Centrosome abnormalities are frequently observed in non-small-cell lung cancer and are associated with aneuploidy and cyclin E overexpression

Centrosome abnormalities are observed in human cancers and have been associated with aneuploidy, a driving force in tumour progression. However, the exact pathways that tend to cause centrosome abnormalities have not been fully elucidated in human tumours. Using a series of 68 non-small-cell lung carcinomas and an array of in vitro experiments, the relationship between centrosome abnormalities, aneuploidy, and the status of key G1 to S-phase transition cell-cycle molecules, involved in the regulation of centrosome duplication, was investigated. Centrosome amplification and structural abnormalities were common (53%), were strongly related to aneuploidy, and, surprisingly, were even seen in adjacent hyperplastic regions, suggesting the possibility that these are early lesions in lung carcinogenesis. Cyclin E and E2F1 overexpression, but not p53 mutation, was observed to correlate with centrosome abnormalities in vivo (p = 0.029 and p = 0.015, respectively). This was further strengthened by the observation that cyclin E was specifically present in the nucleus and/or cytoplasm of the cells that contained centrosome aberrations. The cytoplasmic cyclin E signal may be attributed, in part, to the presence of truncated low-molecular-weight isoforms of cyclin E. In order to isolate the effect of cyclin E on the appearance of centrosome abnormalities, a U2OS tetracycline-repressible cyclin E cell line that has a normal centrosome profile by default was used. With this system, it was confirmed in vitro that persistent cyclin E overexpression is sufficient to cause the appearance of centrosome abnormalities.     

Immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions

Cyclin D1 and E2F-1 proteins are essential for the regulation of the G1/S transition through the cell cycle. Cyclin D1, a product of the bcl-1 gene, phosphorylates the retinoblastoma protein, releasing E2F-1, which in turn activates genes involved in DNA synthesis. Expression patterns of E2F-1 protein in thyroid proliferations have not been reported. This study used monoclonal antibodies for cyclin D1 and E2F-1 proteins to immunostain sections of normal thyroid, hyperplastic (cellular) nodules, follicular adenomas, follicular carcinomas, and papillary carcinomas. The proliferation rate was examined using an antibody specific for the Ki-67 antigen. Fluorescence in situ hybridization (FISH) methods and chromosome 11-specific probes were also employed to determine chromosome copy number and to assess for evidence of amplification at the 11q13 locus in papillary and follicular carcinomas with cyclin D1 overexpression. Concurrent overexpression of Ki-67, cyclin D1, and E2F-1 was found in the majority of benign and malignant thyroid lesions, compared with normal thyroid tissue. Cyclin D1 up-regulation was not due to extra copies of chromosome 11, or bcl-1 gene amplification. Malignant tumours showed the highest expression for all three markers, particularly papillary carcinomas. E2F-1 was detected at the same or slightly lower levels than cyclin D1. It was only found when cyclin D1 was overexpressed. Because cyclin D1 normally activates E2F-1, up-regulation of cyclin D1 may lead to E2F-1 overexpression in benign and malignant thyroid lesions.     

beta-catenin nuclear expression correlates with cyclin D1 overexpression in sporadic desmoid tumours

The immunohistochemical expression of beta-catenin, cyclin D1, Ki-67 and PCNA was Examined in 38 cases of sporadic extra-abdominal or abdominal-wall desmoid tumours without familial adenomatous polyposis (FAP), to evaluate the hypothesis that the accumulated beta-catenin within the nuclei could affect the regulation of the cyclin D1 gene. There was a statistically significant correlation between beta-catenin accumulation and cyclin D1 overexpression (p=0.029). Each group with beta-catenin accumulation or cyclin D1 overexpression showed a higher PCNA-LI than those without, the difference being statistically significant (p=0.007, p=0.004, respectively). Differential PCR was also performed to detect amplification of the cyclin D1 gene and mutational analysis was undertaken for exon 3 of the beta-catenin gene. Amplification of the cyclin D1 gene was observed in 13 out of 22 cases (59.1%). There were nine-point mutations in 7 out of 18 cases (38.9%). The distribution of beta-catenin mutation fell within a wide range, from codon 21 to codon 67. In conclusion, beta-catenin nuclear expression correlated with cyclin D1 overexpression in sporadic desmoid tumours, which could be an in vivo model system for the APC-beta-catenin-Tcf pathway. In addition, beta-catenin mutations in desmoid tumours occurred at an unusually wide range of sites within the gene.     

