Overexpression Of Cyclin Research Articles

Overexpression of cdk4/cyclin D1 induces apoptosis in PC12 cells in the presence of trophic support.

The induction of apoptosis by cell cycle regulator molecules under conditions optimal for exponential growth was examined in rat pheochromocytoma PC12 cells by overexpression of cyclins and cyclin-dependent kinases (cdks). By flow cytometry and by immunofluorescence, only cells overexpressing cdk4 or cyclin D1 underwent apoptosis, which was not associated with G1-arrest. Cdk4 kinase activity was significantly higher in cdk4-, or cyclin D1-expressing cells. Furthermore, induction of apoptosis by cdk4 was abrogated by co-transfection of p16(INK4), or dominant negative cdk4. These results suggest that upregulation of cdk4 kinase activity is a primary and critical mediator of apoptosis in PC12 cells under physiological conditions.

Overexpression of cyclin A in human HeLa cells induces detachment of kinetochores and spindle pole/centrosome overproduction.

The combination of hydroxyurea (HU) and caffeine has been used for inducing kinetochore dissociation from mitotic chromosomes and for causing centrosome/spindle pole amplification. However, these effects on microtubule organizing centers (MTOCs) are limited to certain cell types. It was reasoned that if the biochemical differences in MTOC behavior between cells following HU treatment could be identified, then critical information concerning the regulation of these organelles would be obtained. During these studies, it was determined that cells from hamster, rat, and deer could be induced to enter mitosis with dissociated kinetochores and to synthesize centrosomes during arrest with HU, while cells from human and mouse could not. Comparisons between human HeLa cells and CHO cells determined that cyclin A levels were depressed in HeLa cells relative to CHO cells following HU addition. Overexpression of cyclin A in HeLa cells converted them to a cell type capable of detaching kinetochores from mitotic chromosomes. Ultrastructural analyses determined that the detached human kinetochores exhibited a normal plate-like morphology and appeared capable of associating with microtubules. In addition, HeLa cells overexpressing cyclin A also overproduced spindle poles during HU arrest, demonstrating that cyclin A activity also is important for centrosome replication during interphase. In summary, elevated cyclin A levels are important for the capacity of cells to be driven into mitosis by caffeine addition, for the ability of cells to progress to mitosis with detached kinetochores, and for centrosome/spindle pole replication.

Characterization of a human colorectal carcinoma cell line with acquired resistance to flavopiridol.

Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases (cdks) and represents the first in this anticancer class to enter clinical trials. In anticipation of the likelihood that, as with other cancer drugs, acquired resistance may limit the drug's efficacy, an acquired resistance model has been established by in vitro drug exposure of the human colon carcinoma cell line HCT116. This stably resistant line, possessing 8-fold resistance to flavopiridol, showed a lack of cross-resistance to the anticancer agents etoposide, doxorubicin, paclitaxel, topotecan, and cisplatin, and notably to other chemical classes of cdk inhibitors: the aminopurines roscovitine and purvalanol A, 9-nitropaullone, and hymenialdisine. Resistance did not seem to be related to differences in the levels of multidrug resistance drug efflux proteins, P-glycoprotein, and MRP1. Moreover, there were no changes in overall drug accumulation between the resistant and sensitive cell lines. Flavopiridol induced cell cycle arrest, apoptosis, and inhibition of retinoblastoma gene product phosphorylation on serine 780 in both parental and resistant lines, but the latter required 8-fold higher concentrations to achieve these effects. Cyclin E protein levels and cyclin E-associated kinase activity were increased in the resistant line, suggesting that overexpression of cyclin E may be the mechanism of resistance to flavopiridol. However, transfection of cyclin E to increase expression of this protein did not result in an increase in resistance to flavopiridol. Thus, up-regulation of cyclin E alone does not seem to cause resistance to this cdk inhibitor.

