Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

Inactivation of the tumor suppressor Krüppel-like factor 6 (KLF6) by mutation or decreased expression in hepatocellular carcinomas

Background and aim: The Krüppel-like transcription factor KLF6 is a novel tumor-suppressor gene. It was inactivated in human prostate cancer and other tumors tissue, as the result of frequent mutation and loss of heterozygosity (LOH). However, there is no data reporting the levels of KLF6 both mRNA and protein in hepatocellular carcinomas (HCCs). We therefore detected mutations and expression of KLF6 in HCC tissues and further observed the effect of it on cell growth in HCC cell lines. Methods: We analyzed the exon-2 of KLF6 gene by direct DNA sequencing, and detected the expression of KLF6 by RT-PCR and Western blot in 23 HCC tissues and corresponding nontumorous tissues. Loss of growth suppressive effect of the HCC-derived KLF6 mutant was characterized by in vitro growth curves plotted, flow cytometry and Western blotting. Results: KLF6 mutations were found in 2 of 23 HCC tissues and one of mutations was missense. Expression of KLF6 mRNA or protein was down-regulated in 8 (34.7%) or 9 (39.1%) of 23 HCC tissues. Wild-type KLF6 (wtKLF6) inhibited cellular proliferation and prolonged G1-S transition by inducing the expression of p21WAF1 following stable transfection into cultured HepG2 cells, but tumor-derived KLF6 mutant (mKLF6) had no effects. Conclusion: Our findings suggest that KLF6 may be involved in pathogenesis of HCC.

The False Paradigm of RUNX3 Function as Tumor Suppressor in Gastric Cancer

Gastric cancer (GC) is a major cause of cancer mortality. GC studies that aim to identify relevant oncogenes and tumor suppressor genes (TSGs) are essential for devising effective new therapies. A decade ago, RUNX3, a gene that resides on human chromosome 1p36.1, was claimed to be a major TSG in GC. Since then, hundreds of studies involving thousands of GC patients have attempted to verify and extend the RUNX3 TSG paradigm. However, RUNX3 is not recognized as TSG and not listed in the “Cancer Gene Census” website. To be a TSG that protects normal cells against malignancy, the gene must be expressed in the normal tissue from which the cancer arose and its loss or inactivation should contribute to cancer development. This review summarizes compelling body of evidence challenging the RUNX3-TSG paradigm. Studies show unequivocally that RUNX3 is not expressed in normal gastric epithelium and that it fails to fulfill all other premises of a TSG. RUNX3 mutations and 1p36 deletions are not frequent in GC and RUNX3 is not associated with familial GC or with increased risk of GC. Accordingly, Runx3-/- mice do not develop tumors. RUNX3 promoter methylation, which has been reported to be a frequent event in GC, is not relevant to its alleged TSG function, since the gene is already silent in normal gastric epithelium. In sharp contrast, overexpression of RUNX3 was found in several types of human cancers, including GC, and the 1p36.1 region is amplified in B-cell lymphoma. Thus, it is possible that RUNX3 actually promotes cancer development rather than being a TSG. The true targets for GC therapy are discussed below. Those are genes frequently lost or amplified in GC and are well known for their tumor suppressive or oncogenic activity, respectively.

Integrated transcriptome interactome study of oncogenes and tumor suppressor genes in breast cancer

Breast cancer is the leading cause for mortality among women worldwide.Dysregulation of oncogenes and tumor suppressor genes is the major reason for the cause of cancer.Understanding these genes will provide clues and insights about their regulatory mechanism and their interplay in cancer.In the present study,an attempt is made to compare the functional characteristics and interactions of oncogenes and tumor suppressor genes to understand their biological role.431 breast cancer samples from seven publicly available microarray datasets were collected and analysed using GEO2R tool.The identified 416 differentially expressed genes were classified into five gene sets as oncogenes(OG),tumor suppressor genes(TSG),druggable genes,essential genes and other genes.The gene sets were subjected to various analysis such as enrichment analysis(viz.,GO,Pathways,Diseases and Drugs),network analysis,calculation of mutation frequencies and Guanine-Cytosine(GC)content.From the results,it was observed that the OG were having high GC content as well as high interactions than TSG.Moreover,the OG are found to have frequent mutations than TSG.The enrichment analysis results suggest that the oncogenes are involved in positive regulation of cellular protein metabolic process,macromolecule biosynthetic process and majorly in cell cycle and focal adhesion pathway in cancer.It was also found that these oncogenes are involved in other diseases such as skin diseases and viral infections.Collagenase,paclitaxel and docetaxel are some of the drugs found to be enriched for oncogenes.

