HCV核心蛋白、突变p53和Bcl-2在HCC中的表达及相关性

目的探讨HCC组织中的核心蛋白、突变p53、Bcl-2的表达以及其相关性,探讨HCV核心蛋白是否促进p53突变和Bcl-2的表达.方法收集手术切除HCC组织42例,采用免疫组织化学EnVision法检测HCC组织核心蛋白、突变p53、Bcl-2的表达,用统计学分析三者间的关系.结果C蛋白、p53和Bcl-2在HCC癌组织中的表达率分别为40.5%(17/42)、47.62%(20/42)、83.3%(33/42);3组强度等级资料Kruskal-Wallis秩和检验H=16.33,差异性显著,Mann-Whitney U test:C蛋白和p53、Bcl-2蛋白间的P值分别0.43、0.00;C蛋白与突变p53、Bcl-2阳性强度两者间相关性检验P值分别为0.000、0.914,相关系数rs分别为0.67、0.08;突变p53与Bcl-2两者相关性检验P值为0.27,相关系数rp为0.32.结论3种蛋白的表达有相关性;HCV核心蛋白可能促进野生型p53突变和表达;核心蛋白和突变p53都有可能促进Bcb2蛋白的表达.     

原发性肝细胞癌中P21WAF-1、p53与nm23的表达及意义

目的 探讨p21WAF-1、p53与nm23蛋白在原发性肝细胞癌（HCC）中的表达及意义。方法 应用免疫组化S-P法检测72例HCC和85例非癌肝组织中p21WAF-1、p53与nm23蛋白的表达，并分析它们之间的关系及其与HCC临床、病理特征的关系。结果 HCC组织中的p21WAF-1、p53与nm23蛋白表达率均与非癌组织有显著性差异（P〈0．01）。p21WAF-1及nm23在Ⅰ、Ⅱ期HCC中表达率与Ⅲ、Ⅳ期有明显差异。p53及nm23表达率在HCC无转移组与转移组有明显差异（P〈0．05，〈0．01）。结论 p21WAF-1、p53高表达，nm23低表达均在HCC的发生发展中起作用；联合检测p21WAF-1、p53与nm23蛋白指标有助于判断HCC患者预后。     

细胞凋亡和p53、bc1-2蛋白在肝细胞癌中的表达

目的：探讨HCC中细胞凋亡情况及其与p53和bcl-2蛋白表达的关系。方法：细胞凋亡采用原位细胞凋亡检测法．p53和bcl-2蛋白表达采用免疫组他方法．细胞凋亡与p53、bcl-2蛋白表达的关系采用双标记法进行检测。结果：70例HCC中。癌组织和癌周组织中细胞凋亡指数(1000个细胞中)分别为3．63士1．96和10．63±3.64，bcI-2和突变型p53置自检出率分别为22．9％和42．9％．bcl-2主要表达在癌周组织中，抑制肝细胞凋亡。而p53则仅表过在癌组织，抑制癌细胞凋亡。双染色显示细胞凋亡与p53或bel-2不同时在一个细胞中表选。结论：HCC癌细胞凋亡减弱一突变型p53蛋白是癌细胞凋亡减弱的主要原因．而be-2则可能在维持HCC的肝脏功能方面具有重要作用。     

外源HCV核心基因表达载体在HepG2中蛋白表达的亚细胞定位

目的研究HCV全长（天然）核心蛋白（C蛋白）和成熟C蛋白在HepG2细胞内的亚细胞定位，为进一步研究C蛋白与宿主细胞蛋白因子相互作用提供实验依据。方法设计HCV全长和成熟核心基因引物，以pBRTM／HCV1-3011为真核表达载体为模版行PCR；将PCR扩增产物酶切纯化后定向插入pEGFP-C1的BeglⅡ和EcoRⅠ位点，得到能表达全长HCV核心蛋白（C191aa．）和成熟核心蛋白（C173aa．）的真核表达载体pEGFP-C1-C191、pEGFP-C1-C173；将扩增pEGFP-C1-C191、pEGFP-C1-C173质粒转染到HepG2细胞中，28h、5d在荧光显微镜下观察两种C蛋白的亚细胞定位。5d后再用免疫细胞化学法观察两种C蛋白在HepG2细胞中着色现象。结果HCV核心基因PCR产物和构建的pEGFP-C1-C191、pEGFP-C1-C173经扩增测序证实。pEGDP-C1-C191转染HepG2细胞28h后融合荧光全长C蛋白C191主要出现在核膜和胞质中，少部分出现在胞核及其膜中；而pEGDP-C1-C173转染HepG2细胞28h后主要定位在核中和核膜上。但pEGDP-C1-C191转染HepG2细胞5d后，其表达的融合荧光蛋白主要出现在核内和核膜上，少量在胞质中；而成熟C蛋白C173则仍然在胞核内及核膜上。pEGDP-C1-C转染HepG2细胞5d后进行免疫细胞化学显示，C191蛋白和C173蛋白主要在核中、核膜着色，胞质有少量着色。结论HCV全长C蛋白的亚细胞定位是一动态过程，先出现在胞质中和核膜上，最后定位在核中和核膜上；成熟C蛋白主要定位在核中和核膜上。这种现象为解释瞬时转染HCV不同长度C基因对p53／p21通路作用出现相反结果奠定基础。     