Implication of Malignancy and Prognosis of p27kip1, Cyclin E, and Cdk2 Expression in Epithelial Ovarian Tumors

Objective. The aim of this study was to further evaluate whether the expression of p27kip1, cyclin E, and cdk2 is related to the malignancy of ovarian tumors and whether their expressions, alone or in combination, are associated with prognosis in epithelial ovarian carcinoma.Methods. Immunohistochemical analysis using anti-p27kip1, anti-cyclinE, and anti-cdk2 antibodies was carried out for 103 cases consisting of benign, borderline, and malignant ovarian tumors, and Western blot analysis and cdk2 activity assay were performed in 26 fresh ovarian tumor samples.Results. p27kip1 expression was reduced in ovarian carcinomas in contrast to benign and borderline tumors. The expression of cyclin E and cdk2 gradually increased from benign to borderline to malignant tumors. Kaplan–Meier survival analysis showed that patients with p27kip1 expression had a high overall survival rate. Patients with cyclin E overexpression had a low overall survival rate. When the combination of these proteins was analyzed, patients with the p27kip1 (−)/cyclin E (++)/cdk2 (++) phenotype were significantly associated with the poorest overall survival. In multivariate Cox regression analysis, the combined phenotype of p27kip1 (−)/cyclin E (++)/cdk2 (++) was independently related to poor prognosis.Conclusions. Our results suggest that loss of p27kip1 expression and overexpression of cyclin E or cdk2 were significantly associated with malignancy in ovarian tumors. p27kip1 and cyclin E proteins may be valuable prognostic factors for epithelial ovarian carcinoma patients. Furthermore, the combined evaluation of p27kip1/cyclin E/cdk2 may provide the most important prognostic implication.

Immunohistochemical Expression of Cell Cycle Proteins E2F-1, Cdk-2, Cyclin E, p27kip1, and Ki-67 in Normal Placenta and Gestational Trophoblastic Disease

The role of cell cycle protein expression in gestational trophoblastic disease is poorly understood. In this study we investigated the immunostaining patterns of G1 restriction point and G1-S regulatory proteins E2F-1, Cdk2, cyclin E, p27kip1, and the proliferation marker Ki-67 on routinely processed sections of 29 hydatidiform moles (10 partial moles and 19 complete moles, including 9 persistent moles), 7 choriocarcinomas, and 7 normal placentas. Ki-67 trophoblast staining decreased with increasing gestational age of the placenta, and showed maximal expression in gestational trophoblastic disease. Cyclin-dependent kinase activity, as reflected by Cdk2 expression patterns, also decreased with placental maturation. E2F-1 was uniquely expressed by trophoblasts of moles and choriocarcinoma. Cyclin E was maximally expressed by complete moles and choriocarcinomas, and showed an inverse relationship with the cyclin-dependent kinase inhibitor p27kip1. Abnormal trophoblastic proliferations may be mediated through interactions of Cdk-2, E2F-1, cyclin E, and p27kip1. Overexpression of cyclin E was associated with more aggressive forms of gestational trophoblastic disease. However, we did not find distinguishing features between complete moles that spontaneously resolved after evacuation and persistent moles that required chemotherapy. The different expression patterns of cyclin E and E2F-1 in partial and complete moles may be useful in distinguishing these two entities. Furthermore, loss of p27kip1 in malignant trophoblast may represent a necessary step in the development of choriocarcinoma.

Protection against chemotherapy-induced cytotoxicity by cyclin-dependent kinase inhibitors (CKI) in CKI-responsive cells compared with CKI-unresponsive cells

Inactivation of the retinoblastoma (Rb) protein caused by gene mutation, association with oncoproteins from small DNA viruses, mutational inactivation of p16(Ink4a), or overexpression of cyclin D is a common feature of many human cancer cells and is causally associated with the aberrant proliferation control of cancer cells; whereas normal cells maintain an integrated cell cycle machinery and are subject to cell cycle checkpoint control by cyclin-dependent kinase (CDK) inhibitors (CKIs). To determine whether this difference can be translated into a therapeutic advantage to protect normal cells from adverse cytotoxicity caused by chemotherapy, we established cell model systems for ecdysone-inducible expression of p16(Ink4a), p21(Waf1), and p27(Kip1) in one CKI-responsive cell line (A431 human vulvar epidermoid carcinoma cells with functional Rb) and one CKI-unresponsive cell line (SiHa human cervical cancer cells with nonfunctional Rb). Expression of p16(Ink4a), p21(Waf1), or p27(Kip1) in both SiHa and A431 cells strongly inhibited CDK2 activity, indicating functional expression of the CDK inhibitors in both cell lines. However, only in A431 cells did expression of p16(Ink4a), p21(Waf1), or p27(Kip1) cause Rb dephosphorylation, arrest cell cycle traversal, and potently inhibit cell proliferation. Induction of p16(Ink4a), p21(Waf1), or p27(Kip1) in SiHa cells failed to cause Rb dephosphorylation or to arrest cell cycle traversal, and such induction only minimally inhibited cell proliferation. We then compared the chemosensitivity of clones derived from these two cell lines when the CKIs were and were not induced. Induction of p16(Ink4a), p21(Waf1), or p27(Kip1) conferred strong resistance to paclitaxel- or cisplatin-mediated cytotoxicity on the CKI-responsive A431 cells but not on the CKI-unresponsive SiHa cells. Our results support a novel chemotherapy strategy for treating patients with Rb pathway-impaired cancers by concurrent administration of chemotherapy with CKIs as chemoprotective agents for normal cells.

