Expression of nm23-H1 and nm23-H2 protein in endometrial carcinoma.

nm23 gene expression has been shown to be inversely correlated with tumour metastatic potential in some cancers but not in others. Examination was made of the expression of nm23-H1 and nm23-H2 gene products by immunohistochemistry and immunoblotting in 28 endometrial carcinomas. Immunohistochemistry indicated the cytoplasm of cancer cells to be positive, and myometrium and endometrial stromal cells negative, for nm23-H1 and -H2 protein. The staining intensity for these proteins was significantly stronger in well-differentiated adenocarcinomas (G1) than in those moderately differentiated (G2) (P < 0.05). nm23-H1 and -H2 proteins were shown by immunoblotting to be present at significantly higher levels in G1 than in G2 tumours (P < 0.05). Two of eight cases expressed high nm23-H1 and -H2 protein in poorly differentiated adenocarcinomas (G3). In G3 tumours, nm23 expression may be diverse. In this study, the expression of nm23-H1 and -H2 was not correlated with stage, metastasis, tumour size, myometrial invasion, oestrogen receptor, progesterone receptor or menopause. It follows from the findings presented above that the high expression of nm23-H1 and -H2 is positively correlated with histological differentiation.     

壮骨镇痛胶囊对乳腺癌细胞ｎｍ２３-Ｈ１和ｃ-ｍｙｃ基因表达的影响

目的　观察壮骨镇痛胶囊对高转移人乳腺癌ＭＤＡ-ＭＢ-２３１细胞抑癌基因ｎｍ２３-Ｈ１和原癌基因ｃ-ｍｙｃ表达的影响,探讨壮骨镇痛胶囊抑制ＭＤＡ-ＭＢ-２３１细胞增殖和迁移的可能作用机制。方法　采用ＲＴ-ＰＣＲ法和免疫细胞化学法分别检测不同浓度壮骨镇痛胶囊含药血浆对ＭＤＡ-ＭＢ-２３１细胞ｎｍ２３-Ｈ１和ｃ-ｍｙｃ基因ｍＲＮＡ及其蛋白表达的影响,并分析其对ｎｍ２３-Ｈ１／ｃ-ｍｙｃ蛋白比值的影响。结果　５％壮骨镇痛胶囊含药血浆显著抑制了ＭＤＡ-ＭＢ-２３１细胞ｎｍ２３-Ｈ１和ｃ-ｍｙｃ基因的转录和翻译;１.２５％壮骨镇痛胶囊含药血浆抑制了ｎｍ２３-Ｈ１的转录和翻译以及ｃ-ｍｙｃ基因的翻译,对ｃ-ｍｙｃ基因的转录无显著影响;０.６３％壮骨镇痛胶囊含药血浆对ｎｍ２３-Ｈ１和ｃ-ｍｙｃ基因的转录和翻译均无明显影响;５％和１.２５％壮骨镇痛胶囊含药血浆显著提高了ＭＤＡ-ＭＢ-２３１细胞ｎｍ２３-Ｈ１／ｃ-ｍｙｃ蛋白比值,０.６３％壮骨镇痛胶囊含药血浆对ｎｍ２３-Ｈ１／ｃ-ｍｙｃ蛋白比值无显著影响。结论　不同浓度壮骨镇痛胶囊含药血浆对乳腺癌ＭＤＡ-ＭＢ-２３１细胞ｎｍ２３-Ｈ１和ｃ-ｍｙｃ基因的表达具有不同的作用,该药可能通过降低癌细胞ｃ-ｍｙｃ基因表达继而提高细胞中ｎｍ２３-Ｈ１／ｃ-ｍｙｃ蛋白比值来降低癌细胞的增殖和迁移能力。     

