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.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

幽门螺杆菌感染与P53-Rb肿瘤抑制系统在胃癌发生中的作用

目的 :研究幽门螺杆菌 (helicobacterpylori,Hp)感染与P5 3 Rb肿瘤抑制系统在胃癌发生中的作用。方法 :对 4 2例慢性胃炎 (chronicgastritis,CG) ,71例肠上皮化生 (intestinalmetaplasia ,IM) (其中IMⅠ～Ⅱ 4 6例 ,IMⅢ 2 5例 ) ,75例胃上皮不典型增生 (dysplasia ,Dys) (其中DysⅠ 2 1例 ,DysⅡ～Ⅲ 5 4例 )及 84例胃癌 (gastriccancer,GC)组织采用Warthin Starry细菌染色及S P免疫组化法检测Hp ;HID AB(pH 2 .5 ) PAS粘液染色检测胃粘膜上皮及肿瘤组织中的粘液性质 ;S P免疫组化法检测P5 3和Rb蛋白的表达。结果 :在DysⅠ和GC病变中的Hp感染组 ,Rb蛋白的阳性表达率明显高于非感染组 (P <0 .0 5 ) ;在病变的发展过程中 ,虽然未发现P5 3蛋白的阳性表达与Hp感染有明显相关 (P >0 .0 5 ) ,但从P5 3 Rb肿瘤抑制系统的角度分析 ,该系统异常在Dys和GC病变中与Hp感染则有明显相关性 (P <0 .0 1)。结论 :Hp感染可能通过引起Dys和GC病变中P5 3 Rb肿瘤抑制系统的异常从而导致胃癌发生。

PTEN： a default gate-keeping tumor suppressor with a versatile tail

The tumor suppressor PTEN controls a variety of biological processes including cell proliferation, growth, migration, and death. As a master cellular regulator, PTEN itself is also subjected to deliberated regulation to ensure its proper function. Defects in PTEN regulation have a profound impact on carcinogenesis. In this review, we briefly discuss recent advances concerning PTEN regulation and how such knowledge facilitates our understanding and further exploration of PTEN biology. The carboxyl-tail of PTEN, which appears to be associated with multiple types of posttranslational regulation, will be under detailed scrutiny. Further, a comparative analysis of PTEN and p53 suggests while p53 needs to be activated to suppress tumorigenesis （a dormant gatekeeper）, PTEN is probably a constitutive surveillant against cancer development, thus a default gatekeeper.

A Study of Radiation-Induced Telomere Instability Using Multiplex Ligation-Dependent Probe Amplification (MLPA)

The integrity of the chromosomes for two WIL2-derived lymphoblastoid cell lines (TK6 and WTK1) in the presence and absence of ionizing radiation was analyzed by Multiplex Ligation-Dependent Probe Amplification (MLPA). The TK6 cell line has the native p53 tumor-suppressor gene, whereas WTK1 cells contain a p53 mutation. Each cell line was isolated pre- and post-irradiation (2 and 3 Gy) and analyzed by MLPA. The impact of irradiation on these two cell lines was investigated using probes that target specific regions on chromosomes associated with subtelomeric regions. Results indicate that WTK1 and TK6 are impacted differently after irradiation, and that each cell line presents its own unique MLPA profile. The most notable differences are the appearance of a number of probes in the post-irradiated MLPA profile that are not present in the controls, and two unique probe signals only seen in WTK1 cells. These results build on our previous studies that indicate how different human cell lines can be affected by radiation in significantly different ways depending on the presence or absence of wild type p53.

脑星形细胞瘤P^(53)抗癌基因突变的研究

目的研究Ｐ５３抗癌基因突变与脑星形细胞瘤恶性程度的关系。方法采用非同位素ＰＣＲ－ＳＳＣＰ分析法检测了３１例星形细胞瘤患者Ｐ５３抗癌基因的突变情况。结果（１）９例存在突变，突变区域分布于第５～８外显子中，以第６外显子突变率最高，第９外显子未测得突变病例。（２）２例存在多点突变，其中１例同时存在第５、６外显子突变，另１例同时存在第６、７外显子突变。（３）病理级别为Ⅰ、Ⅱ级组的突变率为０，Ⅲ级组的突变率为３６．４％，Ⅳ级组的突变率为５０％。Ⅲ、Ⅳ级组与Ⅰ、Ⅱ级组的突变率比较，均存在极显著差异（Ｐ＜０．０１）。