Alteration of tumor suppressor gene p16 and Rb in gastric cancinogesis

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In Silico Study Predicts CCDC69 as a Novel Tumour Suppressor Gene in HER2+Breast Cancer

Differential gene expression analysis using databases followed by overall survival(OS)analysis is currently used to identify different oncogenes and tumour suppressor genes.The present study identified coiled coiled domain containing protein 69(CCDC69)as a tumour suppressor gene in breast cancer by differential gene expression analysis using TCGA dataset for breast adenocarcinoma(BRCA)followed by OS and relapse free survival(RFS)analysis using Kaplan Meier(KM)plotter tool.CCDC69 was observed to be down regulated in tumour of breast cancer patients in BRCA.Following OS analysis for different breast cancer sub-types,low expression of CCDC69 has been observed to be associated with poor survival in HER2+breast cancer only.CCDC69 was also found to be down regulated in different HER2+breast cancer cells by analysing Gene Expression Omnibus(GEO)database.Additionally,CCDC69 was found to be under expressed in single cell HER2 positive population,which is evident from the single cell expression ATLAS database.Furthermore,CCDC69 has been observed to be lowly expressed with overexpression of HER2 in breast cancer by co-expression study.The possible mechanism of CCDC69 down regulation in HER2+breast cancer was resolved using P-SCAN tool.P-SCAN analysis suggested a group of transcription factors(TFs)among which androgen receptor(AR)has been selected as the probable TF that could play a role in CCDC69 down regulation in HER2+breast cancer.Moreover,overexpression of AR has been observed in BRCA and HER2+single cell population.AR has also been observed to be co-expressed positively with HER2,but negatively with CCDC69 in breast cancer.Down regulation of CCDC69 can be predicted to stabilize microtubule formation following stimulation of cell growth and cell migration leading to HER2+breast cancer progression and metastasis.

DNA methylation and carcinogenesis in digestive neoplasms

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Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

The events of cell death and the expression of nuclear matrix protein (NMP) have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide. By means of TUNEL assay, the nuclei displayed a characteristic morphology change, and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h. Nucleosomal DNA fragmentation was observed after treatment for 4 h. The morphological change of HL-60 cells, thus, occurred earlier than the appearance of DNA ladder. Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis. Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells. Western blotting was then performed on three nuclear matrix acssociated proteins, PML, HSC70 and NuMA. The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment, while NuMA, a nuclear mitotic apparatus protein, was down regulated. These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

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, PANG1^p53mut, and rat pancreatic cancer cell lines AS^p53wt and DSL6A^p53null were used for in vitro studies. Following infection with different ratios of Adp53-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 vivostudies.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 vitroand in vivoanalyses 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.

