Regulatory Subunit Of Cyclin-dependent Kinase Research Articles

Genome-Wide Identification and Evolutionary and Expression Analyses of the Cyclin B Gene Family in Brassica napus.

Cyclin B (CYCB) is a regulatory subunit of cyclin-dependent kinase (CDK), the concentration of which fluctuates to regulate cell cycle progression. Extensive studies have been performed on cyclins in numerous species, yet the evolutionary relationships and biological functions of the CYCB family genes in Brassica napus remain unclear. In this study, we identified 299 CYCB genes in 11 B. napus accessions. Phylogenetic analysis suggests that CYCB genes could be divided into three subfamilies in angiosperms and that the CYCB3 subfamily members may be a newer group that evolved in eudicots. The expansion of BnaCYCB genes underwent segmental duplication and purifying selection in genomes, and a number of drought-responsive and light-responsive cis-elements were found in their promoter regions. Additionally, expression analysis revealed that BnaCYCBs were strongly expressed in the developing seed and silique pericarp, as confirmed by the obviously reduced seed size of the mutant cycb3;1 in Arabidopsis thaliana compared with Col-0. This study provides a comprehensive evolutionary analysis of CYCB genes as well as insight into the biological function of CYCB genes in B. napus.

Protein-RNA interactions of FMRP and CCND1: Implications of regulation in neurogenesis.

While originally discovered in the 1960s, adult neurogenesis, the production of mature neurons from neuronal stem cells in adults, has become largely accepted by the field only in the 1990s. Thus, the detailed mechanisms of its regulation remain elusive, requiring further characterization of the protein players involved in this process. Cyclin-D1, a regulatory subunit of cyclin-dependent kinases involved in monitoring entry into the cell cycle, has been identified as a key factor in adult neurogenesis and is encoded by the CCND1 gene. Another identified component of this process is the Fragile X messenger ribonucleoprotein (FMRP), an RNA-binding protein associated with translational regulation. Interestingly, FMRP has been shown to regulate cyclin-D1 expression, emphasizing a potential interplay between FMRP function and cyclin-D1 expression in neurons. FMRP has shown high affinity binding for guanine quadruplex (GQ) forming RNAs. We identified that CCND1 mRNA has a G-rich region in its 3’-untranslated region that has the potential to form GQ structures, and thus possibly interacting with FMRP through the recognition of these structures. To elucidate the presence of a GQ in the CCND1 mRNA, we used biophysical methods, such as 1H NMR, UV thermal denaturation, CD spectroscopy, and native polyacrylamide gel electrophoresis (PAGE). This study effectively characterizes a GQ in the 3’UTR of the CCND1 mRNA and its interactions with FMRP, providing insight on additional regulatory mechanisms of adult neurogenesis and guiding development of novel therapeutic methods.

Lack of Cyclin E1 in hepatocytes aggravates ethanol-induced liver injury and hepatic steatosis in experimental murine model of acute and chronic alcohol-associated liver disease

BackgroundCyclin E1 is the regulatory subunit of cyclin-dependent kinase 2 (Cdk2) and one of the central players in cell cycle progression. We recently showed its crucial role for initiation of liver fibrosis and hepatocarcinogenesis. In the present study, we investigated the role of Cyclin E1 in the development of alcohol-associated liver disease (ALD). MethodsMice with constitutive (E1−/−), hepatocyte-specific (Cyclin E1Δhepa), or intestinal-epithelial-cell-specific (Cyclin E1ΔIEC) inactivation of Cyclin E1 and corresponding wild type littermate controls (WT) were administered either a Lieber-DeCarli ethanol diet (LDE) for 3 weeks or acute ethanol binges (6 g/kg) through oral gavage. Serum parameters of liver functionality were measured; hepatic tissues were collected for biochemical and histological analyses. ResultsThe administration of acute EtOH binge and chronic LDE diet to E1−/− mice enhanced hepatic steatosis, worsened liver damage and triggered body weight loss. Similarly, in the acute EtOH binge model, Cyclin E1Δhepa mice revealed a significantly worsened liver phenotype. In contrast, inactivation of Cyclin E1 only in intestinal epithelial cell (IECs)did not lead to any significant changes in comparison to WT mice after acute EtOH challenge. Remarkably, both acute and chronic EtOH administration in E1−/− animals resulted in increased levels of ADH and decreased expression of ALDH1/2. The additional application of a pan-Cdk inhibitor (S-CR8) further promoted liver damage in EtOH-treated WT mice. ConclusionOur data point to a novel unexpected role of Cyclin E1 in hepatocytes for alcohol metabolism, which seems to be independent of the canonical Cyclin E1/Cdk2 function as a cell cycle regulator.

