Powell Charles E, Soto Ana M, Michaelson Cheryl L, Diba Fantahun, Mounier Françoise, Verroust Pierre J, Sonnenschein Carlos
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
Steroids. 2003 Aug;68(6):487-96. doi: 10.1016/s0039-128x(03)00047-3.
Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.
雌激素通过受体介导的途径控制其靶细胞的增殖。最近提出的证据表明，雌二醇消除了人白蛋白（HA）和重组人白蛋白（rHA）对雌激素靶血清敏感细胞施加的增殖抑制作用（间接阴性假说）。我们推测，这一机制需要质膜雌激素受体（mER）和质膜白蛋白结合蛋白（mABP）的存在。最近已经提出了直接证据，证实MCF7细胞中存在mERα。在此，我们现在表明，用C542 ERα特异性单克隆抗体对纯化的T47D膜蛋白进行蛋白质印迹分析，也揭示了特异性的、多个M（r）的mER（67、110和130k M（r））。此外，用ABP抗血清进行的蛋白质印迹分析显示，在MCF7和T47D质膜提取物中均存在一种潜在的60k M（r）ABP。在ER阴性、血清不敏感的MDA-MB231细胞的类似提取物中未观察到此类证据。用牛血清白蛋白对类似质膜提取物进行配体印迹分析，证实MCF7和T47D细胞中存在60k M（r）ABP；同样，在MDA-MB231细胞的可比提取物中未观察到此类证据。通过免疫细胞化学对固定在2.0%多聚甲醛/0.1%戊二醛中的MCF7细胞进行荧光和共聚焦显微镜观察，确定了特异性膜ABP抗原位点。同样固定和标记的血清不敏感的MDA-MB231细胞未表现出这种mABP。这些结果表明，所提出的mABP仅在血清敏感的雌激素靶细胞中表达，而在对HA和rHA的增殖抑制不敏感的细胞中不表达。此外，目前的数据表明，所提出的mABP可能是HA和rHA抑制MCF7和T47D细胞增殖的识别机制。