Expression and prognostic significance of cyclin B1 and cyclin A in non-small cell lung cancer

Aims:  Aberrant expression of cell cycle regulators has been implicated in the pathogenesis of many neoplasms, including non-small cell lung cancer (NSCLC). The aim was to examine the expression and prognostic value of cyclin B1 and cyclin A, key regulators of the G₂/M checkpoint of the cell cycle, in NSCLC and bronchial precursor lesions.
Methods and results:  Immunohistochemical expression of cyclin B1 and A was examined in 90 cases of stage I–II primary NSCLC and bronchial precursor lesions using tissue microarrays. Increased cyclin B1 and A expression was found in 40.9 and 58.9% of NSCLC cases, respectively, and was significantly higher in primary NSCLC, lymph node metastases and some bronchial precursor lesions compared with normal bronchial epithelium. Increased expression of cyclin A and cyclin B1 correlated with tumour type, poorly differentiated tumours and male gender. A significant association was found between increased cyclin B1 expression and reduced survival using Kaplan–Meier survival analysis. On multivariate analysis, cyclin B1 was not an independent prognostic factor ( P  = 0.067). Cyclin A expression was not associated with survival.
Conclusions:  Cyclin B1 and cyclin A are aberrantly expressed in NSCLC and some precursor lesions. Cyclin B1, but not cyclin A, shows some promise as a potential prognostic marker in NSCLC.     

Rsf‐1, a chromatin remodelling protein,interacts with cyclin E1 and promotes tumour development

Chromosome 11q13.5 containing RSF1 (HBXAP), a gene involved in chromatin remodelling, is amplified in several human cancers including ovarian carcinoma. Our previous studies demonstrated requirement of Rsf‐1 for cell survival in cancer cells, which contributed to tumour progression; however, its role in tumourigenesis has not yet been elucidated. In this study, we co‐immunoprecipitated proteins with Rsf‐1 followed by nanoelectrospray mass spectrometry and identified cyclin E1, besides SNF2H, as one of the major Rsf‐1 interacting proteins. Like RSF1, CCNE1 is frequently amplified in ovarian cancer, and both Rsf‐1 and cyclin E1 were found co‐up‐regulated in ovarian cancer tissues. Ectopic expression of Rsf‐1 and cyclin E1 in non‐tumourigenic TP53^mut RK3E cells led to an increase in cellular proliferation and tumour formation by activating cyclin E1‐associated kinase (CDK2). Tumourigenesis was not detected if either cyclin E1 or Rsf‐1 was expressed, or they were expressed in a TP53^wt background. Domain mapping showed that cyclin E1 interacted with the first 441 amino acids of Rsf‐1. Ectopic expression of this truncated domain significantly suppressed G1/S‐phase transition, cellular proliferation, and tumour formation of RK3E‐p53^R175H/Rsf‐1/cyclin E1 cells. The above findings suggest that Rsf‐1 interacts and collaborates with cyclin E1 in neoplastic transformation and TP53 mutations are a prerequisite for tumour‐promoting functions of the RSF/cyclin E1 complex.     