Cyclin E1 in Murine and Human Liver Cancer: A Promising Target for Therapeutic Intervention during Tumour Progression

Simple SummaryThe cell cycle regulator Cyclin E1 is a key mediator and biomarker of liver cancer progression in mice and man independent of its canonical interacting partner Cyclin-dependent kinase 2. Over-expression of Cyclin E1 during hepatocarcinogenesis modulates several distinct biological processes such as proliferation, DNA damage response, stemness, invasion and the tumour microenvironment. Interventional depletion of Cyclin E1 in the course of liver cancer progression significantly reduces tumour burden. In contrast, the expression of Cyclin-dependent kinase 2 is dispensable for the progression of liver cancer in mice and lacked diagnostic or prognostic value in patients. Thus, specific inhibition of Cyclin E1 expression represents a promising strategy for the treatment of liver cancer.Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.

Endothelial overexpression of P25 causes premature vascular aging by promoting SIRT1 protein degradation

Background P25 is a regulatory subunit of cyclin-dependent kinase 5 (CDK5). The CDK5/P25 signaling is implicated in the regulation of SIRT1, a mammalian sirtuin with an important participation in aging. Hypothesis: Overexpression of endothelial p25 leads to premature vascular aging by regulating SIRT1 degradation. Aims: The aims of this study were to determine if overexpression of p25 in the vascular endothelium induces vascular aging by causing degradation of SIRT1 and increasing vascular contraction, oxidative stress and vascular senescence. Methods: The present study established a transgenic mouse model (EC-P25) with selective overexpression of P25 in endothelial cells. Aortas were obtained from 12- or 30-weeks old EC-p25 mice and control littermates for all experiments. Wire myography was performed in quiescent thoracic aorta with Mulvany-Halpern wire myograph system to measure isometric tension in the presence or absence of SIRT1 activators or inhibitors. Vascular senescence was evaluated using senescence-associated β-galactosidase (SA-β-gal) staining in aortae, whereas heamatoxylin-eosine (H & E) staining was performed on aortic sections to evaluate aortic wall hypertrophy. Melondialdehyde (MDA) concentration in aortic homogenates represented a measure of oxidative stress, and protein expression levels were measured by Western blotting. Results: EC-P25 mice showed increased vascular tone responsiveness to alpha-1-adrenergic receptor agonist phenylephrine (PE) compared to the control littermates suggesting that overexpression of p25 in the vascular endothelium has deleterious effects on vascular function. SA-β-gal staining was increased in EC-P25 mice when compared to the control group, demonstrating vascular senescence. H & E staining also showed aortic wall hypertrophy with an increase in aortic wall thickness in EC-P25 mice compared to control mice. MDA concentration was also higher in EC-P25 mice aorta than in the control group, implying increased oxidative stress. SIRT1 protein was found to be fragmented with increased levels of shorter fragments in EC-P25 aorta when compared to their control littermates, suggesting that, over-expression of P25 in the vascular endothelium regulates SIRT1 degradation leading to premature aging, adverse remodeling and loss of functional regulation. Conclusion: Overexpression of endothelial p25 may regulate vascular function by increasing vascular tone, aortic wall hypertrophy, oxidative stress and vascular aging leading to vascular disease. Additionally, a fragment of SIRT1 with enhanced deacetylase activity was detected in arteries of EC-P25 mice, whereas EX-527, a SIRT1 inhibitor, attenuated endothelium-dependent contractions. In conclusion, the results suggest that targeting CDK5/P25/SIRT1 signaling represents a promising strategy for ageing-associated vascular diseases.

