Effect Of Cyclin D1 Overexpression Research Articles

Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Negative effect of cyclin D1 overexpression on recurrence-free survival in stage II-IIIA lung adenocarcinoma and its expression modulation by vorinostat in vitro.

BackgroundThis study was aimed at identifying prognostic biomarkers for stage II-IIIA non-small cell lung cancer (NSCLC) according to histology and at investigating the effect of vorinostat on the expression of these biomarkers.MethodsExpression levels of cyclin D1, cyclin A2, cyclin E, and p16 proteins that are involved in the G1-to-S phase progression of cell cycle were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 372 samples of stage II-IIIA NSCLC. The effect of vorinostat on the expression of these proteins, impacts on cell cycle, and histone modification was explored in lung cancer cells.ResultsAbnormal expression of cyclin A2, cyclin D1, cyclin E, and p16 was found in 66, 47, 34, and 51 % of 372 cases, respectively. Amongst the four proteins, only cyclin D1 overexpression was significantly associated with poor recurrence-free survival (adjusted hazard ratio = 1.87; 95 % confidence interval = 1.12 – 2.69, P = 0.02) in adenocarcinoma but not in squamous cell carcinoma (P = 0.44). Vorinostat inhibited cell cycle progression to the S-phase and induced down-regulation of cyclin D1 in vitro. The down-regulation of cyclin D1 by vorinostat was comparable to a siRNA-mediated knockdown of cyclin D1 in A549 cells, but vorinostat in the presence of benzo[a]pyrene showed a differential effect in different lung cancer cell lines. Cyclin D1 down-regulation by vorinostat was associated with the accumulation of dimethyl-H3K9 at the promoter of the gene.ConclusionsThe present study suggests that cyclin D1 may be an independent prognostic factor for recurrence-free survival in stage II-IIIA adenocarcinoma of lung and its expression may be modulated by vorinostat.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-2001-7) contains supplementary material, which is available to authorized users.

Longer survival in patients with breast cancer with cyclin d1 over-expression after tumor recurrence: longer, but occupied with disease.

PurposeThe effect of cyclin D1 overexpression on breast cancer outcomes and prognosis is controversial, even though amplification of the cyclin D1 gene, CCND1, has been shown to be associated with early relapse and poor prognosis. In this study, we examined the relationship between cyclin D1 overexpression and disease-specific survival (DSS). We also analyzed survival in patients who experienced recurrence.MethodsWe retrospectively analyzed data from patients diagnosed with ductal carcinoma between April 2005 and December 2010. We examined clinicopathologic factors associated with cyclin D1 overexpression and analyzed the influence of cyclin D1 on recurrence-free survival and DSS.ResultsWe identified 236 patients diagnosed with primary breast cancer who completed all phases of their primary treatment. Cyclin D1 overexpression was significantly associated with longer DSS (5-year DSS, 89.9% in patients without cyclin D1 overexpression vs. 98.9% in patients with cyclin D1 overexpression; p=0.008). Multivariate analysis also found that patients with cyclin D1 overexpressing tumors had significantly longer disease-specific survival than patients whose tumors did not overexpress cyclin D1, with a hazard ratio for disease-specific mortality of 7.97 (1.17-54.22, p=0.034). However, in the group of patients who experienced recurrence, cyclin D1 overexpression was not significantly associated with recurrence-free survival. Cyclin D1 overexpression was significantly associated with increased survival after disease recurrence, indicating that cyclin D1 overexpression might be indicative of more indolent disease progression after metastasis.ConclusionCyclin D1 overexpression is associated with longer DSS, but not recurrence-free survival, in patients with breast cancer. Longer postrecurrence survival could explain the apparent inconsistency between DSS and recurrence-free survival. Patients with cyclin D1-overexpressing tumors survive longer, but with metastatic disease after recurrence. This information should spark the urgent development of tailored therapies to cure these patients.

