21waf1/cip1蛋白表达。结论 姜黄素具有明确的抑制VSMC增殖和细胞周期停滞的作用,其与cyclinD1,p21waf1/cip1蛋白变化有关。     

p21<Superscript>WAF1/CIP1</Superscript> is more effective than p53 in growth suppression of mouse renal carcinoma cell line Renca in vitro and in vivo

Purpose Although there are many controversial reports about the effect of p53 and p21^WAF1/CIP1 overexpression in different human tumor cells, the p53 gene is shown to be a more effective candidate for cancer gene therapy because of its more pronounced ability to induce apoptosis. In the present study, we present the effect of p53 and p21^WAF1/CIP1 overexpression on mouse renal carcinoma cells in vitro and in vivo.Methods p53 and p21^WAF1/CIP1 genes were introduced into Renca cells using adenoviral vectors (Ad5CMV-p53 and Ad5CMV-p21). The induction of apoptosis was measured using Annexin V assay and DNA fragmentation analysis. The expression of proteins was examined using immunocytochemistry and Western blot methods. The ability of adenoviral vectors to inhibit tumorigenicity of Renca cells, as well as the growth of pre-established tumors was measured.Results In vitro growth assays revealed higher growth suppression after Ad5CMV-p21 infection. Although both vectors induced apoptosis, Ad5CMV-p53 was slightly more efficient. In vivo studies in Balb/c mice, demonstrated that tumorigenicity was completely suppressed by Ad5CMV-p21. Besides this, Ad5CMV-p21 significantly inhibited the growth of established tumors, while Ad5CMV-p53 did not.Conclusions These data suggest that p21^WAF1/CIP1 is a more potent growth suppressor than p53 of mouse tumor cells Renca. The divergent responses of tumor cells to p21^WAF1/CIP1 overexpression could be due to various networks that differ between species.     

HBC通过NEU1调控肝癌细胞中基因表达的初步研究

目的探讨乙肝病毒核心蛋白(hepatitis B virus C protein,HBC)对唾液酸酶1(neuraminidase 1,NEU1)及相关基因表达的影响。方法通过脂质体将pcDNA3.1和pcDNA3.1-HBC质粒分别转染HepG2细胞和Huh7细胞,采用Quantitative real-time-PCR(q-PCR)及Western blot检测NEU1的表达;PCR扩增NEU1基因并与pcDNA3.1载体质粒连接,构建NEU1过表达质粒pcDNA3.1-NEU1;将pcDNA3.1-NEU1和对照质粒分别转染HepG2细胞,收集细胞,进行转录组测序,获得差异表达基因,用q-PCR对差异表达基因进行验证;将pcDNA3.1-HBC和对照质粒转入肝癌细胞,q-PCR检测HBC对NEU1相关的差异表达基因的影响;在HBC阳性肝癌细胞,利用NEU1的小干扰质粒抑制其表达,q-PCR检测HBC是否通过NEU1调控相关基因的表达。结果与对照组肝癌细胞相比,转染HBC质粒的肝癌细胞NEU1表达显著上调;PCR鉴定pcDNA3.1-NEU1插入片段正确,重组质粒构建成功;转录组测序显示,与对照组比较,过表达NEU1的HepG2细胞有8个基因表达显著不同,其中6种基因表达上调,2种基因表达下调;q-PCR检测转录组测序获得的差异表达基因与转录组测序的结果一致;与对照肝癌细胞相比,NEU1相关的上调差异表达基因在HBC阳性肝癌细胞中高表达,且干扰NEU1表达后以上基因在HBC阳性肝癌细胞中表达下调;与NEU1相关的下调差异表达基因在HBC基因组中低表达,且干扰NEU1表达后相关基因在HBC阳性肝癌细胞中表达上调。结论HBC能够通过NEU1调控肝癌细胞中相关基因的表达。     

Induction of anti-proliferative mechanisms in hepatitis B virus producing cells

BACKGROUND/AIMS: Hepatitis B virus (HBV) preferentially replicates in quiescent cells. It was analyzed whether HBV affects cell cycle control. METHODS: The amount of EGF-receptor (EGFR) and the binding capacity for 125I-EGF was determined. Expression of mdm2 and p21 and relevance of p53 for it were analyzed by reporter gene assays and western blotting. Cyclin A/E associated cdk2 activities were determined by immunocomplex assays. Cell proliferation was quantified by measurement of BrdU incorporation. RESULTS: In HBV producing cells a significant reduction of EGFR expression, diminished 125I-EGF-binding capacity and insensitivity to EGF-stimulation were observed as compared to the control. Moreover, c-Raf-1-dependent induction of mdm2-P2 and p21cip1/waf1-promoter and elevated amounts of the respective proteins were observed in HBV producing cells. Whereas activation of mdm2-P2-promoter requires p53, activation of p21cip1/waf1-promoter is mediated partially by a p53-independent process. Induction of p21cip1/waf1 is reflected by a reduction of cyclin A associated cdk2 activity and an increase of cyclin E associated cdk2 activity. In accordance with this proliferation rate of HBV-producing hepatocytes is reduced as compared to control cells. CONCLUSIONS: These results describe novel cell-cycle inhibitory functions of HBV that correlate well with the general concept of enhanced HBV replication in quiescent cells.     