PTEN和突变型p53蛋白在鼻咽癌组织中的表达及临床意义

采用免疫组化EnVision法,分别检测72例鼻咽癌和32例鼻咽黏膜慢性炎性组织中蛋白酪氨酸磷酸酶基因（PTEN）及突变型p53蛋白的表达情况,分析二者与鼻咽癌临床病理特征的相关性。结果PTEN、突变型p53蛋白阳性表达率在鼻咽癌组织中分别为45．8％、77．8％,在鼻咽黏膜慢性炎性组织中分别为87．5％、9．4％,P均〈0．05;二者在鼻咽癌组织中的表达呈负相关性,且与鼻咽癌临床分期及淋巴结转移相关（P〈0．05）。证实在鼻咽癌组织中存在PTEN蛋白低表达和突变型p53蛋白高表达,且二者呈负相关,此在鼻咽癌发生、发展和转移中起重要作用。     

人脑胶质瘤组织中p63、p53蛋白的表达变化及意义

目的观察人脑胶质瘤组织中p63、p53蛋白的表达变化,并探讨其意义。方法采用免疫组化SP法检测41例人脑胶质瘤和10例非瘤脑组织中的p63、p53蛋白。结果观察组p63、p53蛋白阳性表达21、25例,对照组分别为0、0例;两组比较,P均<0.05。胶质瘤组织中Ⅰ～Ⅱ级者p63、p53蛋白阳性表达7、10例,显著低于Ⅲ～Ⅳ级者的14、15例(P均<0.05)。胶质瘤组织中p63、p53蛋白阳性表达呈正相关(r=0.495,P<0.05)。结论人脑胶质瘤组织中p63、p53蛋白高表达,二者在脑胶质瘤的发生、发展中发挥重要作用。     

肝细胞肝癌组织中p53、E—cadherin、CD34的表达变化及意义

采用免疫组化法检测41例肝细胞肝癌（HCC）组织中的p53、E-cadherin和CD34。结果显示,HCC组织中p53、E-cadherin和CD34阳性表达率分别为63．4％、48．8％和100％。p53的表达与患者血清AFP水平、癌组织Edmondson分级有关（P均〈0．01）;E-cadherin的表达在肿瘤〉5cm和癌组织Edmondson分级Ⅲ、Ⅳ级的HCC患者中明显降低（P均〈0．05）;E-cadherin表达阴性的患者肝切除术后5a生存率明显降低。认为p53在HCC组织中呈高表达,HCC组织中E—cadherin表达阴性的患者预后较差。     

老年胃癌活检标本与手术切除胃癌组织p53、c-erbB-2、p21、nm23基因表达检测的比较研究

目的 探讨胃镜活检标本与手术切除胃癌组织p53、c-erbB-2、p21、nm23联合基因产物表达检测对胃癌诊断与治疗的价值。方法 应用免疫组化技术检测了胃癌胃镜活检标本及手术切除胃癌组织p53、c-erbB-2、p21、nm23等基因产物表达并对胃镜活检标本与手术切除胃癌组织的检测结果进行了比较。结果 p53蛋白表达阳性率38．0％～46．2％，c-erbB-2为36．5％-59．2％，p21为39．4％～69．2％，nm23为34．6％-71．8％，在非胃癌组织（胃、十二指肠溃疡、胃息肉、重度不典型增生胃炎）未见c-erbB-2、p21、nm23等基因表达。c-erbB-2、p21的表达与胃癌的分化程度有关，p21、nm23基因表达与肿瘤浸润深度、肿瘤转移程度有关。p53、c-erbB-2、p21、nm23四种肿瘤蛋白在胃镜活检标本和手术切除标本中表达无显著性差异，胃镜活检标本和手术切除标本四种基因产物表达检测阳性符合率分别为92．2％、85．5％、96．9和94．2％。结论 对胃癌组织尤其胃癌胃镜活检标本p53、c-erbB-2、p21、nm23基因表达检测在胃部良恶性肿瘤鉴别、非手术临床分期的判断及指导临床治疗等方面具有一定价值。     

胰腺癌p53上下游基因Mdm2、p21WAF/CIP1以及p14ARF蛋白的表达及相互作用关系

在肿瘤的形成过程中,有两条通路研究的较为深入,即pRb(p16-pRb-cyclin D1)和p53(ARF-Mdm2-p53-p21WAF/CIP1)通路,由于p53突变在胰腺癌中是一主要事件,我们利用传统的免疫组化技术并结合新兴的组织芯片技术检测p53通路中相关基因:ARF、Mdm2、p53和p21在胰腺癌、癌旁与良性病变组织中的表达情况,以探讨四种蛋白在胰腺癌组织中的改变情况、临床意义及相互作用关系.     

