Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin,abrin, and ricin

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.     

A combined evaluation of biochemical and morphological changes during human neuroblastoma cell differentiation

1. The effects of retinoic acid, gamma-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction. 2. Cell morphology was strongly affected by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and gamma-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects. 3. Acetylcholinesterase activity was significantly stimulated by retinoic acid and by gamma-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold. 4. The cellular catecholamine and related metabolite content was lowered by retinoic acid and gamma-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation. 5. Finally, the cell-associated vasointestinal peptide level was strikingly increased by gamma-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. 6. It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.     

Entry of lethal doses of abrin, ricin and modeccin into the cytosol of HeLa cells

In attempts to assess how many molecules of the toxic lectins abrin, ricin and modeccin are needed in the cytosol to kill HeLa cells the effect of these toxins on protein synthesis and plating efficiency was studied. The incubation time of the cells after a 1 h exposure to the toxins influenced strongly the extent of inhibition of protein synthesis. The full toxic effect was expressed about 20 h of incubation after the exposure. On further incubation, protein synthesis again increased at a rate comparable to that in the control cells. After exposure to increasing concentrations of toxins the inhibition of cellular protein synthesis measured after 20 h showed excellent agreement with the inhibition of plating efficiency, indicating that the inhibition of protein synthesis can be used as a measure of cell killing. The inhibition of protein synthesis by toxins was found to follow first order kinetics, indicating that the cells are killed by an all- or none-effect. Autoradiographic studies indicated that after exposure to intermediate toxin concentrations protein synthesis was completely abolished in some cells, whereas it appeared to proceed at a normal rate in the remaining cells. The results provide evidence that penetration of one molecule of abrin, ricin or modeccin into cytosol is lethal to HeLa cells and that the efficiency of toxin entry into the cytoplasm is very low compared to the rate of bulk toxin uptake.     

Changes in polypeptide synthesis and glycosylation in mouse embryonic fibroblast C3H/10T1/2 Cl 8 cells caused by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate

The mechanism of tumor promotion is not well understood. We have used the transformable, tumor promotable, mouse embryo fibroblast C3H/10T1/2 Cl 8 cells to study tumor promoter specific changes in protein synthesis and protein glycosylation. Two-dimensional gel electrophoresis showed that 12-O-tetradecanoylphorbol 13-acetate caused a significant increase in the synthesis of five cellular and 34 extracellular polypeptides. One of these polypeptides has tentatively been identified as ornithine decarboxylase. One new polypeptide (p 62, Mr 58,000) was found in the medium of 12-O-tetradecanoylphorbol 13-acetate-treated cells. The amounts of several excreted proteins were enhanced 5-10 fold by 12-O-tetradecanoylphorbol 13-acetate. 12-O-tetradecanoylphorbol 13-acetate interfered with glycosylation both by affecting protein synthesis and also directly with glycosylation. At least 15 polypeptides in the medium and two cellular polypeptides decreased after 12-O-tetradecanoylphorbol 13-acetate treatment. Two of the major polypeptides found in the medium (p 8 and 10, Mr approx. 200,000-220,000) have properties similar to fibronectin, while p 9 and 11 both found in the cellular preparations and in the medium (Mr 180,000 and 150,000) were collagenase sensitive and their synthesis was inhibited by 12-O-tetradecanoylphorbol 13-acetate.     

Differential response to retinoic acid of Syrian hamster embryo fibroblasts expressing v-src or v-Ha-ras oncogenes.

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.     

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.     

Functional and phenotypic characterization of two HL60 clones resistant to dimethylsulfoxide

Two HL60 clones (C12 and C13) totally insensitive to differentiation induction by dimethylsulfoxide (Me2SO) are described. They have been growing continuously in the presence of the inducer for more than 6 months. The morphological and cytochemical features of the two populations are quite similar to those of the original HL60 cell line, whereas a different karyotype with marked hyperploidy (modal chromosome number of 86 for C12 and 82 for C13) was detected. An antigenic pattern analogous to that of the native HL60 cell line was found in C12 and C13 populations using three monoclonal antibodies differently reactive to myeloid cells. Both clones can be induced to differentiate by retinoic acid (RA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). The pattern of differentiation was assessed by morphological, cytochemical, phenotypical and functional markers. Differentiation of C12 cells by RA and TPA was similar to that observed with native HL60 cells, whereas C13 cells showed lower degrees of sensitivity to RA and TPA. The data presented suggest the existence of different mechanisms for induction of differentiation by Me2SO, RA and TPA. In addition, they are in accordance with previous observations of different degrees of inducibility to differentiation among leukemic cell populations in culture.     

Inhibitory effect of ammonium chloride and chloroquine on the entry of the toxic lectin modeccin into HeLa cells

Ammonium chloride and chloroquine protected a variety of cell lines against diphtheria toxin and the toxic lectin modeccin. Experiments where the ability of antibody to neutralize the toxin was measured indicate that in the presence of ammonium chloride and chloroquine, modeccin remains at the cell surface and that the two compounds inhibit the uptake of modeccin into the cytoplasm. A cell line tolerating increased concentrations of modeccin was not protected against modeccin by ammonium chloride or chloroquine, whereas the compounds did protect these cells against diphtheria toxin.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

Preparation and properties of a hybrid toxin of modeccin A-chain and ricin B-chain

Hybrid molecules were prepared from the A- and B-chains of the two toxic lectins ricin and modeccin by dialyzing mixtures of isolated chains to allow a disulfide bridge to be formed between them. Whereas the hybrid consisting of ricin A-chain and modeccin B-chain was non-toxic, the converse hybrid, modeccin A-chain/ricin B-chain, was even more toxic to Vero cells than were the parent toxins, native ricin and modeccin. A number of drugs (NH₄Cl, monensin, trifluoperazine, verapamil, ionophore A23187) which protect cells against modeccin, but not against ricin, protected to some extent against the toxic hybrid, but less so than against native modeccin. The possibility is discussed that the modeccin A-chain of the hybrid may enter the cytosol by two routes, one which is highly efficient and identical to that used by native modeccin and another less efficient one which cannot be used by native modeccin.     

