APOPTOSIS OF N-TYPE NEUROBLASTOMA-CELLS AFTER DIFFERENTIATION WITH 9-CIS-RETINOIC ACID AND SUBSEQUENT WASHOUT

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 ye ars, there has been interest in the potential clinical use of drugs ab le to induce differentiation of neuroblastoma cells. Since 9-cis-retin oic acid induces better and more sustained differentiation of neurobla stoma in vitro than other retinoic acid isomers, this may be a more ap propriate retinoid for use in neuroblastoma therapy. Purpose: The purp ose of this work was to compare the long-term effects of all-trans- an d 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 progr ammed 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 presenc e of all-trans- or 9-cis-retinoic acid or for 5 days with retinoic aci d followed by culture in the absence of retinoid for up to 13 days. Mo rphologic changes mere observed using phase-contrast and scanning elec tron microscopy. Apoptosis was determined by flow cytometry of propidi um iodide-stained cells and by using terminal deoxynucleotidyl transfe rase to end-label DNA fragments in situ in apoptotic cells. Results: C ulture of SH SY 5Y cells with all-trans- or 9-cis retinoic acid for 5 days induced morphologic differentiation and inhibited cell growth. Th ese effects were maintained in the continuous presence of each retinoi c acid isomer but were more profound in cells treated with 9-cis-retin oic acid. The differentiation of cells treated with all-trans-retinoic acid was reversible once retinoic acid was removed from the medium. C onversely, 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-retinoi c acid in N- and S-type (substrate-adherent or Schwann-like) neuroblas toma cells: Apoptosis is induced in S-type cells, whereas differentiat ion 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, apoptos is was dependent on removal of 9-cis-retinoic acid from the culture me dium. Implications: Since both differentiation and apoptosis are invol ved in tumor regression, 9-cis-retinoic acid may be a more appropriate retinoid for clinical trials in neuroblastoma. The dependence of apop tosis on treatment and subsequent removal of 9-cis-retinoic acid impli es that drug scheduling may be an important parameter affecting therap eutic efficacy.