Kinetic analysis of choriocarcinoma cell intoxication induced by ricin and ricin A chain immunotoxin.

The kinetics of protein synthesis inhibition was studied in the choriocarcinoma-derived BeWo cell line treated with ricin and an immunotoxin (IT) constructed by linking a specific antibody to the A chain of ricin. The IT was specifically cytotoxic to BeWo and other choriocarcinoma cells. The multistep process underlying this kinetics was explored using two mathematical models where the protein synthesis-inactivation step is preceded by one or two processing rate-limiting steps. Theoretical curves were computed for different concentrations of toxic reagents. Two processing steps were found necessary to predict the duration of the observed latent period before initiation of protein synthesis inhibition. With this model, a satisfactory fit was obtained. A mathematical modeling of the intoxication induced by ricin or ricin A chain IT thus requires two processing steps to account for the data observed. In addition, the data suggest that the cytoplasmic internalization of ricin is a slow process compensated by an extremely fast enzymatic inactivation of ribosomal activity. This implies that in this and similar systems, endocytosis might not be involved in the cytoplasmic internalization of the few molecules of ricin A chain responsible for cell intoxication.

Properties of immunotoxins against a glycolipid antigen associated with Burkitt's lymphoma.

A monoclonal immunoglobulin M (IgM) antibody (38-13) which recognizes Burkitt's lymphoma (BL) cells, by reacting with the neutral glycolipid Gal alpha 1 leads to 4-Gal beta 1 leads to 4-Glc beta 1 leads to 1-ceramide, was recently characterized. This monoclonal IgM was coupled to either ricin A chain or gelonin. The two different immunotoxins obtained retained the apparent immunological specificity of 38-13 IgM, as shown by flow cytofluorometry analysis and complement-dependent cytotoxicity test. The BL Ramos cells and the apparently irrelevant Epstein-Barr virus-containing lymphoblastoid Priess cells were used as targets in in vitro assays of the cytotoxic properties of the two immunotoxins by measuring the inhibition of protein synthesis. Isolated ricin A chain, gelonin, and 38-13 IgM exhibited very low intrinsic cytotoxicity on both target cells. 38-13 ricin A chain and 38-13 gelonin conjugates exerted toxic effects on both target cells which were about 6000-fold and 3000-fold higher than uncoupled ricin A chain and gelonin, respectively. The toxicity of these conjugates almost reached that of intact ricin. On Ramos BL cells, the kinetics of action of the 38-13 ricin A chain conjugate was almost as fast as that of intact ricin, because 50% protein synthesis inhibition was reached after 3 hr. In contrast, the kinetics of action in the non-BL Priess was much slower (50% protein synthesis inhibition after 10 hr). An obviously irrelevant immunotoxin (anti-trinitrophenol IgM-ricin A chain) had no significant cytotoxic effect on BL Ramos and non-BL Priess cells. An excess of D-galactose was shown previously to inhibit the 38-13 IgM from binding to the reactive glycolipid antigen bearing a terminal galactose. An excess of D-galactose (0.1 M) inhibited the cytotoxic effect of the two 38-13 immunotoxins, whereas it did not prevent the cytotoxic effect of the anti-trinitrophenol immunotoxin on the same trinitrophenol labeled target cells. These data suggest that the cytotoxic effect observed with 38-13 immunotoxins on non-BL Priess cells was mediated through their binding to a very low number of antigenic sites undetectable by conventional immunological methods. The main characteristics of 38-13 immunotoxins appear to be their fast kinetics of action and the very low number of antigenic sites required for the expression of their toxic effects. These properties could be related to the glycolipid nature of the reacting antigen. Such glycolipid antigens would represent valuable targets for therapeutic use of immunotoxins.

Decreased accessibility of globotriaosylceramide associated with decreased tumorigenicity in Burkitt's lymphoma variants induced by immunoselection.