Inactivation of the checkpoint kinase Cds1 is dependent on cyclin B-Cdc2 kinase activation at the meiotic G(2)/M-phase transition in Xenopus oocytes.

Checkpoint controls ensure chromosomal integrity through the cell cycle. Chk1 and Cds1/Chk2 are effector kinases in the G(2)-phase checkpoint activated by damaged or unreplicated DNA, and they prevent entry into M-phase through inhibition of cyclin B-Cdc2 kinase activation. However, little is known about how the effector kinases are regulated when the checkpoint is attenuated. Recent studies indicate that Chk1 is also involved in the physiological G(2)-phase arrest of immature Xenopus oocytes via direct phosphorylation and inhibition of Cdc25C, the activator of cyclin B-Cdc2 kinase. Bearing in mind the overlapping functions of Chk1 and Cds1, here we have studied the involvement of Xenopus Cds1 (XCds1) in the G(2)/M-phase transition of immature oocytes and the regulation of its activity during this period. Protein levels of XCds1 remained constant throughout oocyte maturation and early embryonic development. The levels of XCds1 kinase activity were high in immature oocytes and decreased at the meiotic G(2)/M-phase transition. Consistently, when overexpressed in immature oocytes, wild-type, but not kinase-deficient, XCds1 significantly delayed entry into M-phase after progesterone treatment. The inactivation of XCds1 depended on the activation of cyclin B-Cdc2 kinase, but not MAP kinase. Although XCds1 was not directly inactivated by cyclin B-Cdc2 kinase in vitro, XCds1 was inactivated by overexpression of cyclin B, which induces the activation of cyclin B-Cdc2 kinase without progesterone. Thus, the present study is the first indication of Cds1 activity in cells that are physiologically arrested at G(2)-phase, and of its downregulation at entry into M-phase.

Expression of cyclin E and cyclin-dependent kinase 2 correlates with metastasis and prognosis in colorectal carcinoma

The expression of cyclin E and cyclin-dependent kinases 2 (CDK2) in metastatic foci, the relationship of their expression with some clinicopathologic characteristics, and the correlation of their expression with prognosis remain unclear. To examine the roles of their expression in the progression of colorectal carcinoma, 21 normal mucosa, 9 hyperplastic polyps, 58 adenomas, 17 adenocarcinoma in adenomas, 203 primary cancers, 21 lymph node metastases, and 10 hepatic metastases were immunohistochemically stained with anti–cyclin E, anti-CDK2, and anti-Ki67 antibodies. In the carcinogenic process, both cyclin E and CDK2 expressions increased significantly. From the primary to the lymph node–metastatic foci, cyclin E protein remained unchanged, but CDK2 increased significantly. From the primary to the liver-metastatic foci, cyclin E apparently decreased, and CDK2 was reduced almost to zero. In primary carcinomas, the reduction of cyclin E was significantly associated with large tumor size, mucinous type, venous invasion, deep infiltration, lymph nodal metastasis, peritoneal metastasis, advanced stage, and poor prognosis. Decreased CDK2 was obviously correlated with large tumor size, venous invasion, deep infiltration, hepatic metastasis, advanced stage, and poor prognosis. Increased cyclin E protein was related to elevated CDK2, which was further linked to higher Ki67. Thus, CDK2 overexpression could facilitate lymph node metastasis. The overexpression of cyclin E and CDK2 may mainly promote the progression of early cancer. Anti–cyclin E or anti-CDK2 chemotherapy should be targeted to the cancers with such overexpression. HUM PATHOL Copyright © 2001 by W.B. Saunders Company

Mammary Epithelial Cell-Cycle Progression via the α 2β 1 Integrin : Unique and Synergistic Roles of the α 2 Cytoplasmic Domain

The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.

Cell cycle analysis and expression of cell cycle regulator genes in myeloma cells overexpressing cyclin D1.

Among the recently discovered myeloma-specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl-1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin-dependent kinase inhibitors (CDK-Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1-overexpressing lines tended to have a shortened G1 phase compared with the non-expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1-overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation-related genes affect the cell cycle progression in myeloma cells.

TP53 mutations, amplification of P63 and expression of cell cycle proteins in squamous cell carcinoma of the oesophagus from a low incidence area in Western Europe.

In Europe, high incidence rates of oesophageal squamous cell carcinoma (SCCE) are observed in western France (Normandy and Brittany) and in north-eastern Italy. Analysis of TP53 mutations in tumours from these regions has shown a high prevalence of mutations at A:T basepairs that may result from DNA damage caused by specific mutagens. However, the spectrum of TP53 mutations in regions of low incidence is unknown. We report here TP53 mutation analysis in 33 SCCE collected in Lyon, an area of low incidence. These tumours were also examined for MDM2 and P63 amplification, and for expression of p16INK4a/CDKN2a, cyclin E, p27Kipland Cox2. TP53 mutations were detected in 36% of the cases (12/33). In contrast with regions of high incidence, the mutation spectrum did not show a high prevalence of mutations at A:T base pairs. P63 was amplified in 5/32 cases tested (15.5%). No amplification of MDM2 was found. Expression studies revealed frequent loss of p16INK4a/CDKN2a(46%) and p27Kipl(25%) expression, and frequent overexpression of Cyclin E (70%) and Cox2 (42%). Overall, these results indicate that in Europe, SCCE from areas of high and low incidence present a similar pattern of molecular alterations but differ by the type of TP53 mutations. © 2001 Cancer Research Campaign http://www.bjcancer.com

Overexpression of cyclin E and cyclin-dependent kinase 2 is correlated with development of hepatocellular carcinomas

Mammalian cell cycle progression is regulated by the combined action of cyclins/cyclin-dependent kinases (CDKs) and CDK inhibitors. Abnormal expression as well as interaction of these proteins may result in malignant transformation of cells. To further address the role of these cell cycle proteins in hepatocellular carcinomas, we analyzed the expression of cyclin E and CDK2. A panel of livers with human hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis were used as a human experimental system. The inbred LEC (Long–Evans with a cinnamon-like coat color) rats were used as an animal experimental HCC model. Immunohistochemical staining of serial paraffin sections was performed using antibodies to cyclin E and CDK2. The results showed that cyclin E and CDK2 were concurrently overexpressed in hepatocellular carcinomas both in human and rat livers. Western blot analysis and CDK2 kinase assay demonstrated expression levels of cyclin E and CDK2 and CDK2 kinase activity, respectively, and both were shown to increase along with the development of hepatocellular carcinomas. Analysis of the correlation between expression of cyclin E and CDK2 and clinicopathological parameters revealed a significant correlation between expression of cyclin E and tumor grade ( P=0.013), and PCNA index ( P=0.006) as well as CDK2 expression ( P=0.015). Overexpression of CDK2 tended to be associated with poorly differentiated HCCs. The results suggest that overexpression of cyclin E and CDK2 plays an important role in the development of hepatocellular carcinoma.

Expression of cell-cycle regulatory proteins in endometrial carcinomas: correlations with hormone receptor status and clinicopathologic parameters.