Expression of nm23-Hl and nm23-H2 Proteins in Prostate Carcinoma

The nm 23 gene products/nucleoside diphosphate (NDP) kinase expression in prostate carcinomas and benign hyperplasias was evaluated immunohistochemically. Monoclonal antibodies against nm 23-H1 and nm 23-H2 proteins were prepared using the corresponding proteins fused with glutathione S-transferase as immunogens. Of the 80 cases of nonmetastatic prostate carcinoma examined, 74% (59/80) and 60% (48/80) were immunoreactive for nm 23-H1 or nm 23-H2 protein, respectively. Negative staining for nm 23-H1 occurred in 83% of metastatic lesions, while 34% were negative for nm 23-H2. All primary tumors corresponding to the metastases examined showed positive immunostaining for nm 23-H1, indicating an inverse relationship between expression of this protein and metastatic status. nm 23-H2 protein was detected in 83% of primary tumors and its expression appeared to he significantly correlated to the degree of histological differentiation. In contrast, all cases of benign prostatic hyperplasia showed elevated levels of both nm 23-H1 and nm 23-H2 expression. These data suggest that the nm 23/NDP kinase may play a role in suppressing the expression of malignant potential in prostate carcinomas.     

利用定点突变技术构建突变nm23-H1和EGFP融合基因

背景与目的以前的研究已经证明nm23-H1基因是肿瘤转移抑制基因，但其抑制肿瘤转移的分子机制目前还不完全清楚。基因定点突变技术可以精确地改变基因的特定碱基序列，获得突变蛋白质。本研究的目的是应用基因定点突变技术构建突变型nm23-H1-增强型绿色荧光蛋白（EGFP）融合基因，为进一步研究肿瘤抑制基因nm23-H1的功能、生化作用机制提供理论基础和实验依据。方法以逆转录病毒PLXSN-野生型nm23-H1-EGFP质粒为突变模板，应用QuikChange^TM定点突变方法，简单、快速、高效地引入nm23-H1^S44A、nm23-H1^P96S、nm23-H1^H118F、nm23-H1^S120G四个点突变和一个联合位点突变nm23-H1^P96S-S120G，构建了突变型nm23-H1-EGFP融合基因。结果成功构建了nm23-H1^S44A-EGFP、nm23-H1^P96S-EGFP、nm23-H1^H118F-EGFP、rim23-H1^S120G-EGFP、nm23-H1^P96S-S120G-EGFP五个突变型nm23-H1-EGFP融合基因，经DNA序列分析突变的碱基序列与实验设计完全一致。结论成功构建了五个具有不同突变位点的突变型nm23-H1-EGFP融合基因。QuikChange^TM定点突变技术是一种简单、快速、高效的基因定点突变方法。     

突变型nm23-H1基因的磷酸化活性研究

背景与目的 磷酸化活性是nm23-H1重要的生物学活性,本研究旨在探讨不同位点的突变对nm23-H1磷酸化活性的影响.方法 以野生型nm23-H1研究对照,采用放射自显影的方法检测nm23-H1野生型(WT)和4种突变型(P96S,H118F,S120G和S44A)原核表达蛋白的自身丝氨酸与自身组氨酸磷酸化活性,应用反相高效液相色谱技术(RP-HPLC)检测上述蛋白的NDPK激酶活性.结果 野生型和突变型nm23-H1蛋白的自身丝氨酸与自身组氨酸磷酸化活性由大到小依次为P96S、WT、S44A、S120G和H118F,而NDPK活性由大到小依次为:WT、S120G、P96S、S44A和H118F;经线性相关分析,自身丝氨酸与组氨酸磷酸化活性呈正相关(r=0.983,P<0.01),但NDPK活性与自身丝氨酸和自身组氨酸活性均无相关性(分别为r=0.458,P>0.05;r=0.482,P>0.05).结论 不同位点的突变对nm23-H1自身丝氨酸、自身组氨酸及NDPK激酶活性影响不同,NDPK活性与自身磷酸化活性并不完全关联.其中118位组氨酸是nm23-H1激酶活性的关键氨基酸;96位脯氨酸可能与nm23-H1的磷酸转移能力有关;而120位丝氨酸可能为自身丝氨酸和组氨酸磷酸化位点;44位丝氨酸可能为另一具有NDPK激酶的氨基酸.     