缺碘地区甲状腺低分化癌的P^(53)基因变异的研究

本研究组已经报道过缺碘地区甲状腺癌的组织学特征和P^(53)蛋白过表达之间的关系。为进一步明确本地区的少基因变异特点,我们对P^(53)蛋白过表达比较集中的甲状腺低分化癌标本的PCR产物进行单链构象多态性(SSCP)分析,结果为15例低分化癌中有14例P^(53)染色呈阳性,其阳性率为93.3％。P^(53)基因变异共在7例中被检出,检出率为46.7％。变异最多发生于外显子5中。结果显示P^(53)基因变异与甲状腺低分化癌密切相关,并且提示发生变异的甲状腺乳头状癌的恶性程度偏高。

乙型肝炎病毒相关性肝癌与p53基因变化的关系

目的：探讨与ＨＢＶ感染相关的肝癌其ｐ５３基因变化及意义．方法：收集１６例西安地区的肝癌及其癌旁肝组织，应用Ｓｏｕｔｈｅｒｎ杂交检测了组织中ＨＢＶＤＮＡ的状态．免疫组化确定ＨＢＶ的ＨＢｓＡｇ，ＨＢｘＡｇ和肿瘤抑制基因Ｐ５３蛋白的分布，采用ＰＣＲ扩增后直接ＤＮＡ序列分析，检测了ｐ５３基因第５～８外显子的基因序列．结果：１６例中１３例ＨＢＶＤＮＡＳｏｕｔｈｅｒｎ杂交阳性，１０例癌组织和１３例癌旁组织ＨＢｘＡｇ染色阳性，９例Ｐ５３蛋白染色阳性．ｐ５３基因序列分析５例存在点突变，其中仅１例位于２４９编码区．结论：与ＨＢＶ感染密切相关的肝癌，ｐ５３基因第２４９编码区的点突变并不显著，进一步表明细胞内Ｐ５３蛋白积聚可能与ＨＢｘＡｇ的存在有关，二者以结合的形式存在并使ｐ５３功能失活在肝癌的发生机制中具有重要意义．

人肝癌细胞瘤QGY-7703的p53基因及其表达研究

本文利用等位基因分析,PCR-SSCP,RT-PCR/序列分析,Northern印迹,免疫组织化学,Western印迹,免疫沉淀等方法对人肝癌细胞株QGY-7703的p53基因背景进行了研究,发现17号染色体短臂可能存在等位基因缺失,在p53基因的编码序列上没有发现任何突变,但发现其mRNA和蛋白表达水平很低,表明在QGY-7703细胞中p53基因的低水平表达可能同细胞的恶性程度有关。

p53 mutations in colorectal cancer-molecular pathogenesis and pharmacological reactivation

Colorectal cancer(CRC) is one of the most common malignancies with high prevalence and low 5-year survival.CRC is a heterogeneous disease with a complex,genetic and biochemical background.It is now generally accepted that a few important intracellular signaling pathways,including Wnt/β-catenin signaling,Ras signaling,and p53 signaling are frequently dysregulated in CRC.Patients with mutant p53 gene are often resistant to current therapies,conferring poor prognosis.Tumor suppressor p53 protein is a transcription factor inducing cell cycle arrest,senescence,and apoptosis under cellular stress.Emerging evidence from laboratories and clinical trials shows that some small molecule inhibitors exert anti-cancer effect via reactivation and restoration of p53 function.In this review,we summarize the p53 function and characterize its mutations in CRC.The involvement of p53 mutations in pathogenesis of CRC and their clinical impacts will be highlighted.Moreover,we also describe the current achievements of using p53 modulators to reactivate this pathway in CRC,which may have great potential as novel anti-cancer therapy.

Overexpression of P53 and its risk factors in esophageal cancer in urban areas of Xi′an

IM To investigate the risk factors of esophageal cancer (EC) in urban areas of Xi′an and to determine the association between overexpression of P53 and these risk factors.METHODS All cases (89) and controls (97) were permanent residents in urban areas of Xi′an, all cases of primary EC had been histologically confirmed, controls were inpatients with noncancer and nonsmokingrelated disease. Cancer tissues and tissues adjacent to the cancer of 65 cases and 24 available normal esophageal tissues of controls were detected for P53 overexpression by the immunohistochemical method.RESULTS The smoking and familial history of cancer were significantly associated with EC in Xi′an inhabitants. The laboratory assay indicated that P53 positive stain in EC was 500%(34/65) and 61%(4/65) in tissues adjacent to the cancer, but no positive stain was found in normal esophageal tissues of controls. The results showed that P53 overexpression in EC was closely related to smoking and cases with familial history of cancer.CONCLUSION Smoking and familial cancer history were important risk factors for EC, and the alteration of P53 gene may be due to smoking and inheritance factors..

