Involvement of bcl-2 and p53 genes in the survival of human catecholaminergic neuroblastoma SH-SY5Y cells
In the present study, SH-SY5Y cells were used for two primary objectives: (1) to assess the neuroprotective effect of (R)-'N'-(2-heptyl)-' N'-methyl-propargylamine ((R)-2-HMP) and its metabolites against various toxins. Prior to this study, however, SH-SY5Y cells were differentiated and the response of these cells to various insults were assessed; and (2) as a continuation of a previous study (Yu and Zuo, 1997), to investigate biochemical changes that led to the increased resistance of SH-SY5Y cells co-cultured with astrocytes to 6-hydroxydopamine (6-OHDA) toxicity. SH-SY5Y cells were differentiated by either staurosporine or retinoic acid (RA). Even though both staurosporine and RA induce similar morphological differentiation in SH-SY5Y cells, these two groups of differentiated cells exhibited opposite vulnerability to various insults. In the present study, cisplatin, 5-fluorouracil, 'N'-(2-chloroethyl)-'N'-ethyl-2-bromobenzylamine (DSP-4), 6-OHDA, and γ-irradiation, were used to assess the tolerance to toxicity of the differentiated cells. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The staurosporine treated SH-SY5Y cells were more sensitive but the RA-treated cells were more resistant to these toxic insults in comparison to the untreated controls. The changes of cell vulnerability are attributed to the opposite effects on Bcl-2 and p53 proteins in SH-SY5Y cells by RA and staurosporine, based on Western blot and immunocytochemical studies After cells were differentiated by staurosporine, (R)-2-HMP (or one of its metabolites) was added together with the toxic agent. The MTT assay was used to assess cell viability. Results indicated that (R)-2-HMP and its metabolites did not seem to exhibit a protective effect in this system. (R)-2-HMP and its metabolites have been shown to have anti-apoptotic effects in other models. The lack of protection seen here suggests the anti-apoptotic effect of these compounds is selective. To study the biochemical changes in co-cultured SH-SY5Y cells, these cells were grown with astrocytes and treated with 6-OHDA. Trypsin was used to separate SH-SY5Y cells from astrocytes. Western blot analysis showed that 6-OHDA increased p53 protein in monolayer SH-SY5Y cells grown in either regular medium or conditioned medium obtained from astrocyte cultures, but not in those co-cultured with astrocytes. The ribonuclease protection assay indicated that similar changes also occurred at the transcriptional level. The enhanced resistance of the co-cultured SH-SY5Y cells to the toxicity of 6-OHDA is attributed to the ability of astrocytes to prevent the increase of p53 induced by this toxin.