Isolation and characterization of two mouse L cell lines resistant to the toxic lectin ricin.
Two variant mouse L cell lines (termed CL 3 and CL 6) have been selected for resistant to ricin, a galactose-binding lectin with potent cytotoxic activity. The resistant lines exhibit a 50 to 70% decrease in ricin binding and a 300- to 500-fold increase in resistance to the toxic effects of ricin. Crude membrane preparations of CL 3 cells have increased sialic acid content (200% of control), while the galactose, mannose, and hexosamine content is within normal limits. Both the glycoproteins and glycolipids of CL 3 cells have increased sialic acid, with the GM3:lactosylceramide ratios for parent L and CL 3 cells being 0.29 and 1.5, respectively. In contrast, the membranes of CL 6 cells have a decrease in sialic acid, galactose, and hexosamine content with mannose being normal. Both cell lines have specific alterations in glycosyltransferase activities which can account for the observed membrane sugar changes. CL 3 cells have increased CMP-sialic acid:glycoprotein sialyltransferase and GM3 synthetase activities, while CL 6 cells have decrease UDP-GlcNAc:glycoproteinN-acetylglucosaminyltransferase and DPU-galactose:glycoprotein galactosyltransferase activities. The increased sialic acid content of CL 3 cells serves to mask ricin binding sites, since neuraminidase treatment of this cell line restores ricin binding to essentially normal levels. However, the fact that neuraminidase-treated CL 3 cells are still 45-fold resistant to ricin indicates that either a special class of productive ricin binding sites is not being exposed or that the cell line has a second mechanism for ricin resistance. (+info
Induction of lasting complete regression of preformed distinct solid tumors by targeting the tumor vasculature using two new anti-endoglin monoclonal antibodies.
Endoglin (EDG, CD105) is a proliferation-associated antigen on endothelial cells. In this study, two new anti-EDG monoclonal antibodies (mAbs) Y4-2F1 (or termed SN6j) and P3-2G8 (SN6k) were generated and used for treating distinct preformed tumors. These mAbs, both IgG1-kappa antibodies, cross-reacted weakly with mouse endothelial cells but defined epitopes different from the epitope defined by a previously reported anti-EDG mAb K4-2C10 (B. K. Seon et al., Clin. Cancer Res., 3: 1031-1044, 1997). SN6j and SN6k reacted strongly with human endothelial cells and vascular endothelium of malignant human tissues but showed no significant reactivity with tumor cells per se. The deglycosylated ricin A chain (dgRA) conjugates of the two mAbs showed a weak but specific cytotoxic activity against murine endothelial cells in vitro. In the therapeutic studies, severe combined immunodeficient mice were inoculated s.c. with MCF-7 human breast cancer cells and left untreated until palpable tumors of distinct size (4-6 mm in diameter) appeared. Mice with the distinct tumors were treated by i.v. administration of individual anti-EDG conjugates, unconjugated mAbs, or a control conjugate. Long-lasting complete regression of the tumors was induced in the majority of tumor-bearing mice (n = 8 for each conjugate) when 40 microg of the individual conjugates were administered three times via the tail vein. It is remarkable that the tumors remained regressed without further therapy for as long as the mice were followed (i.e., 100 days). Control conjugate did not induce regression of the tumors in any of the treated mice, although weak nonspecific effects were observed in some of the mice (n = 8). The effects of unconjugated mAbs were small with the dose used, i.e., 34 microg three times. The anti-EDG conjugates showed antiangiogenic activity in the dorsal air sac assay in mice. The results suggest good potential of these conjugates for the clinical application. (+info
Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.
The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins. (+info
Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells.
We have found that the antioxidant N-acetylcysteine (NAC) strongly inhibited ricin-induced apoptotic cell death in U937 cells (human myeloid leukemia), as judged by cytotoxicity, nuclear morphological change, and DNA fragmentation. Consistent with these observations, a significant depletion of cellular glutathione was observed in ricin-treated cells, and NAC prevented the decrease in cellular glutathione. On the other hand, among the caspase inhibitors tested, Z-Asp-CH2-DCB, which inhibited ricin cytotoxicity, also suppressed ricin-mediated glutathione depletion, while NAC did not affect the generation of caspase-3 like activity in ricin-treated cells. These results suggest that glutathione loss takes place downstream from caspase activation during the ricin-induced apoptotic process. Treatment with a specific inhibitor of glutathione biosynthesis, buthionine sulfoximine (BSO) failed to induce apoptosis, and had no effect on the overall extent of ricin-induced apoptosis, even though the glutathione level was decreased to less than 5% of the control level. However, NAC still protected against ricin-induced apoptosis in the BSO-treated cells. We conclude that glutathione loss is one of several apoptotic changes caused by ricin, but is not a sufficient factor for the progress of apoptosis. NAC may prevent ricin-induced apoptosis through maintaining an intracellular reducing condition by acting as a thiol supplier. (+info
Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.
