Cyclin D1 and estrogen receptor messenger RNA levels are positively correlated in primary breast cancer

The CCND1 gene, encoding the cell cycle regulatory protein cyclin D1, maps to chromosome 11q13, a locus that is amplified in about 13% of breast cancers. Because several studies have indicated a relationship between 11q13 amplification and markers of phenotype including estrogen receptor (ER) status, we tested the relationship between CCND1 and ER gene expression in 364 primary breast cancers using Northern blot analysis. Seventy-three % of samples were positive for ER mRNA, and cyclin D1 mRNA levels in the ER-positive group were significantly higher than those in the ER-negative group (P = 0.0001). When the samples were divided into quartiles of cyclin D1 expression, 58% of samples were ER positive in the lowest quartile and 87% in the highest quartile. The tumors expressing the highest levels of cyclin D1 (7%) were all ER positive. Furthermore, ER mRNA levels in the half with lower cyclin D1 mRNA were significantly less than in the half with higher cyclin D1 levels (P = 0.0001). Using simple regression analysis, there was a significant positive correlation between cyclin D1 and ER mRNA levels in the total population (P = 0.0001). This study demonstrates that cyclin D1 mRNA and ER mRNA are positively correlated in primary breast cancer, but the functional relationship between these genes remains to be elucidated.     

Cyclin DI amplification is not associated with reduced overall survival in primary breast cancer but may predict early relapse in patients with features of good prognosis

Amplification of chromosome 11q13 is frequently observed in human malignancies, including breast cancers. A candidate oncogene at this locus is the CCND1 gene, which encodes the cell cycle regulatory protein cyclin D1. Because published data on the relationship between 11q13 amplification and prognosis in breast cancer have been controversial, we investigated the clinical significance of CCND1 amplification and its association with established clinicopathological features of prognosis in 1014 primary breast cancer patients. Amplification of the CCND1 gene and the INT-2/FGF-3 gene, which also maps to 11q13, was 10% and 17%, respectively. There were no associations between CCND1 or INT-2 amplification and patient age, tumor size, tumor grade, axillary lymph node status, HER/neu amplification, MIB-1 monoclonal antibody to Ki67 antigen count, or p53 expression. CCND1 amplification was predominantly observed in hormone receptor-positive tumors; at a copy number >/=3, CCND1 amplification was significantly correlated with both estrogen receptor (ER; P = 0.036) and progesterone receptor (P = 0.012) positivity. After a median follow-up period of 66 months, CCND1 or INT-2 amplification was not associated with significant increases in relapse or death from breast cancer. However, in the node-negative and ER-positive subgroups, there was a trend for an increased relapse rate in patients with INT-2 or CCND1 amplification. Thus, in this study, assessment of CCND1 or INT-2 amplification at 11q13 by slot-blot hybridization was of little use in determining phenotype or disease outcome in the whole group of patients but had a potential role in identifying a subset of poor-prognosis patients within the node-negative or ER-positive, good-prognosis groups. Because the prevalence of CCND1 amplification is much lower than the reported prevalence of cyclin D1 overexpression, additional studies are required to determine the true prognostic significance of altered cyclin D1 expression in breast cancer.     

Clinical significance of cyclin D1 expression in patients with node-positive breast carcinoma treated with adjuvant therapy

BACKGROUND: Experimental and clinical studies suggest that cyclin D1 is involved in transformation and tumour progression. However, there is little and contradictory data on the clinical significance of cyclin D1 in human invasive breast carcinoma. PATIENTS AND METHODS: We investigated whether the determination of cyclin D1 has prognostic value in a series of 180 patients with node-positive breast carcinoma and treated with adjuvant therapy with a median follow-up exceeding 6 years. We assessed cyclin D1 expression using the CDS-6 monoclonal antibody and a highly sensitive immunohistochemical technique. RESULTS: We found that most of the evaluable tumours (117 of 167; 70.1%) presented nuclear cyclin D1 staining and that its expression was significantly associated with both the hormone receptors (P = 0.009 and P = 0.005 for ER and PgR, respectively). Furthermore, 29 (17%) of 167 tumours had a weak (15 cases) or strong (9 cases) cytoplasmic cyclin D1 staining. In a subgroup of cases we also studied the amplification of the cyclin D1 gene and a moderate agreement between cyclin D1 nuclear overexpression assessed immunohistochemically and the gene amplification was found. In univariate analysis, cyclin D1 nuclear positivity was significantly associated with improved 6-year relapse-free survival (RFS) (P = 0.004), but not with overall survival (OS) (P = 0.12). The results of the Cox multivariate analysis (final model) indicate that cyclin D1 expression (P = 0.0049) as well as the number of involved nodes (P < 0.001) and tumour size (P = 0.036) are significant prognostic indicators for RFS. Only the number of involved nodes retained significance (P < 0.001) for OS in our series. The joint assessment of the variables considered in the final model of the multivariate analyses had a moderate prognostic capability as determined using the Harrell c statistic (c = 0.66 and 0.64 for RFS and OS, respectively). CONCLUSIONS: The patients with node-positive breast cancer who have a higher likelihood of gaining benefit from adjuvant therapy are those with tumours with cyclin D1 nuclear expression, small size and less than 3 metastatic nodes. Further studies are needed to verify the prognostic value of cyclin D1 in relation to different adjuvant treatments and to deepen the biological pathways that regulate its activation/ suppression in human breast carcinoma.     

Cyclin D1 expression in invasive breast cancer. Correlations and prognostic value

Cyclin D1 overexpression, detected by standard immunohistochemistry, was correlated with other prognostic variables and its prognostic value was evaluated in a group of 148 invasive breast cancers with long-term follow-up. Overexpression of cyclin D1 (59% of cases) was negatively correlated (chi 2 test) with histological grade (P = 0.0001), mean nuclear area (P = 0.004), mean nuclear volume (P = 0.02), and mitotic activity (P = 0.03) and positively correlated with estrogen receptor (P = 0.0001). There was a strong correlation between cyclin D1 overexpression and histological type (P = 0.0001). Positive cyclin D1 staining was seen in 11 of 13 tubular carcinomas, 3 of 3 mucinous carcinomas, 4 of 4 invasive cribriform carcinomas, and 17 of 20 lobular carcinomas. Of 102 ductal cancers, 52 were positive, and all 6 medullary carcinomas were negative. There were no significant correlations with lymph node status, tumor size, or DNA ploidy. In survival analysis, cyclin D1 overexpression did not provide significant univariate or multivariate prognostic value. In conclusion, cyclin D1 is mainly overexpressed in the well differentiated and lobular types of invasive breast cancer and is strongly associated with estrogen receptor positivity. It is negatively correlated with the proliferation marker mitoses count and with the differentiation markers nuclear area and nuclear volume. However, cyclin D1 overexpression does not seem to have prognostic value in invasive breast cancer when no adjuvant treatment is given.     

Expression of cyclin D1 and retinoblastoma protein in colorectal cancer

Abnormal expression of the retinoblastoma protein (pRb) and cyclin D1 have been reported in a variety of malignancies, but the frequencies of these deregulations and their relation to prognosis in colorectal cancer has not been clarified. We characterised 90 colorectal cancers with respect to immunohistochemical expression of cyclin D1, pRb and Ki-67. Two of 90 (2%) tumours lacked nuclear pRb staining, indicating inactivation of the protein, while 10 (11%) expressed high levels of pRb. Abnormal expression of pRb was significantly correlated to low levels of nuclear cyclin D1 observed in 32% of the tumours. Strong nuclear cyclin D1 expression was detected in 12% of the tumours. Cytoplasmic staining of cyclin D1 was observed in 17% of the tumours, showing an inverse relationship (P = 0.006) to the Ki-67 labelling index. Eight of 11 tumours with high nuclear overexpression of cyclin D1 and both tumours with pRb defects were located in the right colon in comparison with zero of 25 in the rectum (P = 0.009). Regarding prognosis, neither pRb nor cyclin D1 expression correlated with patient survival.     

