Expression of cyclin Ds in relation to p53 status in human breast carcinomas

 Cyclin D1 has been reported to be overexpressed in many tumours, including breast carcinomas. Cyclin D1 was first identified as a protooncogene (BCL1/PRAD1), and its overexpression was related to tumour proliferation. The product has also recently been identified as important in mediating cell cycle growth arrest via the p53 pathway in murin fibroblast cell lines. Ninety breast carcinomas previously analysed for p53 status were analysed for amplification of cyclin D1, D2 and D3 genes by Southern blot analysis and for protein expression by immunhistochemistry. In 10 samples gene amplification was detected at the cyclin D1 locus. No gene amplification was detected at the cyclin D2 and D3 loci. Immunoreactivity for cyclin D1 was detected in 38 (42.2%) tumour tissue samples. Fifty samples were immunostained for cyclin D2 and D3. Only 2 samples (4%) showed immunoreactivty for cyclin D2, and 9 samples (18%) for cyclin D3. Cyclin D1 protein overexpression was significantly more often found in tumours with wild type p53 and in tumours with higher grades of differentiation expressing ER. No association was seen between gene amplification of the cyclin D1 gene and p53 status. We conclude there is a relationship between wild type p53 and cyclin D1 protein overexpression in clinical material, indicating that cyclin D1 may be another downstream effector of p53. Received: 25 November 1997 / Accepted: 30 March 1998     

Cyclin D1 gene amplification and overexpression are present in ductal carcinoma in situ of the breast

Cyclin D1 (CCND1) amplification is found in 10–15 per cent of invasive breast carcinomas, but it is not well established whether this gene alteration also occurs in the precursor of invasive breast carcinoma, ductal carcinoma in situ (DCIS). By Southern blot analysis, cyclin D1 gene amplification was detected in 10 per cent (3/32) of DCIS cases. In addition, 15 cases of DCIS were analysed using bright field in situ hybridization (BRISH), of which 11 had already been analysed by Southern blotting. One additional case with gene amplification was found by BRISH. The use of BRISH for the detection of gene amplification is shown to be a novel and reliable in situ method on paraffin-embedded tissue sections. By immunohistochemistry, 147 cases of DCIS were analysed for the expression of cyclin D1. Cyclin D1 overexpression was found in 9 per cent of well-differentiated, 29 per cent of intermediately differentiated, and 19 per cent of poorly differentiated DCIS. No statistically significant association was found between cyclin D1 overexpression and the differentiation grade of DCIS, although 90 per cent of the cases that show overexpression are classified as intermediately and poorly differentiated. An association was found between cyclin D1 overexpression and oestrogen receptor positivity. Cyclin D1 overexpression was found in all four cases with cyclin D1 gene amplification, but was also found in 30 per cent (8/27) of cases without detectable gene amplification. It is concluded that cyclin D1 gene amplification is an early event in the development of breast carcinoma and occurs in poorly differentiated DCIS. Cyclin D1 protein overexpression is also present in tumours without cyclin D1 gene amplification and is seen predominantly in DCIS of intermediately and poorly differentiated histological type and oestrogen receptor positivity. Copyright © 1999 John Wiley & Sons, Ltd.     

Overexpression and localization of cyclin D1 mRNA and antigen in esophageal cancer.

We examined the expression of cyclin D1 mRNA in two human carcinoma cell lines (A431 and TT) and 17 specimens of esophageal cancer with in situ hybridization. Cyclin D1 mRNA was overexpressed in the cytoplasm of cancer cells that showed cyclin D1 gene amplification by Southern blot hybridization. Cyclin D1 antigen was overexpressed in the nucleus of these cancer cells. The distribution of cyclin D1 mRNA-positive cells was similar to that of cyclin D1 antigen-positive cells in the cancer tissues. We then attempted to correlate overexpression of cyclin D1 antigen and prognosis, by using 55 formalin-fixed, paraffin-embedded specimens of esophageal cancer. The overall 5-year survival of patients with strongly staining tumors was significantly lower than that of patients with weakly or nonstaining tumors (7 versus 59%; P < 0.01). There was no significant correlation between cyclin D1 expression and other clinicopathological factors. These results suggest that cyclin D1 may play an important role in carcinogenesis and that cyclin D1 overexpression may be a useful prognostic factor in esophageal cancer.     

