Genetic diagnosis of pancreatic cancer

Genetic analysis of pancreatic juice is a promising aid for the accurate and early diagnosis of pancreatic cancer. K-ras mutation is frequently observed in pancreatic cancer; however, it is not specific for carcinoma because pancreatic adenoma and pancreatitis also show this mutation. Overexpression of p53 protein is solely detected in pancreatic juice from pancreatic cancer patients, but the positivity rate differs among various reports. Telomerase activity in pancreatic juice was detected in 20 of 24 (83.3%) pancreatic cancer patients and in only 1 of 23 (4.3%) pancreatic adenoma patients, while none of 23 (0%) pancreatitis patients showed evident telomerase activity. The relative value for telomerase activity was significantly higher in parcreatic cancer than in adenoma and pancreatitis. Centrosome abnormalities are very frequently seen in pancreas cancer tissues and the detection of these abnormalities is expected to be a potent new diagnostic tool for the genetic analysis of pancreatic juice. Genetic analysis of pancreatic juice will improve the sensitivity and specificity of pancreatic cancer diagnosis. Received: April 23, 2001 / Accepted: May 11, 2001     

宫颈鳞状细胞癌细胞周期蛋白E表达与中心体扩增

目的了解宫颈鳞状细胞癌细胞周期蛋白E（cyclinE）表达与中心体扩增相关性，探讨其中心体扩增的可能分子机制。方法正常宫颈组织12例，不同分化程度的宫颈鳞状细胞癌46例石蜡包埋组织，采用间接免疫荧光双重染色（γ-微管蛋白单克隆抗体及细胞角蛋白多克隆抗体）观察宫颈鳞状细胞癌中心体扩增状况：采用免疫组织化学（SASB法）检测相应组织cyclinE蛋白表达情况，分析cyclinE蛋白表达与中心体扩增之间的相关性。结果中心体扩增可见于80％（37／46）宫颈鳞状细胞癌组织中，而cyclinE蛋白过表达可在65％（30／46）的宫颈鳞状细胞癌组织中见到；中心体扩增发生率在cyclinE阳性组为90．0％（27／30），而在cyclinE蛋白阴性组为62．5％10／16，两组间差异有统计学意义（x^2=5．014，P〈0．05）；Spearman相关分析显示中心体扩增与cyclin E蛋白阳性表达间存在相关关系（r=0．330，P〈0．05；绝对危险度分析OR值为5．400（1．130，25．809）。结论肿瘤细胞中心体循环调控可能是一个多因素参与的复杂过程，cyclin E蛋白表达的高调作为危险因素之一可能在宫颈鳞状细胞癌中心体扩增中起一定作用。     

口腔鳞癌细胞染色体不稳定潜在机制的探讨

目的:探讨口腔鳞癌细胞染色体不稳定形成的潜在机制.方法:对7株非整倍体口腔鳞状细胞癌(OSCC)细胞系细胞,采用荧光活化细胞分类(FACS)及免疫荧光染色(IF)的方法,观察非整倍体OSCC细胞有丝分裂检查点(又称纺锤体检查点)功能及中心体状况.结果:7株非整倍体OSCC细胞系细胞经过0.2μg/ml噻氨酯哒唑(Nocodazole)处理18小时后,都出现了高比例分裂中期(G2/M)细胞的累积,提示这些细胞有丝分裂检查点功能正常:而中心体异常(数目增多及形态的异常)可以在所有非整倍体OSCC细胞系中观察到,异常细胞百分率04%～18.8%.结论:OSCC细胞CIN表型与有丝分裂检查点功能异常之间可能无直接机制上的联系,但中心体异常可能是OSCC细胞染色体不稳定形成的潜在机制之一.     

