Cyclin D1 Gene Amplification Research Articles

Changes in the Cyclin D1 Gene Copy Number in Melanoma Cell Lines Detected by Double-Target Fluorescence In Situ Hybridization.

The cyclin D1 gene is often amplified in solid tumors. Six human melanoma cell lines derived from tumors at various stages of progression were analyzed by double-target fluorescence in situ hybridization (FISH) and dot blot hybridization to detect cyclin D1 gene amplification and deletion. FISH was performed using a probe for cyclin D1 and an alpha satellite of the chromosome 11 probe, to analyze the cyclin D1 copy number relative to the numbers of chromosome 11 in individual cells. Copies of both the cyclin D1 gene and chromosome 11 were observed in the nuclei of all cell lines. However, the copy numbers of the cyclin D1 gene and of chromosome 11 were closely associated and the copy number ratio of cyclin D1 to chromosome 11 (cyclin D1/chromosome 11) was almost one in all cell lines. This finding indicated that the extra copies of cyclin D1 were always accompanied by extra copies of chromosome 11. At the cellular level, cyclin D1 amplification was detected in fewer than 5% of interphase nuclei in only two cell lines, and the gene was deleted in all cell lines to various degrees. Dot blot hybridization revealed no amplification of cyclin D1 in any of the cell lines. We concluded that amplification of the cyclin D1 gene is rare in these melanoma cell lines.

Dysregulated cyclin D1 expression early in head and neck tumorigenesis: in vivo evidence for an association with subsequent gene amplification.

Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Amplification of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of fluorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of amplification at the 11q13 band, we analysed 46 paraffin-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 amplified cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene amplification in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 amplified tumors and five of the 13 (38.4%) non-amplified tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 amplified tumors, and it appeared to precede cyclin D1 gene amplification. In contrast no dysregulated expression was detected in the premalignant lesions of the non-amplified tumors. In conclusion, these findings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene amplification.

Differential polymerase chain reaction and immunohistological studies on amplification and expression of cyclin D1 gene in human glioma tissues

Using differential polymerase chain reaction we studied the amplification of the cyclin D1 gene in formalin‐fixed, paraffin‐embedded surgical specimens of 70 gliomas. We also examined expression of cyclin D1 in these gliomas, and the Ki‐67 labeling indices in 24 glioblastomas by immunohistochemistry. Among 70 gliomas, amplification of cyclin D1 gene was found in only one glioblastoma. The expression of cyclin D1 was immunohistochemically detected in only one glioblastoma with cyclin D1 gene amplification. The highest labeling index of Ki‐67, 48.0%, was noted in one of cyclin D1 positive glioblastoma (the average labeling index of 30.2% in cyclin D1 negative glioblastomas). These results suggest that amplification of cyclin D1 gene could play a role in the development of glioblastoma but it is unlikely to be the main gene alteration in malignant progression of gliomas in the majority of cases.

Deletion of the p16 gene and microsatellite instability in carcinoma arising in pleomorphic adenoma of the parotid gland.

Carcinoma arising in pleomorphic adenoma of the salivary gland is a rare tumor, and its molecular aspects are unknown. Recent studies have revealed that malignant transformation of various human cancers may involve two different genetic alterations: inactivation of the p16 gene, which is a putative tumor suppressor gene, and genetic instability represented by microsatellite instability (MSI). However, so far, molecular investigations including p16 gene alteration and MSI have not been performed on carcinoma arising in pleomorphic adenoma. Both inactivation of the p16 gene and MSI were studied using DNA extracted from paraffin-embedded sections of carcinoma arising in pleomorphic adenoma. Samples also were analyzed for cyclin D1 gene amplification, which is thought to have oncogenic effects similar to those with inactivation of the p16 gene. One case showed the homozygous deletion of the p16 gene in the carcinoma, although hypermethylation of the p16 gene and amplification of the cyclin D1 gene were not observed in any cases. In two of four cases, MSI was observed. One case in two showed MSI in both the pleomorphic adenoma and the carcinoma. Results of this study suggest that two different genetic alterations, the inactivation of the p16 gene and genetic instability, play roles in the malignant transformation of carcinoma in pleomorphic adenoma. The MSI observed in the adenoma suggests that genetic alterations occur in pleomorphic adenoma.

