Inhibition of proliferation of cultured rat liver epithelial cells at specific cell cycle stages by transforming growth factor-beta

Proliferation of early-passage propagable cultured rat liver epithelial cells derived from normal adult rats is markedly inhibited by transforming growth factor-beta (TGF-beta). Inhibition, which is completely reversible, is effected at two distinct points of the cell cycle, the G1/S border and the G0 or early G1 phase. With increasing passages in culture, hepatic epithelial cells progressively become less sensitive to the inhibitory effect of TGF-beta.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.     

Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.     

Endocytosis of EGF-receptor complexes at various cell cycle stages

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.     

Inhibition of cell proliferation by glycerol

The effect of glycerol on proliferation of BHK, CHO, HBL, MCF-7, and human glioma cells was studied. Cell proliferation was significantly decreased in all the cell lines at glycerol concentrations of 2-4% in the culture medium. The inhibition was dose-dependent, complete suppression of proliferation occurring at a glycerol concentration of 4% for the MCF-7 cell line and 6-8% for the BHK, CHO and human glioma cells. Studies on [3H]thymidine incorporation correlate with the effect on cell proliferation. The viability of the cells was not significantly affected until higher concentrations of glycerol (12% +) were present. Recovery studies with BHK cells indicated that replacement of the glycerol medium with glycerol-free medium resulted in full recovery following exposure to 4% glycerol and only partial recovery (65%) of proliferation rate following exposure to 10-12% glycerol. It is concluded that glycerol, a substance that is normally present in tissues, can serve as a potent inhibitor of cell proliferation.     

Inhibition of rat smooth muscle cell adhesion and proliferation by non-anticoagulant heparins

Heparin is a well established growth inhibitor of arterial smooth muscle cells (SMCs) both in animal models and in vitro. Even though the cellular mechanisms involved in the anti-proliferative properties of heparin are being resolved, the structural requirements for the biological effects of heparin are not known in detail. Here, we have studied the effect of chemically modified heparins of different molecular weights and anticoagulant activities on proliferation and adhesion of rat aortic SMCs in vitro. The effects of native heparin (NH) and chemically modified heparins were examined after stimulation with fetal calf serum (FCS), platelet-derived growth factor BB (PDGF BB), basic fibroblast growth factor (bFGF), and heparin-binding epidermal growth factor (hbEGF) with respect to DNA synthesis and expression of phosphorylated and activated mitogen-activated protein kinase (pERK1 and 2). In a similar manner as NH, the modified heparins were capable of inhibiting activation of ERK1 and 2 and DNA synthesis induced by FCS and hbEGF whereas the modified heparins potentiated the mitogenic effect of bFGF and no compound affected PDGF BB-induced ERK activity and SMC growth. In contrast, cell adhesion to fibronectin was inhibited by NH and modified heparins in a size-dependent manner with the lowest effect by the smallest compound. The results show that heparins with varying anticoagulant activities and molecular weights but with similar sulfate content can retain anti-proliferative properties while the effect on some other biological processes such as cell adhesion is lost. Possibly, such chemical alterations may yield useful substances for the prevention of SMC proliferation after arterial injury.     

Inhibition of fibroblast proliferation in cardiac myocyte cultures by surface microtopography

Cardiac myocyte cultures usually require pharmacological intervention to prevent overproliferation of contaminating nonmyocytes. Our aim is to prevent excessive fibroblast cell proliferation without the use of cytostatins. We have produced a silicone surface with 10-µm vertical projections that we term "pegs," to which over 80% of rat neonatal cardiac fibroblasts attach within 48 h after plating. There was a 50% decrease in cell proliferation by 5 days of culture compared with flat membranes (P < 0.001) and a concomitant 60% decrease (P < 0.01) in cyclin D1 protein levels, suggesting a G₁/S₁ cell cycle arrest due to microtopography. Inhibition of Rho kinase with 5 or 20 µM Y-27632 reduced attachment of fibroblasts to the pegs by over 50% (P < 0.001), suggesting that this signaling pathway plays an important role in the process. Using mobile and immobile 10-µm polystyrene spheres, we show that reactive forces are important for inhibiting fibroblast cell proliferation, because mobile spheres failed to reduce cell proliferation. In primary myocyte cultures, pegs also inhibit fibroblast proliferation in the absence of cytostatins. The ratio of aminopropeptide of collagen protein from fibroblasts to myosin from myocytes was significantly reduced in cultures from pegged surfaces (P < 0.01), suggesting an increase in the proportion of myocytes on the pegged surfaces. Connexin43 protein expression was also increased, suggesting improved myocyte-myocyte interaction in the presence of pegs. We conclude that this microtextured culture system is useful for preventing proliferation of fibroblasts in myocyte cultures and may ultimately be useful for tissue engineering applications in vivo. tissue engineering; cell culture; cell cycle     

Inhibition by cocaine of the baroreflex in the rat.

Sprague-Dawley rats were fitted under pentobarbital anesthesia with a catheter in the caudal artery and their carotid arteries were exposed. The pressure signal from the caudal artery was treated on line by a microcomputer for continuous display of blood pressure and heart rate measurements. The animals were administered intraperitoneally either 50 mg/kg of cocaine or an equal volume of saline. Five minutes later, stimulation of the baroreflex was performed by bilateral clamping of the two carotids for a period of 2 min. The same maneuver was repeated at 12, 24, and 31 min. Analysis of variance for repeated measures indicated that before carotid artery clamping, there was no significant difference between blood pressure measurements of the saline- and cocaine-treated groups. A two-factor analysis of variance of the repeated measures of the maximal variation in systolic pressure after each clamping showed a significant difference between control and cocaine-administered groups (P < 0.001), with the former displaying a much greater increment in blood pressure after carotid clamping. Cocaine exerts an inhibitory effect on the baroreflex that may be mediated through the increased angiotension II caused by the alkaloid.     