Expression of cyclin D1 and c-Ki-ras gene product in human epithelial ovarian tumors

The expression of cyclin Dl gene product in human ovarian tumors was studied. We found that cyclin D1 is expressed at high levels in several ovarian cancer cell lines. Immunohistochemical study also showed that a significant proportion of primary ovarian tumor tissues overexpressed cyclin D1 gene product. Clear nuclear staining of cyclin Dl protein was detected in 28% of the cases. We also characterized the expression of c-Ki-ras gene product in ovarian cancer cell lines and tumor tissues. Amplification or overexpression of this protooncogene has been reported in ovarian tumors from Taiwan. These results show that c-Ki-ras is strongly expressed in PA-1 and NIH: OVCAR-3 cells in which cyclin D1 also expressed at high levels. Specific cytoplasmic staining of c-Ki-ras protein was detected in 11 tumors (52%). Statistical analyses show a strong positive correlation between cyclin D1 and c-Ki-ras immunoexpression. Thus, these data support the ideas that cyclin D1 may be involved in the pathogenesis of ovarian cancer, and coactivation of cyclin D1 and c-Ki-ras gene expression may represent one of the major pathways that lead to the development of ovarian cancer in Taiwan.     

肝细胞癌细胞周期蛋白D1基因的扩增和表达

目的　探讨细胞周期蛋白 (cyclin)D1基因 (CCND1)扩增和cyclinD1在肝细胞癌 (HCC)中的表达情况及其与HCC发生发展的关系。方法　分别应用差异聚合酶链反应 (DPCR)、逆转录(RT) PCR和免疫组织化学法检测 2 0例HCC中cyclinD1基因扩增率、mRNA和蛋白表达状况 ,并分析其与HCC组织学分级的关系。结果　cyclinD1基因扩增结果为 6/ 2 0 ,其mRNA和蛋白过表达分别为9/ 2 0和 14 / 2 0。cyclinD1mRNA表达与基因扩增相关 (P <0 .0 5) ,也和蛋白表达水平相关 (P <0 .0 5)。cyclinD1蛋白表达与HCC组织学分级具有相关性 (P <0 .0 5)。结论　cyclinD1基因扩增在HCC中较常见 ,是引起cyclinD1mRNA和蛋白过表达的主要原因之一。cyclinD1蛋白过表达与HCC的组织学分级相关。cyclinD1基因扩增及过表达可能在肝癌的演进和分化中起重要作用     

Expression profile and molecular genetic regulation of cyclin D1 expression in epithelioid sarcoma.

Epithelioid sarcoma is a distinctive, aggressive soft tissue tumor typically presenting as a subcutaneous or deep dermal mass in the distal extremities of young adults. Molecular genetic data of well-characterized cases of epithelioid sarcoma are sparse. A recent cytogenetic study of epithelioid sarcoma by conventional metaphase comparative genomic hybridization reported recurrent gains at chromosome 11q13, a region containing many genes, including the cyclin D1 gene. Cyclin D1 is a positive cell cycle regulator that is overexpressed in a variety of neoplasms, including mantle cell lymphoma and breast carcinoma. The objective of this study was to examine cyclin D1 expression in epithelioid sarcoma. Of 24 cases evaluated, 23 (96%) displayed cyclin D1 nuclear expression using immunohistochemical evaluation. Eight cases, which expressed cyclin D1 by immunohistochemistry, were evaluated by fluorescence in situ hybridization (FISH) and RNA in situ hybridization (RISH) for amplification of the cyclin D1 gene and messenger RNA (mRNA) expression, respectively. Seven of eight cases showed a typical eusomic state. One case showed pseudoamplification due to aneusomy/polysomy. There was no evidence of cyclin D1 gene amplification or messenger RNA overexpression detected by FISH or RNA in situ hybridization analyses, respectively. Our data clearly demonstrate that cyclin D1 protein is upregulated in epithelioid sarcoma, suggesting a role for this cell cycle regulator in the pathogenesis of epithelioid sarcoma. The high level of cyclin D1 protein expression in epithelioid sarcoma appears to be regulated by translational and/or post-translational mechanisms.     