Cyclin D1 in invasive breast carcinoma: favourable prognostic significance in unselected patients and within subgroups with an aggressive phenotype

To study the clinicopathological and prognostic value of cyclin D1 overexpression in patients with breast carcinoma. Immunohistochemistry was performed on paraffin-embedded tissue specimens from 290 invasive breast carcinomas to detect the proteins cyclin D1, oestrogen receptor (ER), progesterone receptor (PR), p53, c-erbB2, and topoisomerase IIα (topoIIα). Cyclin D1 staining was quantified using a computerized image analysis method. Cyclin D1 overexpression characterized smaller, ER-positive and PR-positive tumours (P = 0.017, P < 0.0001, and P < 0.0001, respectively), of a lower histological and nuclear grade (P = 0.011 and P < 0.0001, respectively), and with reduced expression of topoIIα (P = 0.001) and p53 (P < 0.001). Cyclin D1 was found to have an independent favourable impact on the overall survival of both the unselected cohort of patients (P = 0.011) and of patients with ER-negative and lymph node-positive tumours (P = 0.034 and P = 0.015, respectively). In triple-negative tumours, cyclin D1 overexpression was found to have independent favourable impacts on both overall and relapse-free survival (P = 0.002 for both). This is the first immunohistochemical study to dissociate the advantageous prognostic effect of cyclin D1 overexpression from its association with ER expression, and to provide evidence that cyclin D1 overexpression may be a marker of prolonged survival in patient subgroups with aggressive phenotypes.

Abstract CN04-03: Esophageal cancer and the tumor microenvironment

Abstract Squamous cell cancers are the most common type of epithelially derived human cancers and arise from a number of diverse sites. They share a number of common genetic alterations and environmental exposures. Employing oral and esophageal cancers as prototypes, we have developed three-dimensional (3D) or organoptypic models that represent a form of human tissue engineering (Genes and Development 2007; Journal of Clinical Investigation 2008). These approaches underscore the combinatorial roles of EGFR signaling and p53 mutation in fostering tumor cell migration and invasion in the microenvironment. Microarray analyses reveal specific effectors in the adhesion family of genes that might be exploited as biomarkers for detection and targets for new therapeutics. We have also generated genetically engineered mouse models that reveal the effects of cyclin D1 overexpression in concert with p53 loss, as well as the role of p120-catenin loss in the initiation and progression of cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):CN04-03.

Cyclin D1 overexpression increases susceptibility to 4‐nitroquinoline‐1‐oxide‐induced dysplasia and neoplasia in murine squamous oral epithelium

The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus, and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect of cyclin D1 overexpression on susceptibility of mice to carcinogen-induced tumorigenesis, using 4-nitroquinoline-1-oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and nontransgenic littermates were administered 4NQO (20 or 50 parts per million (ppm) in the drinking water) for 8 wk and monitored for an additional 16 wk. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with nontransgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro-esophageal epithelia. Examination of the dysplastic and neoplastic lesions revealed abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen-induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero-digestive cancer.

Tamoxifen Stimulates the Growth of Cyclin D1–Overexpressing Breast Cancer Cells by Promoting the Activation of Signal Transducer and Activator of Transcription 3

De novo or acquired resistance to tamoxifen is a major clinical challenge for the management of estrogen receptor (ER)-positive breast cancers. Although cyclin D1 overexpression is associated with a better outcome for breast cancer patients, its overexpression is also linked to tamoxifen resistance. We previously reported that the beneficial effect of cyclin D1 correlates with its ability to repress the antiapoptotic transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast, molecular pathways linking overexpression of cyclin D1 to tamoxifen resistance have not been established. In the current study, the effect of tamoxifen on the growth of genetically matched high or low cyclin D1-expressing breast cancer cells was characterized and the interactions between cyclin D1, ER, and STAT3 in response to tamoxifen treatment were determined. We show that repression of STAT3 by cyclin D1 inhibits cell growth on Matrigel and in tumors in vivo; however, treatment with tamoxifen abolishes cyclin D1-mediated repression of STAT3 and growth suppression. We show that tamoxifen induces a redistribution of cyclin D1 from STAT3 to the ER, which results in the activation of both STAT3 and the ER. These results offer a molecular mechanism for the dual effect of cyclin D1 overexpression in breast cancer and support the notion that the level of cyclin D1 expression and activated STAT3 are important markers to predict response to tamoxifen treatment.

