A study of polyamine modulation of etoposide-induced apoptosis in HL-60 cells
Etoposide induces apoptosis in HL-60 human promyelogenous leukemic cells as assessed by electron microscopy, agarose gel electrophoresis, flow cytometry and a DNA fragmentation ELISA within 2- 4 h and is essentially complete by 24 h. This system was used to investigate whether the catabolism of polyamines, of which induction of spermidine/spermine N1-acetyltransferase (N1-SSAT) is the rate-limiting step, was a signal for the initiation of apoptosis. This hypothesis was based on the fact that calcium can activate apoptosis and induce N1-SSAT activity. Polyamine depletion using -difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme in the polyamine biosynthetic pathway, and polyamine supplementation through the addition of exogenous polyamines were used to determine how polyamines modulate apoptosis. The relationship between N1-SSAT induction and apoptosis was investigated using polyamine analogues. The H82 (human small-cell lung carcinoma) cell line which lacks a transcriptionally active N1-SSAT gene was used to determine its sensitivity to etoposide. N1-SSAT induction did not correlate with the induction of apoptosis and was more likely to be associated with secondary necrosis in this system. DFMO could reduce the extent of etoposide induced apoptosis as could exogenously supplied spermine. The reason for this contradiction may be that DFMO reduces the activity of ODC, and spermine, through stimulating antizyme, can also reduce ODC activity by promoting its degradation, therefore, both prevent polyamine biosynthesis. Polyamine analogues which were potent inducers of N1-SSAT activity were poor inducers by DNA fragmentation and induced a lower level of apoptosis than atoposide. Exposure of H82 cells to etoposide did not result in N1-SSAT induction and only a small increase in DNA fragmentation. Therefore, N1-SSAT activity may be required for apoptosis, but at a low level as this correlated with highest DNA fragmentation.