Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20057, USA.
Neuropharmacology. 2011 Jun;60(7-8):1292-300. doi: 10.1016/j.neuropharm.2011.01.010. Epub 2011 Jan 15.
In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²+ and cAMP. Hyperthermia increased average amplitudes of Ca²+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
在 CHO-K1 细胞中,热休克强烈激活了由腺病毒和质粒载体中的巨细胞病毒即刻早期(CMV-IE)启动子驱动的报告基因表达。热休克处理(42.5°C 下 2 小时)显著增强了核提取物中的启动子 DNA 结合活性。在受 CMV 启动子控制表达 mGluR1a 和 mGluR5a 受体的 CHO 细胞中,热休克增加了受体蛋白表达、mRNA 水平和通过 PI 水解、细胞内 Ca²+和 cAMP 测量估计的受体功能。热疗增加了 Ca²+反应的平均幅度、反应细胞的数量,并揭示了 mGluR1a 受体的毒性。热休克还有效地增加了 EGFP 的表达。因此,热休克对 CHO 细胞中 mGluR 表达和功能的影响可能归因于 CMV 启动子的激活。此外,这种效应不仅限于 CHO 细胞,因为热休克还增加了 PC-12 和 HEK293 细胞中 EGFP 的表达。热休克处理可能是一种有用的工具,可用于研究在 CMV 启动子控制下异源系统中表达的蛋白质的功能。它在瞬时转染中增加蛋白质表达、在药物筛选应用中增强受体表达以及控制具有毒性的蛋白质的表达方面可能特别有价值。本文是题为“神经药理学趋势:纪念 Erminio Costa”的特刊的一部分。