口腔鳞状细胞癌细胞周期蛋白E表达与中心体扩增相关性

目的了解口腔鳞状细胞癌细胞周期蛋白E（cyclin E）表达与中心体扩增相关性,探讨其中心体扩增的可能分子机制。方法正常口腔黏膜组织12例,不同分化程度的口腔鳞状细胞癌46例石蜡包埋组织,采用间接免疫荧光双重染色（γ-微管蛋白单克隆抗体及细胞角蛋白多克隆抗体）观察口腔鳞状细胞癌中心体扩增状况;采用免疫组织化学（SABC法）检测相应组织cyclin E蛋白表达情况,分析cyclin E蛋白表达与中心体扩增之间的相关性。结果中心体扩增可见于80．4％（37／46）口腔鳞状细胞癌组织中,而cyclin E蛋白过表达可在65．2％（30／46）的口腔鳞状细胞癌组织中见到;中心体扩增发生率在cyclin E阳性组为90．0％（27／30）,而在cyclin E蛋白阴性组为10／16,两组间差异有统计学意义（x^2=5．014,P〈0．05）;Spearman相关分析显示中心体扩增与cyclin E蛋白阳性表达间存在相关关系（r=0．330,P〈0．05）;绝对危险度分析OR值为5．400（1．130,25．809）。结论肿瘤细胞中心体循环调控可能是一个多因素参与的复杂过程,cyclin E蛋白表达的高调作为危险因素之一可能在口腔鳞状细胞癌中心体扩增中起一定作用。     

Nucleophosmin and human cancer

Nucleophosmin (NPM) is a nucleolar phosphoprotein that shuttles between the nucleus and cytoplasm during the cell cycle. NPM has several interacting partners and diverse cellular functions, including the processing of ribosomal RNA, centrosome duplication and the control of cellular processes to ensure genomic stability. Subcellular localization of NPM appears to be strongly correlated with NPM functions and cell proliferation. NPM is phosphorylated mainly at its central acidic domain by several upstream kinases, and its phosphorylation appears to be involved in regulating its functions in ribosome biogenesis and centrosome duplication. Recent studies suggest that NPM may act as a licensing factor to maintain proper centrosome duplication and that the Ran/CRM1 nucleocytoplasmic complex regulates local trafficking of NPM to centrosomes by interacting through its nuclear export sequence motif. Here, we provide a brief overview of NPM functions and its roles in human carcinogenesis, and discuss our recent findings related to the potential mechanisms underlying its regulation of centrosome duplication.     

喉癌细胞系中心体扩增与细胞异常分裂的检测

目的通过对喉鳞状细胞癌(简称喉癌)细胞系中心体表达和细胞分裂形态的研究,探讨中心体通过对细胞分裂过程的调控,在促进喉癌基因不断变异、发展机制过程中的作用。方法6个喉癌细胞系和4例喉癌旁正常组织上皮细胞培养。收获细胞经苏木素和伊红染色,观察分裂中期细胞核形态,计算核多极分裂像细胞百分比。应用抗γ-微管蛋白抗体免疫荧光染色检测细胞中心体,计算含异常中心体数目细胞的百分比。二组数据行直线相关分析。喉癌组和正常对照组两组数据平均值行t检验。结果喉癌细胞系多极核分裂像细胞平均占分裂中期细胞(11．53±4．54)％,含中心体扩增的细胞平均占总体细胞的(7．50±3．13)％。且二者呈显著正相关,r=0．814,P<0．01。而正常对照组细胞仅偶尔出现异常分裂中期形态及异常的中心体数目,其平均值分别为(0．83±0．99)％和(1．26±1．59)％。喉癌组和对照组两组数据平均值行t检验,喉癌组显著高于对照组,t值分别为3．522和3．877,P<0．01。结论喉癌细胞系表现为高度的中心体的扩增及细胞多极分裂,这在促进喉癌基因组不稳定性发展过程中起重要作用。     

中心体异常及P53蛋白过表达在食管癌变过程中的作用

目的了解食管癌变过程中中心体的变化及P53蛋白的表达情况,探讨中心体异常和P53蛋白过表达在食管癌变过程中的作用。 方法选取食管鳞状细胞癌50例,非典型增生22例,正常食管粘膜12例的石蜡包埋组织,采用免疫组织化学染色SP法检测P53蛋白的表达,组织免疫荧光染色法检测中心体的变化。结果食管癌组织中P53蛋白呈过表达（88%）,非典型增生组织中呈弱表达（54%）,而正常组织无表达。 同样,食管癌组织中有明显中心体异常, 非典型增生组织中可见部分中心体异常,而正常组织未见中心体异常。 中心体异常和P53蛋白过表达均与肿瘤分化程度及浸润程度呈正相关(P<0.05)。食管鳞癌组织中突变的P53蛋白表达与中心体异常显著相关(P<0.05)。结论中心体异常和P53蛋白过表达发生于食管癌变的早期阶段,且与预后相关。P53突变可能参与中心体异常的形成,与之共同参与食管癌的发生。     

Chromosome aberrations associated with centrosome defects: a study of comparative genomic hybridization in breast cancer

