HYPERGLUCOSE CIRCUMSTANCE RETARDED EGF-RECEPTOR TURNOVER

一种高表达表皮生长因子受体的人鳞状细胞癌细胞系A431。在含有20g/L葡萄糖的培养液中培养，以使细胞适应高糖条件，与正常浓度葡萄糖培养(倍增时间33h)相比，显示中度生长迟缓(倍增时间为49h)。在高糖条件下培养的A431细胞含有两种形式的泛素：多泛素链，MU，100 ng/mg蛋白和游离泛素，FU，250 ng/mg蛋白，与原始A431细胞(NC)相似。当HC在4ﾟC培养时，用ELISA法测定的EGFR浓度为124U，与NC中的浓度(115U)大致相当。在37ﾟC的常规培养条件下，NC中的EGFR浓度略有升高。相反，在4ﾟC培养时，由于代谢增强和下调…，HC中的受体浓度极大地下降到一半水平免疫沉淀的EGFR可与晚期糖基化内产物抗体反应，但在NC中不反应。通过EGF与细胞孵育，泛素化的EGFR的特异性ELISA以及EGFR免疫沉淀和抗泛素抗体的免疫印迹可以清楚地检测到泛素化的EGFR水平的增加。蛋白质印迹分析显示，EGF刺激的EGFR磷酸化程度在HC较NC明显降低。这一结果表明，高血糖状态，如糖尿病，可能导致EGFR异常下调，分子结构异常。这些结果也提示了一些信号转导途径，即通过受体类型酪氨酸磷酸化的机制。在这样的环境下可能会受到阻碍。研究高糖环境对EGFR-mRNA表达的影响，以及高糖环境下是否存在EGFR的定点糖基化，对于阐明糖尿病的状态和肿瘤的发生是必要的。较少

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