Anderson E, Vinson G P, Puddefoot J R, Raven P W, Gilmore O J
J Steroid Biochem. 1986 Feb;24(2):489-95. doi: 10.1016/0022-4731(86)90110-x.
Homogenates of breast tumours taken at surgery were prepared in phosphate-buffered medium in the presence or absence of the protease inhibitors N-alpha-p-tosyl-L-arginine methyl ester (TAME, 10 mM) or soy bean trypsin inhibitor (STI, 1 mg/ml). Aliquots (0.25 ml) were incubated in 5 ml medium, with the addition of excess trypsin (2 mg/ml) to experimental flasks. Oestrogen was measured by means of a radioreceptor assay (RRA) based on rat or human uterine cytosolic oestradiol receptor. In oestrogen receptor positive (ER +ve) tumour homogenates, TAME decreased while STI increased ethyl acetate extractable oestrogen in these preparations. The addition of trypsin enhanced yields of oestrogen in the TAME, but not in the STI or control (no inhibitor) preparations. None of these treatments affected RRA detectable oestrogen in homogenates of ER -ve tumours. Suspensions of ZR-75-1 cells, prepared in Krebs Ringer bicarbonate (KRBG) incubated with trypsin also gave greatly enhanced yields of extractable oestrogen. Fractionation of oestrogens from both tumour homogenates and from the cell line showed coincidence of RRA detectable steroid with oestradiol and oestrone, and, particularly in trypsin flasks, very non-polar components were also found. In the cell-line extracts, HPLC fractionation combined with specific radioimmunoassays confirmed the presence of both oestradiol and oestrone. The major extracted component was oestrone. The data suggest the existence within breast tumour tissue of sequestered pools of steroid requiring proteolytic action for their release. One possibility, consistent with reports in the literature, is that the steroids may themselves be directly conjugated to protein. Their presence in ER +ve but not ER -ve tumours strongly suggests some relationship to the development of hormone-sensitive disease. Alternatively, the phenomenon may be associated with the rigid compartmentalization of the paracrine function of the tissue.
手术切除的乳腺肿瘤匀浆在含或不含蛋白酶抑制剂N-α-对甲苯磺酰-L-精氨酸甲酯（TAME，10 mM）或大豆胰蛋白酶抑制剂（STI，1 mg/ml）的磷酸盐缓冲培养基中制备。取等分试样（0.25 ml）于5 ml培养基中孵育，向实验瓶中加入过量胰蛋白酶（2 mg/ml）。通过基于大鼠或人子宫胞质雌二醇受体的放射受体分析（RRA）测定雌激素。在雌激素受体阳性（ER +ve）肿瘤匀浆中，TAME使这些制剂中乙酸乙酯可提取的雌激素减少，而STI使其增加。加入胰蛋白酶提高了TAME制剂中雌激素的产量，但在STI或对照（无抑制剂）制剂中未提高。这些处理均未影响ER -ve肿瘤匀浆中RRA可检测到的雌激素。在含有胰蛋白酶孵育的碳酸氢盐林格氏液（KRBG）中制备的ZR-75-1细胞悬液，其可提取雌激素的产量也大大提高。从肿瘤匀浆和细胞系中分离雌激素显示，RRA可检测到的类固醇与雌二醇和雌酮一致，特别是在胰蛋白酶处理的瓶中，还发现了极性非常低的成分。在细胞系提取物中，高效液相色谱分离结合特异性放射免疫测定证实了雌二醇和雌酮的存在。主要提取成分是雌酮。数据表明，乳腺肿瘤组织中存在需要蛋白水解作用才能释放的类固醇隔离池。一种与文献报道一致的可能性是，类固醇本身可能直接与蛋白质结合。它们在ER +ve而非ER -ve肿瘤中的存在强烈提示与激素敏感性疾病的发生有某种关系。或者，这种现象可能与组织旁分泌功能的严格区室化有关。