血吸虫病患者肝癌细胞p53、p16蛋白表达的相关性研究

目的:探讨血吸虫病合并原发性肝细胞癌(hepatocellular carcinoma,HCC)与p53、p16蛋白的基因之间的相关性及其作用机制。方法:58例HCC患者分为两组:1组(HCC合并血吸虫病组)23例和2组(HCC不合并血吸虫病组)35例。采用免疫组织化学方法检测所有患者的p53、p16蛋白表达。结果:p53蛋白阳性表达率在1组中和2组中分别为73.9%(17/23)和31.4%(11/35),两组比较差异有显著性意义(P<0.01);而p16蛋白阳性率在1组和2组中分别为34.8%(8/23)和28.6%(10/35),两组比较差异无显著性意义(χ2=0.25,P>0.05)。p53蛋白及p16蛋白同时阳性仅7例,阳性率为12.1%(7/28);其中1组为13.0%(3/23),2组为11.4%(4/35),两组比较差异无显著性意义(χ2=0.052,P>0.05)。结论:患有日本血吸虫的肝细胞癌患者的癌组织中有较高的p53基因突变率,说明血吸虫感染对肝细胞癌组织细胞中p53突变蛋白的过量表达有促进作用;同时,血吸虫患者肝细胞癌患者肝组织细胞中p16肿瘤抑制基因-p16基因的丢失普遍。p16蛋白表达缺失,其与p53突变蛋白的过量表达同时存在,两者共同促进肿瘤的增长,可能是HCC恶性增殖的原因之一。     

肝胆管癌丙型肝炎病毒感染与p53和p21WAFD/CIP1异常表达的关系

为探讨丙型肝炎病毒（ＨＣＶ）感染与Ｐ５３和Ｐ２１ＴＷＡＦ－／ＣＩＰ１基因的关系。采用 化技术对２９例原发性肝胆管癌中ＨＣＶ抗原（ＮＳ５－Ａｇ）、ｐ５３和ｐ２１＾ＷＡＦＩ＝／ＣＩＰ１蛋白表达进行研究。结果：２９例胆管癌中ＮＳ５－Ａｇ、ｐ５３及Ｐ２１ＴＷＡＦＩ／ＣＩＰＩ蛋白表达进行研究。结果：２９例胆管癌中ＮＳ５－Ａｇ、Ｐ５３display structure     

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.     

The role of XPB in cell apoptosis and viability and its relationship with p53, p21 and c-myc in hepatoma cells

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.     

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.     

Alteration of the <Emphasis Type="Italic">p14</Emphasis><Superscript><Emphasis Type="Italic">ARF</Emphasis></Superscript> gene and p53 status in human hepatocellular carcinomas

Background The INK4a/ARF locus encodes p16^INK4a and p14^ARF, both of which are crucial for two tumor suppressor pathways, retinoblastoma (RB)/p16^INK4a and p53/ARF. Inactivation of RB/p16^INK4a was frequently reported, but alterations of the p14^ARF gene in hepatocellular carcinoma (HCC) in the Japanese population have been insufficiently analyzed.Methods To determine the role of p53/ARF alteration in hepatocarcinogenesis, we examined 44 HCCs for mRNA expression, deletion, mutation, and promoter hypermethylation of the p14^ARF gene; alterations of p53 were also analyzed in the same series of HCCs.Results Homozygous deletion, spanning from exon 1 to exon 2, was found in 1 HCC mutations within exon 2 were found in 2 HCCs, but no promoter hypermethylation was detected. All 3 HCCs with p14^ARF alteration were well differentiated. Twelve of the 44 HCCs (27.2%) showed immunohistochemical evidence of p53 alteration; however, only 1 of the tumors with p53 alteration was well differentiated. TaqMan polymarase chain reaction (PCR) indicated that the expression of p14^ARF in HCCs was higher than in that in all but three of the corresponding non-tumorous tissues (P < 0.0001), and increased expression of p14^ARF seemed to be associated with poorly differentiated phenotype. Absence of p14^ARF expression was seen in only one HCC, with homozygous deletion of the p14^ARF gene.Conclusions Compared with p53 alteration, p14^ARF alteration does not occur frequently, but may play a role in a subset of Japanese HCCs in the early stage of hepatocarcinogenesis. On the other hand, overexpression of p14^ARF was frequently observed in HCC, especially in poorly differentiated tumors, probably reflecting oncogenic stimuli in these tumors.     