Correlation of cyclin D1 and Rb gene expression with apoptosis in invasive breast cancer.

BACKGROUND: In vitro studies have shown that amplification and overexpression of the cyclin D1 gene can accelerate the progress of cells through the G1 phase. Therefore, cyclin D1 may have an apoptosis inhibiting effect. The retinoblastoma (Rb) gene was shown recently to be an important regulator of apoptosis. AIMS: To evaluate whether expression of the cyclin D1 and Rb genes correlated with apoptotic counts in a group of 97 invasive breast cancers. METHODS: Expression of the cyclin D1 and Rb genes was detected by standard immunnohistochemistry using paraffin wax embedded sections. Apoptotic cells were counted according to a strict protocol, in 10 fields of vision systematically spread over the most poorly differentiated area of the tumour, at a magnification of x630. Apoptotic cells counts were expressed as apoptotic cells/mm2. RESULTS: Cyclin D1 overexpression was found in 49% of cases. Loss of Rb expression was found in 44% of cases, and occurred particularly in poorly differentiated tumours. Cyclin D1 and Rb expression showed a positive correlation (p = 0.003). Apoptotic counts varied from 1 to 62/mm2. There were no significant correlations between cyclin D1 overexpression and apoptotic counts in the total group or in the retinoblastoma protein (pRb) positive tumours. Loss of Rb expression also showed no correlation with apoptotic counts. CONCLUSIONS: Cyclin D1 is frequently overexpressed in pRb positive tumours, but no evidence has been found for an anti-apoptotic effect of cyclin D1 overexpression or Rb expression in invasive breast cancer.     

Absence of CCND1 gene amplification in breast tumours of BRCA1 mutation carriers.

BACKGROUND/AIMS: It was recently reported that significantly fewer breast tumours of BRCA1 mutation carriers overexpressed cyclin D1 and HER2 protein than tumours of age matched breast cancer cases unselected for family history. This study aimed to examine the genetic basis of this reduction by determining the frequency of tumours within this cohort showing DNA amplification of these genes. METHODS: Paraffin wax embedded sections of breast tumours from BRCA1 mutation carriers and age, grade, histological type, and tumour size matched non-familial controls that had previously been stained for cyclin D1 and HER2 protein overexpression were analysed for CCND1 and HER2 gene amplification using fluorescence in situ hybridisation. RESULTS: CCND1 amplification was detected in none of the 30 tumours of the BRCA1 mutation carriers and in 19 of 74 tumours of the matched controls. Of those samples previously determined to overexpress the HER2 protein, HER2 amplification was detected in one of three tumours from BRCA1 mutation carriers and in 13 of 17 tumours of the age matched non-familial cases. CONCLUSION: None of the tumours of BRCA1 mutation carriers showed CCND1 amplification and only one tumour showed HER2 amplification. In contrast, a large proportion of cyclin D1 and HER2 overexpression in tumours of non-familial breast cancer cases could be accounted for by amplification of these genes. These data suggest that breast tumorigenesis in BRCA1 mutation carriers occurs by a molecular mechanism distinct from that of age matched non-familial cases.     

Cyclin alterations in giant cell tumor of bone.

