过表达UHRF2通过上调p53及p21表达引起HEK293细胞G1期阻滞

UHRF2（ubiquitin like with PHD and ring finger domains 2）是新近发现的具有多个结构域的核蛋白,在细胞周期调控和表观遗传学中发挥重要作用．近期研究提示,UHRF2是肿瘤抑制蛋白p53的1个E3连接酶,在体内外能与p53相互结合并促进其泛素化,过表达UHRF2能使细胞周期停滞于G1期．然而,UHRF2介导的G1期阻滞及其与p53联系尚不清楚．通过共转染UHRF2质粒及p53特异性小干扰RNA(siRNAs)到HEK293细胞构建细胞模型,探索UHRF2引起细胞周期停滞与p53之间的关系．结果显示,UHRF2能促进HEK293细胞中p53的稳定,从而引起p21 (CIP1/WAF1)基因表达,并使细胞周期停滞于G1期;但在siRNA抑制p53的表达后p21(CIP1/WAF1)表达降低,UHRF2引起的细胞周期阻滞消除．研究结果提示,UHRF2可通过稳定p53,上调p21的表达,从而介导细胞周期阻滞于G1期;同时UHRF2可能参与细胞周期调控及DNA损伤反应（DNA damage response, DDR）．UHRF2稳定p53的具体分子机制及其在DDR中的作用有待进一步研究证明．     

NIRF/UHRF2 occupies a central position in the cell cycle network and allows coupling with the epigenetic landscape

As predicted by systems biology, a paradigm shift will emerge through the integration of information about different layers of cellular processes. The cell cycle network is at the heart of the cellular computing system, and orchestrates versatile cellular functions. The NIRF/UHRF2 ubiquitin ligase is an "intermodular hub" that occupies a central position in the network, and facilitates coordination among the cell cycle machinery, the ubiquitin-proteasome system, and the epigenetic system. NIRF interacts with cyclins, CDKs, p53, pRB, PCNA, HDAC1, DNMTs, G9a, methylated histone H3 lysine 9, and methylated DNA. NIRF ubiquitinates cyclins D1 and E1, and induces G1 arrest. The NIRF gene is frequently lost in tumors and is a candidate tumor suppressor, while its paralog, the UHRF1 gene, is hardly altered. Thus, investigations of NIRF are essential to understand the entire biological systems. Through integration of the enormous information flows, NIRF may contribute to the coupling between the cell cycle network and the epigenetic landscape. We propose the new paradigm that NIRF produces the extreme diversity in the network wiring that helps the diversity of Waddington's canals.     

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.     

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

Down-regulation of cyclic nucleotide phosphodiesterase PDE1A is the key event of p73 and UHRF1 deregulation in thymoquinone-induced acute lymphoblastic leukemia cell apoptosis

Thymoquinone (TQ), the active principle of Nigella sativa black seeds, has anti-proliferative properties on numerous cancer cell types. Others and we have previously reported that TQ acts as agent that triggers cell cycle arrest and apoptosis through either a p53- or p73-dependent pathway. However, the immediate targets recruited upon TQ-induced cytotoxicity have not yet been clearly identified. We therefore asked whether cyclic nucleotide phosphodiesterases (PDEs) could be involved in TQ-triggered pro-apoptotic reactivity; PDEs are regulators of intracellular levels of cyclic nucleotides and therefore can modulate cAMP and cGMP-dependent cell death pathways. Our results showed that TQ specifically repressed PDE1A expression in the acute lymphoblastic leukemia Jurkat cell line. This effect is concomitant with the previously described sequential deregulation of the expression of the tumor suppressor protein p73 and the epigenetic integrator UHRF1 (Ubiquitin-like, PHD Ring Finger 1). Interestingly, RNA-interference knock-down of PDE1A expression as well as decreased PDE1A expression induced growth inhibition of Jurkat cells, cell cycle arrest and apoptosis through an activation of p73 and a repression of UHRF1. Conversely, PDE1A re-expression counteracted the cellular pro-apoptotic effects of TQ in association with a p73 repression and UHRF1 re-expression. Altogether, our results show that TQ induced an initial down-regulation of PDE1A with a subsequent down-regulation of UHRF1 via a p73-dependent mechanism. This study further proposes that PDE1A might be involved in the epigenetic code inheritance by regulating, via p73, the epigenetic integrator UHRF1. Our findings also suggest that a forced inhibition of PDE1A expression might be a new therapeutic strategy for the management of acute lymphoblastic leukemia.     

Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression

The proto-oncogene c-myc is a key player in cell-cycle regulation and is deregulated in a broad range of human cancers and cell proliferation disorders. Here we reported that overexpression of c-myc in human embryonic lung fibroblasts (HEL) that have low endogenous c-myc enriched S phase cells with increased expression of cyclin D3, E, A, Cdk2, and Cdk4, and decreased expression of p21 and p27. To the opposite, using RNAi to downregulate c-myc expression in A549 cells that have high endogenous c-myc enriched G1 phase cells with decreased expression of cyclin D3, E, A, Cdk2, Cdk4, and increased expression of p21 and p27. We found that cyclin A expression was the most susceptive to changes in c-myc levels and essential in c-myc-modulated cell cycle pathway via co-transfection, however, cyclin D1 showed no change between treated and control groups in either HEL or A549 cells. Our results indicated that upregulation of c-myc expression promotes cell cycling in HEL cells, whereas downregulation of c-myc expression causes G1 phase arrest in A549 cells, and the c-myc-mediated cell-cycle regulation pathway was dependent on cyclin A and involved cyclin D3, E, Cdk2, Cdk4, p21, and p27, but not cyclin D1.     

Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells

We studied the effects of apigenin on the cell cycle distribution and apoptosis of human breast cancer cells and explored the mechanisms underlying these effects. We first investigated the antiproliferative effects in SK-BR-3 cells exposed to between 1 and 100 microM apigenin for 24, 48 and 72 h. Apigenin significantly inhibited cell proliferation at concentrations over 50 microM, regardless of exposure time (P<.05), and resulted in significant cell cycle arrest in the G(2)/M phase after 48 h of treatment at high concentrations (50 and 100 microM; P<.05). To investigate the regulatory proteins of cell cycle arrest affected by apigenin, we treated cells with 50 and 100 microM apigenin for 72 h. Apigenin caused a slight decrease in cyclin D and cyclin E expression, with no change in CDK2 and CDK4. In addition, the apigenin-induced accumulation of the cell population in the G(2)/M phase resulted in a decrease in CDK1 together with cyclin A and cyclin B. In an additional study, apigenin also increased the accumulation of p53 and further enhanced the level of p21(Cip1), with no change in p27(Kip1). The expression of Bax and cytochrome c of p53 downstream target was increased markedly at high concentration treatment over 50 microM apigenin. Based on our findings, the mechanism by which apigenin causes cell cycle arrest via the regulation of CDK1 and p21(Cip1) and induction of apoptosis seems to be involved in the p53-dependent pathway.     

Toxoplasma gondii exploits UHRF1 and induces host cell cycle arrest at G2 to enable its proliferation

Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in humans. It is able to infect all nucleated mammalian cells leading to lifelong persistence of the parasite in the host. Here, we studied the effect of T. gondii infection on host cell proliferation and explored the molecular mechanisms involved in host cell cycle progression. We found that T. gondii induced G1/S transition in host cells in the presence of UHRF1, followed by G2 arrest after cyclin B1 downregulation which is probably the major cause of the arrest. Other molecules at the G2/M checkpoint including p53, p21 and Cdk1 were normally regulated. Interestingly, while parasite proliferation was normal in cells that were in the G2 phase, it was suppressed in G1-arrested cells induced by UHRF1-siRNA, indicating the importance of the G2 phase via UHRF1-induced G1/S transition for T. gondii growth.     

Redox control of G(1)/S cell cycle regulators during nitric oxide-mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well-understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate-cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21(cip1/waf1) upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH-sensitive implying that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21(cip1/waf1) and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint.     

Mapping of CIP/KIP inhibitors,G1 cyclins D1, D3, E and p53 proteins in the rat term placenta

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

TSG101 expression in gynecological tumors: relationship to cyclin D1, cyclin E, p53 and p16 proteins.

Recent studies have shown that in vitro steady-state expression of the tumor susceptibility gene TSG101 is important for maintenance of genomic stability and cell cycle regulation. To determine the contribution of TSG101 expression in neoplastic formation, expression of TSG101 protein levels were evaluated in primary ovarian and endometrial adenocarcinoma tumors. Expression of TSG101 was also examined in various tumor cell lines (PA-1, AN3CA, HeLa, HS578T, HCT116). Full-length TSG101 protein was detected in these tumors and cell lines indicating that intragenic deletions were not characteristic of TSG101. In addition, TSG101 protein levels were compared with aberrations of prominent cell cycle regulatory molecules such as cyclin D1, cyclin E, p16 and p53. Reduced TSG101 protein was observed in 36% (8/22) of ovarian and 17% (1/6) of endometrial adenocarcinoma. Aberrant levels of p53, p16, cyclin D or E were comparable to published studies indicating that the clinicopathological distribution of these cases did not favor advanced stage tumors. Altogether, these findings suggest that a down-regulation of TSG101 is associated with tumorigenesis in a subgroup of gynecological tumors.     

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

This study shows that the cell cycle E3 ubiquitin ligase APC/C is a regulator of several chromatin regulatory proteins, including the multivalent epigenetic reader and writer UHRF1. Perturbing UHRF1 ubiquitylation and degradation alters cell cycle and DNA methylation patterning, pointing to a key role for cell cycle degradation in shaping chromatin environments.