title: Induction of apoptosis in human retinoblastoma cells by topoisomerase inhibitors.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry, University of Palermo, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Giuliano
authors.forename: M
authors.initials: M
authors.lastname: Lauricella
authors.forename: E
authors.initials: E
authors.lastname: Vassallo
authors.forename: M
authors.initials: M
authors.lastname: Carabillò
authors.forename: R
authors.initials: R
authors.lastname: Vento
authors.forename: G
authors.initials: G
authors.lastname: Tesoriere
mesh_terms: 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
chemical_list: 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