Induced differentiation affords neuroblastoma cells protection from hypoxic injury

Neuroblastoma (NB) tumors uniquely exhibit heterogeneous cellular differentiation. Advanced histologic differentiation portends favorable clinical outcome, and differentiation therapy with retinoic acid has shown promise clinically. Although cellular hypoxia is a common feature of solid tumors, how this influences differentiation in NB is not known. The authors' hypothesis contends that induced differentiation may alter the susceptibility of NB cells to hypoxic injury.

Human NB cells (SH-SY5Y) were subjected to a self-generated diffusion gradient of oxygen and nutrient deprivation in an in vitro "sandwich" model as previously established. Differentiation was induced by the addition of either all-trans-retinoic acid (RA; 5 micromol/L) or the phorbol ester 12-0-tetradeconoylphorbol-13-acetate (TPA; 16 nmol/L). Cellular survival is quantified as the viable cell border width, the viability of which is confirmed by substrate adherence, MTT assay, and trypan blue exclusion. Differentiation and proliferation were evaluated by tyrosine hydroxylase and proliferating cell nuclear antigen (PCNA) immunocytochemistry, respectively.

Addition of RA or TPA improved NB cell survival under conditions of hypoxia/metabolite deprivation (8.4 +/- 0.3 mm v 4.8 +/- 0.2 mm; RA v control at 24 hours, P <.05). This improvement was evident at all time-points examined (2 hours to 4 days) for both NB cell lines tested. Proliferation was diminished with addition of either TPA or RA. Treated cells displayed advanced differentiation by morphologic and immunocytochemical criteria. Nontreated NB cells did not differentiate under hypoxic conditions alone. Neither RA nor TPA treatment affected survival rate in non-NB cell lines (ie, hepatocyte HepG2 cells).

Pharmacologic differentiation protects NB cells from hypoxia and metabolite deprivation in vitro. Hypoxic conditions do not induce differentiation. Although clinical differentiation therapy may well limit cell proliferation, it also may afford resistance against hypoxia-induced injury and death, antiangiogenesis therapy notwithstanding.