Soluble integrin ligands and growth factors independently rescue neuroblastoma cells from apoptosis under nonadherent conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N- BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment- induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺ levels nor on de novo protein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since (1) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁ and α₁ subunits and (2) the antibody-induced clustering of α₁, α₃, α(v), β₁, and β₃ integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. The de novo protein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁, antibody was associated with the increased expression of bcl-2. In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension- induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.