Effect of Boschniakia rossica on expression of GSTP,p53 and p21^(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats

AIM To investigate the effect of Boschniakiarossica（BR）extract on expression of GST-P,p53 and p21rasproteins in early stage of chemicalhepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS The expression of tumor marker-placental form glutathione S-transferase（GST-P）,p53 and p21rasproteins were investigated byimmunohistochemical techniques and ABCmethod.Anti-inflammatory activities of BR werestudied by xylene and croton oil-induced mouseear edema,carrageenin,histamine and hotscald-induced rat pow edema,adjuvant-inducedrat arthritis and cotton pellet-induced mousegranuloma formation methods.RESULTS The 500 mg/kg of BR-H2O extractfractionated from BR-Methanol extract hadinhibitory effect on the formation of DEN-inducedGST-P-positive foci in rat liver（GST-P stainingwas 78% positive in DEN+AAF group vs 20%positive in DEN+AAF+BR group,P【0.05）andthe expression of mutant p53 and p21rasproteinwas lower than that of hepatic preneoplasticlesions（33% and 22% positive respectively inDEN+AAF group vs negative in DEN+AAF+BRgroup）.Both CH2Cl2 and H2O extracts from BRhad anti-inflamatory effect in xylene and crotonoil-induced mouse ear edema（inhibitory rateswere 26%-29% and 35%-59%,respectively）. BR-H2O extract exhibited inhibitory effect incarrageenin,histamine and hot scald-inducedhind paw edema and adjuvant-induced arthritis inrats and cotton pellet-induced granulomaformation in mice.CONCLUSION BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis. Both CH2CI2 and H2O extracts from BR exerted anti-inflammatory effect in rats and mice.

Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

AIM To evaluate the relationship between expression of ras, p53, bcl 2 gene products, and hepatocarcinogenesis since endotoxemia produced from lipopolysaccharide admi nistration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide. 〖WTH4〗METHODS〓〖WTXFZ〗The hepatocarcinoma model was induced by oral intake of 0 03% thioacetamide for six months. During the induction of hepatocarcinoma model, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied to quantitative analysis of the expression of oncogene proteins. RESULTS In this model system, overexpression of ras p21 protein mainly occurred on precancerous cell population or in early stage of hepatocyte transformation. And the levels of ras p21 declined when nuclear DNA aneuploid increased. Expression of bcl 2 protein slowly and steadily rose with more hepatocytes staying in S+G2M phases as the hepatocarcinoma became more malignant. P53 was moderately expressed during the hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes of ras, p53 and bcl 2 gene products. CONCLUSION Over expression of oncogene ras p21 was likely to be a precursor of the premalignant hepatocytes and it might be responsible for the initiation of hepatocarcinogenesis. Bcl 2 protein expression is proportional to the severity of the malignancies. P53 may be a key pathway on the transformation and development of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expressions of oncogene proteins reflected the properties of the premalignant and malignant cells, but not directly related to endotoxemia statistically.[JP]

Relationship between ras p53 gene RNA and protein expression and HCC metastasis

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Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

To identify the apoptotic cells in gastric MALT lymphoma and its relationship between bcl-2 and p53 gene expression. Methods: TdT-mediated dUTP biotin Nick End labeling (TUNEL) and immuno-histochemistry ABC method were used to display apoptotic cells and the gene protein expression of bcl-2 and p53 independently. Results: Apoptotic indices (AI) in high-grade MALT lymphomas were significantly higher than in mixed-grade group and low-grade group (P<0.05). Bcl-2 was expressed in 83% of low-grade tumors, 61.6% of the median-grade tumors and 43.7% of high-grade tumors. An inverse correlation was observed between the expression of bcl-2 and apoptotic indices. Only 27 cases were p53 positive. The frequency of p53 positivity was significantly increased as the histologic grade advanced (P<0.05). There was also an inverse correlation between the expression of bcl-2 and p53. Conclusion: Apoptosis may be important in tumors development and transmission. p53 and bcl-2 were important regulatory genes of apoptosis and may be associated with transformation from low- grade to high-grade lymphomas.

