Kyakumoto S, Kurokawa R, Ota M
Department of Biochemistry, Iwate Medical University School of Dentistry, Japan.
Biochim Biophys Acta. 1990 Jul 12;1053(2-3):204-12. doi: 10.1016/0167-4889(90)90015-6.
Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.
用10⁻⁶ M曲安奈德处理48小时的人唾液腺腺癌（HSG）细胞，与未处理的HSG细胞相比，[¹²⁵I]人表皮生长因子（hEGF）结合能力增加了1.7至2.0倍。对[¹²⁵I]EGF结合数据进行Scatchard分析表明，未处理细胞中结合位点的数量为83,700（±29,200）个受体/细胞，处理后细胞中为160,500（±35,500）个受体/细胞。未检测到受体亲和力有实质性变化。未处理细胞的EGF受体解离常数为0.78（±0.26）×10⁻⁹ M，而处理后细胞为0.93（±0.31）×10⁻⁹ M。曲安奈德诱导的[¹²⁵I]EGF结合能力增加在10⁻⁹至10⁻⁶ M之间呈剂量依赖性，在10⁻⁶ M时观察到最大结合。通过使用交联剂辛二酸二琥珀酰亚胺酯（DSS），用[¹²⁵I]EGF对HSG细胞上的EGF受体进行亲和标记。交联的[¹²⁵I]EGF占与HSG细胞结合的总[¹²⁵I]EGF的3 - 4%。在SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）后的放射自显影片中，亲和标记的EGF受体被检测为一条特异性的170 kDa条带。光密度分析显示，曲安奈德使这条带的强度比未处理细胞的条带强度放大了2.0倍。还通过用抗hEGF受体单克隆抗体免疫沉淀[³H]亮氨酸标记的EGF受体蛋白来测量EGF受体的合成。当HSG细胞用10⁻⁸ - 10⁻⁶ M曲安奈德处理48小时时，受体合成增加了1.7至1.8倍。当对免疫沉淀的、[³⁵S]甲硫氨酸脉冲标记的EGF受体进行SDS - PAGE和荧光自显影分析时，在170 kDa位置检测到新合成的EGF受体；用曲安奈德处理HSG细胞导致这条170 kDa条带放大了2.0倍。处理和未处理的HSG细胞之间EGF受体的周转率没有显著差异。这些结果表明，曲安奈德诱导的[¹²⁵I]EGF结合能力增加是由于HSG细胞中EGF受体蛋白合成增加所致。
