Zachor D A, Moore J F, Jin J, Theibert A B, Percy A K
Department of Pediatrics, University of Alabama at Birmingham 35294, USA.
Mol Genet Metab. 1998 May;64(1):62-9. doi: 10.1006/mgme.1998.2699.
In utero cocaine exposure can affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion, in nerve growth factor (NGF)-stimulated PC12 cells, without affecting cell viability. NGF activates intracellular signaling proteins, specific immediate-early genes (IEG) including a transient peak of c-fos expression, and induction of late genes expression, leading to the neuronal phenotype. We hypothesized that cocaine interferes with NGF signaling. Therefore, we examined the pattern of c-fos expression in our cellular model. Time course of c-fos expression up to 72 h was determined in cells treated with NGF 20 ng/ml and cocaine 10 microgram/ml (a moderately toxic level) by RT-PCR analysis. Total RNA was isolated from cells, and levels of c-fos mRNA were estimated using gene-specific primers. In both control and experimental conditions, c-fos level was maximal at 0.5 h. In the control cells, c-fos expression declined rapidly to less than 5% of the 0.5h value, while in the cocaine-treated cells, c-fos level persisted through the 72-h exposure. Adding c-fos antisense to cells treated with NGF and cocaine resulted in significant improvement of neurite out-growth, from 28% (NGF + cocaine) to 89% (NGF + cocaine + c-fos antisense) of control differentiation after 72 h of exposure (Dunnet's T < 3.24). Inhibitory effects of cocaine on NGF-induced PC12 differentiation may be attributed to alteration of c-fos expression. Further studies will be required to examine the role of D1 receptor activation in mediating c-fos expression and to explore the effects of cocaine on other IEGs.
子宫内可卡因暴露会影响中枢神经系统发育。先前的研究表明，在神经生长因子（NGF）刺激的PC12细胞中，可卡因以剂量依赖的方式抑制神经元分化，且不影响细胞活力。NGF激活细胞内信号蛋白、特定的即刻早期基因（IEG），包括c-fos表达的短暂峰值，并诱导晚期基因表达，从而导致神经元表型。我们推测可卡因会干扰NGF信号传导。因此，我们在细胞模型中研究了c-fos的表达模式。通过逆转录-聚合酶链反应（RT-PCR）分析，确定了用20 ng/ml NGF和10微克/毫升可卡因（中等毒性水平）处理的细胞中c-fos表达长达72小时的时间进程。从细胞中分离出总RNA，并使用基因特异性引物估计c-fos mRNA的水平。在对照和实验条件下，c-fos水平在0.5小时时最高。在对照细胞中，c-fos表达迅速下降至低于0.5小时值的5%，而在可卡因处理的细胞中，c-fos水平在72小时的暴露过程中持续存在。向用NGF和可卡因处理的细胞中添加c-fos反义寡核苷酸可显著改善神经突生长，暴露72小时后，从对照分化的28%（NGF + 可卡因）提高到89%（NGF + 可卡因 + c-fos反义寡核苷酸）（Dunnet检验T < 3.24）。可卡因对NGF诱导的PC12分化的抑制作用可能归因于c-fos表达的改变。需要进一步研究来检查D1受体激活在介导c-fos表达中的作用，并探索可卡因对其他即刻早期基因的影响。