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Because alterations of several cyclins, especially cyclin D1, have been implicated in the development of many human neoplasms, we examined 32 cases of giant cell tumor of long bones for cyclin D1 gene amplification and protein overexpression using differential polymerase chain reaction and immunohistochemistry, respectively. In addition, the expression of cyclin D3, cyclin B1, and the proliferation-associated antigen Ki-67 (MIB-1) was assessed immunohistochemically. Low-level cyclin D1 gene amplification was detected in 61% of giant cell tumor cases. All tumors showed cyclin D1, cyclin D3, cyclin B1, and Ki-67 (MIB-1) staining; however, the distribution was very characteristic. Cyclin D1 protein expression was seen predominantly in the nuclei of the giant cells, with occasional mononuclear cells staining. There was no correlation between cyclin D1 gene amplification and protein overexpression. Cyclin D3 staining showed a similar distribution, with 88% of cases showing protein overexpression. Cyclin D1 and/or D3 staining in the giant cells was never associated with staining for either cyclin B1 or Ki-67 (MIB-1), as the expression of the latter two proteins was restricted to the mononuclear cells. Cyclin B1 overexpression was seen in 44% of cases. Ki-67 (MIB-1) staining was present in all cases, and between 10 to 50% of the mononuclear cells were positive. These results suggest that alterations in cyclin D1 and/or D3 might play a role in the pathogenesis of giant cell tumor of bone.     

p21WAF1/Cip1 is associated with cyclin D1CCND1 expression and tubular differentiation but is independent of p53 overexpression in human breast carcinoma

p21^WAF1/Cip1 is an inhibitor of cdk/cyclin complexes, and thus regulates the cell cycle. p21 is also related to cell differentiation and is regulated by wild-type p53, although p53-independent regulatory pathways have been proposed. In order to analyse p21 expression as well as its relationship with p53 in human breast cancer, an immunohistochemical analysis was undertaken of 77 breast carcinomas, 16 of them with an in situ component; 30 adjacent normal tissue samples; and five non-neoplastic specimens. Forty-four infiltrating carcinomas (57 per cent) were p21-positive. Expression of p21 was also observed in pre-invasive lesions, whereas normal ducts were negative or focally and weakly positive. p21 expression was associated with high histological grade (II+III) (P-0·017) and poor tubule formation (P-0·002), and was significantly less frequent in lobular carcinomas (P-0·0001). p21 positivity also correlated with increased proliferation, but this seemed to be dependent on the histological grade. Twenty carcinomas (26 per cent) showed p53 overexpression, but this was not associated with p21 negativity, suggesting the existence of p53-independent mechanisms for p21 regulation in vivo. Cyclin D1^CCND1 expression was analysed in the same series and an association between p21 and cyclin D1 expression was found, since 23 of 26 cyclin D1-positive carcinomas were p21-positive (P<0·001 …). In conclusion, p21 is frequently overexpressed in breast carcinomas and this occurs in the early stages of neoplastic progression. This overexpression seems to be independent of p53 status and might be involved in cyclin D1 modulation. © 1998 John Wiley & Sons, Ltd.     

Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients

The amplification of cyclin D1, located on chromosome 11q13, in breast cancer patients has been found to be associated with reduced relapse-free and overall survival; however, there still exists strong controversy about these findings. In order to evaluate the prognostic value of cyclin D1 and other prognostic variables in human breast cancers, we have assessed estrogen receptor (ER) status, cyclin D1, c-erbB2 and p53 overexpression in 175 primary breast carcinomas, and investigated the relationships of prognostic variables to the patient clinical outcome and the association between cyclin D1 overexpression and other prognostic variables. There was some degree of variability in staining intensities and proportions within the same tumor. The overexpression of both cyclin D1 and ER revealed a significantly prolonged survival in univariate analysis (P = 0.020). Among the various prognostic variables, distant metastasis showed a statistically significant association with overall survival. A significant correlation was observed between cyclin D1 overexpression and small size of the primary tumor (P = 0.031), low Bloom and Richardson's histological grade (P = 0.001), and positive ER status (P = 0.000). In contrast to what was previously expected, the present study suggests that the overexpression of cyclin D1 has a tendency to have a positive clinical outcome and a potential role in identifying a subset of patients predicting a good prognosis, particularly when ER is coexpressed.     