Inhibition of endothelial cell proliferation by gamma-interferon

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose- dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor- ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.     

Inhibition of ras-mediated cell proliferation by benzyloxybenzaldehyde

Excessive proliferation of vascular smooth muscle cells (VSMC) in the intima is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. Therefore, control of VSMC growth may be a suitable therapeutic intervention in vascular proliferative disorders. In the present work, we have studied the 2-benzyloxybenzaldehyde (CCY1a)-mediated antiproliferative effect and its mechanisms of action. CCY1a inhibited serum-induced VSMC proliferation in a concentration-dependent manner, as demonstrated using [³H]thymidine incorporation and MTT assays; the IC₅₀ values were calculated to be 7.0 × 10^–6 and 1.2 × 10^–5 M, respectively. Furthermore, it also significantly suppressed serum-induced progression of the cell cycle, as shown by flow cytometric analysis. CCY1a as well as PD98059 almost completely abolished serum-induced activation of p42/44 mitogen-activated protein kinase (MAPK), the downstream effectors of c-fos and c-jun mRNA expression and activator protein-1 (AP-1) DNA binding activity, suggesting the central roles of these signaling cascades. Interestingly, CCY1a also effectively blocked serum-induced IB- phosphorylation, IB- degradation and nuclear factor-B (NF-B) binding activity. Based on these observations, we examined the effect of CCY1a on serum-mediated Ras activity, an upstream regulator of the above signaling events. The data demonstrated a marked inhibition of Ras activation by CCY1a. We conclude that CCY1a blocks cell proliferation via inhibition of the upstream effector of Ras and downstream events, including p42/44 MAPK activation and c-fos and c-jun mRNA expression, as well as NF-B and AP-1 DNA binding activities.     

Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth

In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Delta or pep12Delta in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Inhibition of cell proliferation by an anti-EGFR aptamer

Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K_d = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.     

Inhibition of cell proliferation by D-ribose and deoxy-D-ribose

D-Ribose and deoxy-D-ribose inhibited DNA, RNA, and protein synthesis in a wide variety of cells (dividing and nondividing, normal and neoplastic, freely floating and substrate adhering, human and murine) at concentrations at which other monosaccharides have little or no effect. Inhibition was irreversible and proportional to the sugar concentration and time of contact. However, the first effects were seen only after 24 hr of incubation and progressed slowly to cell death. Whether the two sugars share the same mechanisms of action is not known. In any case, they deeply disturb metabolic processes in both dividing and nondividing cells.     

Inhibition of cell aggregation by specific carbohydrates.

One approach to investigating the potential role of surface carbohydrates in mediating intercellular adhesion is to study cell reaggregation in the presence of defined concentrations of specific saccharides. Fifteen different exogenously added saccharides were tested for their effect on the reaggregation of 24 h sea urchin embryo cells (Strongylocentrotus purpuratus) dissociated by removal of divalent cations. Aliquots (0.2 ml) of cell suspension were rotated at 68 rpm, 17 °C, pH 8.0, with varying concentrations (0.5 × 1^?1?0.5 × 10^?5 M) of the sugars. Relative percents of cell aggregation were determined using an electronic particle counter assay. In all experiments cell viability using trypan blue was over 95.8%. Among the sugars tested, in 15 separate experiments, d-galactose and N-acetyl-d-galactosamine consistently inhibited aggregation to the greatest extent at early time points. d-Galactose, at all concentrations tested, at 10, 20, 30, 40, and 60 min rotation, showed mean decreases of aggregation over control values in the absence of sugar of 59.3, 53.6, 43.2, 35.0 and 36.4%, respectively. N-Acetyl-d-galactosamine also caused mean decreases in aggregation of 73.5, 54.5, 40.8, 42.2 and 45.6%, respectively. Each difference over the control is significant to the p value of less than 0.01. In three experiments, β-galactosidase substantially inhibited reaggregation of these cells. These results suggest that galactopyranosyl-like groups may be implicated in mediating adhesion of 24 h sea urchin embryo cells to each other.     

T-kininogen inhibits fibroblast proliferation in the G(1) phase of the cell cycle

By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblast-derived cell lines that express biologically active T-kininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G(0) cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G(0) through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G(1) and S phases of the cell cycle.     

Inhibition of mitogen and specific antigen-induced human lymphocyte proliferation by cadmium

Cadmium chloride (CdCl2) added to human lymphocyte culture inhibits the proliferative response induced by phytohaemagglutinin (PHA), pokeweed mitogen and allogenic lymphocytes in mixed lymphocyte reaction. Minimally effective concentrations of CdCl2 were 3.3, 1.6 and 1.6 microM, respectively. The inhibition was greatest when CdCl2 was added at initiation of cultures and declined if the addition of CdCl2 was postponed. The presence of CdCl2, regardless of the presence of PHA during the first 24 h of incubation suppressed the proliferative response to subsequent stimulation with PHA, indicating that cadmium affects an early step of blastogenic transformation.     

Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors

TGF beta is a potent (ED50 approximately 10(-11) M) inhibitor of the proliferative activities of both acidic and basic FGF on vascular and capillary endothelial cells in vitro. The inhibition of cell growth is dose-dependent and characteristic of a non-competitive interaction. The results demonstrate that TGF beta and FGF can interact at the cellular level to modulate growth and suggest that many of the biological activities of FGF observed in vitro and in vivo (ie angiogenesis, cell growth, cell differentiation) may be regulated by the presence of TGF beta and related proteins (ie inhibin) in the local cellular milieu. The possible identity of TGF beta with the inhibitors of endothelial cell growth detected in in vitro assays of crude extracts is discussed.