Expression of Calmodulin-Dependent Phosphodiesterase, Calmodulin-Dependent Protein Phosphatase, and Other Calmodulin-Binding Proteins in Human SMS-KCNR Neuroblastoma Cells

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.     

Protein Kinase C and Its 80-Kilodalton Substrate Protein in Neuroblastoma Cell Neurite Outgrowth

A potential role of the protein kinase C (PKC) system in differentiation of human neuroblastoma cell line LA-N-5 was investigated. It was found that neurite outgrowth induced by 12-O-tetradecanoylphorbol 13-acetate (TPA, 81 nM) was associated with a down-regulation of PKC as determined independently by immunocytochemistry, immunoblot, and enzyme activity assay. Down-regulation of PKC in cells induced to differentiate by retinoic acid (1 microM) was less pronounced, whereas it was undetected in cells induced to differentiate by nerve growth factor (100 ng/ml). The in vitro phosphorylation of an 80-kilodalton protein present in control LA-N-5 cells or in cells treated with TPA, retinoic acid, or nerve growth factor for 1 day decreased to various extents at days 4 or 7 concomitant with neuritogenesis. Pretreatment of LA-N-5 cells with a high concentration (1 microM) of TPA to deplete cellular PKC rendered the cells unresponsive to the differentiating effect of the agents. It was observed that CHP-100 cells, another human neuroblastoma line shown to be resistant to differentiation induced by the agents, had a reduced PKC level and the amount of in vitro phosphorylation of the 80-kilodalton protein was greatly reduced in control cells and remained relatively unchanged when the cells were treated with the agents for up to 7 days. The present studies suggested that PKC and its 80-kilodalton substrate protein were likely involved in initiation and/or progression of LA-N-5 cell differentiation induced by TPA and that separate PKC-independent pathways might also be involved in the differentiating effect of retinoic acid or nerve growth factor.     

Toxin-induced cell lysis: Protection by 3-methyladenine and cycloheximide

The protein toxins ricin, abrin, Shiga toxin, and diphtheria toxin were found to induce lysis of several cell lines in a manner characteristic for programmed cell death or apoptosis. The toxins induced DNA degradation, and light and electron microscopical studies revealed that lysis was preceded by reorganization of intracellular vacuoles, cell blebbing, and chromatin condensation both in Vero and in MDCK cells. Cell lysis was efficiently inhibited by cycloheximide and 3-methyladenine (3MA), a specific inhibitor of autophagy. Cycloheximide, which like 3MA inhibits autophagy, protected even when added at a time when the protein synthesis had been blocked by ricin, suggesting that the effect of cycloheximide on cell lysis is independent of its ability to inhibit protein synthesis. Also theophylline and dibutyryl-cGMP had some protective effect, whereas a number of compounds reported to protect against apoptosis in other systems were without protective effects. The data suggest that autophagy is important for the toxin-induced cell lysis.     

Specific stimulation of histone H2B and H4 phosphorylation in mouse lymphocytes by 12-O-tetradecanoylphorbol 13-acetate

This report demonstrates that the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate rapidly stimulates the phosphorylation of histones H2B and H4 in a cell cycle-independent manner. This effect was observed in primary cultures of BALB/c mouse splenocytes, a population of noncycling, G0 cells which are not stimulated to divide by 12-O-tetradecanoylphorbol 13-acetate treatment alone. The biological nature of this cell system allowed the analysis of histone phosphorylation in the absence of a background of cell cycle-dependent changes and in response to a nonmitogenic agent. The phosphorylation of H2B was determined with high resolution through the use of two-dimensional gel electrophoresis. In contrast to 12-O-tetradecanoylphorbol 13-acetate, the mitogen from pokeweed did not induce stimulation of H2B and H4 phosphorylation, but did, however, elicit increases in the phosphorylation of histones H1, H2A, and H3, in parallel with changes in rate of DNA synthesis.     

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.     

Reduction of ricin and other plant toxins by thiol:protein disulfide oxidoreductases

The inhibitory effect of ricin, abrin, and modeccin on protein synthesis by a rabbit reticulocyte lysate is enhanced after preincubation of the toxins with GSH in the presence of a thiol:protein disulfide oxidoreductase purified from bovine liver. The same toxins, as well as the toxin from Viscum album, are reduced also by another thiol:protein disulfide oxidoreductase purified from rat liver cytosol.     

Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of 125I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two types of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Ribosomal protein S6 phosphorylation and morphological changes in response to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate in primary human tumour cells, established and transformed cell lines

The phosphorylation of ribosomal protein S6 in fibroblasts, primary human tumour cells, established and SV40-transformed human cell lines was compared after the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA). In fibroblasts and primary tumour cell cultures, stimulation of S6 phosphorylation was about 4-6-fold. Established and transformed cell lines showed enhanced S6 phosphorylation which was not further stimulated by the addition of TPA. These findings indicated that the influence of TPA on the metabolic pathway, that finally leads to the phosphorylation of protein S6 in cells with a limited lifespan (fibroblasts, primary human tumour cells) can be mimicked by unknown steps also associated with immortalization (establishment function) and the transformed state of the tumour cells. Another interesting observation were morphological changes of the established and SV40-transformed cells which were visible as early as 20 min after the addition of TPA. In fibroblasts and primary tumour cells no changes in morphology were observed, even after 8h incubation.     

Diphtheria toxin entry into cells is facilitated by low pH

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.