HYPERGLUCOSE CIRCUMSTANCE RETARDED EGF-RECEPTOR TURNOVER

表皮生长因子受体（EGFR）高表达的人鳞状细胞癌细胞系A431。用含20 g/L葡萄糖的培养液培养细胞，使其适应高糖条件，与正常浓度葡萄糖培养的细胞（倍增时间为33 h）相比，细胞生长中度迟缓（倍增时间为49 h）。高糖培养的A431细胞含有相对高浓度的两种形式的泛素（多泛素链，MU，100 ng/mg蛋白和游离泛素，FU，250 ng/mg蛋白），这与原始A431细胞（NC）几乎相似。HC在4 ℃培养时，ELISA法测得EGFR浓度为124 U，与NC（115 U）相当。在37 ℃常规培养条件下，NC中EGFR浓度略有升高。相比之下，HC中受体浓度在4 ℃培养时由于代谢增强和下调而急剧下降，达一半水平。 ...更多信息 免疫沉淀的EGFR在HC中与针对晚期糖基化内产物的抗体反应，但在NC中不反应。通过与EGF孵育细胞，泛素化EGFR的水平增加可以使用特异于泛素化EGFR的ELISA以及EGFR免疫沉淀随后通过抗泛素抗体进行蛋白质印迹的程序清楚地检测到。Western blot分析显示，EGF刺激的EGFR磷酸化在HC中的表达强度较NC明显降低。这些结果表明，高糖条件下，如糖尿病条件下，可能诱导EGFR异常下调，并伴有分子结构异常。这些结果也提示了一些信号转导途径，即通过受体型酪氨酸磷酸化机制。在这样的环境下，可能会受到阻碍。确定对EGFR-mRNA表达的影响，以及在高葡萄糖环境下是否存在EGFR的定点糖基化，以阐明糖尿病状态和癌症发病率。少

An epidermal growth factor receptor (EGFR) highly expressing human squamous cell carcinoma cell line, A431. which was cultured with a medium containing 20g/L of glucosefor adaptation of the cells to high glucose condition, showed moderate growth retardation (doubling time, 49h) as compared with that with normo-concentration of glucose (doubling time, 33h). A431 cultured with high glucose condition (HC) contained relatively elevated concentration of two forms of ubiquitin (multiubiquitin chain, MU, 100ng/mg protein and free ubiquitin, FU, 250ng/mg protein), which was almost similar to that in original A431 cells (NC). When HC was cultured at 4ﾟC, EGFR concentration determined with ELISA was 124U, which was approximately equal level to that In NC (115U). Under the conventional culture conditions at 37ﾟC, EGFR concentration in NC slightdccreased. By contrast, the receptor concentration in HC decreased extremely up to half level at 4ﾟC culture because of enhanced metabolism and down regula … More tion of EGFR.Immunoprecipitated EGFR in HC reacted with an antibody against advanced glycation endoproduct, but not in NC.By incubation of cells with EGF, increased levels of ubiquitinated EGFR were detectable clearly using an ELISA specific to ubiquitinated EGFR as well as a procedure of EGFR-immunoprecipitation followed by western blot by an anti-ubiquitin antibody. By western blot analysis, EGF-stimulated EGFR phosphorylation was also reduced in intensity in HC compared to in NC.These results indicated that the hyperglucose condition, such as diabetic condition, might induce the unusual down regulation of the EGFR with structural abnormality in the molecule. These results also suggested that some signal transduction pathways, namely via receptor type tyrosine phosphorylation mechanism. might be obstructed under such environment. To determine the effect on expression of EGFR-mRNA and whether presence or absence of site directed glycation of EGFR under the high glucose environment will be necessary to elucidate the diabetic status and cancer incidence. Less