The normal human endometrium is characterized by hormone-dependent variations in the levels of cell-cycle regulatory proteins during the menstrual cycle. As this tightly controlled system is disturbed in endometrial carcinomas, we analyzed which cell-cycle regulators are involved in endometrial carcinogenesis. We performed Western blot analysis of five cell-cycle stimulating (cyclins D1, E, B1, cdk2, cdk4) and three cell-cycle inhibiting (p16(INK4a), p21(WAF1), Rb) proteins in 41 endometrial carcinoma specimens. In addition, expression of the estrogen and progesterone receptors (ER, PR), Ki67, and, in selected cases, p16, cyclin E, and cyclin B1 was studied by immunohistochemistry. We found upregulation of all analyzed cell-cycle regulators in most tumors compared to normal endometrial tissue samples. Overexpression of cyclin E, cyclin B1, and p21 was associated with a less differentiated phenotype. In addition, high levels of cyclin E, cdk2, and cdk4 correlated with weak/absent ER expression, and p16 and p21 overexpression was significantly associated with low PR immunoreactivity. Cyclin B1 expression correlated with cyclin E, cdk2, cdk4, p21, Rb, and Ki67, and cyclin E expression with cyclin D1 and Rb. We conclude that cyclin E and cyclin B1 might be the major cell-cycle regulators involved in proliferation and reduced differentiation of endometrial carcinomas. In addition, p16, p21, and Rb appear to be uncoupled from their normal cell-cycle inhibiting function in many endometrial carcinomas.

Reciprocal expressions of cyclin E and cyclin D1 in hepatocellular carcinoma

Deregulation of the cell cycle by overexpression of G1 cyclins, cyclin E and cyclin D1 genes, has been demonstrated to be a prerequisite for the development of human cancer. Recently, cyclin E is proposed to be sufficient for the progression of the G1 cell cycle without cyclin D1. Here we show that the proposed model system was specifically present in human hepatocellular carcinoma (HCC) unlike other human cancers. Of 31 HCC tissues analyzed, 21 (67.7%) exhibited an overexpression of cyclin E protein. In contrast to cyclin E gene expression, cyclin D1 expression was strongly downregulated in 19 (61.2%) HCCs. Interestingly, 65% of HCC tissues with overexpression of the cyclin E gene exhibited downregulation of cyclin D1, suggesting reciprocal deregulation of these cyclins in the G1 progression of the cell cycle. Southern blot analysis proved the amplification of cyclin E gene in HCC with a high level of overexpression. The present findings suggest that the reciprocal deregulation of cyclin E lacking cyclin D1 expression might play a role in G1 progression and the development of HCC.

Frequent cyclin D1 gene amplification and protein overexpression in oral epithelial dysplasias.

Amplification of the cyclin D1 gene has been identified in 17-55% of head and neck squamous cell carcinoma. In some tumors, this alteration has been associated with decreased survival and increased recurrence rates. In precancerous lesions of the mouth, the frequency of cyclin D1 gene amplification is not known. In addition, it is unknown whether amplification of the gene translates to overexpressed cyclin D1 protein in these lesions. We examined 59 formalin-fixed, paraffin embedded tissue biopsies of oral epithelial dysplasias (OED) and 25 oral squamous cell carcinoma (SCC) from the floor of the mouth for cyclin D1 gene and protein levels. Genomic DNA was extracted from laser microdissected lesional tissue and a duplex, quantitative PCR assay was used to determine the amplification of the cyclin D1 gene relative to interferon-gamma. Cyclin D1 protein expression was determined using immunohistochemistry and counting positive nuclei by computer image analysis. We found cyclin D1 gene amplification in 41% of mild, 45% of moderate and 24% of severe OEDs. Cyclin D1 was amplified in 36% of SCC. Overexpression of cyclin D1 protein was identified in 29% of mild, 47% of moderate, 29% of severe OED's, and in 32% of SCC. Overexpression of cyclin D1 protein was identified in similar proportions of all grades of dysplasia and SCC. There were statistically significant correlations identified between gene and protein levels in all categories of disease. We concluded that amplification of the cyclin D1 gene is frequent in OED and that duplex, quantitative polymerase chain reaction is a reliable method to detect this change in routinely processed biopsies. The strong correlation between cyclin D1 gene amplification and protein levels suggests that this method may be suitable to assess cyclin D1 gene status in tissues not suitable for protein analysis.

The human cytomegalovirus immediate early 2 protein dissociates cellular DNA synthesis from cyclin-dependent kinase activation.