Effect of indomethacin on cell cycle proteins in colon cancer cell lines

AIM: To study the effect of indomethacin (IN) on human colon cancer cell line SW480 with p53 mutant and SW480 transfected wild-type p53 (wtp53/SW480) in vitro and investigate molecular mechanism of anti-tumor effect of IN on colon cancer. METHODS: SW480 cells and wtp53/SW480 cells were treated with different concentrations of IN respectively, the expressions of CDK2, CDK4 and p21WAF1/CIP1 protein were detected by Western blotting. RESULTS: IN gradually down-regulated the expression of CDK2, CDK4 protein of wtp53/SW480 cells in a dose-dependent manner, and inhibitory effect reached the maximum level at 600 μmol/L; IN up-regulated the expression of p21WAF1/CIP1 protein in a dose-dependent manner at a certain concentration range, and the expression reached the maximum level at 400 μmol/L, and returned to the base level at 600 μmol/L. The expression of CDK2, CDK4 and P21WAF1/CIP1 protein of SW480 cells did not change. CONCLUSION: IN exerts antitumor effect partly through down regulation of the expression of CDK2, CDK4, protein and up regulation of the expression of p21WAF1/PIC1.     

P21/WAF1 is an independent survival prognostic factor for patients with hepatocellular carcinoma after resection.

BACKGROUND/AIMS: The cyclin kinase inhibitor p21/WAF1 is regulated by p53-dependent or independent pathways and inhibits the action of proliferating cell nuclear antigen (PCNA). The prognostic role of p21/WAF1 in hepatocellular carcinoma (HCC) is ambiguous. To further clarify this, we examined the expression of three genes in HCC. METHODS: A total of 122 resected HCC specimens were collected from 1987 to 1998. Expression of p21/WAF1, p53, and PCNA in HCC was analysed by immunohistochemistry. RESULTS: Immunoreactivity was detectable for p21/WAF1 in 37%, and for p53 in 41.8% of HCCs. Positive expression of both genes does not relate to each other, but both are associated with a high PCNA labelling index (LI) (P<0.05) in tumour. p53 (+) is also associated with high serum alpha-foetoprotein (alphaFP) (P<0.001), tumour dedifferentiation (P=0.001) and advanced pathologic stages (P=0.017). However, p21/WAF1 (+) did not show clinicopathologic significance. Survival analysis indicated that poor prognostic factors were p21/WAF1 (-) (P=0.024), p53 (+) (P=0.008), high PCNA (P<0.001), tumour without capsule (P=0.001), poor tumour differentiation (P=0.004), advanced pathologic stage (P<0.001), and high serum alphaFP(P<0.001). Independent factors were p21/WAF1 expression, pathologic stage, and PCNA. CONCLUSION: In HCC, increased proliferation index PCNA is significantly associated with positive p53 and p21/WAF1. But p21/WAF1 expression did not relate to p53 expression. P21/WAF1 (+) is a good event and serves as an independent survival prognostic factor for HCC, which is a novel finding apart from previous reports.     

Does melatonin induce apoptosis in MCF‐7 human breast cancer cells in vitro?

Melatonin inhibits proliferation of the estrogen-responsive MCF-7 human breast cancer cells. The objective of this work was to assess whether melatonin not only regulates MCF-7 cell proliferation but also induces apoptosis. In this experiment we used 1,25-dihydroxycholecalciferol (D3) as a positive control because it inhibits MCF-7 cell proliferation and induces apoptosis. MCF-7 cells were cultured with either I nM melatonin, 100 nM D3 or its diluent to determine their effects on cell proliferation, cell viability, cell-cycle phase distribution, population of apoptotic cells, and expression of p53, p21WAF1, bcl-2, bcl-X(L) and bax proteins. After 24 or 48 hr of incubation, both melatonin and D3-treatment significantly decreased the number of viable cells in relation to the controls, although no differences in cell viability were observed between the treatments. The incidence of apoptosis, measured as the population of cells falling in the sub-G1 region of the DNA histogram, or by the TUNEL reaction, was similar in melatonin-treated and control cells whereas, as expected, apoptosis was higher among cells treated with D3 than in controls. The expression of p53 and p21WAF1 proteins significantly increased after 24 or 48 hr of incubation with either melatonin or D3. No significant changes in bcl-2, bcl-XL and bax mRNAs were detected after treatment with melatonin whereas in D3-treated cells, a significant drop in bcl-XL was observed. These data support the hypothesis that melatonin reduces MCF-7 cell proliferation by modulating cell-cycle length through the control of the p53-p21 pathway, but without clearly inducing apoptosis.