Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

The cell biology of liver fibrogenesis – an imbalance of proliferation, growth arrest and apoptosis of myofibroblasts

Fibrosis following liver damage and factors influencing this process are discussed with special reference to hepatic stellate cells and their transformation to myo- fibroblasts.     

H1 subtype expression during cell proliferation and growth arrest

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analysed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G1 phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G1 as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.     

p53 mediates density-dependent growth arrest

While the stress-response-associated importance of the p53 tumor suppressor is well established, recent studies have also linked p53 with several basic parameters in the normal behavior of cells. Here, we present evidence that basal p53 expression in WI38 human embryonic lung fibroblasts restricts growth rate and mediates density-dependent inhibition of growth and the associated G1 phase arrest of the cell cycle by affecting the density-dependent regulation of p16/INK4a. Additionally, we show that prolonged culturing of hTert-immortalized WI38 cells leads to a loss of density-dependent growth inhibition that correlates with p27/KIP deregulation as well as the previously shown INK4a locus silencing, and to an onset of contact-induced, p53-dependent cell death.     

Lipid hydroperoxide-induced apoptosis in human colonic CaCo-2 cells is associated with an early loss of cellular redox balance.

Apoptosis plays a critical role in maintaining homeostasis of the intestinal epithelium. Dietary oxidants like peroxidized lipids could perturb cellular redox status and disrupt mucosal turnover. The objective of this study was to delineate the role of lipid hydroperoxide (LOOH) -induced redox shifts in intestinal apoptosis using the human colonic CaCo-2 cell. We found that subtoxic concentrations of LOOH increased CaCo-2 cell apoptosis. This LOOH-induced apoptosis was associated with a significant decrease in the ratio of reduced glutathione-to-oxidized glutathione (GSH/GSSG), which preceded DNA fragmentation by 12 to 14 h, suggesting a temporal relationship between the two events. Oxidation of GSH with the thiol oxidant diamide caused significant decreases in cellular GSH and GSH/GSSG at 15 min that correlated with the activation of caspase 3 (60 min) and cleavage of PARP (120 min), confirming a temporal link between induction of cellular redox imbalance and initiation of apoptotic cell death. These kinetic studies further reveal that oxidant-mediated early redox change (within 1 h) was a primary inciting event of the apoptotic cascade. Once initiated, the recovery of redox balance did not prevent the progression of CaCo-2 cell apoptosis to its biological end point at 24 h. Collectively, the study shows that subtoxic levels of LOOH disrupt intestinal redox homeostasis, which contributes to apoptosis. These results provide insights into the mechanism of hydroperoxide-induced mucosal turnover that have important implications for understanding oxidant-mediated genesis of gut pathology.     

Superoxide-hydrogen peroxide imbalance differentially modulates the keratinocytes cell line (HaCaT) oxidative metabolism via Keap1-Nrf2 redox signaling pathway

The purpose of this study was to investigate the effect of a superoxide-hydrogen peroxide (S-HP) imbalance of the superoxide dismutase manganese dependent (SOD2) gene, generated by paraquat and porphyrin exposure, on the keratinocytes cell line (HaCaT) oxidative metabolism. Paraquat acts increasing superoxide (O^·?₂) levels, while porphyrin increases hydrogen peroxide (H₂O₂) levels, acting as VV-SOD2-like and AA-SOD2-like molecules, respectively. First of all, HaCAT cells were treated with different concentrations of paraquat and porphyrin (1; 10; 30, and 70 μM) to determine the concentration of both that causes imbalance. After defining the concentration of paraquat and porphyrin (70 μM), a time curve was performed (1, 3, 6, and 24 h) to evaluate ROS production levels. Other oxidative parameters, such as nitric oxide (NO), lipoperoxidation (TBARS) and protein carbonyl, were evaluated after 24 h of incubation, as well as genotoxic analyses, apoptosis detection, and gene expression. Our findings revealed that paraquat exposure decreased cell viability, increasing lipoperoxidation, DNA damage, and apoptosis. On the other hand, porphyrin treatment increased cell viability and proliferation, ROS and NO production, triggering protein and DNA damage. In addition, porphyrin up-regulated Keap1 and Nrf2 gene expression, while paraquat decreased Nrf2 gene expression. In this sense, we suggested that the superoxide-hydrogen peroxide imbalance differentially modulates oxidative stress on keratinocytes cell line via Keap1-Nrf2 gene expression pathway.