PTEN在HepG2细胞中诱导凋亡发生及上调p53表达

目的 研究肿瘤抑制基因PTEN对HepG2细胞凋亡及p53蛋白表达的影响,并探讨相关机制。方法 将携带有野生型PTEN基因及突变型G129E-PTEN,C124A-PTEN基因的真核表达载体转染HepG2细胞,利用western印迹杂交检测PTEN表达、蛋白激酶B(PKB/Akt)和焦点粘附激酶(FAK)的磷酸化状态,以及野生型p53蛋白表达水平的变化,并应用流式细胞仪,激光共聚焦技术检测细胞周期及细胞凋亡情况。结果 与对照细胞相比,转染野生型PTEN和G129E-PTEN的HepG2细胞中磷酸化FAK(-65％,-65％)与磷酸化Akt(-93％,-35％)表达均存在不同程度的下调,而细胞凋亡率分别增加至19.8％±1.2％和9.2％±0.6％,并且p53蛋白表达上调( 120%,, 50％);然而转染C124A-PTEN的细胞中各项检测指标均无明显变化。 结论 PTEN依赖其蛋白磷酸酶活性抑制FAK的磷酸化;并主要通过脂质磷酸酶活性抑制Akt的磷酸化,并诱导HepG2细胞凋亡和p53蛋白表达上调。     

Role of p42/p44 mitogen-activated-protein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21(waf1/cip1) in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like alpha-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis. S-nitroso-N-acetylpenicillamine or (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 microM, and DFMO elicited effects at concentrations of 100 microM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21(waf1/cip1). This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21(waf1/cip1) depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21(waf1/cip1) were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21(waf1/cip1) were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21(waf1/cip1) and consequent inhibition of cell proliferation.     

Blocking effects of genistein on cell proliferation and possible mechanism in human gastric carcinoma

AIM: To study the blocking effects of genistein on cell proliferation cycle in human gastric carcinoma cells (SGC-7901) and the possible mechanism. METHODS: MTT assay was applied in the detection of the inhibitory effects of genistein on cell proliferation. Flow cytometry was used to analyze the cell cycle distribution. Immunocytochemical technique and Western blotting were performed to detect the protein expression of cyclin D_1, cyclin B_1 and p21~(waf1/cip1). RESULTS: Genistein significantly inhibited the growth and proliferation of human gastric carcinoma cells (SGC-7901). Seven days after treatment with different concentrations of genistein (2.5, 5.0, 10.0, 20.0 μg/mL), the growth inhibitory rates were 11.2%, 28.8%, 55.3%, 84.7% respectively and cell cycles were arrested at the G(2)/ M phase. Genistein decreased cyclin D_1 protein expression and enhanced cyclin B_1 and p21~(waf1/cip1) protein expression in a concentration-dependent manner. CONCLUSION: The growth and proliferation of SGC-7901 cells can be inhibited by genistein via blocking the cell cycle, with reduced expression of cyclin D_1 and enhanced expression of cyclin B_1 and p21~(waf1/cip1) protein in the concentration range of 0-20 μg/mL.     

Expression of p53 and p21WAF1/CIP1 Proteins in Gastric and Esophageal Cancers (Comparison with Mutations of the p53 Gene)

The p53 gene has been shown to be commonlymutated in various human cancers, and mutant p53 can actas a dominant oncogene. The intact p53 protein is alsoknown to induce the cyclin-dependent kinase inhibitor p21^WAF1/CIP1 and is implicated incell cycle arrest. We investigated p53 gene alterationsin gastric adenocarcinoma and esophageal squamous cellcarcinoma to elucidate the association of the nuclearaccumulation of the p53 protein and/orp21^WAF1/CIP1 protein. Abnormalities of thetumor suppressor gene p53 protein and the expression ofp21^WAF1/CIP1 protein were analyzed byimmunohistochemical techniques in 32 cases of gastric adenocarcinoma and 15 cases ofesophageal squamous cell carcinoma. Twenty cases ofgastric cancer and five cases of esophageal cancer werealso analyzed for p53 gene mutation by polymerase chain reaction and direct nucleotide sequencing.Overexpression of p53 protein was found in 13/32 (41%)of gastric cancers and 5/15 (33%) of esophageal cancers.We found immunodetectable p53 in 10/14 cases with mutations and in none of 11 cases withoutmutations in gastric and esophageal cancers. Hence,immunohistochemical and genetic analyses gave concordantresults in 84% of 25 cases, revealing a good correlation between immunostaining of p53 and missensemutation of the p53 gene. p53 immunostaining was notobserved in cases with frameshift or splicing mutation.The expression of p21^WAF1/CIP1 protein wasfound in 9/32 (29%) of gastric cancers and 4/15 (27%) ofesophageal cancers and in 2/14 (14%) cases withalteration of the p53 gene and in 5/11 (45%) without.These results suggest that abnormalities of p53 may be closely associated with the pathogenesisof gastric adenocarcinoma and esophageal squamous cellcarcinoma and that the immunoreactivity of p53 proteinis a general indicator of the tumors with altered p53 function. The expression ofp21^WAF1/CIP1 protein was suppressed in theneoplastic tissues with and without p53 genealteration.     

p16真核表达载体的构建及其对肝癌细胞的作用

研究p16基因对肝癌细胞的作用;构建pcDNA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中,对其p16基因的表达、细胞的生长抑制及机制进行分析;转染细胞p16蛋白免疫组化阳性,MTY法结果显示,50×103/cm2细胞经培养24h～96h后,每组细胞数均有不同程度的增加,但经pcDNA3.0/p16转染的CBRH-7919细胞数比对照明显减少。与空白对照比,细胞在24h,48h,72h和96h的生长抑制率分别为29％,29％,40％和52％,流式细胞仪检测细胞周期显示经pcDNA3.0/p16转染的CBRH-7919细胞有显著的细胞凋亡现象和G0/G1期阻滞。pcD-NA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中能够抑制细胞的增殖活性,p16基因可能通过诱导肿瘤细胞凋亡及G1期阻滞在肿瘤的基因治疗方面发挥作用。