Sjur Olsnes

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

Abstract 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.

Dual mode of signal transduction by externally added acidic fibroblast growth factor

Acidic fibroblast growth factor (aFGF), fused to diphtheria toxin and translocated into cells, stimulated DNA synthesis in toxin-resistant cells lacking functional aFGF receptors while having a high number of diphtheria toxin receptors. In NIH 3T3 cells that lack diphtheria toxin receptors, but have receptors for aFGF, both aFGF and the fusion protein induced tyrosine phosphorylation, but only aFGF as such entered the nuclei and stimulated DNA synthesis. The results indicate that signaling occurs partly through cell surface receptors and partly by transport of the growth factor into the cell.

The ways of endocytosis

Publisher Summary This chapter focuses on certain aspects of the endocytic pathways of the protein import into cells and the routes they subsequently follow. Transport and sorting of proteins are essential processes for the function and differentiation of eukaryotic cells. The cell utilizes two distinct, specialized organelle systems for the transport and sorting of proteins, one for protein export and the other for protein import. The export system comprises the endoplasmic reticulum (ER) and the Golgi complex. The import system comprises a variety of vesicular structures, the most prominent being endosomes and lysosomes. The role of the various intracellular compartments and of the sorting signals that are needed to deliver specifically the right components to the right compartments, are described in the chapter. The existence of a clathrin-dependent endocytic pathway and some ideas and problems of defining the structural equivalent of this pathway are discussed. The clathrinin-dependent endocytic pathway is used to inhibit the coated-pit pathway. Endocytosis from coated pits can also be inhibited by acidification of the cytosol.

Dependence of Ricin Toxicity on Translocation of the Toxin A-chain from the Endoplasmic Reticulum to the Cytosol

Ricin acts by translocating to the cytosol the enzymatically active toxin A-chain, which inactivates ribosomes. Retrograde intracellular transport and translocation of ricin was studied under conditions that alter the sensitivity of cells to the toxin. For this purpose tyrosine sulfation of mutant A-chain in the Golgi apparatus, glycosylation in the endoplasmic reticulum (ER) and appearance of A-chain in the cytosolic fraction was monitored. Introduction of an ER retrieval signal, a C-terminal KDEL sequence, into the A-chain increased the toxicity and resulted in more efficient glycosylation, indicating enhanced transport from Golgi to ER. Calcium depletion inhibited neither sulfation nor glycosylation but inhibited translocation and toxicity, suggesting that the toxin is translocated to the cytosol by the pathway used by misfolded proteins that are targeted to the proteasomes for degradation. Slightly acidified medium had a similar effect. The proteasome inhibitor, lactacystin, sensitized cells to ricin and increased the amount of ricin A-chain in the cytosol. Anti-Sec61α precipitated sulfated and glycosylated ricin A-chain, suggesting that retrograde toxin translocation involves Sec61p. The data indicate that retrograde translocation across the ER membrane is required for intoxication.

Roles of Fibroblast Growth Factor Receptors in Carcinogenesis

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression. Mol Cancer Res; 8(11); 1439–52. ©2010 AACR.

Toxicity, distribution and elimination of the cancerostatic lectins abrin and ricin after parenteral injection into mice

The survival time of mice after i.v. injection of the cancerostatic lectins, abrin and ricin was recorded. The LD50 dose was found to be 10-13 ng and 55-65 ng per mouse for abrin and ricin, respectively. Increasing amounts of toxin reduced the survival time, reaching a minimum of about 10 h. Lactose injected with ricin, provided partial protection against ricin, as measured by the survival time. Abrin and ricin labelled with 125I, and shown to retain their full toxic activity, were injected into mice. Most of the radioactivity found in the organs was present in the form of intact toxins, at least up to 5 h after injection. After i.v. injection the highest concentration/g tissue was found in spleen, followed by kidneys, heart, liver and thymus. The relative concentration in liver was considerably higher for ricin than for abrin. Similar results were found after i.p. injection. When lactose was administered together with ricin, almost 80% of the ricin injected was found in the liver after 30 min, compared to 48% without lactose, and the amount in other organs was concurrently reduced. The elimination of total radioactivity was much faster for ricin than abrin. The radioactivity found in the urine was largely present in non-trichloroacetic acid precipitable form, indicating that the toxins were extensively degraded before excretion.

Ricin — a potent inhibitor of protein synthesis

Ricin, a protein present in castor beans (the seeds of Ricinus communis L.) is extremely toxic to animals and man [ 11. The effect of ricin on living animals has been extensively studied [2--S]. The reduction in serum protein in treated animals has been taken as evidence that ricin might exert its toxic action by inhibiting protein synthesis [4], and recently it has been found that in Ehrlich ascites tumour cell cultures protein synthesis is inhibited more stongly than the RNA and DNA synthesis [6]. In the present study it is shown that in a cell-free system very small amounts of ricin completely inhibit protein synthesis. The results indicate that ricin interferes with the completion of already initiated peptide chains.

Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells

Abstract The effect of temperature on the uptake of abrin and ricin and on the subsequent excretion and degradation of the toxins was measured. Uptake was assessed either by monitoring the amount of cell-bound 125 I-labelled toxin that could not be released with lactose or by measuring the time required for transport of the toxins into a state where they were protected against neutralizing antibodies. The presence of toxin in this state was monitored by measuring inhibition of protein synthesis after a subsequent prolonged incubation period. In the case of abrin, straight lines were found in both cases when the data were plotted according to Arrhenius. The activation energies estimated was 18–21 kcal/mol (75–88 kJ/mol) in the case of uptake of [ 125 I]abrin and 15–19 kcal/mol (63–79 kJ/mol) when the indirect method was used. After internalization of [ 125 I]abrin and ricin a fraction of the radioactive material is released to the medium. Most of this material can be precipitated by trichloroacetic acid (TCA). There is a rapid release during the first 30 min and then over the next few hours the release occurs at a constant, but lower rate. The release of ricin was not affected by addition of colchicine, cytochalasin B (CB), ammonium chloride, sodium azide or bovine serum albumin, whereas the degradation of ricin was reduced by the above mentioned compounds (except albumin). The release of ricin was strongly temperature-dependent with a sharp transition at about 20 °C. The activation energies for the release above and below 20 °C were found to be 2.5 and 31 kcal/mol (10.5 and 172 kJ/mol), respectively.

How protein toxins enter and kill cells

The toxins described in this chapter (Table 1) are produced by certain pathogenic bacteria and certain poisonous plants. So far our knowledge is very limited as to the reason why these organisms produce toxins.

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.