肺鳞癌和肺腺癌中Pin1与cyclin D1的表达及意义

目的探讨Pin1（the peptidy-proly1 isomerase）在肺鳞癌和肺腺癌组织中的表达及其与组织类型、肿瘤分化、分期及淋巴结转移关系,并分析其与细胞周期素（cycinD1）是否存在相关性。方法采用SP免疫组化方法检测69例肺鳞癌和肺腺癌组织中Pin1和cyclin D1的表达。结果免疫组化结果显示：Pin1与cyclinD1在肺鳞癌、肺腺癌中的阳性表达率分别为78．3％（54／69）、52．2％（36／69）。Pin1与cyclinD1在肺癌组织中的表达水平均高于正常肺组织。Pinl的表达与患者的性别、年龄、组织类型、分化、淋巴结转移与否等临床病理学参数无相关。cyclin D1的表达与分化程度负相关（P=0．0274）,而与患者的性别、年龄、组织类型、淋巴结转移与否等临床病理学参数无相关。Pin1和cyclin D1二者的表达呈正相关（P=0．0048）。结论在肺癌中,Pin1的表达高于正常肺组织,但与临床病理学参数无关,而与cyclin D1正相关。其对cyclin D1的影响可能是Pin1发挥作用的主要机制之一。     

Cyclin E overexpression and amplification in human tumours

Cyclin E amplification and overexpression have recently been described in several tumour types. However, many tumour entities have never been examined for cyclin E alterations. Numerous and time-consuming experiments were previously required to determine the significance of potential oncogenes across different tumour types. To overcome this problem, tissue microarrays (TMAs) consisting of 3670 primary tumours from 128 different tumour types, 709 metastases, and 354 normal tissues were generated. Cyclin E alterations were then analysed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Cyclin E gene amplification was observed in 15 different tumour types and subtypes, ie rhabdomyosarcoma, urinary bladder cancer (three subtypes), ovarian cancer (two subtypes), malignant fibrous histiocytoma, adenocarcinoma of the small intestine, medullary breast cancer, gall bladder adenocarcinoma, phaeochromocytoma, gastric adenocarcinoma, squamous cell carcinoma of the uterine cervix, colonic adenocarcinoma, and endometrial carcinoma. Cyclin E protein accumulation was found in 48 different tumour types. The use of TMA technology has enabled us to expand considerably our knowledge of cyclin E alterations in human tumours. The occurrence of amplification and overexpression in many different tumour types suggests that cyclin E plays an important role in tumour biology.     

Expressions of cyclin E, A, and B1 in Hodgkin and Reed–Sternberg cells: Not suppressed by cyclin-dependent kinase inhibitor p21 expression

p21 Is involved in the control of the mammalian cell cycle through the binding and inhibition of cyclin-dependent kinases. The cyclins are dependent on the phases of the cell cycle, and divided into two classes: mitotic cyclins (A, B1, B2) and G1 cyclins (C, D1, D2, D3, E). The product of the p21 gene is a potent downstream effector of the p53 tumor-suppressor gene function. The Hodgkin and Reed- Sternberg (H & RS) cells in Hodgkin's disease are reported to frequently express p53, p21, and nuclear proliferative activity (Ki-67). To clarify the relationship of p21, p53 and cyclins, we performed the immunohistochemistry of p53, p21, Ki-67, cyclin D1, cyclin E, cyclin A and cyclin B1, using 11 cases with Hodgkin's disease. In addition, we performed p53 gene sequencing of exon 5-8, and in situ hybridization of Epstein-Barr virus (EBV) EBER-1 region, whose products have reported to induce the expression of cyclin D. In this study, in all cases, Ki-67 was expressed in almost all H & RS cells, and p53 and p21 were expressed in H & RS cells. No p53 gene mutations were detected in any case, and p53 protein overexpression did not correlate with p53 gene mutations. The number of p21-positive H & RS cells was significantly related with that of the p53-positive cells. The cyclins E, A, B1 and D1 were also expressed in H & RS cells. Unexpectedly, the expression of the cyclins was not suppressed by p21 and p53 expression. In addition, the existence of EBV was not related to the expression of cyclins. It is considered that H & RS cells are, indeed, in cell cycle and commonly express the cell cyclins, and that the cell cycle of H & RS cells may not be specifically fixed in the G1, S, G2 or M phases.     