The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-beta-cyclodextrin (MbetaCD) to selectively extract cholesterol from the plasma membrane. MbetaCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MbetaCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MbetaCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MbetaCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles. (+info
A laboratory model of toxin-induced hemolytic uremic syndrome.
BACKGROUND: Verocytotoxin-producing (Shiga-like toxin-producing) Escherichia coli infection is the principal cause of hemolytic uremic syndrome (HUS). The pathogenesis is unclear, and there is a need for animal models. These are impeded by the different distribution of verocytotoxin receptors between species. We have circumvented this restriction using ricin, which gains entry into cells via various galactose receptors. Like verocytotoxin, ricin specifically cleaves a single adenine from ribosomal RNA. METHODS: Rats were given ricin at a dose of 6.7 micrograms/100 g body wt, with or without lipopolysaccharide at 10 micrograms/100 g body wt. Lipopolysaccharide alone or saline were used as controls. Changes in glomerular filtration rate, hematological parameters, histology, and plasma cytokine concentrations were measured. RESULTS: Extensive glomerular thrombosis, pyknotic nuclei, and an infiltration of ED1-positive cells into glomeruli were observed eight hours after an injection of ricin. Other vascular beds were unaffected. Histologic changes were preceded by oliguric renal failure, hemolysis, and thrombocytopenia. Ricin produced a rise in plasma concentrations of monocyte chemotactic protein-1, > tumor necrosis factor-alpha, > interleukin-1 beta, > interleukin-6. Interferon-gamma showed a small increase at the end of the experiment. CONCLUSIONS: Ricin induces glomerular thrombotic microangiopathy, closely resembling that which occurs in verocytotoxin-producing E. coli-induced HUS. As in HUS, high concentrations of proinflammatory cytokines are present, which are probably a result of cytokine superinduction by the toxin. (+info
Galectin-3 and polarized growth within collagen gels of wild-type and ricin-resistant MDCK renal epithelial cells.
Previous studies (Q. Bao and R. C. Hughes (1995) J. Cell Sci., 108, 2791-2800) showed that the beta-galactoside-binding protein, galectin-3, is secreted onto the basolateral surface domains of Madin-Darby canine kidney MDCK cells growing as polarized cysts within a collagen gel. The growth and enlargement of such cysts were shown to be increased significantly when cultured in the presence of antibodies directed against the lectin and were slowed down by addition of exogenous galectin-3. These results suggested a role for galectin-3, interacting with appropriately glycosylated surface receptors, as a negative growth regulator in the development of MDCK cysts, a well-known model for renal epithelial morphogenesis. In the present report we have tested this proposal by use of a ricin-resistant mutant of MDCK cells that is unable to transfer galactose residues during biosynthesis of cellular glycoconjugates and hence lacks extracellular receptors for galectin-3. We find that when grown within collagen gels, the mutant cell cysts grow significantly faster than wild-type cell cysts. Furthermore, they form nonspherical and tubular cysts that are induced in wild-type cell cysts only under the influence of the morphogen, hepatocyte growth factor (HGF). (+info
Promotion of ATP and S-140 to ribosome inactivation with camphorin, cinnamomin, and other RNA N-glycosidases.
AIM: To study the effect of ATP and extra-ribosomal factors (S-140) on type I and type II RNA N-glycosidases in inactivating ribosome. METHODS: The activity of ATP and S-140 was determined by characterization of R-fragment in gel. An improved two-step method of cell-free protein synthesis system was used to quantitate the requirements of S-140 in ribosome inactivation. RESULTS: IC50 ratios of camphorin, gamma-momorcharin, luffin S, luffin A, trichosanthin (type I); and ricin, ricin A-chain; cinnamonin, cinnamomin A-chain (type II) between the absence and presence of ATP and S-140 were 3108, 151, 51, 45, 15; and 47, 7, 26, 12, respectively. CONCLUSION: The ribosome-inactivating activity of type II ribosome-inactivating proteins, including intact protein and its A-chain, was promoted by ATP and S-140. Camphorin showed a significant difference from cinnamomin in need of ATP and S-140 for such promoting. (+info