肿瘤相关基因N-myc、Fas、MTA1、nm23-H1对子宫内膜癌细胞增殖转移的影响

目的探讨原癌基因N-myc、抑癌基因Fas、转移促进基因MTA1及转移抑制基因nm23-H1对子宫内膜癌细胞增殖转移的影响。方法采用实时荧光定量RT-PCR检测正常子宫内膜组织、子宫内膜腺癌未转移患者及转移患者标本各30例中N-myc、Fas、MTA1、nm23-H1的基因表达情况,并行比较分析。结果 (1)子宫内膜癌组织中N-myc、MTA1基因扩增水平均高于正常子宫内膜组织,差异有统计学意义(均P<0.05);(2)子宫内膜癌组织中Fas、nm23-H1基因扩增水平均低于正常子宫内膜组织,差异有统计学意义(均P<0.05);(3)转移子宫内膜癌组织中MTA1基因扩增水平均高于未转移组,nm23-H1基因扩增水平均低于未转移组,差异均有统计学意义(均P<0.05);(4)子宫内膜癌组织转移组与未转移组中N-myc、Fas基因扩增水平均具有差异性,差异有统计学意义(均P<0.05)。结论 N-myc、MTA1基因能促进子宫内膜癌的细胞增殖,Fas、nm23-H1基因能抑制子宫内膜癌的细胞增殖;MTA1基因可促进子宫内膜癌的细胞转移,nm23-H1基因可抑制子宫内膜癌的细胞转移。     

人类nm23-H1基因重组腺病毒的构建及鉴定

目的构建人肿瘤转移抑制基因nm23-H1的重组腺病毒载体。方法通过PCR方法以pcDNA3．1-nm23-H1为模板扩增出编码152个氨基酸的nm23-H1基因,并连接入穿梭质粒 pShuttle-CMV。将含有目的基因的重组穿梭质粒pShuttleCMV-nm23-H1经Pme I线性化后转化入 AdEasier-1感受态细胞,用含硫酸卡那霉素的LB培养基筛选重组子,并用PCR及酶切方法鉴定。鉴定正确的pAd-nm23-H1质粒经Pac I线性化后转染293细胞,包装重组腺病毒Adeno-nm23- H1,并进行PCR鉴定及病毒滴度测定。结果 PCR、酶切鉴定及测序结果证实pShuttleCMV-nm23- H1及pAd-nm23-H1质粒正确构建,收获病毒后的PCR鉴定及DNA测序结果证明Adeno-nm23- H1包被成功且无野生型腺病毒产生。重组腺病毒Adeno-nm23-H1滴度为4．3×109／ml。结论成功构建了重组腺病毒载体Adeno-nm23-H1,为进一步研究nm23-H1抑制肿瘤转移分子机制及基因治疗奠定了基础。     

人类nm23-H1基因重组腺病毒的构建及鉴定

目的构建人肿瘤转移抑制基因nm23-H1的重组腺病毒载体.方法通过PCR方法以pcDNA3.1-nm23-H1为模板扩增出编码152个氨基酸的nm23-H1基因,并连接入穿梭质粒pShuttle-CMV.将含有目的基因的重组穿梭质粒pShuttleCMV-nm23-H1经Pme Ⅰ线性化后转化入AdEasier-1感受态细胞,用含硫酸卡那霉素的LB培养基筛选重组子,并用PCR及酶切方法鉴定.鉴定正确的pAd-nm23-H1质粒经Pac Ⅰ线性化后转染293细胞,包装重组腺病毒Adeno-nm23-H1,并进行PCR鉴定及病毒滴度测定.结果PCR、酶切鉴定及测序结果证实pShuttleCMV-nm23-H1及pAd-nm23-H1质粒正确构建,收获病毒后的PCR鉴定及DNA测序结果证明Adeno-nm23-H1包被成功且无野生型腺病毒产生.重组腺病毒Adeno-nm23-H1滴度为4.3×109/ml.结论成功构建了重组腺病毒载体Adeno-nm23-H1,为进一步研究nm23-H1抑制肿瘤转移分子机制及基因治疗奠定了基础.     

