The D-type cyclin CYCD3;1 is limiting for the G1-to-S-phase transition in Arabidopsis

The G1-to-S-phase transition is a key regulatory point in the cell cycle, but the rate-limiting component in plants is unknown. Overexpression of CYCLIN D3;1 (CYCD3;1) in transgenic plants increases mitotic cycles and reduces endocycles, but its effects on cell cycle progression cannot be unambiguously determined. To analyze the cell cycle roles of plant D-type cyclins, we overexpressed CYCD3;1 in Arabidopsis thaliana cell suspension cultures. Changes in cell number and doubling time were insignificant, but cultures exhibited an increased proportion of G2- over G1-phase cells, as well as increased G2 arrest in response to stationary phase and sucrose starvation. Synchronized cultures confirm that CYCD3;1-expressing (but not CYCD2;1-expressing) cells show increased G2-phase length and delayed activation of mitotic genes such as B-type cyclins, suggesting that CYCD3;1 has a specific G1/S role. Analysis of putative cyclin-dependent kinase phosphorylation sites within CYCD3;1 shows that mutating Ser-343 to Ala enhances CYCD3;1 potency without affecting its rate of turnover and results in a fivefold increase in the level of cell death in response to sucrose removal. We conclude that CYCD3;1 dominantly drives the G1/S transition, and in sucrose-depleted cells the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by ectopic CYCD3;1 expression. Ser-343 is likely a key residue in modulating CYCD3;1 activity in response to sucrose depletion.     

cAMP-dependent positive control of cyclin A2 expression during G1/S transition in primary hepatocytes.

cAMP positively and negatively regulates hepatocyte proliferation but its molecular targets are still unknown. Cyclin A2 is a major regulator of the cell cycle progression and its synthesis is required for progression to S phase. We have investigated whether cyclin A2 and cyclin A2-associated kinase might be one of the targets for the cAMP transduction pathway during progression of hepatocytes through G1 and G1/S. We show that stimulation of primary cultured hepatocytes by glucagon differentially modulated the expression of G1/S cyclins. Glucagon indeed upregulated cyclin A2 and cyclin A2-associated kinase while cyclin E-associated kinase was unmodified. In conclusion, our study identifies cyclin A2 as an important effector of the cAMP transduction network during hepatocyte proliferation.     

c-myc can induce expression of G0/G1 transition genes.

The human c-myc oncogene was linked to the heat shock-inducible Drosophila hsp70 promoter and used to stably transfect mouse BALB/c 3T3 cells. Heat shock of the transfectants at 42 degrees C followed by recovery at 37 degrees C resulted in the appearance of the human c-myc protein which was appropriately localized to the nuclear fraction. Two-dimensional analysis of the proteins of density-arrested cells which had been heat shock treated revealed the induction of eight protein species and the repression of five protein species. All of the induced and repressed proteins were nonabundant. cDNA clones corresponding to genes induced during the G0/G1 transition were used as probes to assay for c-myc inducibility of these genes. Two anonymous sequences previously identified as serum inducible (3CH77 and 3CH92) were induced when c-myc was expressed. In response to serum stimulation, 3CH77 and 3CH92 were expressed before c-myc mRNA levels increased. However, in response to specific induction of c-myc by heat shock of serum arrested cells, 3CH77 and 3CH92 mRNA levels increased after the rise in c-myc mRNA. Therefore, we hypothesize that abnormal expression of c-myc can induce genes involved in the proliferative response.     

Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1

We have previously shown that the mitogenic effect of endothelin-1 (ET-1) in primary astrocytes is dependent on activation of both extracellular signal-regulated kinase (ERK)- and cytoskeleton (CSK)-dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET-1-induced expression of cyclins D1 and D3 is dependent on the ERK- and CSK-dependent pathways, respectively; moreover, a decrease in the levels of the cyclin-dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell-cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D-type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D-type cyclin-associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET-1, are regulated by distinct signaling pathways.     

Dysregulated expression of cyclin D1 in normal human mammary epithelial cells inhibits all-trans-retinoic acid-mediated G0/G1-phase arrest and differentiation in vitro.

Overexpression of cyclin D1 protein is observed in the majority of breast cancers, suggesting that dysregulated expression of cyclin D1 might be a critical event in breast cancer carcinogenesis. We investigated whether retroviral-mediated expression of cyclin D1 might affect all-trans-retinoic acid (ATRA)-mediated growth inhibition and differentiation of normal cultured human mammary epithelial cells (HMECs). HMECs treated with 1.0 microM ATRA undergo irreversible growth inhibition starting at 24 h and complete G0/G1-phase arrest by Day 3. Cyclin D1 protein levels are observed to decrease in association with the initiation of growth arrest starting at 24 h and then increase by approximately 35% on Day 3. Concomitant with this observed increase in cyclin D1, HMECs undergo morphologic changes consistent with progression to a more differentiated phenotype, including an increase in cell size, increased cell spreading, increased tonofilaments, and accumulation of cytoplasmic vesicles containing lipid. Dysregulated expression of cyclin D1 in HMECs results in inhibition of G0/G1-phase arrest mediated by ATRA. In addition, HMECs expressing exogenous cyclin D1 are resistant to differentiation by ATRA. Our results suggest that coordinated expression of cyclin D1 may be critical for normal mammary epithelial cell homeostasis, and dysregulated expression of cyclin D1 might result in retinoid resistance and promote mammary carcinogenesis.     

NF-kappaB transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth

A novel function of NF-kappaB in the development of most ectodermal appendages, including two types of murine pelage hair follicles, was detected in a mouse model with suppressed NF-kappaB activity (c(IkappaBalphaDeltaN)). However, the developmental processes regulated by NF-kappaB in hair follicles has remained unknown. Furthermore, the similarity between the phenotypes of c(IkappaBADeltaN) mice and mice deficient in Eda A1 (tabby) or its receptor EdaR (downless) raised the issue of whether in vivo NF-kappaB regulates or is regulated by these novel TNF family members. We now demonstrate that epidermal NF-kappaB activity is first observed in placodes of primary guard hair follicles at day E14.5, and that in vivo NF-kappaB signalling is activated downstream of Eda A1 and EdaR. Importantly, ectopic signals which activate NF-kappaB can also stimulate guard hair placode formation, suggesting a crucial role for NF-kappaB in placode development. In downless and c(IkappaBalphaDeltaN) mice, placodes start to develop, but rapidly abort in the absence of EdaR/NF-kappaB signalling. We show that NF-kappaB activation is essential for induction of Shh and cyclin D1 expression and subsequent placode down growth. However, cyclin D1 induction appears to be indirectly regulated by NF-kappaB, probably via Shh and Wnt. The strongly decreased number of hair follicles observed in c(IkappaBalphaDeltaN) mice compared with tabby mice, indicates that additional signals, such as TROY, must regulate NF-kappaB activity in specific hair follicle subtypes.     

Migratory localization of cyclin D2-Cdk4 complex suggests a spatial regulation of the G1-S transition

The association of the cyclin D-Cdk (DC) complex with retinoblastoma protein (pRb) is required for the G1-S transition of the cell cycle. Cyclin synthesis, nuclear localization and degradation are control mechanisms for the transition, but regulation of the DC complex nuclear import also contributes to the transition. Analysis of the timing of the G1-S transition in mammalian cell lines revealed acceleration with overexpression of cyclin D2 and Cdk4. Immunolocalization assays revealed that cyclin D2 and Cdk4 formed a complex in the cytoplasm and approached the nucleus. They accumulated on the cytosolic surfaces of the nuclear pores and then were arrested at the nuclear membrane before the nucleus reached a critical size. Finally, the complex was released into the nucleus and colocalized with pRb there, which led to pRb phosphorylation and DNA synthesis. The translocalization depended on the G1-S transition. In contrast, a truncated cyclin D2 that was not able to fully associate with Cdk4 lost the ability for release into the nucleus. This pattern of translocalization suggests a spatial separation of the cyclin D-Cdk complex from pRb and DNA in the nucleus to regulate the G1-S transition.     

Epstein-Barr virus nuclear antigen 3C facilitates G1-S transition by stabilizing and enhancing the function of cyclin D1

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.     

Synchronization of goat fibroblast cells at quiescent stage and determination of their transition from G0 to G1 by detection of cyclin D1 mRNA

A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G0/G1. Additionally, detection of cyclin D1 mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G1 a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G0 to G1 transition within 5 h of serum stimulation and were at the mid-G1 stage within 10 h of serum stimulation.     

CUL-2 is required for the G1-to-S-phase transition and mitotic chromosome condensation in Caenorhabditis elegans

The human cullin protein CUL-2 functions in a ubiquitin-ligase complex with the von Hippel-Lindau (VHL) tumour suppressor protein. Here we show that, in Caenorhabditis elegans, cul-2 is expressed in proliferating cells and is required at two distinct points in the cell cycle, the G1-to-S-phase transition and mitosis. cul-2 mutant germ cells undergo a G1-phase arrest that correlates with accumulation of CKI-1, a member of the CIP/KIP family of cyclin-dependent-kinase inhibitors. In cul-2 mutant embryos, mitotic chromosomes are unable to condense, leading to unequal DNA segregation, chromosome bridging and the formation of multiple nuclei.     

Up-regulation of cyclin D1 by HBx is mediated by NF-kappaB2/BCL3 complex through kappaB site of cyclin D1 promoter

Cyclin D1 is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis Bx (HBx) protein is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Here we show that HBx up-regulates cyclin D1 and that this process is mediated by the NF-kappaB2(p52)/BCL-3 complex. Our experiments indicate that HBx up-regulates BCL-3 in the mRNA level, which subsequently results in the up-regulation of the NF-kappaB2(p52)/BCL-3 complex in the nucleus. Moreover, impaired HBx-mediated BCL-3 up-regulation by small interfering RNA for BCL-3 reduced HBx-mediated cyclin D1 up-regulation. Down-regulation of the HBx protein level by p53 also reduced HBx-mediated cyclin D1 up-regulation. From these results, we conclude that the up-regulation of cyclin D1 by HBx is mediated by the up-regulation of NF-kappaB2(p52)/BCL-3 in the nucleus. This HBx-mediated-cyclin D1 up-regulation might play an important role in the HBx-mediated HCC development and progression.     

Genetic replacement of cyclin D1 function in mouse development by cyclin D2

D cyclins (D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. It is unclear whether each of the D cyclins performs unique, tissue-specific functions or the three proteins have virtually identical functions and differ mainly in their pattern of expression. We previously generated mice lacking cyclin D1, and we observed that these animals displayed hypoplastic retinas and underdeveloped mammary glands and a presented developmental neurological abnormality. We now asked whether the specific requirement for cyclin D1 in these tissues reflected a unique pattern of D cyclin expression or the presence of specialized functions for cyclin D1 in cyclin D1-dependent compartments. We generated a knock-in strain of mice expressing cyclin D2 in place of D1. Cyclin D2 was able to drive nearly normal development of retinas and mammary glands, and it partially replaced cyclin D1's function in neurological development. We conclude that the differences between these two D cyclins lie mostly in the tissue-specific pattern of their expression. However, we propose that subtle differences between the two D cyclins do exist and they may allow D cyclins to function in a highly optimized fashion. We reason that the acquisition of multiple D cyclins may allow mammalian cells to drive optimal proliferation of a diverse array of cell types.     

PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition

Oncogenic RAS mutants such as v-Ha-RAS induce cell cycling, in particular the G1 to S transition, by upregulating cyclin D1 and downregulating p27, an inhibitor for cyclin-dependent kinases (CDKs). PI-3 kinase appears to be involved in the regulation of both cyclin D1 and p27. In this report, using two distinct inhibitors specific for PAK1-3 (CEP-1347 and WR-PAK18), we present the first evidence indicating that the PIX/Rac/CDC42-dependent Ser/Thr kinases PAK1-3, acting downstream of PI-3 kinase and upstream of the Raf/MEK/ERKs kinase cascade, is essential for RAS-induced upregulation of cyclin D1, but not downregulation of p27. Since these PAK-inhibitors block selectively the malignant growth of RAS transformants, in which PAK1 is constitutively activated, but not normal cell growth, it is suggested that RAS transformants are addicted to the high levels of PAK1 for their malignant entry to S phase.