(1/25) 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. (+info)
(2/25) Control of cyclin G2 mRNA expression by forkhead transcription factors: novel mechanism for cell cycle control by phosphoinositide 3-kinase and forkhead.
Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G(0), with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of phosphoinositide 3-kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the phosphoinositide 3-kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for phosphoinositide 3-kinase and FoxO transcription factors in the control of cell cycle entry. (+info)
(3/25) Effect of cyclin G2 on proliferative ability of SGC-7901 cell.
AIM: To study the effect of cyclin G2 on proliferation of gastric adenocarcinoma cell line-SGC-7901 cell in vitro. METHODS: By use of cation lipofectamine transfection reagent, the pIRES-G2 and pIRESneo plasmids were transferred into SGC-7901cell line. Anticlones were selected by G418. Positive clones were observed and counted using Giemsa staining. Cell proliferative ability was assayed by MTT. RESULTS: (1) The clone number of pIRES-G2 group decreased, clone volume reduced. The number of cell clones in pIRESneo group was 87+/-3, that of pIRES-G2 group was 53+/-4, occupying 60.1% of pIRESneo group, there was significant difference obviously (P<0.01, t=15.45). (2) The average absorbance of clone cell obtained by stable transfection of pIRES-G2 at 570 nm was 1.6966+/-0.2125, the average absorbance of clone cell obtained by stable transfection of pIRESneo at 570 nm was 2.1182+/-0.3675, there was significant difference between them (P<0.01, t=3.412). CONCLUSION: Cyclin G2 can inhibit SGC-7901cell proliferative ability obviously, it may be a negative regulator in cell cycle regulation. (+info)
(4/25) Cyclin G2 dysregulation in human oral cancer.
Using expression microarray, we have previously shown that human cyclin G2 (hCG2) is significantly down-regulated in laser capture microdissected oral cancer epithelia. Western analysis showed detectable hCG2 protein in normal (2 of 2) but not in malignant (4 of 4) oral keratinocyte cell lines. Immunohistochemistry analysis done on oral cancers showed that normal oral mucosa (100%, 12 of 12) and 69.1% (47 of 68) of dysplastic oral epithelia expressed readily detectable hCG2 in the nuclei. However, only 11.1% of oral cancer epithelia (14 of 126) showed mild hCG2 nuclear staining. Interestingly, of the oral cancers devoid of nuclear hCG2 (112 cases), 58 cases (52%) showed cytoplasmic hCG2 immunostaining, whereas the other 54 cases (48%) exhibited neither nuclear nor cytoplasmic hCG2 staining. In vitro functional study by ectopic restoration of hCG2 expression in the human malignant squamous cell carcinoma (SCC) line SCC15 resulted in a significant inhibition of cellular proliferation (P < 0.001) and colony formation (P < 2 x 10(-5)) with increased population of G(1) phase and decreased in S phase (P < 0.01). Furthermore, stable down-regulation of hCG2 by short interference RNA-based gene silencing in immortalized normal oral keratinocytes resulted in enhanced cell growth with increase in S and prominently in G(2) phase. Because hCG2 has been implicated as a negative regulator in cell cycle progression, our results support that hCG2 dysregulation may play an important role in epithelial transformation and the early stages of human oral cancer development. (+info)
(5/25) Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA.
Inhibition of the up-regulated telomerase activity in cancer cells has previously been shown to slow cell growth but only after prior telomere shortening. Previously, we have reported that, unexpectedly, a hairpin short interfering RNA specifically targeting human telomerase RNA rapidly inhibits the growth of human cancer cells independently of p53 or telomere length and without bulk telomere shortening (Li, S., Rosenberg, J. E., Donjacour, A. A., Botchkina, I. L., Hom, Y. K., Cunha, G. R., and Blackburn, E. H. (2004) Cancer Res. 64, 4833-4840). Here we have demonstrated that such telomerase RNA knockdown in cancer cells does not cause telomere uncapping but rather induces changes in the global gene expression profile indicative of a novel response pathway, which includes suppression of specific genes implicated in angiogenesis and metastasis, and is distinct from the expression profile changes induced by telomere-uncapping mutant template telomerase RNAs. These cellular responses to depleting telomerase in human cancer cells together suggest that cancer cells are "telomerase-addicted" and uncover functions of telomerase in tumor growth and progression in addition to telomere maintenance. (+info)
(6/25) FOXO transcription factors cooperate with delta EF1 to activate growth suppressive genes in B lymphocytes.
Forkhead transcription factors regulate many aspects of lymphocyte development and function. The FOXO subgroup of Forkhead factors opposes proliferation and survival, and FOXO inactivation is an important outcome of Ag receptor signaling. FOXO activity at target promoters is modulated by other transcription factors in a manner dependent on cell type and external stimulus. We have investigated the mechanisms by which FOXO proteins activate the promoters of two target genes in murine B lymphocytes, Ccng2 (encoding cyclin G2) and Rbl2 (p130), each of which has been implicated in cell cycle arrest. FOXO proteins bound directly to both promoters in vitro and in vivo, augmented transcriptional activity in reporter assays, and increased expression of the endogenous genes. Each of the promoter sequences has consensus binding sites for the deltaEF1 transcription factor, previously shown to either repress or activate different promoters. deltaEF1 bound to the Ccng2 and Rbl2 promoters in vitro and in vivo and increased reporter activity as well as endogenous mRNA levels for these genes. Strikingly, deltaEF1 synergized with FOXO proteins to strongly activate transcription from both promoters. Coexpression of deltaEF1 enhanced FOXO-induced cell cycle arrest in B lymphoma cells. These findings establish a novel mechanism of FOXO function at target promoters: cooperation with deltaEF1. (+info)
(7/25) Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter.
Estrogens, acting through their nuclear receptors have a broad impact on target cells, eliciting a transcriptional response program that involves gene repression as well as gene stimulation. While much is known about the mechanisms by which the estrogen-occupied estrogen receptor (ER) stimulates gene expression, the molecular events that lead to gene repression by the hormone-ER complex are largely unknown. Because estradiol represses expression of the cyclin G2 gene, which encodes a negative regulator of the cell cycle, our aim was to understand the mechanism by which cyclin G2 is repressed by estrogen. We show that cyclin G2 is a primary ER target gene in MCF-7 breast cancer cells that is rapidly and robustly down-regulated by estrogen. Promoter analysis reveals a responsive region containing a half-estrogen response element and GC-rich region that interact with ER and Sp1 proteins. Mutation of the half-ERE abrogates hormone-mediated repression. Mutational mapping of receptor reveals a requirement for its N-terminal region and DNA binding domain to support cyclin G2 repression. Following estradiol treatment of cells, chromatin immunoprecipitation analyses reveal recruitment of ER to the cyclin G2 regulatory region, dismissal of RNA polymerase II, and recruitment of a complex containing N-CoR and histone deacetylases, leading to a hypoacetylated chromatin state. Our study provides evidence for a mechanism by which the estrogen-occupied ER is able to actively repress gene expression in vivo and indicates a role for nuclear receptor corepressors and associated histone deacetylase activity in mediating negative gene regulation by this hormone-occupied nuclear receptor. (+info)
(8/25) Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest.
Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G(1)/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the mature centriole present at microtubule foci, indicates that cyclin G2 resides primarily on the mother centriole. Copurification of cyclin G2 and PP2A subunits with microtubules and centrosomes, together with the effects of ectopic cyclin G2 on cell cycle progression, nuclear morphology and microtubule growth and stability, suggests that cyclin G2 may modulate the cell cycle and cellular division processes through modulation of PP2A and centrosomal associated activities. (+info)