PPM1D silencing by RNA interference inhibits proliferation and induces apoptosis in breast cancer cell lines with wild-type p53

PPM1D is an oncogene that is amplified and overexpressed in many human tumors, including breast cancer. It functions as a negative regulator of the p38 MAP kinase-p53 signaling pathway and is also proposed to participate in other critical cell survival pathways. To define the functional significance of PPM1D specifically in breast cancer, we used RNA interference to inhibit PPM1D expression in BT-474, MCF7, and ZR-75-1 breast cancer cell lines harboring amplification and increased expression of PPM1D. Efficient downregulation of PPM1D resulted in significantly reduced cell proliferation in MCF7 and ZR-75-1 cells carrying wild-type p53 but not in BT-474 carrying mutant p53, which indicates that the antiproliferative effect of PPM1D silencing is dependent on the p53 status of the cells. This result is in excellent agreement with the notion that PPM1D activation is an alternative mechanism for p53 inactivation. Additionally, our data indicate that the reduced cell growth observed after PPM1D silencing is due at least in part to increased apoptotic cell death. Our findings demonstrate that PPM1D is involved in the regulation of cell proliferation in breast cancer in a p53-dependent manner and that overexpression of PPM1D contributes to malignant phenotype by promoting sustained cell growth and cell survival.     

Deletion of 11q23 and cyclin D1 overexpression are frequent aberrations in parathyroid adenomas

Hyperparathyroidism may result from parathyroid hyperplasia or adenoma, or rarely from parathyroid carcinoma. Pericentromeric inversion of chromosome 11 that results in activation of the P:RAD1/cyclin D1 gene and tumor suppressor gene loss have been described as genetic abnormalities in the evolution of parathyroid neoplasms. We studied tissue samples taken from primary parathyroid hyperplasia, parathyroid adenoma, and histologically normal parathyroid tissue by comparative genomic hybridization, fluorescent in situ hybridization, and immunohistochemistry for cyclin D1. DNA copy number changes were infrequent in primary hyperplasia (4 of 24, 17%), but common in adenomas (10 of 16, 63%; P: = 0.0059). The most common change was deletion of the entire chromosome 11 or a part of it, with a minimal common region at 11q23. This change was present in five (31%) adenomas and two (8%) primary hyperplasias. Fluorescent in situ hybridization confirmed the presence of both MEN1 alleles located at 11q13 despite deletion of 11q23 in all three cases studied. Cyclin D1 was overexpressed in six (40%) of the 15 adenomas studied, whereas none of the 27 hyperplasias (P: = 0.0010) nor the five histologically normal tissue samples overexpressed cyclin D1. Either DNA copy number loss or cyclin D1 overexpression was present in 13 (81%) of the 16 adenomas. We conclude that DNA copy number loss and cyclin D1 overexpression are common in parathyroid adenomas. The region 11q23 is frequently lost in parathyroid adenomas and occasionally in parathyroid hyperplasias, and this suggests the possibility that a tumor suppressor gene that is important in their pathogenesis is present on 11q23.     

Expression of cyclin D1 and retinoblastoma protein in Paget’s disease of the vulva and breast: an immunohistochemical study of 108 cases

Aims: Loss of retinoblastoma protein expression and overexpression of cyclin D1 have been implicated in the development and progression of some cancers. Paget’s disease of the vulva (PDV) and Paget’s disease of the breast (PDB) are uncommon conditions and the pathogenesis of these diseases is still unclear. The aim was to examine the expression of the retinoblastoma and cyclin D1 proteins in PDV and PDB and to correlate any differences between PDV and PDB, and in the presence or absence of an underlying carcinoma. Methods and results: Seventy‐two archival cases of PDV including 10 with invasive disease and 36 cases of PDB were evaluated immunohistochemically for the expression of cyclin D1 and retinoblastoma protein. Forty‐four percent (32/72) of cases of PDV showed loss of expression of the retinoblastoma protein, compared with 67% (24/36) of PDB cases. Fifty‐nine percent (41/69) of PDV overexpressed cyclin D1. In PDB, 8% (3/34) overexpressed cyclin D1. There were no significant differences in the expression of retinoblastoma and cyclin D1 in PDV cases with or without underlying invasive disease. There were significant differences between the expression of retinoblastoma (P = 0.03) and cyclin D1 (P < 0.001) in PDV compared with PDB. Conclusions: The differences in the expression of cyclin D1 and retinoblastoma may indicate the differences in the pathogenesis of PDV and PDB.     

