title: Apoptosis of N-type neuroblastoma cells after differentiation with 9-cis-retinoic acid and subsequent washout.
abstract: BACKGROUND
  The overall survival rate for patients with neuroblastoma has improved over the past two decades, but long-term survival for the subgroup of patients with high-risk disease remains low. In recent years, there has been interest in the potential clinical use of drugs able to induce differentiation of neuroblastoma cells. Since 9-cis-retinoic acid induces better and more sustained differentiation of neuroblastoma in vitro than other retinoic acid isomers, this may be a more appropriate retinoid for use in neuroblastoma therapy.
  
  
  OBJECTIVE
  The purpose of this work was to compare the long-term effects of all-trans- and 9-cis-retinoic acid on neuroblastoma differentiation using an N-type (neuroblastic) cell line, SH SY 5Y, as an in vitro model. In addition, we wanted to find out whether 9-cis-retinoic acid would induce programmed cell death (apoptosis) in these N-type neuroblastoma cells and to determine whether the effects of either 9-cis- or all-trans-retinoic acid are dependent on their continued presence in the culture medium.
  
  
  METHODS
  SH SY 5Y cells were incubated in either the continued presence of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic acid followed by culture in the absence of retinoid for up to 13 days. Morphologic changes were observed using phase-contrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodide-stained cells and by using terminal deoxynucleotidyl transferase to end-label DNA fragments in situ in apoptotic cells.
  
  
  RESULTS
  Culture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. These effects were maintained in the continuous presence of each retinoic acid isomer but were more profound in cells treated with 9-cis-retinoic acid. The differentiation of cells treated with all-trans-retinoic acid was reversible once retinoic acid was removed from the medium. Conversely, apoptosis was induced in cells treated with 9-cis-retinoic acid for 5 days and cultured for 9 days (4 days after washout) but not in cells cultured in the continuous presence of 9-cis-retinoic acid. This effect was specific to 9-cis-retinoic acid.
  
  
  CONCLUSIONS
  Previous studies have demonstrated differential responses to all-trans-retinoic acid in N- and S-type (substrate-adherent or Schwann-like) neuroblastoma cells: Apoptosis is induced in S-type cells, whereas differentiation occurs in N-type cells. The present results show that, unlike all-trans-retinoic acid, 9-cis-retinoic acid induces both differentiation and apoptosis in N-type SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9-cis-retinoic acid from the culture medium.
  
  
  CONCLUSIONS
  Since both differentiation and apoptosis are involved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apoptosis on treatment and subsequent removal of 9-cis-retinoic acid implies that drug scheduling may be an important parameter affecting therapeutic efficacy.
authors.affiliation: Department of Medicine, University of Newcastle Upon Tyne, U.K.
authors.forename: P E
authors.initials: PE
authors.lastname: Lovat
authors.forename: H
authors.initials: H
authors.lastname: Irving
authors.forename: M
authors.initials: M
authors.lastname: Annicchiarico-Petruzzelli
authors.forename: F
authors.initials: F
authors.lastname: Bernassola
authors.forename: A J
authors.initials: AJ
authors.lastname: Malcolm
authors.forename: A D
authors.initials: AD
authors.lastname: Pearson
authors.forename: G
authors.initials: G
authors.lastname: Melino
authors.forename: C P
authors.initials: CP
authors.lastname: Redfern
mesh_terms: D000077556:Alitretinoin; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D004253:DNA Nucleotidylexotransferase; D005434:Flow Cytometry; D007124:Immunoenzyme Techniques; D009447:Neuroblastoma / Q000145:classification / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology*; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D000077556:Alitretinoin; D014212:Tretinoin; D004253:DNA Nucleotidylexotransferase