nm23—H1蛋白和c-erbB-2蛋白在人肝细胞癌中的表达

应用免疫组化检测68例肝细胞癌（HCC）及其癌旁肝组织中nm23－H1蛋白和c－erbB－2蛋白的表达。结果显示nm23－H1蛋白在癌旁肝组织中均呈强阳性表达，肝细胞癌组织中38／68（56％）例呈阳性表达。阳性产物主要定位于肿瘤细胞胞浆，nm23－H1蛋白表达与HCC肿瘤体积、组织病理学分型及分级无关，而与肝内或肝外转移呈负相关。结果表明nm23-H1在抑制HCC肝内或肝外转移中起着重要作用。有可能成为评价HCC病人预后的一项指标。     

Reduced expression of nm23-H1, but not of nm23-H2, is concordant with the frequency of lymph-node metastasis of human breast cancer

The nm23 gene is a potential metastasis-suppressor gene originally identified in a murine melanoma line. Several investigators have reported the probable inverse association of nm23 expression with disease prognosis and/or metastasis. Since there are now 2 known isotypes of human nm23, namely nm23-H1 and -H2, we immunohistochemically examined expression of these isotypes in human breast-cancer tissues using monoclonal antibodies (MAbs) specific for each isotype protein. We also analyzed expression of c-erbB-2 in the same collection of cancer tissues, in order to examine the significance of nm23 expression in comparison with c-erbB-2 expression. Of 130 tumors from breast-cancer patients, 73 (56%) and 69 (53%) positively expressed nm23-H1 and -H2 respectively. Expression of c-erbB-2 was positive in 36 (28%). Expression of nm23-H1, but not nm23-H2, was inversely associated with lymph-node metastasis (p < 0.01). Expression of c-erbB-2 was associated with Tnm stage, tumor size and lymph-node metastasis (p < 0.01, p < 0.05 and p < 0.05 respectively). Overall survival was better (p = 0.014) in patients in whom expression of nm23-H1 was positive than in those in whom it was negative. In multivariate analyses using a Cox's proportional-hazards regression model with 9 variables, nm23-H1 showed the fourth greatest contribution to patient survival following lymph-node metastasis, Tnm stage and menopausal status. No significant contribution was shown for c-erbB-2 expression. nm23-H1, but not nm23-H2, may perform a role in disease prognosis in addition to its participation in cancer metastasis. It may have value for predicting long-term survival of human breast-cancer patients. © 1993 Wiley-Liss, Inc.     

膀胱癌中MMP-2与nm23的表达及其临床意义

目的　探讨膀胱癌组织中的MMP - 2与nm2 3-H1蛋白的表达及其临床意义。方法　采用免疫组化S -P法分析 6 3例手术切除膀胱癌中MMP - 2与nm2 3-H1蛋白的表达情况。结果　MMP - 2在膀胱移行细胞癌与乳头状瘤中的表达有显著性差异 (P <0 0 5 ) ,其表达与肿瘤分级与临床分期呈正相关 ;nm2 3-H1蛋白表达与肿瘤分期呈负相关 ,在肿瘤组织中随TCCB临床病理分期升高 ,MMP - 2表达增高趋势有显著性差异 ,而nm2 3-H1的表达随TCCB分级及临床分期的升高而呈现降低趋势 ,且有显著性差异 (P <0 0 5 )。结论　检测膀胱癌转移相关基因表达并分析其表达的相互关系 ,有助于判断膀胱癌的转移潜能及指导术后干预治疗     

膀胱癌中nm23-H1基因突变及表达的意义

目的：探讨ｎｍ２３－Ｈ１基因在膀胱癌中突变及表达的意义。方法：应用半定量逆转录聚合酶链反应（ＲＴ－ＰＣＲ）和银染单链构象多态性（ＳＳＣＰ）方法检测ｎｍ２３－Ｈ１基因在２５例膀胱癌组织及１５例对照组织中突变和表达情况。结果：对照粘膜中未检测出ｎｍ２３－Ｈ１基因突变,而在２５例膀胱癌组织中发现６例出现ＰＣＲ产物单链泳动状态异常,异常率为２４％。癌组织和对照组织均有ｎｍ２３－Ｈ１基因ｍＲＮＡ的表达,８８     