Expression and amplification of Her2, EGFR and cyclin D1 in breast cancer: immunohistochemistry and chromogenic in situ hybridization

Determination of Her2, epidermal growth factor receptor (EGFR) and cyclin D1 status is now of major clinical importance due to the development of molecule-targeting drugs in anticancer therapy. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) are the most simple and convenient methods for evaluating gene alterations and their protein consequences. The purpose of the present study was to investigate the status of Her2, EGFR and cyclin D1 on both IHC and CISH in 95 primary breast carcinomas. There was substantial consistency between the IHC and CISH results of Her2 and EGFR, showing fair agreement between protein overexpression and gene amplification. However, cyclin D amplification was not related to protein overexpression. Moreover, there was no correlation between Her2, EGFR and cyclin D1. Her2 protein overexpression and amplification were positively associated with histological grade, nuclear grade and inversely correlated with the expression of estrogen receptor (ER) and progesterone receptor (PR). In ER-negative and postmenopausal patients, EGFR gene amplification was strongly associated with worse recurrence-free survival (P = 0.0087, P = 0.0149, respectively). Overall, the present findings suggest that EGFR gene amplification is important in predicting prognosis and this should be evaluated in breast carcinoma in addition to Her2 status in routine pathological practice.     

Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53

Cyclin B1, which plays a key role in the control of cell cycle progression from G(2) through M phase, was recently identified by us as a tumor antigen recognized by human T-cells. To understand what makes this normal molecule antigenic, we compared its expression in malignant versus normal cells. Immunohistology showed overexpression of cyclin B1 protein in tumors compared to surrounding normal tissue and localization in the cytoplasm rather than the nucleus. Cyclin B1 is overexpressed at protein and mRNA level in many tumor cell lines including breast, lung, colorectal carcinoma, lymphoma and leukemia. While overexpressed in tumor cells at all stages of the cell cycle, its expression still peaks at G(2)/M phase, as it does in normal cells. We compared cyclin B1 expression in two cell clones derived from the same colorectal tumor cell line, one wild type for p53 (HCT116p53(+/+)) and one with deleted p53 (HCT116p53(-/-)). HCT116p53(+/+) cells had undetectable (normal) level of cyclin B1 protein, while HCT116p53(-/-) cells showed overexpression. When reconstituted with p53, HCT116p53(-/-) cells reverted to normal cyclin B1 expression. We conclude that p53 plays an important role in cyclin B1 regulation and that tumors with mutated p53 will be good candidates for cyclin B1 based immunotherapy.     

表达反义细胞周期蛋白cyclinD1载体对胰腺癌细胞生长和凋亡的影响

目的观察反义细胞周期蛋白ｃｙｃｌｉｎＤ１对胰腺癌细胞生长的影响。方法采用Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ方法检测了５株人胰腺癌细胞系中ｃｙｃｌｉｎＤ１的扩增及表达情况，发现ＰＣ７细胞中基因有扩增及过度表达。我们构建了反义（ＡＳ）ｃｙｃｌｉｎＤ１的表达载体，采用ｌｉｐｏｆｅｃｔｉｎ转染方法转染ＰＣ７细胞，获得转化细胞系ＰＣ７／ＡＳｃｙｃｌｉｎＤ１。经Ｎｏｒｔｈｅｒｎｂｌｏｔ、Ｗｅｓｔｅｒｎｂｌｏｔ检测转化细胞系，表明有外源反义ｃｙｃｌｉｎＤ１的表达，而内源性ｃｙｃｌｉｎＤ１ｍＲＮＡ表达及蛋白合成下调，并进行了细胞凋亡分析。结果转化细胞系细胞恶性生物学行为及表型部分逆转，细胞生长速度、ＤＮＡ合成及细胞增殖和代谢能力均明显下降，软琼脂集落形成能力减低。流式细胞术分析发现细胞被阻滞于Ｇ１期，ＤＮＡ凝胶电泳、原位凋亡检测发现凋亡细胞增多。结论通过反义ＲＮＡ技术，下调在细胞周期调控中起重要作用的ｃｙｃｌｉｎＤ１的表达，可使胰腺癌细胞恶性表型部分逆转。     

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.