Uno K, Shimada S, Tsuruta J, Matsuzaki H, Tashima S, Ogawa M
Department of Surgery II, Kumamoto University School of Medicine, Honjo, Japan.
Histochem J. 1998 Aug;30(8):553-9. doi: 10.1023/a:1003239302471.
Our previous reports have demonstrated frequent and strong expression of glycogen phosphorylase (EC 2.4.1.1) activity mainly in the cytoplasm of gastric carcinoma. Although previous studies have suggested the phosphorylase glycosyltransferase system to be in the nucleus from enzyme histochemical analyses, intranuclear localization of the phosphorylase has not been fully established. The aims of the present study are to investigate the nuclear localization of glycogen phosphorylase and to identify the isoform of phosphorylase in the nucleus of gastrointestinal carcinoma. The activity of glycogen phosphorylase in carcinoma cells corresponding to the nucleus was demonstrated using enzyme cytochemical analysis. The phosphorylase activity coincided with localization revealed by immunocytochemistry using affinity-purified specific anti-human brain-type glycogen phosphorylase antibody. The isoform expressed in the nuclei of carcinoma cells was identified as being only the brain type according to a polymerase chain reaction-based assay using RNA obtained from gastric carcinoma cells and primers specific to muscle, liver and brain types of glycogen phosphorylase. The intranuclear localization of the brain-type isoform was confirmed by immunoelectron microscopical analyses. Further investigation to examine the nuclear localization in human carcinoma tissue (145 and 25 specimens with gastric and colonic carcinoma respectively) was carried out by immunohistochemistry using specific anti-brain-type antibody. Nuclear immunostaining was observed in seven cases out of 145 gastric carcinoma. The present study is the first to clarify the nuclear localization of glycogen phosphorylase with enzymatic activity in gastrointestinal carcinoma. The isoform of the enzyme expressed in the carcinoma was identified as the brain type. These results warrant further studies on the mechanisms for transporting the large molecule of brain-type glycogen phosphorylase to nuclei and its function in the nucleus of carcinoma cells.
我们之前的报告表明，糖原磷酸化酶（EC 2.4.1.1）活性频繁且强烈表达，主要存在于胃癌细胞的细胞质中。尽管先前的研究通过酶组织化学分析表明磷酸化酶糖基转移酶系统存在于细胞核中，但磷酸化酶的核内定位尚未完全确定。本研究的目的是调查糖原磷酸化酶的核定位，并鉴定胃肠道癌细胞核中磷酸化酶的同工型。使用酶细胞化学分析法证实了癌细胞中与细胞核相对应的糖原磷酸化酶活性。磷酸化酶活性与使用亲和纯化的特异性抗人脑型糖原磷酸化酶抗体进行免疫细胞化学所显示的定位一致。根据使用从胃癌细胞获得的RNA以及肌肉、肝脏和脑型糖原磷酸化酶特异性引物进行的基于聚合酶链反应的分析，确定癌细胞核中表达的同工型仅为脑型。通过免疫电子显微镜分析证实了脑型同工型的核内定位。使用特异性抗脑型抗体通过免疫组织化学对人癌组织（分别为145例胃癌和25例结肠癌标本）中的核定位进行了进一步研究。在145例胃癌中有7例观察到核免疫染色。本研究首次阐明了胃肠道癌中具有酶活性的糖原磷酸化酶的核定位。癌细胞中表达的该酶同工型被鉴定为脑型。这些结果值得进一步研究脑型糖原磷酸化酶大分子转运至细胞核的机制及其在癌细胞核中的功能。