Clinical significance of CCNE2 protein and mRNA expression in thyroid cancer tissues

PurposeThyroid carcinoma (TC) is the most common endocrinal malignancy worldwide. Cyclin E2 (CCNE2), a member of the cyclin family, acts as a regulatory subunit of cyclin-dependent kinases (CDKs). It controls the transition of quiescent cells into the cell cycle, regulates the G1/S transition, promotes DNA replication, and activates CDK2. This study explored the role and potential molecular mechanisms of CCNE2 expression in TC tissues. Material/methodsImmunohistochemistry was used to evaluate the CCNE2 protein expression levels in TC. High-throughput data on CCNE2 in TC were obtained from RNA sequencing (RNA-seq), microarray, and literature data. The CCNE2 expression levels in TC were comprehensively assessed through an integrated analysis. Analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein–protein interaction (PPIs) data facilitated the investigation of the relative molecular mechanisms of CCNE2 in TC. ResultsThe immunohistochemical experiment showed a significant increase in the expression of CCNE2 in the TC tissues. For 505 TC and 59 non-cancerous samples from RNA-seq data, the area under the curve (AUC) was 0.8016 (95% confidence interval [CI] 0.742–0.8612; p<0.001). With another 14 microarrays, the pool standard mean difference [SMD] was 1.01 (95% CI [0.82–1.19]). The pooled SMD of CCNE2 was 1.12 (95% CI [0.60–1.64]), and the AUC was 0.87 (95% CI [0.84–0.90]) for 1157 TC samples and 366 non-cancerous thyroid samples from all possible sources. Nine hub genes were upregulated in TC. ConclusionsA high expression of CCNE2 may lead to carcinogenesis and the development of TC.

Low-Molecular-Weight Cyclin E in Human Cancer: Cellular Consequences and Opportunities for Targeted Therapies.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (CDK2), is central to the initiation of DNA replication at the G1/S checkpoint. Tight temporal control of cyclin E is essential to the coordination of cell-cycle processes and the maintenance of genome integrity. Overexpression of cyclin E in human tumors was first observed in the 1990s and led to the identification of oncogenic roles for deregulated cyclin E in experimental models. A decade later, low-molecular-weight cyclin E (LMW-E) isoforms were observed in aggressive tumor subtypes. Compared with full-length cyclin E, LMW-E hyperactivates CDK2 through increased complex stability and resistance to the endogenous inhibitors p21CIP1 and p27KIP1 LMW-E is predominantly generated by neutrophil elastase-mediated proteolytic cleavage, which eliminates the N-terminal cyclin E nuclear localization signal and promotes cyclin E's accumulation in the cytoplasm. Compared with full-length cyclin E, the aberrant localization and unique stereochemistry of LMW-E dramatically alters the substrate specificity and selectivity of CDK2, increasing tumorigenicity in experimental models. Cytoplasmic LMW-E, which can be assessed by IHC, is prognostic of poor survival and predicts resistance to standard therapies in patients with cancer. These patients may benefit from therapeutic modalities targeting the altered biochemistry of LMW-E or its associated vulnerabilities. Cancer Res; 78(19); 5481-91. ©2018 AACR.

Reduction of glutamate-induced excitotoxicity in murine primary neurons involving calpain inhibition

Excessive glutamate secretion leads to excitotoxicity, which has been shown to underlie neurodegenerative disorders. Excitotoxicity is in part exerted by overactivation of calpains, which promote neuronal cell death via induction of limited proteolysis of the cellular proteins p35, regulatory subunit of cyclin-dependent kinase 5, and αII-spectrin. We used primary murine neuronal cells in a model of glutamate toxicity. The protease inhibitor α1-antitrypsin was able to prevent glutamate toxicity as determined by MTT assay and immunofluorescence. Calpain and caspase 3 activity were reduced following α1-antitrypsin treatment, as assessed by calpain and caspase 3 activity assays. In addition we could observe a modulation of cleavage of the calpain/caspase substrates αII-spectrin and p35 in Western blots. In summary, α1-antitrypsin shows inhibitory effects on excitotoxicity of primary neurons involving the inhibition of calpain activity. The advantage of using α1-antitrypsin is that the substance is already in clinical use for the treatment of patients with hereditary α1-antitrypsin deficiency. Further experiments are required in animal models of neurodegenerative disorders to assess the suitability of this substance in patients suffering from Alzheimer's disease or Parkinson's disease.