Changes of p53 and Waf1p21 and cell proliferation in esophageal carcinogenesis

AIM To study the correlationship between the changes of p53, Waf1p21 and the cell proliferation determined by PCNA at different stages of human esophageal carcinogenesis. METHODS Biopsied and resected esophageal tissues from a high risk population for esophageal cancer in northern China were used in this study. All the specimens were fixed with 85% alcohol and further processed with routine histology. The avidin biotin peroxidase complex (ABC) method was used for the detection of p53, Waf1p21 and PCNA. RESULTS The strong nuclear staining for p53, Waf1p21 and PCNA was observed in the normal esophageal epithelium and the epithelia with different severities of lesions. As the lesions progressed to dysplasia (DYS) and to esophageal squamons cell carcinoma (SCC), the percentage of Waf1p21 immunoreactivity decreased. The number of Waf1p21 immunostaining positive cells increased slightly from normal to basal cell hyperplasia (BCH), but there was no further increase in DYS and in SCC. The total number of positive cells for Waf1p21 stain appeared to be lower than that of p53 in normal and BCH esophageal epithelia and much lower in DYS and SCC. The Waf1p21 positive immunostaining cells were located at the third and forth cell layers in half of the samples examined, which was 2～4 cell layers higher than that of PCNA and p53 in the same histological categories of normal, BCH and DYS. CLNCLUSION The low levels of Waf1p21 at the stage of DYS may be related to a functional loss of p53. Other mechanisms may also be responsible to the lack of Waf1p21 expression in DYS and SCC.

Function of apoptosis and expression of the proteins Bcl-2,p53 and C-myc in the development of gastric cancer

INTRODUCTIONIn China ,the incidence and mortality of gastric cancer rank the second among all cancers. Recent development of cancer [1-20].The aim of this study was investigat the insight of apoptosis and bcl-2, p53 and C-myc protein expression in the development of gastric cancer .

P53 immunohistochemical scoring:an independent prognostic marker for patients after hepatocellular carcinoma resection

AIM: To confirm if p53 mutation could be a routine predictive marker for the prognosis of hepatocellular carcinoma (HCC) patients. METHODS: Two hundreds and forty-four formalin-fixed paraffin-embedded tumor samples of the patients with HCC receiving liver resection were detected for nuclear accumulation of p53. The percent of P53 immunoreactive tumor cells was scored as 0 to 3+ in P53 positive region (【10% -, 10-30% +, 31-50% ++, 】50% +++). Proliferating cell nuclear antigen (PCNA) and some clinicopathological characteristics, including patients' sex, preoperative serum AFP level, tumor size, capsule, vascular invasion (both visual and microscopic), and Edmondson grade were also evaluated. RESULTS: In univariate COX harzard regression model analysis, tumor size, capsule status, vascular invasion, and p53 expression were independent factors that were closely related to the overall survival (OS) rates of HCC patients. The survival rates of patients with 3+ for P53 expression were much lower than those with 2+ or + for P53 expression. Only vascular invasion (P【0.05) and capsule (P【0.01) were closely related to the disease-free survival (DFS) of HCC patients. In multivariate analysis, p53 overexpression (RI 0.5456, P【0.01) was the most significant factor associated with the OS rates of patients after HCC resection, while tumor size (RI 0.5209, P【0.01), vascular invasion (RI 0.5271, P【0.01) and capsule (RI-0.8691, P【0.01) were also related to the OS. However, only tumor capsular status was an independent predictive factor (P【0.05) for the DFS. No significant prognostic value was found in PCNA-LI, Edmondson's grade, patients' sex and preoperative serum AFP level. CONCLUSION: Accumulation of p53 expression, as well as tumor size, capsule and vascular invasion, could be valuable markers for predicting the prognosis of HCC patients after resection. The quantitative immunohistochemical scoring for P53 nuclear accumulation might be more valuable for predicting prognosis of patients after HCC resection than the common qualitative analysis.