Overexpression of cyclin D1 correlates with early recurrence in superficial bladder cancers

Cyclin D1 is a cell cycle regulator essential for G1 phase progression and is frequently overexpressed in several human tumour types as a consequence of gene amplification or chromosomal rearrangements. We analysed the expression of cyclin D1 in 75 patients with transitional cell carcinoma (TCC) to investigate the possible relationship between its expression and clinical outcome as well as histopathological findings using the immunohistochemical method. We observed strong staining (++, > 50% positive cells) for cyclin D1 in 19 cases (25.3%) and weak staining (+, 5-50% positive cells) in 19 cases (25.3%). Overexpression of cyclin D1 was not associated with tumour invasion. No significant association was found between overexpression of cyclin D1 and tumour grade (P > 0.05). We assessed the differences of disease-free interval in superficial tumours and actuarial survival probability in invasive tumours according to the status of cyclin D1 expression. Tumours with (++) staining for cyclin D1 recurred much more rapidly than (-) and/or (+) staining tumours (P < 0.01 for - vs ++; P < 0.05 for + vs ++). However, overexpression of cyclin D1 was not associated with a shortened overall survival of patients with invasive tumours (P < 0.1). These results suggest that genetic alteration of cyclin D1 appears to be an early event in the tumorigenesis of bladder TCC and is associated with early recurrence in superficial tumours.     

Immunohistochemical detection and gene amplification of cyclin D1 in mammary infiltrating ductal carcinoma

The deregulation of cyclin D1 (BCL-1, PRAD1, CCND1) protein, normally synthesized in the G1 phase of the cell cycle, has been implicated in the pathogenesis of some malignant neoplasms, including invasive mammary carcinomas. We used rabbit polyclonal antibody 19 to detect cyclin D1 in 55 infiltrating ductal carcinomas and compared the findings to six important clinicopathologic parameters and cyclin D1 gene amplification. Nuclear immunoreactivity of variable intensity for cyclin D1 was present in 35% of the neoplasms, whereas immunoreactivity of normal mammary epithelial nuclei was absent. No significant correlations were observed between immunoreactivity and patient age, axillary lymph node status, estrogen receptors, progesterone receptors, histologic grade, or any of its three components. There was a correlation between cyclin D1 immunostaining and tumor size (P = 0.013). Fourteen of 15 tumors 2 cm or less were negative, whereas 7 of 12 neoplasms larger than 4 cm were immunopositive. Fifteen percent of the invasive carcinomas had cyclin D1 gene amplification. Of these eight tumors, six showed cyclin D1 immunoreactivity (P = 0.017). In this study, cyclin D1 was detected immunohistochemically in approximately one-third of infiltrating ductal carcinomas; approximately one-third of these had detectable cyclin D1 gene amplification. These results further implicate cyclin D1 in breast tumorigenesis and are additional evidence for the role of cell cycle regulatory proteins in invasive mammary carcinoma.     

Predicted anti-oestrogen resistance in BRCA-associated familial breast cancers

There is controversy concerning the prognosis of breast cancers arising in women carrying loss of function mutations in the breast cancer susceptibility genes BRCA1 and BRCA2. This study was carried out to assess the likely hormone dependence of this group of tumours in comparison with an age and grade matched group of control sporadic tumours. We used quantitative immunohistochemical analysis for the oestrogen receptor (ER), progesterone receptor (PgR), cyclin D1 and pS2 on sections of primary tumours and ductal carcinoma in situ (DCIS). Expression of PgR (P < 0.05) and cyclin D1 (P < 0.01) was low in the BRCA1- and BRCA2-associated cancers compared with sporadic cases. The low frequency of expression of ER (9/40), PgR (2/40) cyclin D1 (5/36) and pS2 (5/36) in the familial tumours indicates that the majority of such tumours will be oestrogen insensitive and unlikely to respond to hormonal manipulation even at the in situ stage in their evolution. The low level of PgR (2/40 cases) suggests that there may be some abnormality of transactivating function of the ER in these tumours.     