Regulation of N-type Voltage-Gated Calcium Channels and Presynaptic Function by Cyclin-Dependent Kinase 5

N-type voltage-gated calcium channels localize topresynaptic nerve terminals and mediate key eventsincluding synaptogenesis and neurotransmission.While several kinases have been implicated in the modulation of calcium channels, their impact on presynaptic functions remains unclear. Here we report that the N-type calcium channel is a substrate for cyclin-dependent kinase 5 (Cdk5). The pore-forming α(1) subunit of the N-type calcium channel is phosphorylated in the C-terminal domain, and phosphorylation results in enhanced calcium influx due to increased channel open probability. Phosphorylation of the N-type calcium channel by Cdk5 facilitates neurotransmitter release and alters presynaptic plasticity by increasing the number of docked vesicles at the synaptic cleft. These effects are mediated by an altered interaction between N-type calcium channels and RIM1, which tethers presynaptic calcium channels to the active zone. Collectively, our results highlight a molecular mechanism by which N-type calcium channels are regulated by Cdk5 to affect presynaptic function.

Cyclin A Is Redundant in Fibroblasts but Essential in Hematopoietic and Embryonic Stem Cells

Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation.

Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol

BackgroundCyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers.MethodsIn order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models.ResultsDownregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo.ConclusionOur data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.

Arabidopsis cyclin D2 expressed in rice forms a functional cyclin-dependent kinase complex that enhances seedling growth

D-class cyclins play important roles in controlling the cell cycle in development and in response to external signals by forming the regulatory subunit of cyclin-dependent kinase (CDK) complexes. To evaluate the effects of D-class cyclins in transgenic rice plants, Arabidopsis cyclin D2 gene (CycD2) was linked to the maize ubiquitin1 promoter (Ubi1) and introduced into rice by the Agrobacterium-mediated transformation method. Genomic deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Western blot hybridizations of the Ubi1:CycD2 plants revealed copy number of transgene and its increased expression in leaf and callus cells at messenger RNA (mRNA) and/or protein levels. The H1 kinase assay using the immunoprecipitates of protein extracts from the Ubi1:CycD2 plants and nontransgenic controls demonstrated that the introduced Arabidopsis CycD2 forms a functional CycD2/CDK complex with an unidentified CDK of rice. Shoot and root growth was enhanced in the Ubi1:CycD2 seedlings compared with nontransgenic controls, together, suggesting that Arabidopsis cyclin D2 interacts with a rice cyclin-dependent kinase, consequently enhancing seedling growth.

WAVE1 controls neuronal activity-induced mitochondrial distribution in dendritic spines

Mitochondrial fission and trafficking to dendritic protrusions have been implicated in dendritic spine development. Here, we show that Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1) controls depolarization-induced mitochondrial movement into dendritic spines and filopodia and regulates spine morphogenesis. Depolarization-induced degradation of the p35 regulatory subunit of cyclin-dependent kinase 5 (Cdk5), with the resultant decreased inhibitory phosphorylation on WAVE1, depend on NMDA receptor activation. Thus, WAVE1 dephosphorylation and activation are likely associated with mitochondrial redistribution and spine morphogenesis.

Regaining the Lost Past

People with Alzheimer’s disease or related neurodegenerative disorders may progress from absentmindedness and difficulty learning new things to a heartbreaking state in which they forget their own past and no longer recognize the faces of people they love. Expression of p25 (a truncated form of the p35 regulatory subunit of cyclin-dependent kinase 5) is associated with various neurodegenerative disorders, and the CK-p25 Tg mouse, in which p25 expression--and thereby neuronal loss--can be conditionally regulated, is a model for these disorders. Fischer et al . found that exposing CK-p25 Tg mice to an enriched environment (after neuronal loss had occurred and learning was impaired) improved their associative and spatial learning. Moreover, environmental enrichment led to the recovery of long-term memories that had apparently been lost. Experiments in wild-type mice revealed that exposure to an enriched environment stimulated histone acetylation in the hippocampus and cortex. The histone deacetylase (HDAC) inhibitor sodium butyrate (SB) improved associative and spatial learning in wild-type and CK-p25 Tg mice (after p25 induction) and, like environmental enrichment, enhanced dendritic and synaptic growth in CK-p25 Tg mice without affecting brain atrophy or neuronal loss. Furthermore, SB enabled the recovery of lost memories. The authors conclude that their data raise the possibility that HDAC inhibitors may provide a means to enable people with neurodegenerative disorders associated with memory loss to recover lost memories. Sweatt provides thoughtful commentary, noting (as do the authors) that HDAC inhibitors may affect the acetylation status of proteins other than histones. A. Fischer, F. Sananbenesi, X. Wang, M. Dobbin, L. H. Tsai, Recovery of learning and memory is associated with chromatin remodelling. Nature 447 , 178-182 (2007). [PubMed] J. D. Sweatt, Down memory lane. Nature 447 , 151-152 (2007). [PubMed]

Mitotic Checkpoint Slippage in Humans Occurs via Cyclin B Destruction in the Presence of an Active Checkpoint

In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast , and likely Drosophila, this occurs through an "adaptation" process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, we conducted fluorescence microscopy studies on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. We find that in the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. We also find that cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, our data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis.

Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo

Cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and is critical for the initiation of mitosis. Accumulating data indicate that the deregulation of cyclin B1 is tightly linked to neoplastic transformation. To study the phenotype and the potential preclinical relevance, we generated HeLa cell lines stably transfected with the plasmids encompassing short hairpin RNA (shRNA) targeting cyclin B1. We demonstrate that the reduction of cyclin B1 caused inhibition of proliferation by arresting cells in G2 phase and by inducing apoptosis. Cells, entering mitosis, were impaired in chromosome condensation and alignment. Importantly, HeLa cells with reduced cyclin B1 were more susceptible to the treatment of small interfering RNA targeting Polo-like kinase 1 (Plk1) and to the administration of the chemotherapeutic agent taxol. Finally, HeLa cells with reduced cyclin B1 showed inhibited tumor growth in nude mice compared to that of control cells. In summary, our data indicate that cyclin B1 is an essential molecule for tumor cell survival and aggressive proliferation, suggesting that the downregulation of cyclin B1, especially in combination with other molecular targets, might become an interesting strategy for antitumor intervention.

Identification of the p28 subunit of eukaryotic initiation factor 3(eIF3k) as a new interaction partner of cyclin D3

Cyclin D3 is found to play a crucial role not only in progression through the G1 phase as a regulatory subunit of cyclin-dependent kinase 4 (CDK 4) and CDK 6, but also in many other aspects such as cell cycle, cell differentiation, transcriptional regulation and apoptosis. In this work, we screened a human fetal liver cDNA library using human cyclin D3 as bait and identified human eukaryotic initiation factor 3 p28 protein (eIF3k) as a partner of cyclin D3. The association of cyclin D3 with eIF3k was further confirmed by in vitro binding assay, in vivo coimmunoprecipitation, and confocal microscopic analysis. We found that cyclin D3 specifically interacted with eIF3k through its C-terminal domain. Immunofluorescence experiments showed that eIF3k distributed both in nucleus and cytoplasm and colocalized with cyclin D3. In addition, the cellular translation activity in HeLa cells was upregulated by cyclin D3 overexpression and the mRNA levels are constant. These data provide a new clue to our understanding of the cellular function of cyclin D3.

