Concanavalin A induced apoptosis in fibroblasts: the role of cell surface carbohydrates in lectin mediated cytotoxicity.

Abstract

Cell surface carbohydrates play important regulatory roles during development and in tissue homeostasis by mediating cellular signalling. Concanavalin A (con A) cross links mannose residues on cell surfaces. We used con A to examine the role of cell surface carbohydrates in apoptosis. Balb/c 3T3 (3T3) and diploid human gingival fibroblasts (HGF) incubated with con A (50 micrograms/mL) exhibited rounding, reduction of cell size, loss of cytoskeletal definition, nuclear condensation, and ultrastructural changes consistent with apoptotic cell death. However oligonucleosomal DNA fragmentation was not observed. The sugar methyl-alpha-D-mannopyranoside (MADM) competes for binding sites with con A but failed to induce apoptosis. Cell survival assays after con A treatment demonstrated a concentration dependent (5-500 micrograms/mL) loss of cell viability in 3T3 and HGF cells which was blocked by MADM. The relationship between cell surface binding of con A and cell death was studied in 3T3 cells labelled with varying concentrations of FITC-con A. Flow cytometry and cell survival analyses revealed that smaller cells with lower total cell surface FITC-con A binding sites were approximately 8 times more susceptible to cell death than larger cells with higher number of binding sites. This suggests a positive relationship between cell size, number of con A binding sites, and cell death. Flow cytometric cell cycle analysis of HGF cells showed a 46% reduction in the proportion of G1 phase cells but there was little change in the relative proportions of S and G2+M phase cells. 3H-thymidine labelling of con A treated cells showed a five-fold decrease (chi sq; P < 0.05) in the percentage of labelled cells, indicating blockade of cell cycle transit into S. Thus, cell death occurred predominantly in G1, possibly due to inhibition of protein synthesis which in turn prevented entry of cells into S phase. 35S-methionine uptake in con A treated cells was significantly reduced (approximately 42% con A 50 micrograms/mL; approximately 67% con A 500 micrograms/mL; P < 0.05) compared to untreated controls indicating inhibition of de novo protein synthesis. SDS-PAGE of total cellular proteins confirmed reduction of predominantly lower molecular mass proteins after con A treatment. Con A treatment (50 micrograms/mL) induced a 25% reduction of free intracellular calcium ion concentration over 30 min suggesting that calcium dependent enzymes and translational mechanisms may be inhibited. Collectively, these results indicate that con A binding of cell surface carbohydrates can induce apoptotic cell death in fibroblastic cells due in part to protein synthesis inhibition.