miR-146a在结肠癌中的表达及其作用研究

目的探究miR-146a在结肠癌中的表达及其作用。方法选取26只体重15~20 g、周龄6周的雄性C57BL/6小鼠作为实验对象,将其中13只为miR-146a基因的小鼠作为对照组,另外13只则为miR-146a基因敲除的小鼠作为观察组。比较两组小鼠结肠肿瘤情况(肿瘤数、肿瘤直径)、排便情况(便血程度及粪便形状评分)及增殖细胞核抗原(PCNA)相对表达量、肿瘤组织中促炎性因子的信使核糖核酸(mRNA)相对表达量。结果观察组小鼠的肿瘤数(12.48±3.35)个明显多于对照组的(5.23±1.31)个,肿瘤直径(3.57±0.83)mm明显大于对照组的(2.02±0.76)mm,差异具有统计学意义(P<0.05)。观察组小鼠的便血程度及粪便形状评分分别为(2.04±0.31)、(3.02±0.48)分,明显高于对照组的(1.03±0.12)分和(2.01±0.33)分,差异具有统计学意义(P<0.05)。观察组小鼠的PCNAmRNA相对表达量(2.74±0.79)明显高于对照组的(1.03±0.31),差异具有统计学意义(P<0.05)。观察组小鼠肿瘤组织中促炎性因子的mRNA相对表达量白细胞介素-6(IL-6)(1.67±0.69)、环氧合酶2(COX2)(6.58±1.25)、巨噬细胞炎性蛋白2(MIP2)(7.12±1.18)均明显高于对照组的(0.91±0.34)、(1.34±0.43)、(1.21±0.54),差异具有统计学意义(P<0.05)。结论miR-146a的表达可以对结肠癌细胞的增殖及生长起到显著的抑制效果,其可作为一种抑癌基因,通过对肿瘤细胞凋亡的影响实现对肿瘤病理变化的作用,通过对miR-146a水平的检测可以对患者预后效果等进行评估,具有重要价值。

BRD7基因转染对鼻咽癌细胞生长的抑制作用

目的：探讨鼻咽癌负相关基因ＢＲＤ７对鼻咽癌细胞系ＨＮＥＩ生长的影响。方法：构建ＢＲＤ７基因真核表达载体ｐｃＤＮＡ３.１（＋）／ＢＲＤ７重组体，采用脂质体介导转染技术，将ＢＲＤ７真核表达重组质粒和空载体质粒分别导入鼻咽癌细胞系ＨＮＥ１，Ｓｏｕｔｈｅｒｎ杂交和ＲＴ－ＰＣＲ分别检测外源性ＤＮＡ的整合和ＢＲＤ７基因的表达，并借助细胞生长曲线、软琼脂集落形成试验、流式细胞计数和裸鼠接种方法对转染细胞的生物学行为进行了检测。结果：转染ＢＲＤ７基因的 ＨＮＥ１生长倍增时间为５３ ｈ，较ＨＮＥ１（２３．９ｈ）和空载体转染 ＨＮＥ１（２４.１ｈ）明显延长，流式细胞仪表明，ＢＲＤ７表达升高延缓细胞由Ｇ０－Ｇ１期进人Ｓ期，ＢＲＤ７转染ＨＮＥ１在软琼脂中集落形成率较对照组显著下降（Ｐ＜０．０１），裸鼠接种试验显示ＢＲＤ７基因转染细胞ＨＮＥ１生长速度受到抑制。结论：ＢＲＤ７基因重表达有助于ＨＮＥ１的恶性表型的逆转；ＢＲＤ７是一个鼻咽癌相关的抑瘤基因良好的候选者。

新大肠癌相关HSU 17714基因在大肠癌及其它肿瘤组织中的表达研究

HSU17714是应用减式杂交技术筛选出来的大肠癌相关基因，为了进一步了解此基因在大肠癌及其它肿瘤组织中的表达情况。采用RNAdotblot进行了研究。结果显示：50例大肠腺癌中，表达降低的有16例，占32％，而且与是否伴有淋巴结转移有关。15例乳腺癌中有3例表达降低，占20％。另外，在急淋、急粒、肺腺癌、食管鳞癌各1例中亦有表达降低现象。说明HSU17714在大肠癌组织中可能存在着不同程度的表达缺陷，可能是大肠癌晚期的分子事件。同时，HSU17714基因亦可能是其它肿瘤发生发展的一个分子标志。

UBAP1基因真核表达载体的构建及对人鼻咽癌细胞生长的影响

为研究鼻咽癌相关新基因UBAP1的功能,探讨其对鼻咽癌细胞生长特性的影响,构建了UBAP1真核表达载体并转染到鼻咽癌细胞株HNE1中,借助细胞生长曲线、软琼脂集落形成试验、裸鼠接种和流式细胞计数方法对转染细胞的生物学行为进行了检测.结果显示,UBAP1基因转染细胞生长速度明显减慢,在软琼脂中集落形成率较对照组显著下降,裸鼠接种试验显示,UBAP1基因转染细胞HNE1生长速度受到抑制,流式细胞计数分析发现,UBAP1基因表达升高能延缓细胞由G0-G1期进入S期.因此,UBAP1基因的表达有助于HNE1恶性表型的逆转,初步证明UBAP1是一个鼻咽癌相关的抑瘤基因.

衰老过程中原癌基因及抑癌基因的表达谱

细胞衰老时增殖能力逐渐减退 ,实质细胞不断减少 ,是脏器功能衰退的原因之一。原癌基因及抑癌基因与细胞增殖调控密切相关 ,因而它们与细胞衰老的关系早已引起人们的关注。本文就此进行了概括介绍。总的看来 ,衰老时多数原癌基因表达降低 ,抑癌基因表达增强 。

一种新的抑癌基因PTEN在肾细胞癌中的表达及生物学意义(英文)

背景与目的:PTEN基因是新发现的一种抑癌基因,定位于人染色体10q23。它的缺失和突变可能构成一个新的信号转导途径,与人类恶性肿瘤的发生普遍相关。资料报道,肾细胞癌有抑癌基因PTEN的缺失和突变。但目前尚未见到关于抑癌基因PTEN在肾细胞癌中表达的研究,本实验研究抑癌基因PTEN在肾细胞癌的表达和生物学意义。方法:应用免疫组织化学SP法检测5例正常肾组织、18例癌旁肾组织和40例肾细胞癌组织中抑癌基因PTEN的表达;分析抑癌基因PTEN的表达与肾细胞癌病理类型、淋巴结转移之间的关系。结果:5例正常肾组织和18例癌旁肾组织均有较强的PTEN蛋白的表达,正常肾组织和癌旁肾组织PTEN蛋白的表达强度、阳性细胞的分布形式无明显差异。肾小管上皮细胞的胞质呈PTEN蛋白阳性,免疫组化染色程度较强,肾小球无PTEN蛋白的表达。抑癌基因PTEN在肾细胞癌中的表达不同于正常肾组织和癌旁肾组织,12.5%呈PTEN蛋白阴性,17.5%呈弱阳性,70%呈阳性或强阳性。PTEN蛋白阴性、弱阳性和阳性的肾细胞癌,肾门淋巴结转移率分别为80%,51.74%,10.71%。PTEN蛋白阴性和弱阳性肾细胞癌的肾门淋巴结转移率与PTEN阳性者比较,差异有显著性(P<0.05)。结论:本实验提示肾细胞癌中存在着抑癌基因PTEN的表达缺失和异常,可能与肾细胞癌的发生。

两个鼻咽癌负相关新基因的分离与特性

8个通过 c DNA代表差异分析法 ( c DNA representational difference analysis,c DNA RDA)分离的新 c DNA序列中 ,经 RT- PCR验证 ,发现其中一 c DNA序列 (登录号 :AF0 91 51 7)在 40 %的鼻咽癌活检组织中存在表达缺失和下调 .Northern杂交显示 ,AF0 91 51 7代表转录本为 1 .1 kb和 1 .4kb大小的两个基因 ,进而采用文库筛选 ,成功分离出 3′端完全不同的两个基因 ,命名为 NAG1 1和 NAG 1 2 (登录号分别为 AF 1 70 30 7和 AF 1 94971 ) .经过计算机预测 ,NAG 1 1编码 87个氨基酸组成的跨膜蛋白 ,NAG1 2编码 1 36个氨基酸组成的可溶性的核蛋白 ,两者无任何同源性 .NAG 1 1蛋白含有 3个 ATP结合区、两个蛋白激酶 C磷酸化位点和两个 N-肉豆寇酸化位点 ,NAG 1 2含有POU结构域和多个功能位点 .结果说明 NAG1 1和 NAG1 2的表达的缺失与下调可能参与了鼻咽癌的进程 ;NAG1 1基因产物可能与 ATP的跨膜转运有关 ;NAG1 2基因产物可能与转录翻译有关 .

