Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.
When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, 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 treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.
Carlberg
M
M
Department of Tumor Pathology, Karolinska Institutet, Stockholm, Sweden.
Dricu
A
A
Blegen
H
H
Kass
G E
GE
Orrenius
S
S
Larsson
O
O
D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002461:Cell Line, Transformed; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects; D006031:Glycosylation; D006801:Humans; D013539:Simian virus 40; D019284:Thapsigargin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology*
D014415:Tunicamycin; D019284:Thapsigargin; D004247:DNA; D002118:Calcium