DNA amplifications at 20q13 and MDM2 define distinct subsets of evolved breast and ovarian tumours

DNA amplification seems to be particularly frequent in human breast tumours and has been associated with cancer evolution and aggressiveness. Recent data indicate that new events should be added to the list, such as the amplifications at chromosome 20q13 or the MDM2 gene. The present work aimed at determining the incidence and clinicopathological signification of these amplifications in a large series of breast and ovarian tumours. We tested 1371 breast and 179 ovarian tumours by Southern blotting and observed amplification of 20q13 in 5.4% breast and 2.8% ovarian carcinomas, whereas MDM2 was found amplified in 5.3% and 3.8% of breast and ovarian tumours respectively. MDM2 RNA expression levels were analysed in a subset of 57 breast tumours and overexpression was observed in 4/57 (7%) of the tumours. Elevated expression levels coincided with amplification of the gene. In breast cancer, 20q13 and MDM2 amplifications seem to define subsets of aggressive tumours. Indeed, 20q13 was correlated to axillary nodal involvement and occurred preferentially in younger patients (< 50 years). Furthermore, 20q13 correlated, as did MDM2 amplification, to aneuploidy. In parallel, we had also tested our tumour DNAs for amplification of CCND1, ERBB-2 and MYC, which made it possible to test for correlations with 20q13 or MDM2 amplifications. Whereas 20q13 showed a very strong correlation to CCND1 amplification, that of MDM2 was prevalent in MYC-amplified tumours. Interestingly, 20q13 and MDM2 amplifications showed some degree of correlation to each other, which may possibly be owing to the fact that both events occurred preferentially in aneuploid tumours. In ovarian cancer, no statistically significant correlation was observed. However, 20q13 amplification occurred preferentially in stage 3 tumours and MDM2 was correlated to ERBB-2 amplification. This may suggest that in ovarian tumours also, 20q13 and MDM2 amplifications occur in late or aggressive cancers.     

Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients

Tumours develop through the accumulation of genetic alterations associated with a progressive increase of the malignant phenotype. In lung cancer, chronic exposure of bronchial epithelium to carcinogens in cigarette smoke may lead to multiple dysplastic and hyperplastic lesions scattered throughout the tracheobronchial tree. Little is known about the genetic alterations in such lesions. This study was carried out to examine cyclin D1 (CCND1) and retinoblastoma (RB1) gene expression in the bronchial epithelium of patients with lung cancer. Lung tumours and their corresponding tumour-free resection margins from 33 patients who underwent resection of non-small-cell lung cancer (NSCLC) were examined by immunostaining with monoclonal antibodies against cyclin D1 (DCS-6; Novocastra) and pRb (NCL Rb-1; Novocastra). Examination of the resection margins revealed four carcinomas in situ, 19 hyperplasias and ten sections showing apparently normal bronchial epithelium. A control group of patients, without lung tumours and who had never smoked, revealed no or weak cyclin D1 and positive pRb staining within bronchial epithelia. Increased cyclin D1 and diminished pRb expression were found in 76% (n = 25) and 27% (n = 9) of the resection margins respectively, and in 12% (n = 4) both cyclin D1 and pRb expression were altered. In the corresponding tumours, 48% (n = 16) were normal, while altered expression was found for cyclin D1 in 33% (n = 11), pRb in 27% (n = 9) and both in 9% (n = 3) of cases. It appears that altered expression of cyclin D1 and pRb is an early event in NSCLC development in almost half of cases analysed. Further investigations are needed to determine the significance of immunostaining of bronchial specimens in individuals at risk of lung cancer, with the possibility that the observations are of importance in the early diagnosis of NSCLC.     

Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb.     

Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

BACKGROUND/AIMS: A checkpoint mechanism in late G1, whose regulation via loss of retinoblastoma protein (pRB) or p16, or overexpression of cyclin D1 or cyclin dependent kinase 4 (CDK4), has been proposed to constitute a common pathway to malignancy. The aims of this study were (a) to compare markers of cell cycle G1-S phase transition in an intraocular tumour with known pRB deficiency (retinoblastoma) and compare it with one with an apparently functional pRB (uveal melanoma); (b) to determine if one of these markers may have a role in the pathogenesis of uveal melanoma; and (c) to determine if there is a difference in cell cycle marker expression following treatment of uveal melanoma and retinoblastoma. METHODS: 90 eyes were enucleated from 89 patients for retinoblastoma (n = 24) or for choroidal or ciliary body melanoma (n = 66). Conventional paraffin sections were assessed for cell type and degree of differentiation. Additional slides were investigated applying standard immunohistochemical methods with antibodies specific for cyclin D1 protein, pRB, p53, p21, p16, BCL-2, and MIB-1. RESULTS: Cyclin D1 protein and pRB were negative in retinoblastoma using the applied antibodies. In contrast, cyclin D1 protein expression was observed in 65% of uveal melanomas; a positive correlation between cyclin D1 cell positivity and tumour cell type, location, growth fraction, as well as with pRB positivity was observed. p53, p21, and p16 could be demonstrated in both tumours. An inverse relation between p53 and p21 expression was demonstrated in most choroidal melanomas and in some retinoblastomas. Apart from a decrease in the growth fractions of the tumours as determined by MIB-1, a significant difference in the expression of G1-S phase transition markers in vital areas of uveal melanoma and retinoblastoma following treatment with radiotherapy and/or chemotherapy was not observed. CONCLUSION: Retinoblastomas and uveal melanomas, two tumours of differing pRB status, differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.     

Aberrant expression of cyclin A and cyclin B1 proteins in oral carcinoma

Cyclins play an important role in regulating the passage of dividing cells through critical checkpoints in the cell cycle. Aberrant expression of cyclin proteins has been found in a number of human cancers, including carcinomas of the head and neck, where amplification of the cyclin D1 gene is a common finding. The objective of this study was to examine cell cycle kinetics in oral carcinomas by determining the expression of the S phase protein cyclin A and the M phase protein cyclin B1. Routinely processed tissue sections of 50 oral squamous cell carcinomas from the floor of the mouth were stained by immunohistochemistry for cyclin A, cyclin B1 and Ki-67 proteins. Ten specimens of normal epithelium from the floor of the mouth were used as controls. The number of cells showing nuclear staining for cyclin A, cyclin B1 and Ki-67 proteins was determined by computer image analysis. There were 17 well-differentiated, 25 moderately differentiated and 8 poorly differentiated tumours. Mean counts for cyclin A (29.50+/-4.10, mean+/-95% CI), cyclin B1 (2.05+/-0.30) and Ki-67 (49.46+/-5.91) proteins in the carcinomas were significantly higher than counts for the normal epithelial controls (cyclin A: 9.30+/-1.72; cyclin B1: 1.01+/-0.36; Ki-67: 17.40+/-4.17). For cyclin A, cyclin B1 and Ki-67, mean staining scores for all tumour grades were significantly higher than controls. There was a strong correlation between Ki-67 and cyclin A scores in all tumour groups (r2=0.68); however, the correlations between cyclin B1 and cyclin A scores (r2=0.35) and between cyclin B1 and Ki-67 scores (r2= 0.39) were weak. We conclude that there is overexpression of cyclin A and cyclin B1 proteins in oral carcinoma. Furthermore, the poor correlations for cyclin B1 scores with other cell cycle indices suggest that there may be aberrant cell cycle progression at the G2/M checkpoint in oral carcinomas.     