Cyclin D1 gene amplification in esophageal carcinosarcoma shown by differential polymerase chain reaction

We studied cyclin D1 gene amplification in four esophageal carcinosarcomas using the differential polymerase chain reaction. Three cases showed amplification in the sarcomatous components, and one of these also showed amplification in the carcinomatous component. No amplification was detected in the other case. We also examined p53 expression in these four tumors by immunostaining. Both components of all four cases showed diffuse overexpression of p53 protein in the nuclei. Our results showed that the cyclin D1 gene is frequently amplified in esophageal carcinosarcoma, especially in the sarcomatous component. Two cases showed amplification only in the sarcomatous component, suggesting that amplification may have a role in the pathogenesis of that component of this malignancy. The diffuse overexpression of p53 observed in both components suggests that such overexpression, which could be the result of somatic mutation, might be an early event in the pathogenesis of esophageal carcinosarcoma.

Amplification and overexpression of cyclin D1 in aggressive human esophageal cancer.

Cyclin D1 is a cell-cycle regulator essential for G1 phase progression and a candidate proto-oncogene implicated in pathogenesis of several human tumor types including esophageal cancers. Association between cyclin D1 gene amplification and clinical outcome was examined using southern blotting in 45 patients with primary esophageal cancer. In cases showing gene amplification, expression levels of mRNA and gene product were further examined by northern blotting, western blotting and immunohistochemical analyses. Amplification of cyclin D1 gene was found in 14 of 45 patients (31. 1%). There was no association between gene amplification and clinicopathological parameters but the frequency of hematogenous recurrence in cases with gene amplification was significantly higher than that in cases without amplification in 30 patients undergoing curative surgery (70.0% vs 25.0%, p<0.05). Furthermore, cumulative survival rate of cases with gene amplification was significantly lower than that of cases without amplification (p<0.05). Overexpression of cyclin D1 mRNA was observed in 12 of 14 cases (85. 7%) showing gene amplification but overexpression of its protein was found in only four cases (28.6%). Amplification of the cyclin D1 gene is a reliable prognostic factor and post-translational modification of this gene may play a functionally important role in development of primary esophageal cancer.

Inverse association between cyclin D1 overexpression and retinoblastoma gene mutation in thyroid carcinomas.

Cyclin D1 plays a key role in the regulation of the G1/S transition through the cell cycle. Deregulation of cyclin D1, most often leading to overexpression of the gene, has been reported in many tumor types. It has been suggested that cyclin D1 overexpression could be an alternative mechanism for pRb inactivation. We have previously found Rb gene mutations in 55% of malignant thyroid tumors. In the present study, we examined the cyclin D1 gene expression and amplification in 24 tumor samples (two of them are benign goiters) randomly selected from the same series of thyroid tumors, to see whether cyclin D1 overexpression is present in those specimens without Rb gene mutations. We found a four- to fivefold increase in cyclin D1 expression in 7 of 22 thyroid carcinomas as compared with that in benign nodular goiters. Six of them were found in carcinomas without Rb gene mutations. Among the remaining 15 thyroid carcinoma samples, 11 were found previously to have Rb gene mutations. The association between increased cyclin D1 expression and absence of Rb mutation is statistically significant (p < 0.05). We found no evidence of the cyclin D1 gene amplification or rearrangement to account for such an increase in cyclin D1 expression. We conclude that cyclin D1 overexpression may be relevant to thyroid carcinogenesis. Two mechanisms may be involved in the inactivation of pRb: one is through Rb gene mutations, and the other is by cyclin D1 overexpression.

P16MTS1/CDK4I mutations and concomitant loss of heterozygosity at 9p21-23 are frequent events in squamous cell carcinoma of the larynx.

