Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation.

The insulin receptor substrates (IRSs)-1-4 play important roles in signal transduction emanating from the insulin and insulin-like growth factor (IGF)-I receptors. IRS-4 is the most recently characterized member, which has been found primarily in human cells and tissues. It interacts with SH2-containing proteins such as phosphatidylinositol 3'-kinase (PI3K), Grb2, Crk-II, and CrkL. In this study, we transfected IRS-4 in mouse NIH-3T3 cells that overexpress IGF-I receptors. Clones expressing IRS-4 showed enhanced cellular proliferation when cells were cultured in 1% fetal bovine serum without added IGF-I. Addition of IGF-I enhanced cellular proliferation in cells overexpressing the IGF-I receptor alone but had an even greater proliferative effect in cells overexpressing both the IGF-I receptors and IRS-4. When etoposide and methylmethane sulfonate (MMS), both DNA damaging agents, were added to the cells, they uniformly induced cell cycle arrest. Fluorescence-activated cell sorter analysis demonstrated that the arrest of the cell cycle occurred at the G(1) checkpoint, and furthermore no significant degree of apoptosis was demonstrated with the use of either agent. In cells, overexpressing IGF-I receptors alone, IGF-I addition enhanced cellular proliferation, even in the presence of etoposide and MMS. In cells overexpressing IGF-I receptors and IRS-4, the effect of IGF-I in overcoming the cell cycle arrest was even more pronounced. These results suggest that IRS-4 is implicated in the IGF-I receptor mitogenic signaling pathway.     

Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

The ubiquitin (Ub)-dependent proteolytic pathway may function in selective elimination of cellular proteins during erythroid differentiation. Murine erythroleukemia (MEL) cells, which can be induced to differentiate to reticulocytes in culture, may provide a convenient system for studying the role of Ub-dependent proteolysis in erythroid differentiation. The following observations indicate that MEL cells possess an active Ub-dependent proteolytic pathway. (i) Addition of purified Ub to MEL cell fraction II (Ub-depleted lysate) stimulated ATP-dependent degradation of radioiodinated proteins. (ii) Covalent conjugation of carboxyl termini of Ub molecules to substrate protein amino groups is a necessary step in Ub-dependent degradation. Des-glygly-Ub (Ub lacking its carboxyl-terminal glygly moiety) did not stimulate protein degradation in MEL cell fraction II. (iii) The Ub-dependent component of protein degradation in MEL cell fraction II was specifically inhibited by amino acid derivatives that are inhibitors of Ub-protein ligase. (iv) MEL cell fraction II contained apparent homologs of all of the rabbit reticulocyte Ub carrier proteins (E2's) except E2(20K) and E2(230K). Ub-dependent proteolysis was seen only in MEL cell lysates prepared in the presence of leupeptin; an enzyme of the proteolytic pathway was inactivated if leupeptin was omitted.     

Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan

The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log-phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum- and insulin-deficient medium, HSPG prepared from log-phase cells stimulated the growth rate of these slow-growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG-treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG-treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1 phase of the cell cycle, prior to the G1/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

植物泛素/26S蛋白酶体途径研究进展

泛素/26S蛋白酶体途径是最重要的,有高度选择性的蛋白质降解途径,由泛素激活酶、泛素结合酶、泛素蛋白连接酶和26S蛋白酶体组成,参与调控植物生长发育的多个方面。泛素蛋白酶体途径参与植物体内的众多生理过程,如植物激素信号,光形态建成、自交不亲和反应和细胞周期等。本文就泛素/26S蛋白酶体途径以及在植物生长发育中的作用的研究近况做一综述。     

Aurora-A kinase interacting protein 1 (AURKAIP1) promotes Aurora-A degradation through an alternative ubiquitin-independent pathway

Mitotic Aurora-A is an oncogene, which undergoes a cell-cycle-dependent regulation of both its synthesis and degradation. Overexpression of Aurora-A leads to aneuploidy and cellular transformation in cultured cells. It has been shown that the cell-cycle-dependent turnover of Aurora-A is mediated by Cdh1 (CDC20 homologue 1) through the anaphase-promoting complex/cyclosome (APC/C)-ubiquitin-proteasome pathway. We have described previously the identification of an Aurora-A kinase interacting protein, AURKAIP1 (formerly described as AIP), which is also involved in the destabilization of Aurora-A through the proteasome-dependent degradation pathway. In an attempt to investigate the mechanism of AURKAIP1-mediated Aurora-A degradation, we report here that AURKAIP1 targets Aurora-A for degradation in a proteasome-dependent but Ub (ubiquitin)-independent manner. AURKAIP1 inhibits polyubiquitination of Aurora-A. A non-interactive AURKAIP1 mutant that cannot destabilize Aurora-A restores ubiquitination of Aurora-A. An A-box mutant of Aurora-A, which cannot be targeted for proteasome-dependent degradation by Cdh1, can still be degraded by AURKAIP1. Inhibition of cellular ubiquitination either by expression of dominant negative Ub mutants or by studies in ts-20 (temperature sensitive-20) CHO (Chinese-hamster ovary) cell line lacking the E1 Ub activating enzyme at the restrictive temperature, cannot abolish AURKAIP1-mediated degradation of Aurora-A. AURKAIP1 specifically decreases the stability of Aurora-A in ts-20 CHO cells at the restrictive temperature, while cyclinB1 and p21 are not affected. This demonstrates that there exists an Ub-independent alternative pathway for Aurora-A degradation and AURKAIP1 promotes Aurora-A degradation through this Ub-independent yet proteasome-dependent pathway.     

The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice.

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3(-/-) mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57(Kip2). These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. beta-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3(-/-) cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.     

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A + Ras-transformed cells to proapoptotic stimuli

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome – apoptotic cell death or cell cycle arrest – depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A + Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A + Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A + Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A + Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.     

Role of p21WAF1 in the Cellular Response to UV

UV or gamma irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin-dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21 function.     

Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.     

Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation

Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks.     

Effect of covalent photoconjugation of affibodies to epidermal growth factor receptor (EGFR) on cellular quiescence

Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the nondividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor-mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to epidermal growth factor receptors (EGFR) on EGFR-positive MDA-MB-468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two-fold and reduced ERK activity by 30% as compared to cells treated with an FDA-approved anti-EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5-ethynyl-2-deoxyuridine and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

Apoptosis occurs via the ceramide recycling pathway in human HaCaT keratinocytes

Keratinocytes contain abundant ceramides compared to other cells. However, studies on these cells have mainly focused on the barrier function of ceramide, while their other roles, such as those in apoptosis or cell cycle arrest, have not been well addressed. In this study, we investigated the apoptosis-inducing effect of exogenously added cell-permeable ceramides in HaCaT keratinocytes. We found that N-hexanoyl sphingosine (C6-ceramide) induced apoptosis efficiently through the accumulation of long chain ceramides. On the other hand, N-acetyl sphingosine (C2-ceramide) induced neither apoptosis nor accumulation of long chain ceramides. We also found that exogenously added C6-ceramide was hydrolyzed to sphingosine and then reacylated in long chain ceramides (ceramide recycling pathway), but that C2-ceramide was not hydrolyzed and thus not recycled. We propose that this is the basis for the chain length-specific heterogeneity observed in ceramide-induced apoptosis in these cells. These results also imply that keratinocytes utilize exogenous sphingolipids or ceramides to coordinate their own ceramide compositions.     

Cdh1-anaphase-promoting complex targets Skp2 for destruction in transforming growth factor beta-induced growth inhibition

As a subunit of a ubiquitin ligase, Skp2 is implicated in facilitating cell cycle progression via degradation of various protein targets. We report here that Skp2 is rapidly degraded following cellular stimulation by the cytokine transforming growth factor beta (TGF-beta) and that this degradation stabilizes the cell cycle arrest protein p27. The Skp2 degradation is mediated by Cdh1-anaphase-promoting complex (APC), as shown by depletion of Cdh1 with small interfering RNA, and by reconstitution of ubiquitylation reactions in a purified system. Blockage of Skp2 degradation greatly reduces TGF-beta-induced cell cycle arrest, as does expression of a nondegradable Skp2 mutant. Furthermore, we demonstrate that TGF-beta-induced Skp2 degradation is mediated by the Smad cascade. The degradation of Skp2 stabilizes p27, thereby ensuring TGF-beta-induced cell cycle arrest. These results identify a novel mechanism for tumor suppression by TGF-beta and explain why dysfunction of APC in the TGF-beta pathway in responsive cells is associated with cancer.     

Regulation of Raf-1-dependent signaling during early Xenopus development.

The Raf-1 gene product is activated in response to cellular stimulation by a variety of growth factors and hormones. Raf-1 activity has been implicated in both cellular differentiation and proliferation. We have examined the regulation of the Raf-1/MEK/MAP kinase (MAPK) pathway during embryonic development in the frog Xenopus laevis. We report that Raf-1, MEK, and MAPK activities are turned off following fertilization and remain undetectable up until blastula stages (stage 8), some 4 h later. Tight regulation of the Raf-1/MEK/MAPK pathway following fertilization is crucial for embryonic cell cycle progression. Inappropriate reactivation of MAPK activity by microinjection of oncogenic Raf-1 RNA results in metaphase cell cycle arrest and, consequently, embryonic lethality. Our findings demonstrate an absolute requirement, in vivo, for inactivation of the MAPK signaling pathway to allow normal cell cycle progression during the period of synchronous cell divisions which occur following fertilization. Further, we show that cytostatic factor effects are mediated through MEK and MAPK.     

Cell Death of MCF-7 Human Breast Cancer Cells Induced by EGFR Activation in the Absence of Other Growth Factors

The Epidermal Growth Factor (EGF) Receptor (EGFR) plays an important role in the growth and progression of breast cancer. Overexpression of EGFR or the high activity of EGFR signal pathway has been related with increases in cell proliferation and a poor prognosis in patients with breast cancer. Several human breast cancer cell lines depend on estrogen for their proliferation. EGF may bypass the requirement of estrogen for the proliferation of breast cancer cells. To evaluate this hypothesis, MCF-7 breast cancer cells were stimulated with EGF and the effects on cell proliferation, signal pathways, and cell cycle progression were determined. The results demonstrate that EGF stimulation in the absence of others growth factors induced a modest effect on cell proliferation and the induction of a cellular arrest in the G1 phase of the cell cycle. Although phosphorylation of AKT and ERK proteins were detected, this phosphorylation was insufficient to support of cell cycle progression. Cellular arrest in G1 phase was accompanied by an increase in p21CIP1 protein, down regulation of the BCL-2 protein, induction of caspase-8, and ARHI/NOEY2 an imprinted tumor suppressor gene. These results indicate that EGFR activation by itself is not sufficient for the proliferation of breast cancer cells and suggest the existence of a mechanism that induces apoptosis upon EGFR activation.     

Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis

Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S-phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S-phase was preceded by a 6- to 8-hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 microM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S-phase by an additional 2 to 4 hr. The rate of cellular progression through S-phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 microM) that prevented isoproterenol-induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S-phase. These findings demonstrate the ability of the beta-adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor-mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP-dependent phenomenon.