双氢青蒿素对Lewis肺癌小鼠的抑瘤作用及其机制探讨

目的探讨双氢青蒿素对Lewis肺癌小鼠的抑瘤效应及其作用机制。方法C57BL/6J小鼠皮下接种3LL细胞(2×106)50只,随机分为5组,分别为生理盐水组、阳性对照顺铂组、双氢青蒿素高、中、低剂量组,检测各组小鼠的体重变化和抑瘤率,应用流式细胞术进行TUNEL分析和凋亡相关基因及其表达改变的检测。结果双氢青蒿素中、高剂量组体重较生理盐水组增加明显,其抑瘤率分别为53.5%及59.2%;流式细胞术TUNEL检测结果显示,其作用细胞凋亡和坏死同时存在。FCM法分析其对肺癌组织凋亡相关基因检测表明中、高剂量组Fas、p53等凋亡基因上调,Bcl-2抑制凋亡基因下调。结论双氢青蒿素对Lewis肺癌小鼠肺癌生长具有一定的抑制作用,能促进肿瘤细胞凋亡,其机制可能与调控细胞凋亡相关基因的表达有关。

检测KAI1/CD82蛋白在结肠腺癌中表达的临床意义

目的 KAI1是一种特异性抑制肿瘤转移的基因 ,其蛋白产物为KAI1/CD82。通过检测 6 4例结肠腺癌石蜡切片的KAI1/CD82蛋白表达 ,探讨其与预后等临床因素的相关性。方法 肿瘤经福尔马林固定 ,石蜡包埋。免疫组化方法检测石蜡切片中KAI1/CD82蛋白表达水平。生物学统计采用 χ2 检验法。生存曲线采用Kaplan Meier软件绘制。结果 KAI1/CD82蛋白呈现棕褐色、细颗粒状物 ,弥漫性分布结肠腺癌细胞膜上。KAI1/CD82蛋白在结肠腺癌组织中表达阳性率为 5 1.5 6 %。生物学统计结果表明 ,KAI1/CD82蛋白表达与淋巴结转移、远处转移及肿瘤临床分期等密切相关 ,有统计学意义 ;而与肿瘤患者的年龄和性别、肿瘤大小、分化程度及肿瘤部位等无关。KAI1/CD82蛋白阳性结肠腺癌患者的术后生存期明显高于阴性患者 ,前者平均术后生存期为 5 4 .2 7± 2 1.5 1月 ,而后者仅为 37.5 5± 15 .17月 ,具有统计学意义。结论 KAI1/CD82表达水平与结肠腺癌分期、淋巴结转移及远处转移关系密切 ,可能和其它指标一起作为判断结肠腺癌预后的指标之一 ,对术后进一步治疗具有一定的指导意义。

8一甲氧基补骨脂素对MEC-1细胞癌基因/抑癌基因表达的影响

目的：研究8-甲氧基补骨脂素（8－MOP）对MEC－1细胞部分基因表达的影响。方法：细胞贴壁后用30％细胞生长抑制浓度的药物处理120h，常规制备标本，ABC法免疫组织化学杂色。结果：药物处理后细胞P16和nm23－H1蛋白表达强度增强，而CDK4和H－ras蛋白表达强度减弱。结论：8－MOP可以调节MEC－1细胞部分基因的表达，从而可能影响细胞相关的生物学特性。

肝门部胆管癌外科治疗的临床与实验研究

肝管汇合部癌曾一直被认为是一少见病,患者生前很少能确诊.由于其位置深在,曾以为不可能手术切除.Altemeier et al1957年报告3例主要肝管的硬化型癌,其中1例在3 a 时间内曾手术7次而未得到确诊,直至最后肝门发生腺癌转移才明确诊断。