SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells

p130 is a member of the retinoblastoma family of pocket proteins, which includes pRB and p107. Unlike pRB and p107, p130 protein levels decrease dramatically following its hyperphosphorylation starting in the mid-G1 phase of the cell cycle. However, the mechanism leading to p130 downregulation is unknown. We have found that the proteasome inhibitor, lactacystin, inhibited p130 downregulation in T98G cells progressing through the G1/S transition and S phase and that p130 is multiubiquitylated in 293 cells. We have previously shown that ectopic expression of both cyclin D and E induces phosphorylation and downregulation of p130. Since the SKP1/Cul1/SKP2 E3 ubiquitin ligase complex mediates ubiquitylation of substrates previously phosphorylated by cyclin-dependent kinases, we investigated the potential role of this ubiquitin ligase in mediating p130 downregulation. We found that p130 interacts with SKP1, Cul-1 and SKP2 in human 293 cells. We also found that ectopic coexpression of SKP2 and p130 leads to dose-dependent downregulation of p130, reduces p130 protein half-life and induces p130 ubiquitylation in these cells. Moreover, adenoviral-mediated expression of SKP2 accelerates downregulation of endogenous hyperphosphorylated p130 in mitogen-stimulated T98G cells and primary WI38 fibroblasts. We conclude that p130 is a substrate of the SCF(SKP2) ubiquitin ligase and this E3 ligase regulates p130 abundance during the cell cycle.     

The retinoblastoma protein is partially phosphorylated during early G1 in cycling cells but not in G1 cells arrested with alpha-interferon.

The retinoblastoma protein (pRB) is thought to act as a tumour suppressor which is inactivated by phosphorylation. In quiescent (G0) cells pRB exists in a hypophosphorylated form (pRB110), but proliferating cells in G1 contain a significant proportion of phosphorylated pRB (pRB112-114). Studies of synchronized or elutriated cells have suggested that the phosphorylated forms of pRB disappear as cells pass from G2/M to G0/G1 and that pRB is phosphorylated again to pRB114 at the G1/S border. In this study we used two-parameter flow cytometry and cell sorting to isolate cycling cells in early and late G1 (G1A and G1B), and we show that partially phosphorylated pRB is present in cycling human lymphoid cells even in G1A. These G1A cells contain intermediate forms of pRB which become further phosphorylated to pRB112-114 as cells pass into G1B. Therefore pRB is at least partially phosphorylated from early G1 onwards. Cell cycle arrest by alpha-interferon (alpha-IFN) results in an accumulation of cells in both G1A and G1B, and these cells contain mainly pRB110. Since pRB110 is thought to prevent cell proliferation, the cytostatic effect of alpha-IFN may therefore occur by preventing the initial phosphorylation of pRB during or prior to G1A.     

pRB and p107 have distinct effects when expressed in pRB-deficient tumor cells at physiologically relevant levels

A key difference among the three structurally similar pRB family members is that only pRB is a tumor suppressor. Identification of distinctive functional differences between pRB and p107/p130 therefore holds promise for a better understanding of the tumor suppression mechanisms of pRB. Enigmatically, pRB and p107 have been shown to have indistinguishable growth suppression activities when studied in the pRB-deficient Saos-2 cell system. In this study, we discovered that, when expressed at physiologically relevant levels, pRB and p107 had distinctive effects in causing growth suppression. pRB induced cellular p130 levels while p107 repressed them. p107, but not pRB, blocked cells inside S phase in addition to G1 arrest. In contrast, no qualitative differences were identified in their abilities to repress the expression of a set of suspected pRB/E2F repression target genes. These results indicate that pRB and p107 possess different growth suppression effects, despite the fact that they have similar E2F repression effects.     