nm23-H1与Tiam1在睑板腺癌中的表达及意义

目的:检测睑板腺癌组织中nm23-H1及Tiam1的表达,探讨其与临床病理特征的关系,评价其临床意义.方法:睑板腺癌标本34例及癌旁正常组织34例,应用Western blot法及qRT-PCR检测nm23-H1和Tiam1蛋白及mRNA的表达,观察其蛋白水平表达与临床病理特征的相关性,观察其与睑板腺癌术后复发的关系.结果:nm23-H1蛋白及mRNA在睑板腺癌组织中表达高于癌旁组织(P<0.05),Tiam1蛋白及mRNA在睑板腺癌组织中表达高于癌旁组织,差异有统计学意义(P<0.05).nm23-H1 及Tiam1蛋白表达水平与肿瘤直径、TNM分期、淋巴结转移及肿瘤分化程度有相关性(P<0.05),nm23-H1 及Tiam1与睑板腺癌术后复发率具有相关性,在复发的睑板腺癌中nm23-H1及 Tiam1表达高于未复发者(P<0.05).结论:nm23-H1和Tiam1表达水平与睑板腺癌的临床病理特征具有相关性,nm23-H1和Tiam1高表达提示睑板腺癌患者不良预后.     

nm23-H1与造血系统肿瘤关系的研究进展

nm23-H1作为肿瘤转移抑制基因,在多种实体肿瘤中能抑制肿瘤细胞的转移和植入,与预后呈正相关,但在造血系统肿瘤研究中却发现,nm23-H1是一种分化抑制因子,具有抑制造血细胞的分化成熟,参与白血病和淋巴瘤的发生、发展过程,与预后呈负相关。     

E-cadherin及nm23-H1在滋养细胞疾病中的表达及意义

目的:探讨E-cadherin及nm23-H1基因在妊娠滋养细胞疾病发生发展中的作用.方法:采用免疫组化法检测24例葡萄胎(随访2年以上未发生恶变)、15例侵蚀性葡萄胎、15例绒毛膜上皮癌、18例正常绒毛组织的石蜡包埋标本E-cadherin和nm23-H1基因的表达状况.结果:E-cadherin的表达在正常早孕绒毛高于侵蚀性葡萄胎和绒毛膜癌(P＜0.01),葡萄胎高于侵蚀性葡萄胎和绒毛膜癌(P＜0.05).nm23-H1的表达正常早孕绒毛明显高于恶性滋养细胞疾病(P＜0.01),葡萄胎高于侵蚀性葡萄胎(P＜0.01)和绒毛膜癌(P＜0.05).在葡萄胎和恶性滋养细胞疾病中E-cadherin和nm23-H1基因的表达均为正相关.结论:滋养细胞疾病E-cadherin和nm23-H1表达与其侵袭性相关,二者可能成为葡萄胎预后的标志物.在侵袭转移过程中细胞滋养细胞较合体滋养细胞更为重要.     

Prune cAMP phosphodiesterase binds nm23-H1 and promotes cancer metastasis

We identify a new enzymatic activity underlying metastasis in breast cancer and describe its susceptibility to therapeutic inhibition. We show that human prune (h-prune), a phosphoesterase DHH family appertaining protein, has a hitherto unrecognized cyclic nucleotide phosphodiesterase activity effectively suppressed by dipyridamole, a phosphodiesterase inhibitor. h-prune physically interacts with nm23-H1, a metastasis suppressor gene. The h-prune PDE activity, suppressed by dipyridamole and enhanced by the interaction with nm23-H1, stimulates cellular motility and metastasis processes. Out of 59 metastatic breast cancer cases analyzed, 22 (37%) were found to overexpress h-prune, evidence that this novel enzymatic activity is involved in promoting cancer metastasis.     

