乳腺细胞周期蛋白E保持高分子量与钙激活中性蛋白酶稳态有关

A Primary Study of the Mechanism whereby Cyclin E in Breast Epithelial Cells Maintains Its High Molecular Weight Form

目的 :探讨正常乳腺上皮细胞周期蛋白E保持高分子量 (HMW )的机制。方法 :以乳腺肿瘤细胞为对照 ,采用蛋白质印迹法分析正常乳腺培养细胞裂解液经Ca2 + 处理后周期蛋白E分子量的变化 ,观察纯化的外源性钙离子激活中性蛋白酶 (CANP)对周期蛋白E的作用 ,同时观察和分析CANP小亚基在Ca2 + 作用下自我水解的程度。结果 :(1)正常乳腺细胞周期蛋白E主要以HMW形式表达 (分子量约 5 0kDa) ,而乳腺肿瘤细胞则表达大量 4 5kDa～ 35kDa小分子量 (LMW)周期蛋白E ;(2 )正常乳腺细胞匀浆经Ca2 + 处理后仅有很少量高分子量蛋白E水解为小分子片断 ,同时CANP小亚基也呈低水平水解 ;(3)预先用Ca2 + 激活的外源性CANP能使正常细胞HMW蛋白E全部转变成LMW蛋白 ,并伴随小亚基大量小分子水解片断的形成 ,而其内源性CANP小亚基仍保持原有分子量 ;(4 )Ca2 + 对正常乳腺细胞CANP活性产生负反馈式抑制效应。结论 :正常乳腺细胞周期蛋白E保持HMW形式与细胞内CANP活性保持稳态有关。

Objective: To study the elementary mechanism by which the high molecular weight (HMW)form of cyclin E in normal breast cells is maintained. Methods: Western blotting was employed to analyze the molecular weight variations of cyclin E in breast cells after treatment of their lysates with calcium chloride of different concentrations or with purified exogenerous calcium-activated neutral protease(CANP). Results: 1)Cyclin E in normal breast cells was expressed mainly in the HMW form(50 kDa), while in tumor breast cells, plenty of cyclin E in low molecular weight (LMW,45 kDa～35 kDa) was observed; 2)Ca 2+ treatment of the lysates of normal breast cells resulted in only formation of very small amount of LMW cyclin E with limited proteolysis of small subunit of CANP; 3) pre-activated purified exogenerous CANP cleaved all cyclin E in normal cells into LMW forms, accompanied by large amount of proteolyzed CANP small subunit, and 4) Ca 2+ of higher concentrations gave rise to a dose-dependent inhibition of CANP activity in normal breast cells, which was not detected in breast tumor cells. Conclusion: The maintenance of HMW cyclin E in normal breast cells correlates with the homeostasis of Ca 2+ activity.