Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation.

Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13-acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I.

12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation of human neuroblastoma cells is not accompanied by an increase in the intracellular concentration of cyclic AMP.

Cultured human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically followed by growth inhibition, by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The cells showed a limited differentiation when treated with substances known to increase the intracellular concentration of cyclic AMP. When these substances were combined with TPA, morphological differentiation and growth inhibition of the cells were potentiated. In contrast, these substances inhibited the TPA-induced increase in noradrenaline concentration and the relative activity of neuron-specific enolase. Both the intracellular concentration of cyclic AMP and the cytosolic level of cyclic AMP-binding components were similar in control and TPA-treated cells. It is suggested that cyclic AMP has a limited and non-regulatory role in the initiation of differentiation of SH-SY5Y cells. The effect of cyclic AMP is probably coupled mainly to the polymerization of microtubules, thus enhancing the morphological differentiation of the cells.

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells.

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.

Retinoic acid-induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced differentiation.

Cultured human SH-SY5Y neuroblastoma cells differentiated in the presence of retinoic acid (RA) or 12-0-tetradecanoyl-phorbol-13-acetate (TPA). In both cases, the cells acquired long cell processes and the cell growth was partially inhibited. Treatment with RA or TPA resulted in an increased neuron-specific enolase activity, relative to the total cellular enolase activity. At the optimal concentration, TPA induced a 200-fold increase in the concentration of noradrenalin, whereas in RA-treated cells the corresponding increase was only fourfold. Cells treated with a combination of RA and TPA were morphologically differentiated and growth inhibited and had a high relative activity of neuron-specific enolase. The increase in the concentration of noradrenalin induced by TPA was inhibited by RA in a concentration-dependent fashion. However, despite this result there seemed to be no general antagonistic effect of RA on the TPA-induced differentiation. The phenotypes of the cells treated by RA, TPA, or the combination of RA and TPA, did, on the other hand, differ from each other. Our results suggest that RA and TPA induce the SH-SY5Y cells to differentiate along different pathways.

Kinetics and concentration effects of TPA-induced differentiation of cultured human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells differentiate morphologically and biochemically in the presence of 12-0-tetradecanoylphorbol-13-acetate (TPA). The degree of differentiation, as demonstrated by the appearance of cell surface projections, growth inhibition and increase in noradrenalin concentration, was dependent on the TPA concentration and had an optimum at 1.6 X 10(-8) M of TPA. At that concentration neuron specific enolase (NSE) increased to a maximum level after 10 days of culture with no further changes in the NSE level during additional culture for 10 days. In contrast, the noradrenalin concentration reached a maximum after 4 days of TPA treatment and decreased during longer exposures to TPA. Based on the facts that the phorbolester induced differentiation shows stereo specificity and was optimal at the same concentration range as common polypeptide hormones, a putative TPA-hormone receptor interaction is discussed. An opposite effect of TPA on the SH-SY5Y cells, antagonizing the differentiation effect, is further suggested to explain the decrease in differentiation observed at TPA concentrations higher than 1.6 X 10(-8) M.