SHORT EXPOSURES TO TUNICAMYCIN INDUCE APOPTOSIS IN SV40-TRANSFORMED BUT NOT IN NORMAL HUMAN FIBROBLASTS

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunica mycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts, A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive, A 2-h pulse tr eatment with tunicamycin was found to be almost as effective as a cont inuous 48-h treatment in killing the cells, Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). Th e morphology of the dying tunicamycin-treated 90VAVI cells suggested t hat they were undergoing apoptosis. This was also supported by the app earance of nuclear condensation, as assayed by propidium iodide uptake , which was detectable within 2 h after tunicamycin addition, Furtherm ore, analysis of DNA from tunicamycin-treated 90VAVI cells by field in version gel electrophoresis revealed DNA degradation into 50 kbp fragm ents within 2 h, and conventional agarose gel electrophoresis showed ' DNA laddering', indicating internucleosomal DNA cleavage, detectable a fter 36 h, Together with the finding that tunicamycin within seconds c aused an elevation of [Ca2+](i), a well documented early feature of ap optosis in many experimental systems, these results strongly suggest t hat tunicamycin-induced cell death in 90VAVI is due to apoptosis, The short tunicamycin exposure required to trigger cell death in 90VAVI in dicates that the apoptotic process is irreversibly induced soon after its addition, It seems unlikely that the pool of one or several specif ic N-linked glycoproteins could be depleted during such a short period , Instead the overall accumulation of unglycosylated proteins in ER mi ght contribute to the apoptotic response in 90VAVI, Tunicamycin also k illed and induced DNA degradation in the breast cancer cell line MDA-2 31.