Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2)

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604 bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.–F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.     

Gene structure and chromosomal localization of mouse cyclin G2 (Ccng2)

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604 bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.–F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.     

Cyclin I: A New Cyclin Encoded by a Gene Isolated from Human Brain

A new member of the cyclin family has been isolated from an equalized cDNA library derived from human forebrain cortex. This putative cyclin, designated cyclin I, contains a typical cyclin box near the N-terminus and a PEST sequence near the C-terminus. Cyclin I shows the highest sequence similarity in the cyclin box to cyclins G and E, while the similarity between cyclins I and G also extends toward the C-terminus from the cyclin box. Cyclin I mRNA was expressed at high levels in postmitotic tissues, including skeletal muscle, heart, and brain, and was expressed constantly during cell cycle progression. The expression of cyclin I mRNA does not correlate directly to the cell cycle, and therefore cyclin I may be a novel cyclin member that functions independently of the cell cycle control.     

Cyclin mRNA and protein expression in recombinant interleukin 2-stimulated cloned murine T lymphocytes

Expression of cyclin, a non-histone nuclear protein, during recombinant interleukin 2 (rIL2)-driven cell-cycle progression of cloned T lymphocytes has been assessed. We found that expression of cyclin protein, as detected by immunofluorescence, is tightly associated with proliferation, and not merely S-phase, of L2 cells stimulated with rIL2. Cyclin immunofluorescence was detected in all cell-cycle phases (G1/S/G2/M, as detected by flow cytometry) of proliferating L2 cells. Accumulation of cyclin mRNA levels was induced as early as 1 h after stimulation, was maximal at 25-49 h, and remained elevated throughout stimulation, as detected by Northern blot analysis. A cDNA-encoding murine cyclin was cloned from a cDNA library prepared from IL2-stimulated cloned T cells. The sequence of the 5' end of the murine cyclin cDNA was determined and found to be 88% and 82% similar to the sequences of cDNA clones encoding rat and human cyclin, respectively. The present studies demonstrate that cyclin protein and mRNA accumulation are highly regulated during IL2-induced proliferation of a cloned T cell. These data provide a framework for addressing the molecular mechanisms regulating cyclin gene expression during cellular proliferation.     

Securin regulates entry into M-phase by modulating the stability of cyclin B

Timely progression into mitosis is necessary for normal cell division. This transition is sensitive to the levels of cyclin B, the regulatory subunit of the master mitotic kinase, Cdk1. Cyclin B accumulates during G2 and prophase when its rate of destruction by the anaphase promoting complex (APC) is low. Securin is also an APC substrate and is known for its role in inactivating the cohesin-cleaving enzyme, separase, until the metaphase to anaphase transition. Here we show that securin has an additional role in cell-cycle regulation, that of modulating the timing of entry into M-phase. In mouse oocytes, excess securin caused stabilization of cyclin B and precocious entry into M-phase. Depletion of securin increased cyclin B degradation, resulting in delayed progression into M-phase. This effect required APC activity and was reversed by expression of wild-type securin. These data reveal a role for securin at the G2-M transition and suggest a more general mechanism whereby physiological levels of co-competing APC substrates function in modulating the timing of cell-cycle transitions.     

ATM is required for rapid degradation of cyclin D1 in response to γ-irradiation

The cellular response to DNA damage induced by γ-irradiation activates cell-cycle arrest to permit DNA repair and to prevent replication. Cyclin D1 is the key molecule for transition between the G1 and S phases of the cell-cycle, and amplification or overexpression of cyclin D1 plays pivotal roles in the development of several human cancers. To study the regulation of cyclin D1 in the DNA-damaged condition, we analyzed the proteolytic regulation of cyclin D1 expression upon γ-irradiation. Upon γ-irradiation, a rapid reduction in cyclin D1 levels was observed prior to p53 stabilization, indicating that the stability of cyclin D1 is controlled in a p53-independent manner. Further analysis revealed that irradiation facilitated ubiquitination of cyclin D1 and that a proteasome inhibitor blocked cyclin D1 degradation under the same conditions. Interestingly, after mutation of threonine residue 286 of cyclin D1, which is reported to be the GSK-3β phosphorylation site, the mutant protein showed resistance to irradiation-induced proteolysis although inhibitors of GSK-3β failed to prevent cyclin D1 degradation. Rather, ATM inhibition markedly prevented cyclin D1 degradation induced by γ-irradiation. Our data indicate that communication between ATM and cyclin D1 may be required for maintenance of genomic integrity achieved by rapid arrest of the cell-cycle, and that disruption of this crosstalk may increase susceptibility to cancer.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G1/S phase cell cycle arrest

Cyclin G2, together with cyclin G1 and cyclin I, defines a novel cyclin family expressed in terminally differentiated tissues including brain and muscle. Cyclin G2 expression is up-regulated as cells undergo cell cycle arrest or apoptosis in response to inhibitory stimuli independent of p53 (Horne, M., Donaldson, K., Goolsby, G., Tran, D., Mulheisen, M., Hell, J. and Wahl, A. (1997) J. Biol. Chem. 272, 12650-12661). We tested the hypothesis that cyclin G2 may be a negative regulator of cell cycle progression and found that ectopic expression of cyclin G2 induces the formation of aberrant nuclei and cell cycle arrest in HEK293 and Chinese hamster ovary cells. Cyclin G2 is primarily partitioned to a detergent-resistant compartment, suggesting an association with cytoskeletal elements. We determined that cyclin G2 and its homolog cyclin G1 directly interact with the catalytic subunit of protein phosphatase 2A (PP2A). An okadaic acid-sensitive (<2 nm) phosphatase activity coprecipitates with endogenous and ectopic cyclin G2. We found that cyclin G2 also associates with various PP2A B' regulatory subunits, as previously shown for cyclin G1. The PP2A/A subunit is not detectable in cyclin G2-PP2A-B'-C complexes. Notably, cyclin G2 colocalizes with both PP2A/C and B' subunits in detergent-resistant cellular compartments, suggesting that these complexes form in living cells. The ability of cyclin G2 to inhibit cell cycle progression correlates with its ability to bind PP2A/B' and C subunits. Together, our findings suggest that cyclin G2-PP2A complexes inhibit cell cycle progression.     

Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.     

CELL CYCLE REGULATORS IN THE FLORIDA RED TIDE DINOFLAGELLATE, GYMNODINIUM BREVE

The diel cycle is a key regulator of the cell cycle in many dinoflagellates, and may play a rate limiting role in bloom formation. Diel phasing of the cell cycle in the Florida red tide dinoflagellate, Gymnodinium breve Davis was previously described in our laboratory. In cultures grown on a 16:8 light:dark cycle, S-phase began 6–8 h into the light phase, and mitosis followed 12–14 h later. The dark/light "dawn" transition was found to provide the diel cue that serves to entrain the G. breve cell cycle. However the cell cycle mechanisms and regulators acted upon by this cue are poorly understood in dinoflagellates. The cell cycle regulatory complex, CDK1-cyclinB, is therefore currently being investigated. Cyclin dependent kinase (CDK) was first identified in G. breve using two approaches: (1) identification of a 34 kDa protein immunoreactive to an antibody raised against a conserved amino acid sequence unique to the CDK protein family (PSTAIR) and (2) inhibition of the cell cycle by olomoucine, a selective CDK inhibitor. Several approaches are currently being employed in order to describe its partner, cyclin B: (1) PCR on genomic DNA with primers deduced from known cyclin box sequences, (2) G. breve expression library screening with an antibody raised against the fission yeast cyclin B (3) western blot analysis on whole protein extracts and cyclin B immunoprecipitated proteins. Current work focuses on the differential expression of the cyclin B homologue in G. breve during its cell cycle and its relation to diel cycle control.     

Cell cycle regulators in bladder cancer: relationship to schistosomiasis

Dysregulation of cell cycle control may lead to genomic instability, neoplastic transformation and tumor progression. In terms of the particular roles in regulation of the cell-cycle, p21(WAF1) causes growth arrest through inhibition of cyclin-dependant kinases required for G1/S transition. P16 (INK4A) and p15 (INK4B) are thought to act as tumor suppressors, since their inactivation and/or deletion are observable in various types of malignancies. Cyclin D1 is hypothesized to control cell cycle progression through the G1-S check point. The present study evaluated p21 expression, p16 and p15 gene deletion and cylin D1 expression in bladder carcinoma among Egyptian patients, in relation to different clinicopathological features of the tumors and presence or absence of bilharziasis. Tissue specimens were obtained from 132 patients with bladder carcinoma and 50 normal tissue samples from the same patients served as control. P21 was determined by Western blot (WB) and enzyme immunoassay (EIA), p16 and p15 gene deletions were examined by polymerase chain reaction (PCR) and Cyclin D1 was detected by WB. Levels of p21 were lower in malignant tumors than in normal tissues. Lower expression of p21 was evident in lymph node positive, well differentiated tumors and squamous cell carcinoma (SCC) than in lymph node negative, poorly differentiated tumors and transitional cell carcinoma (TCC). In all normal samples, p15 and p16 genes were detected while cyclin D1 was not detected. P16 and p15 genes were deleted in 38.7% (41/106) and 30.2% (32/106) of bladder tumors respectively. The deletion of both genes was associated with poor differentiation grade and presence of bilharziasis. P16 deletion was also correlated to advancing tumor stage. Cyclin D1 was expressed in 57.5% of bladder tumors (69/120), where its expression was correlated to early stage, well differentiation grade, schistomiasis, and low levels of p21. Cell cycle is dysregulated in bladder carcinoma. This was evident from the increased expression of cyclin D1, the decreased levels of p21 and the deletion of p15 and p16 genes. Moreover, p16 and p15 gene deletion was related to tumor progression and might have a role in bilharzial bladder carcinogenesis. Cyclin D1 over-expression appears to be an early event in bladder cancer and might explain bilharzial associated bladder carcinogenesis.     

Patterns of expression of cyclins A, B1, D, E and cdk 2 in preimplantation mouse embryos

Cell cycle regulatory proteins have been characterized in somatic cells and exhibit phase-specific expression patterns. Changes in expression of these regulatory proteins have not been clearly characterized in early preimplantation mouse embryos. This study utilized indirect immunofluorescence to determine the expression pattern of G1/S phase cyclins D and E; S, G2/M phase cyclins A and B1, and cdk 2 during the first three cell cycles of mouse embryo development. Cyclin D demonstrated low expression throughout the first cell cycle but had a somatic-like pattern of expression in cycles 2 and 3 with peak expression at G1 declining through S phase to a low during G2. Cyclin E was present at peak levels in G1 declining through S to a low in G2 during both the first and third cell cycles, but remained at moderate levels during the entire second cell cycle. Cyclin A was maintained at moderate levels throughout the first two cell cycles but showed a somatic-like pattern with a low level in G1 increasing during S phase with peak levels during G2 of the third cell cycle. Cyclin B consistently demonstrated a pattern opposite to a somatic G2/M cyclin, with peak levels in G1 declining through S phase to a low in G2 during each of the three cell cycles examined. Cdk 2 was present at consistent levels during G1 and S phases of all three cell cycles declining slightly in G2.     

Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase

Cell cycle progression is tightly regulated by cyclins, cyclin-dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D-type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up-regulated in the mid-G1 phase (4-6 h) while cyclin D3 expression emerged only in late G1 (9-12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid-G1 phase. Exposure of C6 glioma to VPA induced a marked up-regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid-G1 phase (3-5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA-induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.     

Genetic reevaluation of the role of F-box proteins in cyclin D1 degradation

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.