Cyclin D1 Expression in Pre-cancerous and Cancerous Lesions of the Canine Mammary Gland

Overexpression of cyclin D1, the regulatory subunit of cyclin-dependent kinases (cdk4 and cdk6) involved in cell cycle control, has often been found in breast cancer and other types of human cancer. Increased expression, or stability, of cyclin D1 molecules may cause sufficient cdk4 activation to produce retinoblastoma protein phosphorylation independently of mitogenic signals; this results in commitment of cells to the G1 phase at mitosis. In the present study, cyclin D1 expression was investigated in pre-cancerous and cancerous lesions of the canine mammary gland by a complex experimental approach, which included Western blot and immunohistochemical analysis of cyclin D1 and the related molecular system. Furthermore, to define relationships between cell growth and expression of cyclin D1, proliferative activity was studied by the AgNOR technique. The study provided the following information. Cyclin D1 overexpression was largely independent of the type of proliferative anomaly. Indeed, cyclin D1 was expressed in 60% of the pre-cancerous lesions and in 44% of cancerous lesions. Mitotic activity and cyclin D1 expression were related: mammary lesions that expressed cyclin D1 showed a high proliferative ratio, the opposite being true of cyclin D1-negative cell populations. This study may contribute to the establishment of an animal model for anti-cancer research based on cyclin D1 suppression or cdk inactivation, or both.

Ras regulation of cyclin D1 promoter

Publisher Summary This chapter focuses on the Ras regulation of Cyclin D 1 promoter. Cyclin D l, the regulatory subunit of cyclin-dependent kinases (CDK) 4 and 6, is required for, and capable of, shortening, the G j phase of the cell cycle through the formation of holoenzyme complexes (cyclin-CDK) that phosphorylate retinoblastoma protein (pRB). Ras transformation is inhibited by antisense to cyclin D1 mRNA. The Ras-related proteins, Rac and Rho, also induce the cyclin D1 promoter. Racl regulates several distinct pathways; no single Rac effect is necessary or sufficient for transformation. Platelet-derived growth factor (PDGF) activates the extracellular signal-regulated kinase (ERK) pathway, which has been shown to induce DNA synthesis, increase cyclin D1 protein levels, and stimulate the transcription of the cyclin D1 promoter. Therefore, Rac 1 is neither necessary nor sufficient to activate the ERK pathway, suggesting that Rac regulation of cyclin D1 is ERK independent. The cyclin DI promoter is used as a molecular probe of the signal transduction pathways involved in Ras signaling. The valid assessment of promoter activation by Ras requires the use of a reporter system that is not itself responsive to components of the Ras signaling pathway.

High-Throughput Tissue Microarray Analysis of Cyclin E Gene Amplification and Overexpression in Urinary Bladder Cancer

Studies by comparative genomic hybridization revealed that the 19q13 chromosomal region is frequently amplified in bladder cancer. The cyclin E gene (CCNE), coding for a regulatory subunit of cyclin-dependent kinase 2, has been mapped to 19q13. To investigate the role of cyclin E alterations in bladder cancer, a tissue microarray of 2,317 specimens from 1,842 bladder cancer patients was constructed and analyzed for CCNE amplification by fluorescence in situ hybridization and for cyclin-E protein overexpression by immunohistochemistry. Fluorescence in situ hybridization analysis showed amplification in only 30 of the 1,561 evaluable tumors (1.9%). Amplification was significantly associated with stage and grade (P: < 0.0005 each). Immunohistochemically detectable cyclin E expression was strong in 233 (12.4%), weak in 354 (18.9%), and negative in 1, 286 of the 1,873 interpretable tumors. The majority (62.1%) of CCNE-amplified tumors were strongly immunohistochemistry-positive (P: < 0.0001). The frequency of protein expression increased from stage pTa (22.2%) to pT1 (45.5%; P: < 0.0001) but then decreased for stage pT2-4 (29.4%; P: < 0.0001 for pT1 versus pT2-4). Low cyclin E expression was associated with poor overall survival in all patients (P: < 0.0001), but had no prognostic impact independent of stage. It is concluded that cyclin E overexpression is characteristic to a subset of bladder carcinomas, especially at the stage of early invasion. This analysis of the prognostic impact of CCNE gene amplification and protein expression in >1,500 arrayed bladder cancers was accomplished in a period of 2 weeks, illustrating how the tissue microarray technology remarkably facilitates the evaluation of the clinical relevance of molecular alterations in cancer.