HYPERGLUCOSE CIRCUMSTANCE RETARDED EGF-RECEPTOR TURNOVER

一种高表达的人鳞状细胞癌细胞A431的表皮生长因子受体（EGFR）。为使细胞适应高糖环境，在含葡萄糖20g/L的培养基中培养，与正常葡萄糖浓度下（加倍时间33h）相比，出现中度生长迟缓（加倍时间49h）。高糖条件下培养的A431细胞（HC）中两种形式的泛素（多泛素链，MU， 100ng/mg蛋白和游离泛素，FU， 250ng/mg蛋白）浓度相对升高，与原始A431细胞（NC）基本相似。当HC在4℃培养时，ELISA测定的EGFR浓度为124U，与In NC （115U）基本相等。在37℃的常规培养条件下，NC中的EGFR浓度略有下降。与此相反，由于代谢增强和EGFR的下调，在4次培养时，HC中的受体浓度下降了一半。免疫沉淀的EGFR在HC中与抗晚期糖基化内源性产物的抗体反应，但在NC中不发生反应。通过用EGF孵育细胞，使用特异的酶联免疫吸附法（ELISA）以及EGFR免疫沉淀法（随后用抗泛素抗体进行western blot）可以清楚地检测到泛素化EGFR水平的升高。通过western blot分析，与NC相比，HC中egf刺激的EGFR磷酸化强度也有所降低。上述结果提示，糖尿病等高血糖状态可能导致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阻。确定对EGFR- mrna表达的影响，以及在高糖环境下EGFR定向糖基化是否存在，对于阐明糖尿病状态和癌症发病率是必要的。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