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Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs., (Copyright 2000 Academic Press.)

Keratinocytes proliferate, stratify, and differentiate in vitro but if the calcium concentration of the medium is reduced to 0.07 mM Ca2+ (low calcium medium) the cells proliferate but do not stratify or differentiate. Keratinocytes proliferating in low calcium medium synthesized DNA at a higher rate than cultures of stratifying keratinocytes and this correlated with increased phosphorylation of a membrane-associated Mr 23,200 phosphoprotein (pp23) relative to cytosolic phosphoproteins of Mr 26,500 (pp27a and pp27b). In both normal and low calcium medium, all-trans-retinoic acid increased phosphorylation of pp23 relative to that of pp27 and increased DNA synthesis after 12-24 h. These results suggest that the phosphoproteins pp23 and pp27 are cell-cycle regulated and that the changes in phosphorylation were a consequence of a stimulation of cell proliferation by retinoic acid. The half-life of all-trans-retinoic acid in these cultures was about 6 h; increased DNA synthesis and concomitant changes in phosphorylation patterns also resulted from a 6-h pulse of retinoic acid followed by an 18-h washout period.