Passage through the restriction point late in G1 normally commits cells to replicate their DNA. Here we show that the previously reported cell cycle block mediated by the human cytomegalovirus (HCMV) immediate early 2 (IE2) protein uncouples this association. First, IE2 expression leads to elevated levels of cyclin E-associated kinase activity via transcriptional activation of the cyclin E gene. This contributes to post-restriction point characteristics of IE2-expressing cells. Then these cells fail to undergo substantial DNA replication although they have entered S phase, and the induction of DNA replication observed after overexpression of cyclin E or D can be antagonized by IE2 without impinging on cyclin-associated kinase activities. These data suggest that IE2 secures restriction-point transition of cells before it stops them from replicating their genome. Our results fit well with HCMV physiology and support the view that IE2 is part of a viral activity which, on the one hand, promotes cell cycle-dependent expression of cellular replication factors but, on the other hand, disallows competitive cellular DNA synthesis.

Modulation of Sp1 activity by a cyclin A/CDK complex

Transcription factors of the Sp1 family are targets of several regulatory pathways and can induce or inhibit gene expression. Here we show that Sp1 is associated with a histone 1 kinase activity. This activity is growth regulated and correlates with the expression of cyclin A. Co-immunoprecipitation experiments demonstrate, that Sp1 interacts with cyclin A and can be phosphorylated by a cyclin A associated kinase. The interaction is direct and requires the zinc-finger region of Sp1 and the amino-terminal domain of cyclin A. Over-expression of cyclin A enhances the expression of a reporter gene controlled by an Sp1 responsive promoter. Addition of olomoucine, a specific inhibitor of CDK2 and CDC2 activity on the other hand reduces the expression of the reporter. Electrophoretic mobility shift assays suggest that this is due to a reduction of the DNA-binding ability of Sp1 family members. Our results indicate that phosphorylation of Sp1 and other members of the family by a cyclin A/CDK complex may play a role in the growth and cell cycle regulation of its transcriptional activity.

Expression of cyclin E and cyclin D1 in non-small cell lung cancers

The relationships between overexpression of cyclin D1 or cyclin E and clinicopathological factors were investigated in 157 patients with non-small cell lung cancers (NSCLCs) using immunohistochemical analysis. Fifty-eight cases of NSCLCs (58/157, 37%) showed the overexpression of cyclin D1, and 64 cases (64/157, 41%) were positive for cyclin E. Cyclin E and cyclin D1 were infrequently concurrently overexpressed (17/157, 10.8%). Overexpression of cyclin E was more frequently observed in squamous cell carcinoma (29/57, 51%) compared with that in adenocarcinoma (28/86, 33%) ( P<0.05). In addition, overexpression of cyclin E was more frequently observed in poorly or moderately differentiated NSCLCs (52/103, 50%) than in well-differentiated ones (12/54, 22%) regardless of their histological types ( P<0.01). On the contrary, there was no statistically significant relationship between cyclin D1 overexpression and histological types or grade of tumor differentiation. These findings suggest that expression of cyclin E was frequently independent of that of cyclin D1 and played some roles in the grade of tumor differentiation in NSCLCs.

Regulation of RNA Polymerase III Transcription during Cell Cycle Entry

Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G(0) and early G(1), but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid- to late G(1) phase, when RB becomes phosphorylated by cyclin D- and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.

Over-expression of cyclin A is highly associated with early relapse and reduced survival in patients with primary breast carcinomas.

Progression through the mammalian cell cycle is facilitated by cyclin-cyclin-dependent kinase (cdk) complexes, which are activated at specific points during the cell cycle. Alteration in cyclin-cdk complexess may lead to altered cell cycle and tumorigenesis. In this study, we analyzed expression of cyclins A, D1, D3 and E in tumor tissue from 170 patients with primary invasive breast carcinomas. Immunohistochemical methods were used to detect protein expression of these cyclins. We detected positive immunoreactivity in 55 (32%), 22 (13%), 38 (22%) and 37 (21.8%) of the samples for cyclins A, D1, D3 and E, respectively. A highly statistically significant association was observed between expression of cyclin A and early relapse (p = 0.001 univariate analysis, p = 0.006 multivariate analysis) as well as cancer-specific death (p < 0.0001) during the follow-up time. No association was observed between cyclin D1 or cyclin E, respectively, and relapse of disease or survival, while cyclin D3 over-expression was associated with development of metastases during follow-up (p = 0.005 univariate analysis, p = 0.01 multivariate analysis). However, cyclin D3 did not show any statistically significant association when cancer-specific death was examined in a multivariate analysis (Cox regression for survival function).