Granulocytic differentiation of myeloid progenitor cells by p130, the retinoblastoma tumor suppressor homologue

The retinoblastoma protein (pRB) and the related pocket proteins, p107 and p130, play crucial roles in mammalian cell cycle control. Recent studies indicate that these pocket proteins are also involved in cellular differentiation processes. We demonstrate in this work that the pRB-related p130 selectively accumulates during the in vitro differentiation of the myeloid progenitor cell, 32Dcl3, into granulocyte in response to granulocyte-colony stimulating factor (G-CSF). This G-CSF-dependent granulocytic differentiation is blocked by the adenovirus E1A oncoprotein, which binds to and inactivates the pRB family of pocket proteins including p130. Furthermore, enforced overexpression of p130 but not pRB inhibits the myeloid cell proliferation that is concomitantly associated with granulocytic differentiation morphologically characterized by nuclear segmentation. However, simple G1-cell cycle arrest induced by cytokine deprivation or ectopic overexpression of the p27 cyclin-dependent kinase inhibitor, or inhibition of E2F activities by dominant negative DP-1 is not sufficient to trigger granulocytic differentiation. The differentiation-promoting activity of p130 in myeloid cells requires both the pocket domain and the spacer domain. Our results indicate that the pRB-related p130 plays a critical role in myeloid cell differentiation and suggest that coupling of cell cycle exit with the cellular differentiation program may be specifically achieved by p130.     

E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes

We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.     

Cell cycle and transcriptional control of human myeloid leukemic cells by transforming growth factor beta

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.     

Pocket proteins and cell cycle control

The retinoblastoma protein (pRB) and the pRB-related p107 and p130 comprise the 'pocket protein' family of cell cycle regulators. These proteins are best known for their roles in restraining the G1-S transition through the regulation of E2F-responsive genes. pRB and the p107/p130 pair are required for the repression of distinct sets of genes, potentially due to their selective interactions with E2Fs that are engaged at specific promoter elements. In addition to regulating E2F-responsive genes in a reversible manner, pocket proteins contribute to silencing of such genes in cells that are undergoing senescence or differentiation. Pocket proteins also affect the G1-S transition through E2F-independent mechanisms, such as by inhibiting Cdk2 or by stabilizing p27(Kip1), and they are implicated in the control of G0 exit, the spatial organization of replication, and genomic rereplication. New insights into pocket protein regulation have also been obtained. Kinases previously thought to be crucial to pocket protein phosphorylation have been shown to be redundant, and new modes of phosphorylation and dephosphorylation have been identified. Despite these advances, much remains to be learned about the pocket proteins, particularly with regard to their developmental and tumor suppressor functions. Thus continues the story of the pocket proteins and the cell cycle.     

Mechanisms controlling cell cycle arrest and induction of apoptosis after 12-lipoxygenase inhibition in prostate cancer cells

Extensive studies have implicated the role of dietary fatty acids in prostatecancer progression. Platelet-type 12-Lipoxygenase (12-LOX) has beenshown to regulate growth, metastasis, and angiogenesis of prostate cancer. The effect of two 12-LOX inhibitors, Baicalein and N-benzyl-N-hydroxy-5-phenylpentamide (BHPP), on the mechanisms controlling cell cycle progression and apoptosis were examined in two prostate cancer cell lines, PC3 and DU-145. Treatment with Baicalein or BHPP resulted in a dose-dependent decrease in cell proliferation, as measured by BrdUrd incorporation. This growth arrest was shown to be because of cell cycle inhibition at G0/G1, and was associated with suppression of cyclin D1 and D3 protein levels. PC3 cells also showed a strong decrease in phosphorylated retinoblastoma (pRB) protein, whereas the other retinoblastoma-associated proteins, p107 and p130, were inhibited in DU-145 cells. Treatment with 12-hydroxyeicosatetraenoic acid in the presence of Baicalein blocked loss of pRB, whereas 12(S)-HETE alone induced pRB expression. Treatment with either Baicalein or BHPP resulted in significant apoptosis in both cell lines as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. DU-145 cells underwent apoptosis more rapidly than PC-3 cells. The mechanisms involved were decreased phosphorylation of Akt, loss of survivin and subsequent activation of caspase-3 and caspase-7 in each cell line, decreased Bcl-2 and Bcl-X(L) expression in DU-145, and a shift in Bcl-2/Bax levels favoring apoptosis in PC-3 cells. Addition of 12(S)-HETE protected both cell lines from Baicalein-induced apoptosis, whereas other LOX metabolites, 5(S)-HETE, or 15(S)-HETE did not. These results show that the 12-LOX pathway is a critical regulator of prostate cancer progression and apoptosis, by affecting various proteins regulating these processes. Therefore, inhibition of 12-LOX is a potential therapeutic agent in the treatment of prostate cancer.     

