INDUCTION OF APOPTOSIS IN HUMAN RETINOBLASTOMA CELLS BY TOPOISOMERASEINHIBITORS

PURPOSE. To examine the apoptotic effect induced in human retinoblasto ma Y79 cells by camptothecin, etoposide, and amsacrine, to examine the effect of these drugs on the expression of many apoptosis-related mod ulators, and to test the antiapoptotic effect exerted by insulin-like growth factor-I (IGF-I). METHODS. Morphologic features of apoptosis we re demonstrated using acridine orange-ethidium bromide staining and el ectron microscopy. DNA fragmentation was determined by means of an in situ cell detection procedure (TdT-dUTP terminal nick-end labeling [TU NEL]) or by electrophoresis on agarose gels and was quantified by enzy me-linked immunosorbent assay. The expression of apoptosis-related mod ulators was studied by western blot analysis. The processing of latent p53 was examined by means of pulse-chase analysis. RESULTS. Camptothe cin, 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 aft er a lag of 8 hours and increased for as long as 24 hours. It was lowe r in cells treated with ICE-I, a potent mitogenic factor. Camptothecin and etoposide increased the p53 level after 4 hours of treatment, bef ore 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 p2 1, 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 ce lls pretreated with IGF-I. CONCLUSIONS. Topoisomerase inhibitors induc e apoptosis in Y79 cells. This event is accompanied by a decrease in t he expression of Bcl-2, a death antagonist, and an increase in that of Bax, a death agonist. A probable consequence of these modifications i s the activation of ICE-like activity with degradation of poly-(adenos ine diphosphate [ADP] ribose)-polymerase. Insulin-like growth factor-I exerts an antiapoptotic action in Y79 cells, and this function is mos t likely reduced by the overexpression of IGF-BPS that is induced by d rug treatment.