Effect of Wild-type p53 Gene Transfection on the Growth and Radiotherapeutic Sensitivity of Human Glioma Cells

Summary： To evaluate the effect of wild type p53 gene on the growth and radiotherapeutic sensitivity of human glioma cells, plasmid PC53 SN3 carrying wild type p53 gene was transfected into U251 cells, p53 gene expression in transfected cells was detected by RT PCR, and the cell growth inhibition and apoptosis in the absence or presence of irradiation were assessed by MTT and flow cytometry. The transfection of p53 gene into U251 cells was confirmed by RT PCR. MTT showed that p53 gene alone induced strong inhibitory effect on the growth of U251 cells （inhibition rate （IR）： （79.60±5.69）%）, The killing effect of irradiation alone on U251 cells was not strong （IR： （17. 06±4.35）%, （17.39±1.67）%, （18.73±4.68）%） and increased with the irradiation doses （3, 6, 9 Gy）. When combined treatment of wild-type p53 gene transfection and irradiation was used, the effect was significantly increased （IR：（80.60±5.35）%, （90.30＋1.67） %, （91.30±2.01） %）. Theapoptosis rate of U251 cells induced by p53 gene transfectionwas 17.38 %. The rate induced by irradiation increased （4. 61%, 4. 84%, 5. 40 %） with the irradiation doses （3, 6, 9 Gy）. Theapoptosis rate was also significantly increased （17.80%, 20.03%, 22.34%） after combined treatment of p53 and irradiation with different doses （3, 6, 9 Gy）. It is concluded that wildtype p53 gene and irradiation could result in synergistic inhibitory effect on the growth of human glioma cells.

Overexpression and mutations of tumor suppressor gene p53 in hepatocellular carcinoma

AIMS To examine the prevalance of p53 mutations in hepatocellular carcinoma (HCC) from Chongqing area and the relationship between the p53 mutations and clinicopathological features of HCC,as well as the risk factors. METHODS The overexpression and point mutations of tumor suppressor gene p53 in 38 cases of HCC were detected by a sensitive antigen retrieval fluid (ARF) immunohistochemical method and polymerase chain re- action(PCR)-restriction fragment length polymorphism (RFLP),and single strand conformation polymorphism (SSCP)-silver staining analysis. RESULTS The results showed that 16 of 38 HCCs had positive p53 protein (42.1%),7 HCCs had p53 mutation at 249 (18.4 % ) and 2 HCCS had point muta- tion within exon 7 other than 249. Among 9 cases of HCC with mutations,8 cases demonstrated positive p53 protein,its coincidental rate was 88.9%. The overexpression and mutations of p53 were significantly related to the differentiation and metastasis of HCCs. The frequency of p53 mutations was consistent with high prevalence of HBV and a moderate aflatoxin B1 (AFB1) exposure in our area. CONCLUSIONS The results suggest that AFB1 acts synergistically with HBV in the generation of p53 mutations. Furthermore,dietary exposure to AFB1 may mainly contribute to the tumor specific mutation at codon 249,while HBV may account for other scattered mutations in HCC.

Infrequent p53 gene mutation and expression of the cardia adenocarcinomas from a high-incidence area of Southwest China

INTRODUCTIONAdenocarcinomas of the cardia are the lesionsarising from the proximal stomach or within 3 cm ofthe gastroesophageal junction.These cancerstended to be advanced at the time of presentation,usually with poor prognosis.In recent decade,the incidence of adenocarcinoma of gastric eardiaand esophagus are increasing steadily,while therehas been a decrease in the proportion of the cancersarising from the distal stomach.The

Correlation of p53 over-expression and alteration in p53 gene detected by polymerase chain reaction-single strand conformation polymorphism in adenocarcinoma of gastric cancer patients from India

AIM： To study the alterations in p53 gene among Indian gastric cancer patients and to correlate them with the various clinicopathological parameters. METHODS： A total of 103 gastric cancer patients were included in this study. The p53 alterations were studied by both immunohistochemical method as well as polymerase chain reaction （PCR）-single strand conformation polymorphism （SSCP） analysis. We only studied four （exon 5, 6, 7, and 8） of the 11 ,p53 exons. The alterations in p53 were also correlated with respect to various clinicopathological parameters. RESULTS： Among 103 cases, p53 over-expression and alteration were detected in 37 （35.92%） and 19 （18.44%） cases, respectively. Most of the ,p53 alterations were found at exon 5 （31.54%）, followed by exon 6 （26.31%）, exon 7 （21.04%） and exon 8 （21.04%）. A significant correlation of p53 overexpression was found with p53 alteration （P = 0.000）. Concordance between ,p53 alteration （as detected by SSCP） and over-expression [as detected by immunohistochemistry （IHC）] was found in 75% cases. We found that IHC-positive/SSCP-negative cases accounted for 21% of cases and IHC-negative/SSCP- positive cases accounted for remaining 4% cases. CONCLUSION： Our results show that p53 gene mutations are significantly correlated with p53 protein over-expression, with 75% concordance in over-expression and alteration in the p53 gene, but 25% disconcordance also cautions against the assumption that p53 over-expression is always associated with a gene mutation. There may be other mechanisms responsible for stabilization and accumulation of p53 protein with no evidence of gene mutation that reflect an accumulation of a non-mutated protein, or a false negative SSCP result.

Changes of p53 protein blood level in esophageal cancer patients and normal subjects from a high incidence area in Henan,China

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Expression and significance of VEGF and p53 in rat degenerated intervertebral disc tissues

Objective:To discuss the expression and significance of vascular endothelial growth factor (VEGF) and p53 expression in rat degenerated intervertebral disc(IVD) tissues.Methods:A total of 78 even-aged Sprague-Dawley(SD) rats(observation group) were chosen to establish a rat IVD degeneration model with anterior limbs and tail-removing method.Three months after modeling, rats were executed and degenerated IVD tissues were obtained for pathological biopsy so as to observe VEGF and p53 expression thereof.Meanwhile,IVD tissues from 8 healthy rats were gained for comparison.Results:Capillary infiltration was found in 62.8%of cases in observation group. Positive expression of VEGF and p53 was seen in 74.4%and 59.0%of cases respectively with a co-expression rate of 53.8%.VECF and p53 expression in degenerated IVD tissues with capillary infiltration was higher than that in non-infiltration group(P【0.05).Conclasion:A coordinate expression of VEGF and p53 in rat degenerated IVD tissues is demonstrated,indicating that they are both involved in neovascularization and infiltration,which accelerates IVD degeneration.

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Clinical significance of immunohistochemica study of P53 protein in colorectal carcinoma

AIMS To determine the clinical significance of P53 protein ex-pression in colorectal carcinoma.METHODS The expression of P53 protein in 92 colorectal car-cinomas was examined using the monoclonal antibody PAb 1801.Correlation between P53 protein expression and prognosis in col-orectal carcinoma was analyzed using log-rank test.RESULTS The frequency of P53 protein expression was 57.61%,corresponding with Dukes’ staging.Analysis of survivordata demonstrated that the survival rate of colorectal carcinomawith positive P53 protein group was lower than that of negativeP53 protein group.CONCLUSIONS We conclude that the expression of P53 pro-tein is correlated with poor prognosis in colorectal carcinoma.