Differential regulation of cyclin D1 and cell death by bile acids in primary rat hepatocytes

Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids modulate apoptosis and regulate cell-cycle effectors, including cyclin D1. In contrast, deoxycholic acid (DCA) induces cell death and cyclin D1. In this study, we explored the role of cyclin D1 in DCA-induced toxicity and further elucidated the antiapoptotic function of UDCA and TUDCA in primary rat hepatocytes. Cells were incubated with DCA and with or without UDCA or TUDCA for 8-30 h. In addition, hepatocytes were transfected with either an adenovirus expressing cyclin D1 or with a cyclin D1 reporter plasmid with or without bile acids. Finally, cells were cotransfected with short interfering RNA targeting p53. Unlike DCA, both UDCA and TUDCA reduced cyclin D1 expression and transcriptional activation, confirming our previous DNA microarray data. Furthermore, UDCA and TUDCA prevented DCA-induced cyclin D1 and cell death. Cyclin D1 overexpression increased DCA-induced Bax translocation, cytochrome c release, and apoptosis. However, UDCA and TUDCA were less efficient at decreasing cyclin D1 levels as well as DCA-induced changes with overexpression. Finally, after p53 silencing, the effects of cyclin D1 overexpression were almost completely abrogated, whereas UDCA and TUDCA cytoprotective potential was reestablished. In conclusion, cyclin D1 is a relevant player in modulating apoptosis by bile acids, in part through a p53-dependent mechanism.

Cyclin D1 Overexpression Increases the Susceptibility of Human U266 Myeloma Cells to CDK Inhibitors through a Process Involving p130-, p107- and E2F-Dependent S-Phase Entry

Dysregulation of cyclin D1 expression is one of the most common genetic aberrations found in hematopoietic malignancies, including multiple myeloma. To address the effects of cyclin D1 overexpression might have on the response of malignant hematopoietic cells to CDK inhibitors, the impact of ectopic cyclin D1 overexpression on the response of human multiple myeloma U266 cells to various cyclin-dependent kinase (CDK) inhibitors was examined. Cyclin D1 overexpression markedly increased the apoptotic response of cells to the CDK inhibitors flavopiridol, roscovitine, and R-roscovitine. Ectopic expression of cyclin D1 resulted in p21CIP1 accumulation, an effect that was diminished by CDK inhibitor exposure. In pRb-null U266 cells, enforced overexpression of cyclin D1 diminished CDK inhibitor-mediated dephosphorylation of the pocket proteins p130 and p107, reduced binding of E2F1 and E2F4 to p130 and p107, and attenuated inhibition of E2F activity. Notably, CDK inhibitors failed to reduce the S-phase fraction in cyclin D1/U266 cells in contrast to effects in their wild-type counterparts. Finally, cyclin D1/U266 cells exhibited diminished basal NF-?B activity compared to controls, which was essentially completely abrogated by CDK inhibitor exposure. Together, these findings suggest that dysregulation of cyclin D1 sensitizes human myeloma cells to the actions of CDK inhibitors through mechanisms involving interference with p21CIP1 expression, dephosphorylation of pocket proteins and inactivation of E2Fs culminating in S-phase entry, as well as inactivation of NF-?B, leading to apoptosis rather than growth arrest.

Cyclin D1 overexpression induces epidermal growth factor-independent resistance to apoptosis linked to BCL-2 in human A431 carcinoma

Overexpression of EGF receptors and constitutive cyclin D1 expression are frequently associated with human squamous carcinomas. We have now investigated whether these parameters influence susceptibility to okadaic acid induced cell death in EGF-receptor overexpressing mutant p53 A431 human carcinoma. Exposure of these cells to 20 nM okadaic acid induced apoptosis-associated caspase 3 activation, DNA fragmentation, cleavage of Poly ADP-Ribose Polymerase (PARP), p53-independent expression of pro-apoptotic bax, and loss of proliferation-promoting cyclin D1. All these alterations were antagonized by concurrent addition of exogenous EGF. Ectopic overexpression of the cyclin D1 gene in A431 carcinoma conferred resistance to 20 nM okadaic acid irrespective of exogenous EGF, associated with a parallel induction of anti-apoptotic bcl-2. Treatment with a subtoxic concentration of a bispecific bcl-2/bcl xL antisense oligonucleotide cooperated with okadaic acid to down-regulate bcl-2 and sensitize cyclin D1-overexpressing cells to okadaic acid. Although EGF protects EGF-R proficient epithelial cells from diverse apoptotic stimuli through Mcl-1, this is the first report demonstrating that cyclin D1 overexpression provides an EGF independent protection from okadaic acid-induced cell death through induction of bcl-2. We also show that this anti-apoptotic effect of cyclin D1 overexpression, can be partly antagonized with antisense strategies that down-regulate anti-apoptotic bcl-2 family members.