To investigate a role for globotriaosylceramide (Gb3) as a tumor-associated antigen, variant cells resistant to treatment with complement and monoclonal antibody 38-13, which recognizes Gb3, were selected from a Burkitt's lymphoma cell line, Ramos. Variant cells displayed a clear decrease of antibody-binding capacity whereas the amount of Gb3 at their plasma membrane was not significantly different from that of Ramos parental cells. This demonstrated a reduced accessibility of Gb3 at the surface of variant cells. In parallel, no changes in other surface antigens were recorded as compared to those in Ramos cells. No changes of proliferative properties in suspension culture or of c-myc expression were observed although variant cells showed a decreased colony-forming capacity in agar. Variant cells showed a significant reduction in tumorigenic potential when injected s.c. into nude mice. The decreased tumorigenicity appeared related to the low antibody-binding capacity because both tumorigenicity and Gb3 antigenicity were recovered in parallel in revertant cells growing in suspension culture. In vivo, after two transplantations of variant cells into mice, cells isolated from the few induced tumors still retained the low antibody-binding capacity.

Determination of antibody-complement mediated cytotoxicity using ATP release induced by a monoclonal antibody against the Burkitt lymphoma associated globotriaosylceramide antigen.

The kinetics of ATP release from Burkitt lymphoma target cells following complement activation by rat monoclonal IgM antibody have been used to develop a standard assay for antibody-complement mediated cytotoxicity. This assay permitted the study of the reactivity of the monoclonal IgM with Burkitt and non-Burkitt cell lines and to perform IgM-complement mediated cytotoxicity inhibition experiments with oligosaccharides. This led to a precise definition of the antigenic determinant recognized by the monoclonal IgM on globotriaosylceramide.

Sponge glycoconjugates: immunological properties and localization by fluorescent antibodies and lectins.

By immunodiffusion and immunoelectrophoresis, glycoconjugates previously isolated from a sponge, Spongia officinalis, and fractionated on lectins, showed identical immunological behaviour which was species specific. By fluorescent antiserum and lectins, these glycoconjugates were located on sections of two sponges, S. officinalis and Chondrosia reniformis. A strong pericellular staining occurred together with a diffused labeling.

Antitumor activity against L1210 mouse leukemia of immunotoxins containing a monoclonal antibody to the glycolipid globotriaosylceramide.

Since immunotoxin (IT) containing the antiglobotriaosylceramide monoclonal antibody was found to be cytotoxic in murine L1210 leukemia cells, its potential antitumor activity could be evaluated in animals using the L1210 model. In vitro, L1210 cells incubated IT before grafting in DBA/2 mice failed to induce leukemia. All tumor cells were neutralized by IT. In animals, a significant but limited therapeutic effect on leukemic mice was obtained when IT was injected i.p. shortly after L1210 cell grafting. In contrast, no toxic effect of IT was observed in non-leukemic mice at doses far above those used in our therapeutic treatment. The potentiation effect of chloroquine on IT was moderated when a cloning efficiency assay was used, but 70% of the mice grafted with in vitro chloroquine-treated L1210 cells were cured with IT treatment.

Genistein resistance in human leukaemic CCRF-CEM cells: selection of a diploid cell line with reduced DNA topoisomerase II beta isoform.

Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo. From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly cross-resistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2'-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold cross-resistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximately 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II beta isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.

Cockroach collagen: isolation, biochemical and biophysical characterization.

Collagen fibrils from the mesenteric connective sheath of the adult cockroach Periplaneta americana were extracted by enzymatic digestion with pepsin and were purified. Chromatographic studies and sodium dodecylsulfate electrophoresis revealed the presence of a single chain. It was demonstrated that the structure of this collagen could be represented by the formula (alpha)3. The amino acid composition is typical of collagens (one-third glycine, and a high imino acid content) and similar to that of type II. The carbohydrate content was high (8.8%), and the cyanogen bromide pattern was different from that of known collagens. The chains were linked by the stable intermolecular bond dihydroxylysinonorleucine. The banding patterns of the segment-long-spacing crystallites and of the reconstituted fibrils were similar to type I collagen. The molecular weight (Mr 280,000) and length (285 nm) were typical, but the denaturation temperature was high (38.5 degrees C). It was concluded that cockroach mesenteric collagen showed the characteristic features of invertebrate mesodermal collagens, except that of the thermal stability of the triple-helical structure.

