Induction of apoptosis in human retinoblastoma cells by topoisomerase inhibitors.
OBJECTIVE
To examine the apoptotic effect induced in human retinoblastoma Y79 cells by camptothecin, etoposide, and amsacrine, to examine the effect of these drugs on the expression of many apoptosis-related modulators, and to test the antiapoptotic effect exerted by insulin-like growth factor-I (IGF-I).


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 IGF-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-BP3 that is induced by drug treatment.
Giuliano
M
M
Institute of Biological Chemistry, University of Palermo, Italy.
Lauricella
M
M
Vassallo
E
E
Carabillò
M
M
Vento
R
R
Tesoriere
G
G
D000677:Amsacrine / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002166:Camptothecin / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis / Q000187:drug effects*; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D019572:Retinal Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D012175:Retinoblastoma / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D018972:Insulin-Like Growth Factor Binding Protein 3; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D059004:Topoisomerase I Inhibitors; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D000677:Amsacrine; D003609:Dactinomycin; D005047:Etoposide; D003513:Cycloheximide; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin