METHODS. Morphologic features of apoptosis were demonstrated using acridine orange-ethidium bromide staining and electron microscopy. DNA fragmentation was determined by means of an in situ cell detection procedure (TdT-dUTP terminal nick-end labeling [TUNEL]) or by electrophoresis on agarose gels and was quantified by enzyme-linked immunosorbent assay. The expression of apoptosis-related modulators was studied by western blot analysis. The processing of latent p53 was examined by means of pulse-chase analysis.

RESULTS. Camptothecin, etoposide, and amsacrine induced apoptosis in Y79 cells in a dose-dependent manner; camptothecin was the most efficacious compound. The effect, which was dependent on macromolecular synthesis, appeared after a lag of 8 hours and increased for as long as 24 hours. It was lower in cells treated with ICE-I, a potent mitogenic factor. Camptothecin and etoposide increased the p53 level after 4 hours of treatment, before the onset of apoptosis. This effect seemed to be a consequence of the conversion of latent p53 to one that is transcriptionally active. The drugs also induced an increase in p53-related proteins, such as p21, Bax, and IGF binding protein-3 (IGF-BP3), and caused a significant reduction of the Bcl-2 level. The latter effect was less evident in cells pretreated with IGF-I.

CONCLUSIONS. Topoisomerase inhibitors induce apoptosis in Y79 cells. This event is accompanied by a decrease in the expression of Bcl-2, a death antagonist, and an increase in that of Bax, a death agonist. A probable consequence of these modifications is the activation of ICE-like activity with degradation of poly-(adenosine diphosphate [ADP] ribose)-polymerase. Insulin-like growth factor-I exerts an antiapoptotic action in Y79 cells, and this function is most likely reduced by the overexpression of IGF-BPS that is induced by drug treatment.