We have examined the presence of p16MTS1/CDK4I gene deletions, mutations and methylation status, and 9p21-23 deletions in a series of 46 squamous cell carcinomas of the larynx and paired normal mucosa previously characterized for cyclin D1 gene amplification and overexpression. pRb expression was also examined by immunohistochemistry. p16MTS1/CDK4I mutations were found in 10/46 (22%) carcinomas and hypermethylation in 2/31 (7%). Loss of heterozygosity at 9p21-23 was found in 24 out of 42 (57%) carcinomas examined. All p16MTS1/CDK4I mutated cases and the two hypermethylated carcinomas showed 9p21-23 loss of heterozygosity. The loss of heterozygosity correlated with advanced local invasion (P=0.0045), lymph node metastases (P=0.0326), stage IV of the tumors (P=0.0058), and existence of cyclin D1 amplification/overexpression (P < 0.03). Only one out of 37 carcinomas was negative for pRb expression. No alterations in p16 gene or 9p21-23 loss of heterozygosity were detected in this case. These findings indicate that p16MTS1/CDK4I is frequently inactivated by gene mutation, hypermethylation, and allelic deletions in a significant subset of squamous cell carcinomas of larynx. Since 9p21-23 loss of heterozygosity was more frequently detected than p16MTS1/CDK4I mutations, and mutated carcinomas invariably had loss of heterozygosity, allelic losses probably precede the p16MTS1/CDK4I mutations. Their association with cyclin D1 deregulation in advanced carcinomas could indicate a possible cooperative effect in the progression of these neoplasms.

Quantitative analysis of cyclin D1 messenger RNA expression in head and neck squamous cell carcinomas.

Cyclin Dl is thought to play a critical role in the G1/S phase transition of the cell cycle. Amplification of this gene has been reported in several types of human neoplasms including breast, lung, esophageal, and head and neck tumors. In this study, we have analyzed the relative level of expression of cyclin D1 messenger RNA (mRNA) in fresh specimens of head and neck squamous cell carcinoma (HNSCC), and investigated the concordance of the overexpression of cyclin D1 mRNA with gene amplification. Levels of cyclin D1 mRNA were analyzed hy a modified method of competitive reverse transcription‐polymerase chain reaction and levels of cyclin D1 gene amplification were evaluated by Southern blot hybridization in a series of 23 matched normal mucosas and HNSCC. Overexpression of cyclin D1 mRNA was observed in 10 of 23 cases (43.5%) of HNSCC, ranging from 2 to 50‐fold higher than the normal control. Twelve of 23 cases could be evaluated by Southern blot hybridization, and gene amplification was found in only 2 of 12 cases (16.7%), These findings suggest that cyclin D1 plays an important role in tumorigenesis of HNSCC, and gene amplification is not one of the major mechanisms for overexpression of cyclin D1.

Amplification and expression of the cyclin D1 gene in anal and esophageal squamous cell carcinomas

Cyclin Dl is a cell-cycle regulator and candidate proto-oncogene implicated in the pathogenesis of numerous tumor types. Amplification of the cyclin Dl gene occurs commonly in esophageal squamous cell carcinomas. However, no studies have examined the role of cyclin D1 in anal carcinogenesis. We examined 20 esophageal squamous cell carcinomas and 24 anal carcinomas for cyclin Dl alterations. Protein expression was evaluated by immunohistochemistry using the cyclin D1GM antibody (Novocastra, Newcastle upon Tyne, UK). Cyclin D1 amplification was examined by fluorescent in situ hybridization (FISH), using a cyclin D1 probe obtained from Toshiya Inaba at St. Jude Children's Research Hospital, Memphis, TN. The FISH sections were analyzed using a Leica (Deerfield, IL) confocal microscope. By immunohistochemistry, 75% of esophageal carcinomas showed evidence of cyclin D1 expression. Cyclin Dl amplification was detected by FISH in 65% of esophageal cancers. There was good correlation between cyclin D1 protein expression and gene amplification, although some tumors showed protein overexpression in the absence of gene amplification. Among the 24 anal carcinomas studied, 8% showed weak cyclin D1 immunoreactivity in rare tumor cells. None of the anal tumors showed cyclin D1 amplification. We conclude that cyclin Dl alterations are common in esophageal carcinomas but do not appear to be important in anal carcinogenesis. Immunohistochemical detection of cyclin D1 protein overexpression is a good predictor of cyclin D1 amplification.