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.     

Lack of relationship between CDK activity and G1 cyclin expression in breast cancer cells

The G1 cyclins, cyclin D1 and E, are rate limiting for progression through G1 phase of the cell cycle in breast epithelial cells and are oncogenic when expressed in the mammary epithelium of transgenic mice. These genes are frequently overexpressed in clinical breast cancer where overexpression appears to be associated with specific disease phenotypes, altered responsiveness to therapeutic intervention and patient survival. In order to investigate the functional correlates of cyclin D1 and cyclin E overexpression we employed a panel of normal, immortalized and neoplastic breast epithelial cell lines to examine the relationships between cyclin gene expression, cyclin-CDK complex formation and CDK activity. In agreement with earlier studies cyclin D1 and E expression varied over an approximately tenfold range among the 18 cell lines studied. There was no apparent relationship, however, between cyclin D1 expression and the in vitro activity of its major kinase partner, Cdk4, although MDA-MB-134 cells displayed the highest level of both cyclin D1 expression and Cdk4 activity. Similarly, there was no significant relationship between cyclin E expression and cyclin E-Cdk2 activity. Fractionation of whole cell lysates by gel filtration chromatography revealed that approximately 90% of the cyclin E protein was present in inactive complexes containing the CDK inhibitors p21 and p27. Much of the small fraction of active cyclin E protein was of very high apparent molecular mass, >400 kDa, suggesting that formation of these complexes is a more important determinant of cyclin E-Cdk2 activity than cyclin E abundance. These data suggest that properties of cyclins D1 and E in addition to their ability to activate Cdk4 and Cdk2 may contribute to the effects of overexpression on the breast cancer phenotype.     

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The gene for Cyclin D1 (CCND1) lies within chromosomal region 11q13 and codes for a cell cycle regulator essential for G1 phase progression. This G1-cyclin is a putative protooncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias, including head and neck squamous cell carcinomas. Data from the literature suggest that amplification and overexpression of the CCND1 gene could lead to destabilisation of cell cycle control mechanisms and uncontrolled cell proliferation. We developed a differential PCR system for the determination of CCND1 gene amplification in head and neck squamous cell carcinomas. A 115 bp CCND1 fragment and a 150 bp gamma-interferon fragment are amplified simultaneously in the same reaction tube under optimized conditions. Statistical analysis of amplification data obtained by differential PCR revealed excellent correlation with amplification data obtained by conventional Southern hybridization.     

Expression of cyclins D1 and E in human colon adenocarcinomas

Cyclins D1 and E play critical roles in the progression of cells through the G1 phase of the cell cycle. Amplification and/or overexpression of the cyclin D1 gene and aberrant expression of cyclin E have been described in several forms of human cancer. In the present study, we examined the expression of these two genes by Western, Northern and Southern blot analyses in a series of primary human colon carcinomas of various stages and degrees of differentiation and in paired adjacent normal mucosa samples, and also in a series of human colon carcinoma cell lines. About 50% of the colon carcinomas displayed a two to five fold increase in the expression of cyclin D1 mRNA and protein, when compared with the paired normal mucosa samples. Six out of eight carcinomas examined showed a four to nine fold increase in cyclin E mRNA and about 50% of the carcinomas displayed a two to three fold increase in cyclin E protein. Low molecular weight cyclin E-related proteins were observed in four out of ten carcinomas. These changes in cyclins D1 and E occurred in both early and late stage tumors. Three of the six cell lines examined displayed a high expression of cyclin D1 mRNA and protein. A very high level of cyclin E mRNA expression was seen in HCT116 cells and this was associated with the presence of low molecular weight cyclin E-related proteins. None of the primary colon carcinomas nor the six cell lines examined displayed amplification of either the cyclin D1 or cyclin E genes. Thus, an aberrant expression of both cyclins D1 and E occurs in a significant fraction of human colon carcinomas.