Retinoic acid alters the mechanism of attachment of malignant astrocytoma and neuroblastoma cells to thrombospondin-1

Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti-tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP-1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors, TSP-1-adherent U-251MG; cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins alpha 3 beta 1 and alpha 5 beta 1 in clusters in lamellipodia and filopodia, The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36, Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins alpha 3 beta 1 and alpha 5 beta 1 mediated TSP-1 attachment of SK-N-SH cells, and integrins alpha 3 beta 1, alpha 5 beta 1, and alpha v beta 3 mediated TSP-1 attachment of U-251MG: cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1, Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG; cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells. (C) 1999 Academic Press.