The effect of cyclin D1 overexpression in human head and neck cancer cells.

Overexpression of cyclin D1 in head and neck cancer has been suggested to be a poor prognostic factor. To understand the role of cyclin D1 expression in head and neck cancer, we overexpressed cyclin D1 in TU182 (a cell line derived from pharyngeal cancer) using a retroviral vector. Stable transfectants were isolated by neomycin (G418) selection. Compared to the parental and control-vector transfected cells, the cyclin D1 transfected cells revealed a decrease of the G1/G0 population and resulted in continuous proliferation under low serum conditions. Proliferation assays revealed an increase in resistance to cisplatin in cyclin D1 overexpressing cells. These observation suggest that deregulation of cyclin D1 may reduce growth factor requirements and contribute to the resistance to some chemotherapeutic agents among head and neck cancer patients.

Ectopic cyclin D1 expression blocks STI571-induced erythroid differentiation of K562 cells

Bcr-Abl tyrosine kinase inhibitor induces apoptosis and erythroid differentiation of K562 cells. During this erythroid differentiation, c-Myc and cyclin D1 transcripts are transiently downregulated. Accordingly, we studied the effect of cyclin D1 overexpression on erythroid differentiation. After treatment with 250 nM STI571, 90% of K562 and 25% of K562/D1 cells underwent erythroid differentiation. The basal expression of glycophorin A in K562/D1 cells was markedly diminished compared with that by parental cells. STI571 treatment failed to induce glycophorin A expression in K562/D1 cells. During STI571 treatment, ERK activity was downregulated in parental cells, while it was constantly activated in K562/D1 cells. These results suggest that ectopic expression of cyclin D1 causes the resistance of K562 cells to erythroid differentiation by modulating ERK regulation.

Investigation of gene expression correlating with centrosome amplification in development and progression of breast cancer

Abstract : We previously demonstrated that centrosome amplification correlates with chromosomal instability and loss of differentiation in breast tumors. The goal of this research is to identify genes important in breast cancer due to their association with amplified centrosomes, We determined that centrosomes are amplified prior to invasion and amplification is maintained during progression. We identified candidate genes for further investigation. Last year, we 1) measured chromosomal instability in tumors for which we have gene expression and centrosome amplification data, 2) analyzed the relationships between lymph node and estrogen receptor status, chromosomal instability and centrosome amplification, and 3) developed a collaborative study to look at centrosome amplification, chromosomal instability, and gene expression in a rat model of estrogen-induced mammary cancer. During this last reporting period we 1) demonstrated that expression of Aurora-A, a centrosome-associated kinase, correlates to Nottingham Prognostic Index in human breast tumors, 2) demonstrated that estrogen exposure leads to centrosome amplification and aurora-A over-expression prior to invasion in a rat mammary tumor model, and 3) developed a collaboration to study the effects of cyclin D1 overexpression in a mouse model and demonstrated that cyclin D1 induces centrosome amplification and aneuploidy.

Overexpression of cyclin D1 in nonmelanocytic skin cancer.

Although the overexpression of cyclin D1 has been believed to play important roles in neoplastic transformation of some tumors, little is known about the function of cyclin D1 protein in carcinogenesis in human skin. A total of 307 patients with nonmelanocytic skin cancer, being 46 with Bowen's disease (BOD), 134 with squamous cell carcinoma (SCC) and 127 with basal cell carcinoma (BCC), were investigated immunohistochemically using monoclonal antibody to cyclin D1 by the LSAB method, to assess the expression of cyclin D1 in skin cancer including its precursors. The positive rates of cyclin D1 immunostaining in BOD, SCC and BCC were 63.0%, 69.4% and 54.3%, respectively. The positive rates in dysplasia adjoining BOD, SCC and BCC were 43.6%, 67.9% and 59.8%, respectively. In morphologically normal skin, however, only 2 cases, 1 of SCC and 1 of BCC, exhibited positive staining. These findings suggested that overexpression of cyclin D1 is an early event in dysplastic lesions of skin. Overexpression of cyclin D1 was related to sun exposure, especially in dysplasia of SCC. The score for cyclin D1 expression in dysplasia of BCC was correlated with age. Expression of cyclin D1 markedly increased from normal skin through dysplasia to BOD, but was not significantly related to the degree of SCC differentiation. These findings demonstrate that the effect of cyclin D1 overexpression is restricted to proliferation of cells, so that they gain a growth advantage, but their differentiation is not increased. Comparison with the results for p53 protein expression in these tumors, a significant correlation with cyclin D1 expression was found in dysplasia in BOD and SCC, and in patients with BCC who were less than 74 years old. These findings suggested the hypothesis that prior aberrant p53 expression may affect or regulate the overexpression of cyclin D1.