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.     

UCN-01去除放射后肿瘤细胞G2期阻滞及其相关机制

背景与目的:辐射对肿瘤细胞的影响常表现为细胞周期的改变。本研究观察X线照射后肿瘤细胞G2期阻滞及药物UCN鄄01去除此阻滞的情况,并进一步研究其相关机制。方法:选用已知p53突变的人鼻咽癌CNE鄄1细胞系和人肺腺癌973细胞系进行研究,并以p53功能正常的人纤维母细胞瘤HT鄄1080细胞系作为对照。采用细胞培养和流式细胞仪技术分析X线照射对以上细胞系的细胞周期的影响,并观察UCN鄄01对放射后细胞G2期阻滞的影响;应用蛋白印迹法(Westernblot)测定在UCN鄄01去除CNE鄄1细胞G2期阻滞的过程中,细胞内磷酸化CDC2鄄Tyr15含量的相应变化。结果:X线照射明显导致CNE鄄1细胞和973细胞G2阻滞,照射2Gy后两组的G2期细胞比例分别由18.4%和14.8%上升至43.6%和42.8%,两组细胞的G1期阻滞不显著,衡量G1期阻滞的指标“S期消耗率”均较低,分别为14.8%和-1.2%,明显低于p53功能正常的HT鄄1080细胞(57.0%)。UCN鄄01能够去除放射后CNE鄄1细胞和973细胞G2阻滞,两组的G2期细胞比例分别从单纯照射组的63.5%和35.4%降至16.1%和16.3%。CNE鄄1细胞照射后随着细胞G2期阻滞增加,磷酸化CDC2鄄Tyr15的含量相应增加,而UCN鄄01能够降低照射后细胞内磷酸化CDC2鄄Tyr15的含量,并与该药去除细胞G2期阻滞的过程相一致。结论:对于p53突变的人鼻咽癌CNE     

Extracellular signal-regulated kinase induces cyclin D1 and Cdk-2 expression and phosphorylation of retinoblastoma in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Hyperphosphorylation of retinoblastoma (pRB) by cyclin/CDKs in G1/S transition is required for its inactivation and cell cycle progression. In the present study, we report that phosphorylation of pRB at Ser780 and Ser795 was detected in 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively. pRB protein was undetectable in 13% (6 of 46) of HCCs examined. Phosphorylated pRB was localized in the nuclei of hepatocarcinoma cells. Benign hepatocytes exhibited very weakly or no nuclear staining for phosphorylated pRB. Over-expression of E2F-1, cyclin D1, Cdk-2, Cdk-4 and cyclin A was found in 64% (30 of 46), 43% (26 of 46), 28% (11 of 46), 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively and this was correlated with elevation of ERK. Treatment of HepG2 cells with MEK1/2 inhibitor U0126 resulted in cell cycle arrest, downregulation of cyclin D1 and Cdk-2 expression and inhibition of pRB phosphorylation at Ser780 and Ser795. Ectopic expression of activated MEK1 in HepG2 cells increased cyclin D1 and Cdk-2 expression, phosphorylation of pRB at Ser780 and Ser795, and percentage of cells in S phase. Our data indicate that activated ERK plays an important role in cyclin D1 and Cdk-2 expression and phosphorylation of pRB at Ser780 and Ser795 in liver cancer cells.     

Growth Inhibition of A549 Human Lung Adenocarcinoma Cells by L-Canavanine Is Associated with p21/WAF1 Induction

L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53–dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.