Reduced selenium-binding protein 1 expression is associated with poor outcome in lung adenocarcinomas

The effects of selenium, an essential nutrient with anti-carcinogenic properties, are mediated by selenium-binding proteins. The protein expression status of human selenium-binding protein 1 (SBP1) in human tumours and the exact function of this protein are not known. In this study, quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used on 93 lung adenocarcinomas and ten uninvolved lung samples. Two likely isoforms of a 56 kD protein that showed a significantly decreased abundance in lung adenocarcinomas were observed. Tandem mass spectrometry and 2-D western blot analysis identified these two proteins as human SBP1. Tumour tissue microarrays were utilized to examine the cellular expression patterns of SBP1 using immunohistochemistry. The same tissue samples were examined for SBP1 mRNA expression using oligonucleotide microarrays. Two major SBP1 isoforms were detected, with an acidic isoform (457) being significantly down-regulated in lung adenocarcinomas compared with normal lung (p = 0.02). Two additional more acidic SBP1 isoforms were only observed in normal lung. SBP1 protein isoforms and SBP1 mRNA levels were significantly decreased in poorly differentiated (versus moderately and well-differentiated), T2-T4 (versus T1), and bronchus-derived (versus bronchioloalveolar) tumours. Low levels of SBP1 protein (native form, 460) correlated significantly with poor survival (p = 0.007). The lack of SBP1 expression was not due to gene deletion. Treatment of A549 lung adenocarcinoma cells with the methylation inhibitor 5-azacytidine did not affect expression of the SBP1 protein. Analysis of the tumour proliferation status using Ki-67 suggests that down-regulated expression of SBP1 may reflect increased cell proliferation and decreased differentiation in lung adenocarcinomas.     

乳腺浸润性导管癌中p57kip2、cyclin D1和cyclin E的表达及意义

目的探讨p57kip2、cyclin D1及cyclin E蛋白在乳腺癌发生、发展中作用.方法用免疫组化S-P法检测64例乳腺浸润性导管癌(invasive ductal carcinoma,IDC)、15例乳腺导管原位癌(ductal carcinoma in situ,DCIS)和15例癌旁正常乳腺组织中p57kip2、cyclin D1和cyclin E蛋白的表达情况.结果p57kip2、cyclin D1和cyclin E蛋白在IDC的阳性率与在乳腺不同组织之间相比,cyclin D1、cyclin E蛋白在DCIS的阳性率与癌旁正常乳腺组织之间相比差异均有显著性(P≤0.05,P＜0.01).在IDC中,三者表达均与腋窝淋巴结转移有关(P≤0.05,P＜0.01),cyclin D1蛋白的表达与组织学分级有关(P＜0.01),cyclinE蛋白的表达与肿块大小有关(P＜0.01);p57kip2与cyclin D1之间、p57kip2与cyclin E之间的表达均呈负相关(P＜0.01)、cyclinD1与cyclin E之间的表达呈正相关(P＜0.01).结论p57kip2蛋白低表达、cyclin D1和cyclin E蛋白高表达可能是乳腺组织恶性转变以及乳腺癌发生淋巴结转移的重要生物学标志,cyclin D1和cyclin E蛋白异常表达是乳腺癌发生的早期事件.联合检测p57kip2、cyclin D1及cyclin E蛋白对预测乳腺癌淋巴结转移有重要意义.     

Limited prognostic value of tissue protein expression levels of cyclin E in Danish ovarian cancer patients: from the Danish 'MALOVA' ovarian cancer study

The primary objective of this study was to assess the expression of cyclin E in tumour tissues from 661 patients with epithelial ovarian tumours. The second was to evaluate whether cyclin E tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays (TA), we analysed the cyclin E expression levels in tissues from 168 women with borderline ovarian tumours (BOT) (147 stage I, 4 stage II, 17 stage III) and 493 Ovarian cancer (OC) patients (127 stage I, 45 stage II, 276 stage III, 45 stage IV). Using a 10% cut-off level for cyclin E overexpression, 20% of the BOTs were positive with a higher proportion of serous than mucinous tumours. Sixty-two per cent of the OCs were positive for cyclin E expression with the highest percentage found in clear cell carcinomas. Results based on univariate and multivariate survival analyses with a 10% cut-off value showed that cyclin E had no independent prognostic value. In conclusion, we found cyclin E expression in tumour tissue to be of limited prognostic value to Danish OC patients.     