[Regulation of the biosynthesis of macromolecules of the intercellular corneal matrix]. / Etude de la régulation de la biosynthèse des macromolécules de la matrice intercellulaire de la cornée

The influence of a 1M CaCl2 extract and of a collagenase digest of corneal stroma of the biosynthesis of the macromolecules of corneal stroma was investigated. Calf corneas were incubated "in vitro" with radioactive tracers (14C-L-proline; 3H-D-glucosamine or 35SO4) in the presence or absence of the above extracts. After incubation the corneas are submitted to a fractional extraction in order to separate the major macromolecular fractions of the stroma. An increase in incorporation of all tracers is observed in the 1M CaCl2 (CTC), TCA and urea-extracts (containing resp. the diffusible macromolecules, proteoglycans, polymeric collagen and structural glycoproteins) in the presence of the macromolecular extracts and also with the collagenase-hydrolysate of cornea. These results show the existence of a stimulation of the biosynthesis of intracellular matrix macromolecules of the cornea by corneal extracts, probably through positive "feedback" type of mechanism.

Comparison of inhibitory effect of galactose analogs on the binding and cytotoxicity of an anti-globotriaosylceramide monoclonal antibody coupled or not coupled to pokeweed antiviral protein.

The results described here provided an example of a human IgM monoclonal antibody against a tumor-associated glycolipid and of the unusual properties of its corresponding immunotoxin (IT). The monoclonal antibody referred to as 38-13 has been previously described and reacted with the globotriaosylceramide [Gb3:Gal(alpha 1----4)-Gal(beta 1----4)-Glc(beta 1----1)ceramide] specifically expressed on surface membrane of Burkitt's lymphoma (BL) cells. An immunotoxin (38-13 IT) combined with the pokeweed antiviral protein (PAP) toxin via S-S bridges showed paradoxically a lower cytotoxic effect in BL Ramos cells than in non-BL cells such as leukemic mouse L1210 cells, while these cells appeared not to be involved by flow cytometric analysis and complement-dependent cytotoxicity. Consequently, the inhibitory effect of selective galactose analogs on the binding and the cytotoxicity of 38-13 antibody conjugated or not to PAP toxin was compared on BL and non-BL cells. Only the galactose blocked in alpha configuration provided a fine inhibition of 38-13 binding on BL Ramos cells and both alpha and beta-galactose allowed us to establish a clear distinction between the pathway entry of 38-13 IT in BL and non-BL cells; in close correlation with the 38-13 binding specificity the 38-13 IT cytotoxic effect in Ramos BL cells could also be prevented by alpha-Gal only, suggesting that this toxic action is probably mediated through the IT binding to Gb3 antigenic sites. In contrast, on apparently irrelevant L1210 cells, 38-13 IT showed a cytotoxic effect which was inhibited preferentially by lactose (Gal in beta configuration). It was discussed that IT binding alone to either antigenic sites which are inhibited by the hapten alpha-Gal, or nonspecific sites which can compete with the hapten beta-Gal is unable to induce efficient killing of cells. But cooperation of both bindings might give an attractive explanation of IT cytotoxic effect. It was concluded that the unexpected activity of 38-13 IT in non-BL cells probably could be mediated through an active macromolecular transport process which could implicate a beta-galactoside-binding protein (lectin).

Sugar-specific endocytosis of glycoproteins by Lewis lung carcinoma cells.

Lewis lung carcinoma cells from tumors, metastasis nodules, or from culture bind fluorescent derivatives of neoglycoproteins containing alpha-D-glucose residues: This binding is competitively inhibited by neoglycoproteins containing alpha-D-glucose, by mannan, and by several other neoglycoproteins. Cell binding and uptake of the fluorescent derivatives of the neoglycoproteins was quantified by lysing the cells with an alkylpolyol (MAC 19 or MAC 18) and measuring the fluorescence intensity of the supernatant. The amount of cell-associated neoglycoprotein was higher at 37 degrees C than at 4 degrees C with LLC from tumor. The binding and uptake were inhibited by glycoconjugates containing alpha-D-glucose. These results suggest the presence of sugar specific receptors in Lewis lung carcinoma cells which are involved in a sugar-specific binding and endocytosis phenomenon. The implication of the existence of a carbohydrate-binding protein on the surface of Lewis lung carcinoma cells are discussed with regard to the in vivo behaviour of these cells, especially in relation to their metastatic properties and to the possibility of using neoglycoproteins as specific carriers of cytotoxic drugs. Hybrid molecules of gelonin and neoglycoprotein containing alpha-D-glucose were used as targetted toxin: The targetted toxin was found to bind to and to enter the intact cells and was 100 times more toxic than free drug.