Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cell was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2–100 μg/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. Theses results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells whihc represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the ¹⁴C-labelled asialofetuin degradation. We can conclude that Ricinus lectic present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.     

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixtyfold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G₀/G₁ cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling. © 1994 Wiley-Liss, Inc.     

A redox signaling mechanism for density-dependent inhibition of cell growth

Reactive oxygen species (ROS) have recently drawn significant attention as putative mitogenic mediators downstream of activated growth factor receptors and oncogenic Ras; however, the possibility that a redox-related mechanism also operates in the negative control of cell proliferation by inhibitory signals has not been investigated thus far. Here we show that the arrest of growth induced by cell confluence ("contact inhibition") is due, at least in part, to a decrease in the steady-state levels of intracellular ROS and the consequent impairment of mitogenic redox signaling. In confluent fibroblast cultures, the decrease in the concentration of oxygen species was associated with diminished activity of the small GTPase Rac-1, a signal transducer directly involved in the ligand-dependent generation of oxygen-derived molecules, and was effectively mimicked by exposure of sparse cultures to dithiothreitol (DTT) and inhibitors of enzymes (phospholipase A2 and lipoxygenase) acting in the arachidonic acid cascade downstream of growth factor receptors and Rac-1. Sparse fibroblasts treated with nontoxic amounts of DTT underwent growth arrest, whereas a low concentration of hydrogen peroxide significantly increased thymidine incorporation in confluent cultures, demonstrating a causal link between redox changes and growth control by cell density. Removal of oxygen species from sparse cultures was accompanied by a drastic decrease of protein tyrosine phosphorylation after epidermal growth factor stimulation, which, at a biochemical level, reproduced the signaling hallmarks of contact inhibition. Moreover, the cytosolic tyrosine phosphatase SHP-2 was identified as a putative target for redox signaling by cell density because the enzyme itself and the associated substrates appear markedly dephosphorylated in both confluent and reductant-treated cells after exposure to epidermal growth factor, and SHP-2 enzymatic activity is strongly activated by DTT in vitro. Taken together, these data support a model in which impaired generation of ROS and increased protein tyrosine phosphatase activity impede mitogenic signaling in contact-inhibited cells.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Lymphocyte commitment to DNA replication induced by concanavalin A

Commitment of lymphocytes to DNA replication induced by ConA was investigated by adding α-MM (a saccharide that competes with the lectin's lymphocyte binding sites) at various times to lymphocyte cultures that had been treated with a wide range of ConA concentrations. After 24 h of culture, α-MM addition has essentially no effect on cells treated with optimal concentrations of ConA, results in a marked blastogenic response of cells treated with supra-optimal (non-mitogenic) concentrations of ConA, and severely inhibits blastogenesis of cells treated with suboptimal concentrations of the lectin. Furthermore, the time of commitment is progressively shortened as the concentration of mitogen is increased. Thus, cells were committed to DNA replication as early as 5 h after incubation with supra-optimal concentrations of ConA. Induction of commitment does not occur when cells are incubated with ConA at 4 °C. Extensive crosslinking of membrane sites appears to be associated with the induction of early commitment by ConA, since the bivalent succinyl ConA failed to exhibit this effect. These findings indicate the need for re-evaluation of previous studies utilizing competing saccharides to determine kinetics of cell commitment to DNA replication.     

Effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 in human hepatoma cell lines

The effects of phorbol ester on cell growth inhibition by transforming growth factor beta 1 (TGF-beta 1) in human hepatoma cell lines, Mahlavu and PLC/PRF/5, were investigated. TGF-beta 1 (2.5 to 10 pM) alone could not inhibit the growth of Mahlavu cells, whereas in the presence of 12-O-tetradecanoyl phorbol 13-acetate (TPA) at 1 ng/ml, TGF-beta 1 could suppress their growth in a dose-dependent manner. The growth of PLC/PRF/5 cells could be inhibited by addition of TGF-beta 1 (2.5 to 10 pM) alone in a dose-dependent manner, and this action was not affected by TPA (1 ng/ml). The TGF-beta 1 inhibition induced by TPA in Mahlavu cells could not be cancelled by addition of protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) (10 microM) or staurosporin (1 nM). Thus, TPA could induce TGF-beta 1 inhibition of cell growth in Mahlavu cells which did not respond to TGF-beta 1 alone, and activation of protein kinase C does not seem to be behind this TPA action.     