Experimental research for specific down-regulated expression of p53 gene by individual antisense RNA in vitro

Objective： To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro. Methods： The single strand antisense transcription system containing mt-p53 exon 8 sequence （pGEM3zf（＋/-）p53exon8） was constructed. The ligation of antisense RNAwith mt-p53 gene was confirmed by in situ hybridization; MDA-MB-231 human breast cancer cells were transfected with ASp53exon8＇RNA cotionic liposome-mediated. Expression of mt-p53 protein was examined by immunocytochemical staining and Western blot. Cell proliferation was evaluated by MTT assay; Cell cycle distribution was determined by flow cytometry （FCM）; Apoptosis was observed by TUNEL. Results： In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. ASp53exon8＇RNA transfection induced inhibition of cell proliferation, G2/M phase arrest and increasing apoptotic rates. In addition, expression of p53 protein was down-regulated. Conclusion： pGEM3zf（＋/-）p53exon8 was well constructed and ASp53exon8＇RNA can block mt-p53 gene expression specifically and then inhibit MDA-MB-231 cell proliferation in vitro, which may serve as therapeutic means for human malignancy.

CIN和子宫颈鳞癌的HPV16/18感染与p53、MDM2蛋白表达的关系

目的研究子宫颈鳞癌及C IN的HPV感染、p53蛋白和M DM 2蛋白的表达及三者之间的相互关系。方法选取子宫颈鳞癌61例、C IN 47例、正常子宫颈组织54例,分别采用原位杂交技术检测HPV 16/18感染,用免疫组化技术S-P法检测p53蛋白和M DM 2蛋白。结果HPV 16/18感染率鳞癌组为62.3%(38/61),C IN组为70.2%(33/47),对照组为16.7%(9/54)。C IN组和鳞癌组的HPV 16/18的感染率均显著高于对照组(P值均<0.05)。而C IN组与鳞癌组的HPV 16/18感染率差异无显著性(P>0.05)。C IN组、鳞癌组、对照组的p53阳性率分别为19.1%(9/47)、26.2%(16/61)、3.7%(2/54)。C IN组、鳞癌组、对照组的M DM 2阳性率分别为14.9%(7/47)、26.2%(16/61)、7.4%(4/54)。C IN组和鳞癌组的p53阳性率均显著高于对照组(P值均<0.05),C IN组与鳞癌组比较差异无显著性(P>0.05)。M DM 2阳性率鳞癌组显著高于对照组(P<0.05),而在C IN组和鳞癌组之间、C IN组和对照组之间差异均无显著性(P值均>0.05)。在本实验各组中,p53与M DM 2之间的阳性率均无统计学相关(P值均>0.05)。p53阳性率和HPV 16/18感染之间差异无显著性(P值均>0.05),而M DM 2的阳性率与HPV 16/18感染仅在子宫颈鳞癌组呈正相关(P<0.05)。结论C IN及子宫颈鳞癌与HPV 16/18感染关系密切;p53和M DM 2均参与子宫颈癌及癌前病变的发生,但p53的突变、M DM 2的扩增和过表达均不占主导地位。

急性白血病细胞bcl-2、c-myc和p53基因表达的研究

目的 :研究bcl 2、c myc、p53基因在急性白血病 (AL)细胞中的表达及其与AL的分型、临床特征、疗效及预后因素的关系。方法 :采用免疫组化方法检测 54例初治AL骨髓细胞bcl 2、c myc、p53蛋白表达情况 ,分析其与FAB分型、临床特征、疗效及预后因素的关系。结果 :AL骨髓细胞中bcl 2、c myc及 p53基因的表达显著高于正常对照组 (P <0 .0 5)。bcl 2、c myc、p53基因表达在急性髓系白血病 (AML)与急性淋巴细胞白血病(ALL)差异不显著 ;在AML亚型中 ,bcl 2在M4 及M5中的表达高于M1 、M2 和M3(P <0 .0 5) ;而c myc及p53的表达在各亚型中差异无显著性意义 (P >0 .0 5)。bcl 2的表达与初诊时的白细胞数呈正相关 (r =0 .44,P <0 .0 5) ,与疗效呈负相关 (r =- 0 .40 ,P <0 .0 5) ,与c myc的表达呈正相关 (r =0 .51 ,P <0 .0 5) ;c myc的表达与初诊时的白细胞数呈正相关 (r =0 .50 ,P <0 .0 5)。结论 :bcl 2、c myc、p53基因的紊乱可能在AL的发病中起着重要作用 ,还与白血病的某些临床特征。

