Apoptosis of N-Type Neuroblastoma Cells After Differentiation With 9-cis-Retinoic Acid and Subsequent Washout
Authors: P. E. Lovat, H. Irving, A. D. J. Pearson, C. P. F. Redfern, A. J. Malcolm, M. Annicchiarico-Petruzzelli, F. Bernassola, G. Melino
Journal: JNCI Journal of the National Cancer Institute (1997)

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. Purpose: 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 phasecontrast and scanning electron microscopy. Apoptosis was determined by flow cytometry of propidium iodidestained 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 Ntype SH SY 5Y neuroblastoma cells. However, apoptosis was dependent on removal of 9- cis -retinoic acid from the culture medium. Implications: 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.