     

Early redox imbalance mediates hydroperoxide-induced apoptosis in mitotic competent undifferentiated PC-12 cells

Our recent study has demonstrated that cellular redox imbalance can directly initiate apoptosis in a mitotic competent PC-12 cell line without the involvement of reactive oxygen species (ROS). However, whether cell apoptosis induced by ROS is, in fact, mediated by a loss of redox balance caused by the oxidant is unresolved. The linkage between oxidant-mediated apoptosis and the induction of cellular redox was examined in PC-12 cells using the oxidant, tert-butylhydroperoxide (TBH). TBH caused cell apoptosis in 24 h that was preceded by an early increase (30 min) in oxidized glutathione (GSSG). Pretreatment with N-acetyl cysteine prevented TBH-induced GSSG increases and cell apoptosis. Altered Bax/BcL-2 expression and release of mitochondrial cytochrome c occurred post-redox imbalance and was kinetically linked to caspase-3 activation and poly ADP-ribose polymerase cleavage. Moreover, cell apoptosis was attenuated by inhibition of caspase-9, but not caspase-8, and blockade of mitochondrial ROS generation and permeability transition pore attenuated caspase 3 activation and cell apoptosis. Collectively, these results show that TBH-induced GSSG elevation is associated with the disruption of mitochondrial integrity, activation of caspase-3 and cell apoptosis. This redox induction of the apoptotic cascade was dissociated from cellular GSH efflux.     

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP⁺/reduced NADP and NAD⁺/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD⁺/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples. cell proliferation; contact inhibition; glutathione     

Cyclooxygenase-2 mediates hydrogen peroxide-induced wound repair in human endothelial cells

Cyclooxygenase-2 (Cox-2) metabolites produced by endothelial cells, particularly prostacyclin and prostaglandin E₂, profoundly affect vascular tone, regional blood flow, and angiogenesis. We have previously shown that reactive oxygen species induce Cox-2 expression in human endothelial cells (HUVEC), either on their own or as components of the signaling pathway triggered by TNFα, the prototypical inflammatory cytokine. Here we investigated the role of Cox-2 induced by hydrogen peroxide (H₂O₂), either exogenous or endogenously generated by TNFα, in the repair of a mechanically wounded HUVEC monolayer and probed the sources of H₂O₂ that are involved in TNFα signaling and the pathways through which H₂O₂ modulates Cox-2 expression. Results indicate that H₂O₂-induced Cox-2 activity participates in the repair of wounded monolayers. Both NADPH oxidase and the mitochondrial electron transport chain are involved in H₂O₂ generation. Signaling triggered by H₂O₂ for Cox-2 induction acts by increasing the protein tyrosine kinase phosphorylation that follows inhibition of protein phosphatase activity. The activation of p38 MAPK and its interaction in the inhibition of serine/threonine phosphatase activity are both critical steps in this event. We conclude that Cox-2 induced by H₂O₂ plays an important role in promoting endothelial wound repair after injury, so that the cardioprotective effect of Cox-2 is due at least in part to its power of healing damaged endothelium.     

Discoidin domain receptor 2 mediates tumor cell cycle arrest induced by fibrillar collagen

During malignant invasion tumor cells establish contact with extracellular matrix proteins, including fibrillar collagen. In addition to providing a physical barrier against invasion, fibrillar collagen also restricts cell proliferation. It has been assumed that the growth regulatory activity of fibrillar collagen is the result of an indirect restrictive effect on cell spreading and cytoskeletal organization. Here we provide evidence for a direct inhibitory effect of fibrillar collagen on proliferation of human melanoma and fibrosarcoma cells that involves activation of the tyrosine kinase discoidin domain receptor 2 and is independent of effects on cell spreading. Cells plated in the presence of fibrillar collagen were growth arrested in the G0/G1 phase of the cell cycle. However treatment with the tyrosine kinase inhibitor genistein, down-regulation of discoidin domain receptor 2, or collagen deglycosylation that prevents discoidin domain receptor 2 activation allowed cells to enter the cell cycle in the presence of fibrillar collagen without a requirement for spreading and actin organization. Our data provide evidence for a novel direct mechanism by which cell contact with fibrillar collagen restricts proliferation.     

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGβ/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGβ/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFβ/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.     

p90/CIP2A mediates breast cancer cell proliferation and apoptosis

Cancerous inhibitor of PP2A (p90/CIP2A) was recently characterized as an innovative oncoprotein in human malignancies. p90/CIP2A inhibited c-Myc-associated PP2A phosphatase activity to promote cell proliferation and tumor growth. A growing number of studies have demonstrated that the overexpression of p90/CIP2A in various human malignancies. But the function of p90/CIP2A in cancer progression is still poorly understood. In the current research, we aim to explore the biological function of p90/CIP2A in breast cancer. shRNA knockdown was performed in MDA-MB-231 and LM2-4 cell lines. Cell proliferation assay, colony formation assay and flow cytometry were carried out to evaluate the role of p90/CIP2A in cell proliferation and apoptosis. p90/CIP2A depletion in breast cancer cells inhibited proliferation and increased paclitaxel-induced apoptosis. Furthermore, p90/CIP2A silencing down-regulated the expression of c-Myc and the level of p-ERK1/2. Taken together, our data suggest that p90/CIP2A as a crucial oncoprotein has been involved in cell proliferation and apoptosis, which may serve as a therapeutic target in breast cancer treatment.     

Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.