Changes of NF-kB,p53,Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells:role of reactive oxygen species

AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Lack of correlation between p53 codon 72 polymorphism and anal cancer risk

AIM:To investigate the potential role of p53 codon 72 polymorphism as a risk factor for development of anal cancer. METHODS:Thirty-two patients with invasive anal carcinoma and 103 healthy blood donors were included in the study.p53 codon 72 polymorphism was analyzed in blood samples through polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing. RESULTS:The relative frequency of each allele was 0.60 for Arg and 0.40 for Pro in patients with anal cancer, and 0.61 for Arg and 0.39 for Pro in normal controls. No significant differences in distribution of the codon 72 genotypes between patients and controls were found. CONCLUSION:These results do not support a role for the p53 codon 72 polymorphism in anal carcinogenesis.

An experimental study on the expression of SV40 large tumor antigen in human brain tumors

Objective: To study the role of SV40 early region gene coding product large tumor antigen(Tag) expression and the interaction between Tag and tumor suppressors p53 and pRb in human brain tumorigenesis. Methods: Tag was investigated by immunoprecipitation followed by silver staining and Western blot in 65 cases of human brain tumors and 8 cases of normal brain tissues. Tag-p53 and Tag-pRb complexes were screened by immunoprecipitation and Western blot in 18 and 15 Tag positive tumor tissues respectively. Results: SV40 Tag was expressed generally in human brain tumors, its positive rate was 66. 2% (43 /65). However, Eight normal brain tissues were all negative for Tag, there was significant difference between them(P < 0. 05). Tag-p53 complex was detected in all of 18 Tag positive tumors as well as Tag-pRb complex in all of 15 Tag positive tumors. Conclnsion: SV40 Tag expression is associated with human brain tumorigenesis. The inactivation of p53 and pRh due to the formation of Tag-p53 and Tag-pRb complexes is possibly an important mechanism in the etiopathogenesis of human brain tumors.

银染PCR-SSCP分析膀胱癌P^(53)基因突变

为探讨P53基因突变与膀胱癌病理分级和临床分期的关系，应用银染PCR-SSCP技术分析46例膀胱癌石蜡标本中P53基因突变情况，结果显示膀胱癌中P53基因突变率为30．4％，分化不良的Ⅲ级膀胱癌P53基因突变率显著高于Ⅰ、Ⅱ级分化较好者。发生浸润和转移的T3－4期膀胱癌P53基因突变率显著高于Tia－T1期表浅性膀胱癌。提示：P53基因突变是膀胱癌恶性程度的生物学标志之一，可用来评估患者的预后。

长链非编码RNA-LA16c-313D11.11与突变型p 53在子宫内膜癌中的表达与临床意义

目的通过检测子宫内膜癌组织中长链非编码RNA(lncRNA)-LA16c-313D11.11和突变型p 53的表达以探讨其与子宫内膜癌发生、发展之间的关系。方法收集复旦大学附属妇产科医院2013年9月-2016年9月收治的54例子宫内膜癌患者的临床资料。采用实时荧光定量PCR和免疫组织化学染色法,分别检测lncRNA-LA16c-313D11.11和突变型p 53的表达。以lncRNA-LA16c-313D11.11相对表达量的中位数(1.168)为标准,将患者分为低表达组(≤1.168)与高表达组(>1.168)。对患者lncRNA-LA16c-313D11.11和突变型p 53的表达情况与临床和病理学特征进行相关性分析。结果lncRNA-LA16c-313D11.11高表达组和低表达组患者在国际妇产科联盟(FIGO)分期Ⅰ期、Ⅱ期、Ⅲ或Ⅳ期患者中的占比分别为66.67%和33.33%、4/7和3/7、3/11和8/11,差异有统计学意义(P<0.05)。高表达组和低表达组在发生淋巴结转移患者中的占比分别为3/11和8/11,在未发生淋巴结转移患者中的占比分别为65.12%和34.88%,差异有统计学意义(P<0.05)。突变型p 53阳性患者在子宫内膜样腺癌、浆液性腺癌和透明细胞癌患者中的占比分别为19.51%、7/9、2/4,在无和有淋巴转移患者中的占比分别为23.26%、7/11,差异均有统计学意义(P值均<0.05)。高表达组和低表达组突变型p 53阳性的患者占比分别为6/7和11/17,差异有统计学意义(P<0.05)。结论lncRNA-LA16c-313D11.11的高表达与子宫内膜癌分期早、无淋巴结转移相关,lncRNA-LA16c-313d11.11可能通过p 53在子宫内膜癌的调控中发挥重要作用。