Evidence for interaction between human PRUNE and nm23-H1 NDPKinase

We have isolated a human and murine homologue of the Drosophila prune gene through dbEST searches. The gene is ubiquitously expressed in human adult tissues, while in mouse developing embryos a high level of expression is confined to the nervous system particularly in the dorsal root ganglia, cranial nerves, and neural retina. The gene is composed of eight exons and is located in the 1q21.3 chromosomal region. A pseudogene has been sequenced and mapped to chromosomal region 13q12. PRUNE protein retains the four characteristic domains of DHH phosphoesterases. The synergism between prune and awdK-pn in Drosophila has led various authors to propose an interaction between these genes. However, such an interaction has never been supported by biochemical data. By using interaction-mating and in vitro co-immunoprecipitation experiments, we show for the first time the ability of human PRUNE to interact with the human homologue of awd protein (nm23-H1). In contrast, PRUNE is impaired in its interaction with nm-23-H1-S120G mutant, a gain-of-function mutation associated with advanced neuroblastoma stages. Consistently, PRUNE and nm23-H1 proteins partially colocalize in the cytoplasm. The data presented are consistent with the view that PRUNE acts as a negative regulator of the nm23-H1 protein. We discuss how PRUNE regulates nm23-H1 protein and postulate possible implications of PRUNE in neuroblastoma progression.     

The 17q23 amplicon and breast cancer

A novel region of amplification in breast tumors was recently identified on chromosome 17q23. Extensive mapping of the amplicon by Southern blotting and fluorescence in situ hybridization (FISH) in breast cancer cell lines determined that the amplicon can be up to 4 Mbp in size and may contain 50 genes. Copy number analysis at 50–75 kb resolution in breast cancer cell lines and breast tumors identified several independently amplified regions within the amplicon, suggesting that a number of genes are selected for amplification because they independently contribute to tumor formation and progression. Support for this hypothesis comes from studies demonstrating that many of the amplified genes are over-expressed in breast cancer cell lines and tumors, and that the RPS6KB1, TBX2, and PPM1D genes from the region, that are amplified and over-expressed in breast tumors and cell lines, contribute to tumor formation and/or tumor progression. In this review we summarize the structural studies of the amplicon that have been carried out, we outline the evidence implicating the RPS6KB1, TBX2, and PPM1D genes as oncogenes, and we describe some of the other candidate oncogenes from the region.     

Survivin nm23-H1在非小细胞肺癌中的表达及相关性研究

目的:研究肿瘤相关基因survivin、nm23-H1在非小细胞肺癌﹙NSCLC﹚组织中的表达及相关性.探讨其与临床病理特征之间的关系.方法:采用免疫组化SP 法检测survivin和nm23-H1在52例NSCLC组织、20例对应癌旁组织和15例正常组织中的表达情况.结果:survivin的表达癌组织明显高于癌旁组织的表达,差异具有显著性(P<0.05),正常组织无survivin表达;nm23-H1的表达癌组织明显低于癌旁组织的表达,差异具有显著性(P<0.05);Survivin在NSCLC组织中的表达与年龄、性别、组织类型、淋巴结转移无关(P>0.05), nm23-H1在伴有淋巴结转移的NSCLC组织中表达的阳性率为19.0%(4/21),在未伴有淋巴结转移的NSCLC组织中表达的阳性率为48.4%(15/31),两者差异有显著性;survivin在TNM(Ⅰ+Ⅱ)期NSCLC组织中表达低于在TNM(Ⅲ+Ⅳ)期NSCLC组织中表达;nm23-H1在TNM(Ⅰ+Ⅱ)期NSCLC组织中表达高于在TNM(Ⅲ+Ⅳ)期NSCLC组织中表达,差别有显著意义(P<0.05);survivin在高分化NSCLC中表达低于在低分化NSCLC中表达;nm23-H在高分化NSCLC组织中表达高于在低分化NSCLC组织中表达,差别有显著意义(P<0.05);survivin、nm23-H1在NSCLC中表达负相关(P<0.05).结论:survivin 和nm23-H1 的表达与NSCLC发生、临床进展及预后密切相关.Survivin可以作为NSCLC的早期诊断指标,并可以根据survivin与nm23-H1的联合检测来判断恶性程度及预后.