Ubiquitination of Free Cyclin D1 Is Independent of Phosphorylation on Threonine 286

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.

Cyclin D1 in breast cancer.

Cyclin D1 protein plays an important part in regulating the progress of the cell during the G1 phase of the cell cycle. The cyclin D1 gene, CCND1, is amplified in approximately 20% of mammary carcinomas, and the protein is over-expressed in approximately 50% of cases. This has led to intensive study to ascertain whether cyclin D1 is a biological marker in breast cancer; however, the clinical work has produced unexpected results. Work in cell lines and in transgenic mice indicate that CCND1 is a weak oncogene and it was expected that, like c-erbB-2, over-expression of cyclin D1 protein would be associated with a poor prognosis. Early immunohistochemical prognostic studies produced equivocal results but we, and others, have recently shown that strong staining for cyclin D1 is more likely to be seen in well differentiated, estrogen receptor positive carcinomas. Furthermore, we have found that over-expression of cyclin D1 is actually associated with a good outcome, both in terms of prognosis and response to endocrine treatment. Cyclin D1 is frequently over-expressed in ductal carcinoma in situ but not in benign breast disease, including atypical ductal hyperplasia; hence its expression appears to be closely linked with carcinogenesis. In order to help explain the apparent beneficial effects of cyclin D1 over-expression, a number of closely associated cell cycle proteins have also been evaluated, including the cyclin dependent kinase inhibitor p27, which blocks the activating effects of cyclin D1. Initial reports show that high levels of p27 are associated with a good prognosis and we have shown a positive association between p27 and cyclin D1 expression. These clinical results of cyclin D1 are an example of how information obtained from basic cell biology studies needs to be complemented by clinical studies to ascertain the true worth of a prognostic marker.

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1.

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

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...

Effects of Cyclin D1 Overexpression on G1 Progression-Related Events

In previous studies (W. Jiang, S. M. Kahn, P. Zhou, Y.-J. Zhang, A. M. Cacace, A. S. Infance, Y. Doi, R. M. Santella, and I. B. Weinstein 1993,Oncogene8, 3447–3457) we reported that stable overexpression of cyclin D1 in R6 rat embryo fibroblasts shortens the G1 phase and impairs growth control. In the present study we examined the effects of cyclin D1 overexpression on other events involved in the G1 to S progression, utilizing the overexpressor cell line R6-ccnD1. We found that when compared to R6 control cells, serum-starved quiescent R6-ccnD1 cells had not only increased levels of the cyclin D1 protein but also increased levels of the cyclin E protein. The latter protein was complexed to phosphorylated cyclin-dependent kinase 2 (CDK2). However, in quiescent serum-starved R6-ccnD1 cells this cyclin E–CKD2 complex lackedin vitrokinase activity due to the presence of a heat-stable inhibitory activity, apparently reflecting the inhibitory effects of the CDK inhibitors (CDKIs) p21WAF1and p27KIP1. Serum stimulation of the quiescent R6-ccnD1 cells was associated with a loss of this inhibitory activity and a decrease in the levels of the latter two proteins, as the cells progressed through the G1 phase. On the other hand, serum stimulation of the control R6 cells was associated with both induction of cyclin E and increased levels of phosphorylated CDK2 proteins and decreased levels of p21WAF1and p27KIP1, as the cells progressed through the G1 phase. Thus, even though overexpression of cyclin D1 can induce the expression of cyclin E and phosphorylated CDK2, premature activation of cyclin E–CDK2 kinase activity in quiescent cells or during progression through G1 appears to be blocked by CDKIs. Nevertheless, the R6ccnD1 cells have a shorter G1 phase than the control cells presumably due to the high levels of both cyclin D1 and cyclin E. Taken together, these results indicate that overexpression of cyclin D, which is frequently seen in human tumors, can have complex effects on the expression of other genes that control cell cycle progression.

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells.

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-transcribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregulation of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells.