cyclin E

Liver receptor homolog (LRH-1) is an orphan nuclear receptor (NR5A2) that regulates cholesterol homeostasis and cell plasticity in endodermal-derived tissues. Estrogen increases LRH-1 expression conveying cell protection and proliferation. Independently, estrogen also protects isolated human islets against cytokineinduced apoptosis. Herein, we demonstrate that LRH-1 is expressed in islets, including b-cells, and that transcript levels are modulated by 17b-estradiol through the estrogen receptor (ER)a but not ERb signaling pathway. Repression of LRH-1 by siRNA abrogated the protective effect conveyed by estrogen on rat islets against cytokines. Adenoviral-mediated overexpression of LRH-1 in human islets did not alter proliferation but conferred protection against cytokines and streptozotocin-induced apoptosis. Expression levels of the cell cycle genes cyclin D1 and cyclin E1 as well as the antiapoptotic gene bcl-xl were unaltered in LRH-1 expressing islets. In contrast, the steroidogenic enzymes CYP11A1 and CYP11B1 involved in glucocorticoid biosynthesis were both stimulated in transduced islets. In parallel, graded overexpression of LRH-1 dosedependently impaired glucose-induced insulin secretion. Our results demonstrate the crucial role of the estrogen target gene nr5a2 in protecting human islets against-stressed-induced apoptosis. We postulate that this effect is mediated through increased glucocorticoid production that blunts the pro-inflammatory response of islets.

The progression of the cell cycle is a tightly regulated process, regulated by the activities of cyclin dependent kinases. Phosphorylation of target proteins regulates key transitions in the cell cycle contributing to accurate DNA replication and cell division. Here we evaluate the regulation of the protein Ciz1 by cyclin A-CDK2. Ciz1 promotes localisation of cyclin A-CDK2 to chromatin sites at the G 1 /S phase transition and this ternary complex promotes initiation of DNA replication. However, the mechanisms by which Ciz1 is regulated are not fully understood. Here, Ciz1 protein levels are shown to be reduced after inhibition of CDK activity, suggesting that CDK-mediated phosphorylation of Ciz1 could contribute to increased protein stability. As Ciz1 is known to be a substrate of cyclin A-CDK2, enzyme kinetic studies were performed to evaluate whether there is preferential phosphorylation at specific sites within Ciz1. We characterise new phospho-specific antibodies that will be of benefit in future in in vivo studies evaluating the role of phosphorylation at specific sites in regulation of Ciz1 function. Using His-ubiquitin, we show that Ciz1 is covalently linked to ubiquitin. These complexes are stabilised after inhibition of the proteasome, consistent with degradation by the proteasomal. Further evidence presented demonstrates that an E3 ligase (Cdh1) interacts with Ciz1 via a conserved destruction box in the N-terminus. Taken together, the data presented here suggest that Ciz1 is regulated at multiple levels post-transitionally that contribute to ensuring it accumulates precisely at the G 1 /S phase transition. This may have important implications for its role as a driver mutation in cancer biology.

The progression of the cell cycle is a tightly regulated process, regulated by the activities of cyclin dependent kinases. Phosphorylation of target proteins regulates key transitions in the cell cycle contributing to accurate DNA replication and cell division. Here we evaluate the regulation of the protein Ciz1 by cyclin A-CDK2. Ciz1 promotes localisation of cyclin A-CDK2 to chromatin sites at the G1/S phase transition and this ternary complex promotes initiation of DNA replication. However, the mechanisms by which Ciz1 is regulated are not fully understood. Here, Ciz1 protein levels are shown to be reduced after inhibition of CDK activity, suggesting that CDK-mediated phosphorylation of Ciz1 could contribute to increased protein stability. As Ciz1 is known to be a substrate of cyclin A-CDK2, enzyme kinetic studies were performed to evaluate whether there is preferential phosphorylation at specific sites within Ciz1. We characterise new phospho-specific antibodies that will be of b...

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP ) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220 NPAT is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220 NPAT coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point. Here, we show that the downstream CDK-dependent cell cycle effector HINFP is genetically required and, in contrast to the CDK2/cyclin E complex, cannot be compensated. We constructed a mouse Hinfp -null mutation and found that heterozygous Hinfp mice survive, indicating that 1 allele suffices for embryogenesis. Homozygous loss-of-function causes embryonic lethality: No homozygous Hinfp -null mice are obtained at or beyond...

Cancer cells are known to display up-regulation of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), the key enzymes in the biosynthesis of polyamines that are essential for cellular proliferation. We have shown previously that overexpression of ODC or AdoMetDC alone can induce tumorigenic transformation of rodent fibroblasts. Because the subversion of normal cell cycle control is thought to be a crucial event in cancer development, we examined ODC- and AdoMetDC-transformed fibroblasts for alterations in the cell cycle components. The level of cyclin D1 and cyclin D1-dependent kinase and total cyclin-dependent kinase (CDK) 4 activities were elevated in the ODC transformants and particularly in the AdoMetDC transformants. Cyclin E content was not elevated, but a moderate increase in cyclin E-dependent kinase activity was seen in both cells. Total CDK2 activity was increased only in the ODC-transformed cells. The amount of the p27Kip1 CDK inhibitor was greatly decreased in both transformants. Nevertheless, p27Kip1 was present in the active cyclin D1/CDK4 complexes in the cells but absent from the cyclin E/CDK2 complexes. Restoration of p27Kip1 expression in the ODC- and AdoMetDC-transformed cells by transfection resulted in growth inhibition, but not in morphological reversion. An elevation in the level of hyperphosphorylated retinoblastoma protein was observed mainly in the ODC-transformed cells. These results suggest that the expression of ODC or AdoMetDC may affect cell cycle regulation in many ways. However, the largest common effect, which is therefore potentially relevant to some aspects of transformation, appears to be the constitutive down-regulation of p27Kip1 and its loss from the cyclin/CDK2 complexes.

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G 2 /M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G 2 /M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/ cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity.

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.