Cellular immunity in pyelonephritis: inhibition of the spleen cell response to the mitogen concanavalin A

Spleen cells from rats given experimental pyelonephritis using Escherichia coli and Proteus mirabilis as infecting organisms were evaluated for their ability to respond to the mitogen Concanavalin A. Results indicate an 80% reduction in the response of animals with gross suppuration in the kidney, but no inhibition is observed in animals with kidney infection, but not renal abscesses. The inhibition is not, apparently, due to serum factors.     

Proteolytic degradation of nuclear matrix proteins in the rat liver, Zajdela's hepatoma and hepatoma 22A in the presence of ATP

An intense proteolytic degradation of both proteins and phosphoproteins has been observed in isolated nuclear matrices from rat liver, Zajdela Hepatoma and Hepatoma 22a, incubated with NP-40, DTT and gamma-[32P] ATP being most intense in Hepatoma 22a. Practically all phosphoproteins of Hepatoma 22a nuclear matrix degraded. This implies either an extremely high proteolytic activity in the preparation or the presence of a specific to phosphoproteins protease absent from rat liver and Zajdela Hepatoma nuclear matrices.     

衣原体抗宿主细胞凋亡调控机制研究进展

衣原体为完成发育周期以及逃避宿主免疫,进化形成一整套机制以实现持续性感染并对宿主细胞进行调控,抑制宿主细胞凋亡。细胞感染衣原体后,早期可以抑制Caspase酶系,抑制相关信号转导途径,且胞内线粒体发生一系列结构和功能的变化,抑制凋亡因子释放,一系列因子协同作用,抑制宿主细胞凋亡。本文从凋亡途径、凋亡蛋白、凋亡信号通路三个主要方面作了衣原体抑制宿主细胞凋亡机制概述,对进一步了解衣原体发育及其致病机制提供了新的研究思路。     

Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.     

Nuclear glycoproteins of hamster liver and Kirkman-Robbins hepatoma recognized by concanavalin A.

1. Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma. 2. The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively. 3. A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed.     

Changes in the blood level and affinity to concanavalin A of rat plasma glycoproteins during acute inflammation and hepatoma growth.

Plasma concentrations of ten individual proteins were measured by electroimmunoassay in young male Buffalo rats following injection of turpentine oil or implantation of Morris hepatoma 7777. The highest relative responses to inflammation and tumour growth were found for alpha 2-macroglobulin, alpha 1-acute-phase globulin and alpha 1-acid glycoprotein. As shown by crossed immuno-affinoelectrophoresis the concanavalin A-reactive fractions of the latter two glycoproteins were predominantly increased in plasma from injured and tumour-bearing rats.     

Explanation for the apparent inactivation of tyrosine aminotransferase in hepatoma cells by concanavalin A

Brief treatment of hepatoma cells in monolayer culture with concanavalin A causes a decrease in tyrosine aminotransferase specific activity that is thought to be a rapid, reversible inactivation of the enzyme (T.V. Gopalakrishnam and E.B. Thompson 1977 J. Biol. Chem. $\text{252}$, 2717–2725). We confirm this decrease, but attribute it to an increased leakage of cellular protein from concanavalin A-treated monolayer cultures during the harvesting procedure. If the cells are washed free of medium and lysed $\text{in}$,$\text{situ}$ by freezing and thawing them, or by treating them with buffer containing a nonionic detergent, equal amounts of tyrosine aminotransferase are found in concanavalin A-treated and untreated cells. If cells are harvested by scraping them from the substrate, some tyrosine aminotransferase is lost into the buffer used to collect the cells. Treatment of cells with concanavalin A markedly increases the amount of enzyme lost during this procedure, and results in a low enzyme content in the washed cells. No inactivation occurs, however, because the total amount of tyrosine aminotransferase present in the cell pellet and the wash buffer is equal for treated and untreated cells.     

Changes in gene expression in the brain of rats with Zajdela's ascitic hepatoma

Expression of some genes in the brain of ascitic hepatoma of Zajdela bearing rats was compared with that of control animals using Northern blot hybridization technique. The differences revealed were: an increased expression of actin gene and decreased expression of hsp70 gene in the brain of tumor-bearing animals.     

Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids

Monovalent antibodies directed against N-acetylhematoside are growth inhibitory for BALB/3T3 and NIL hamster fibroblasts but not their transformed counterparts. Within a similar dose range antibodies directed against globoside have no effect on cell growth. Inhibition of 3T3 cell growth by anti-hematoside correlates with a specific change in the metabolism of hematoside within the cell membrane. Following antibody treatment the radiolabeling of hematoside is elevated for cell in logarithmic growth but reduced relative to control at final saturation density. This effect is not observed for 3T3 cells transformed by Kirsten murine sarcoma virus. It is suggested that cell surface glycolipids may play a role in the control of normal cell growth in vitro.