Differential expression of telomerase activity in neuroendocrine lung tumours: correlation with gene product immunophenotyping

Telomerase activity was examined by the telomeric repeat amplification protocol (TRAP) assay in 38 neuroendocrine (NE) lung tumours. A significantly (p = 0.001) different frequency of telomerase positivity was observed among different histological tumour types. Specifically, a positive TRAP signal was observed in 14 of 15 (93%) small cell lung cancers (SCLCs), 7 of 8 (87%) large-cell NE carcinomas (LCNECs), and only 1 of 15 (7%) typical carcinoid tumours. When telomerase activity was correlated with the gene product-based immunophenotypic profile of individual tumours, it was found that the absence of telomerase activity was associated with a lack of bcl-2, p53, and c-kit expression, and characterized by a low proliferation index consistent with the absence of cdk-4 expression and the presence of the cdk inhibitor Rb. Such a phenotype was appreciable in most of the carcinoid tumours. Conversely, telomerase-positive tumours generally showed an immunophenotype consistent with gene product alterations (including high expression of bcl-2, p53, and c-kit, and loss of Rb) and were characterized by a high proliferation index. These telomerase data support the previously reported evidence for two genetically unrelated groups of NE lung tumours (SCLC, and to some extent LCNEC, versus carcinoid tumours) that have distinct phenotypic profiles.     

细胞周期蛋白E2在人鼻咽癌组织中的表达及临床意义

目的:研究细胞周期蛋白E2(Cyclin E2)在人鼻咽癌(NPC)组织中的表达及临床意义。方法:应用免疫组织化学法和RT-PCR法检测NPC组织及鼻咽非肿瘤组织Cyclin E2蛋白和mRNA的表达。结果:Cyclin E2蛋白在NPC和鼻咽非肿瘤组织中的表达率分别为75%(45/60)和9.5%(2/21),两者相比有统计学意义(P<0.01);鼻咽癌组织中Cyclin E2蛋白的过表达对同年龄、性别的患者无统计学差异(P>0.05),但与淋巴结转移、分期和5年生存率有相关性(P<0.01)。鼻咽癌组与对照组相比,Cyclin E2 mRNA表达亦有统计学差异(P<0.01)。结论:Cyclin E2表达对判断鼻咽癌病变进展、生存率及转移有重要参考价值。     

Expression of cyclin D1, Ki-67 and PCNA in non-small cell lung cancer: prognostic significance and comparison with p53 and bcl-2

Uncontrolled cell proliferation is the hallmark of malignant tumours. Thus, the proliferative potential of tumour cells is an important prognostic factor. However, evaluation of the prognostic significance of the expression of proteins involved in regulation of cell proliferation remains controversial. In the present study, expression of Ki-67, PCNA and cyclin D1 was estimated in a group of 89 surgically resected non-small cell lung carcinomas using immunohistochemistry. The results were compared with expression of bcl-2 and p53 and with clinicopathological parameters including patients' survival. Ki-67 and PCNA were found to be moderately and highly expressed in 39% and 44% of the tumours, respectively. There was a strong correlation between Ki67 and PCNA expression. Forty five of 88 tumours (51%) showed overexpression of cyclin D1. Surprisingly, cyclin D1 was mainly localized in the cytoplasm and only a small group of tumours (9/88, 10%) showed nuclear staining as well. Bcl-2 and p53 expression was observed in 69% and 30% of the tumours, respectively. All these markers were found to be independent of clinicopathological parameters, except for Ki-67 and bcl-2 expression, which was associated with squamous cell carcinomas. It is concluded that none of the markers that were studied can be used as an independent prognostic factor, whereas the following combinations of markers may have favourable prognostic value: p53 positivity and low Ki-67 expression, p53 positivity and lack of cyclin D1 expression, bcl-2 positivity and low Ki-67 expression, and lack of cyclin D1 expression and low Ki-67 expression.