原位杂交法检测MDR1基因在食管癌中的表达及与p53的关系

目的:了解食管癌组织MDR1基因表达的临床意义及其与p53的关系。方法:用原位杂交法检测了57例食管癌组织标本中MDR1基因的表达,免疫组化法检测了其中44例p53蛋白的表达并进行临床病理分析及相关性分析。结果:57例食管癌组织中MDR1mRNA阳性表达率为35%(20/57),其中食管癌无淋巴结转移的患者阳性表达为29%(8/29),有淋巴结转移患者阳性表达分别为41%(12/28),高于无淋巴结转移患者的表达。临床Ⅱ期食管癌患者阳性表达分别为27%(8/30),临床Ⅲ-Ⅳ期阳性表达为44%(12/27),高于临床Ⅱ期患者的表达。食管癌患者MDR1mRNA表达与肿瘤大小、部位、分化程度、肿瘤浸润的程度无明显相关。44例食管癌组织中p53蛋白阳性表达率为73%(32/44),其中食管癌浸润至肌层,p53表达率为45%(5/11),肿瘤浸润至外膜,p53表达率为82%(27/33),差异有显著性,P<0.01。食管癌患者p53表达与肿瘤大小、部位、分化程度、有无淋巴结转移、临床分期无明显相关。32例p53蛋白表达阳性的组织中有10例MDR1mRNA表达阳性,它们之间的表达在统计学上无明显相关。结论:MDR1基因及突变型p53在食管癌组织中均有过表示,其表达可能与食管癌进展相关。在食管癌组织中MDR1基因表达可能并非受p53的调控。

挥发性有机化合物对p53、rasp21、p16表达影响

目的探讨苯、甲醛混合物对NIH/3T3细胞中p53、rasp21、p16表达的影响。方法采用免疫组化法（SABC）观察甲醛、苯混合物致NIH/3T3细胞p53、rasp21、p16的表达情况。结果经苯、甲醛混合物处理后的第9～15d,p53、rasp21、p16表达阳性率分别为26%～45%,50%～60%,阳性率远远高于阴性对照组,差异有统计学意义（P〈0.01）;p16的阳性率为64%～40%,阳性率低于阴性对照组,差异有统计学意义（P〈0.01）。结论（VOCs）可使突变型的p53蛋白表达率增高;上调ras癌基因家族的表达产物rasp21;下调肿瘤抑制基因p16的表达。这可能是VOCs致癌的原因之一。

p53基因在乳腺肿瘤中的表达及其意义

[目的]探讨乳腺肿瘤组织中p 53基因的表达情况及其意义.[方法]利用免疫组织化学技术观察62例乳腺癌及14例乳腺纤维腺瘤中突变型p 53基因的表达情况.[结果]乳腺癌组织中的p53基因表达率明显高于乳腺纤维腺瘤;Ⅲ期乳腺癌组织中p53基因的表达率高于I期及Ⅱ期,且Ⅲ期与I期及Ⅱ期之间均各有显著性差异,与TNM分期呈正相关;p53基因的表达与乳腺肿瘤的组织学类型、组织学分级、淋巴结转移、ER及PR状况、年龄及家族史之间均无相关关系;朝鲜族与汉族之间亦无显著性差异.[结论]p 53基因的表达可作为判断乳腺癌及TNM分期的指标.

大肠癌组织中p53基因的表达及意义

采用S-P免疫组织化学的方法,配对检测了37例大肠癌组织及同一病人的正常粘膜上皮中p53蛋白的表达情况。结果表明,p53蛋白表达阳性19例,阴性18例,阳性率为51.4％,而配对的大肠正常粘膜检测结果均呈p53阴性;p53蛋白表达与病人性别、年龄、Dukes分期等因素无明显相关性,而与淋巴结转移、浸润深度和不同分化程度等存在明显相关性。提示p53蛋白的检测可以作为大肠癌病人病情诊断和预后分析的辅助手段。