Increased expression of cyclin D1 is an early event in multistage colorectal carcinogenesis

BACKGROUND & AIMS: Cyclin D1 gene amplification and/or overexpression occurs in several human cancers. The level of expression of cyclin D1 protein during the multistage process of human colon carcinogenesis was determined. METHODS: Cyclin D1 protein abundance was determined by immunostaining samples of normal colonic mucosa(n=23), transitional normal mucosa adjacent to adenomas or adenocarcinomas (n=41), hyperplastic polyps (n=8), adenomatous polyps (=35), and adenocarcinomas (n=27), using a polyclonal anti-human cyclin D1 antibody. RESULTS: Cyclin D1 nuclear staining occurred in 30% of adenocarcinomas and 34% of adenomatous polyps but not in hyperplastic polyps or normal or transitional mucosa. Nuclear staining did not correlate with sex, age, size, or dysplasia of the adenomatous polyps or with differentiation and Dukes' staging of the adenocarcinomas. Left- sided colon neoplasms showed nuclear staining more frequently than those right-sided lesions. Diffuse or supranuclear cytoplasmic staining occurred in about one third of hyperplastic polyps, adenomas, and adenocarcinomas and in transitional mucosa adjacent to adenocarcinoma. CONCLUSIONS: Increased nuclear expression of cyclin D1 occurs in around one third of colonic tumors as an early event during multistage process of colon carcinogenesis. Increased expression of cyclin D1 may perturb cell-cycle control in benign adenomas and thereby enhance tumor progression. (Gastroenterology 1996 Mar;110(3):669-74)

Amplification and overexpression of the cyclin D1 gene in head and neck squamous cell carcinoma

Aims-To determine cyclin D1 gene amplification and expression levels in head and neck squamous cell carcinoma (HNSCC) patients.Methods-Total RNA and genomic DNA were isolated from 40 samples of HNSCC tissue and matched normal tissue and were hybridised with a cyclin D1 cDNA probe. Northern and Southern analyses were used to detect mRNA overexpression and cyclin D1 gene amplification, respectively.Results-15 of the 40 HNSCC samples examined (38%) showed cyclin D1 gene amplification. Of these 15 samples, all 13 from which RNA was available showed increased cyclin D1 expression.Conclusions-HNSCC patients with both amplification and overexpression of the cyclin D1 gene are at greater risk than patients who showed no cyclin D1 gene amplification; amplification and over-expression of the cyclin D1 gene may play an important role in the progression of HNSCC and in clinical outcome.

Differential Polymerase Chain Reaction Assay of Cyclin Dl Gene Amplification in Esophageal Carcinoma

The cyclin D1 gene, located on chromosome 11q13, is frequently rearranged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin D1 and other markers on chromosome 11q13 (evaluated by Southern or slot blot assays) in approximately 25-50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differential polymerase chain reaction to the evaluation of cyclin D1 gene amplification in squamous cell carcinomas of the esophagus. Cyclin D1 was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin D1 amplification (70%) than in those without amplification (37%). In 36 patients with follow-up, cyclin D1 amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin D1 gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

Altered expression of the cyclin D1 and retinoblastoma genes in human esophageal cancer.

We have examined DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas obtained from China, Italy, and France for amplification of the cyclin D1 gene. We also examined 36 of these 50 carcinomas for expression of the cyclin D1 and retinoblastoma (RB) proteins by immunohistochemistry. We found a 3- to 10-fold amplification of the cyclin D1 gene in 16 of the 50 (32%) tumors and in two of the four cell lines. Cyclin D1 protein was overexpressed in 12 of 13 tumors and the two cell lines that showed gene amplification when compared to normal controls. Studies on RB protein expression indicated that 6 of the 36 (17%) tumor samples examined and one cell line did not show detectable expression of this protein. The tumors and cell lines that had cyclin D1 gene amplification and overexpression exhibited normal levels of expression of RB protein. By contrast, the tumors and cell line that did not appear to express the RB protein did not show amplification of the cyclin D1 gene and expressed only low levels of the cyclin D1 protein (P = 0.03). These results suggest that the inhibitory effect of RB on cell cycle progression can be abrogated during tumor development either by loss of expression of the RB gene or by increased expression of the cyclin D1 gene.