Centrosome abnormalities occur frequently in various tumors and can cause chromosomal instability and eventually promote cancer development. We investigated the chromosome aberrations associated with centrosome abnormalities in 30 cases of breast cancer, combining immunohistochemical staining and comparative genomic hybridization. Except for some common chromosome alterations (including gains of 1q, 8q, 17q, 20q, and Xq and losses of 8p, 11q, 13q, 14q, 16q, 17p, 22q, and Xp) that have also been seen more frequently in other studies, we discovered some new changes that have rarely been reported, including gains at 2p, 5p, 10p, 15q, 16p, 18q, 21q, and 22q and losses at 6p, 8p23, 11p13-pter, 13q34, and 14q32-qter. We also identified some changes (such as gains of 17q, 20q, and Xq and losses of 17p, 13q, and 14q) harboring candidate genes. We also explored the expression of centrosome protein in different molecular subtypes of breast cancer. Our findings provide a new way to explore the molecular mechanisms of breast tumorigenesis and accordingly potential new targets for therapy for this disease.     

Expression of GAP-43 in fibroblast cell lines influences the orientation of cell division

The orientation (vertical or horizontal) of cell division is known to be critical for neural cell fate determination during neurogenesis. At the onset of neurogenesis, neurogenic progenitor cells are dividing with the cleavage plane parallel to the ventricular surface (horizontal division), which would lead to critical apical components being unequally distributed to both their two daughter cells. The daughter cells lack of inheritance is going to differentiate into the neuron. Recent studies have shown that GAP-43 is highly expressed in horizontally dividing neural progenitor cells in the forebrain of mammals. Based on findings from in vivo studies, GAP-43 is locally associated with the centrosome and is required for centrosome positioning, suggesting that GAP-43 may be involved in neurogenesis through regulating the orientation of cell division. With a fibroblast cell model, our results show that both GFP expressing and control cells had the same potential (p > 0.05) with regard to dividing orientation (either vertical or horizontal to the cells long axis). On the other hand, we found that GAP-43 was localized on the membrane instead of the centrosome during all phases of mitosis within GAP-43 transgenic cells, but expressing of GAP-43 could make the cells dividing more likely along their long axis (p < 0.05). Our observations suggest that GAP-43 might link the cell membrane and spindle pole and consequently participate in controlling cleavage orientation during cell division.     

p21~(waf1)-p27~(kip1)基因联合转染对乳腺癌细胞增殖和中心体复制的影响

目的 探讨外源性 p21~(waf1)-p27~(kip1)基因联合转染对人乳腺癌细胞细胞增殖及中心体复制的影响和意义.方法 应用脂质体介导法分别将构建的plRES-p27~(waf1)、p27~(kip1)、pIRES-p21~(waf1)-p27~(kip1)真核表达载体,转染入常规培养的人乳腺癌细胞系MCF-7,未转染组、转染空质粒组细胞做对照,Western免疫印迹法验证转染前后p21~(waf1)、p27~(kip1)基因蛋白表达情况;噻唑蓝(MTT)法绘制细胞生长曲线;流式细胞仪检测细胞周期DNA分布变化;免疫荧光技术检测转染前后中心体复制的变化情况.结果 Western免疫印迹法证实p27~(waf1)、p27~(kip1)、p27~(waf1)基因转染24 h后,其蛋白表达量明显增多(P<0.01),与对照组比较,各转染实验组细胞生长明显受抑制(P<0.01),细胞周期大部分停滞于G1期,各实验组G.期和S期细胞比例依次为:(69.52±3.21)%、(18.38±2.51)%,(60.83±3.02)%、(24.52±1.89)%,(78.37±2.83)%、(15.27±1.74)%,对照组则为(47.28±2.25)%、(33.52±1.97)%,中心体复制异常的乳腺癌细胞数较转染前(13.47±0.33)%明显减少:(5.07±0.38)%,(6.28±0.35)%,(3.47±23)%,两两比较差异有统计学意义(P<0.01).结论 外源性p21~(waf1)和p27~(kip1)基因转染可抑制人乳腺癌细胞的增殖及中心体的异常复制,二者联合转染时效果显著.提示,乳腺癌的发生、发展是多基因、多因素共同参与的过程,p21~(waf1)和p27~(kip1)在调控细胞增殖及中心体复制方面具有协同作用,对于p21~(waf1)、p27~(kip1)基因失活、低表达或不表达的乳腺癌,联合转染外源性p21~(waf1)、p27~(kip1)基因不失为一种有效的治疗手段.