Ornstein D K, Englert C, Gillespie J W, Paweletz C P, Linehan W M, Emmert-Buck M R, Petricoin E F
Urologic Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Clin Cancer Res. 2000 Feb;6(2):353-6.
The proportion of unbound serum prostate-specific antigen (PSA; percent-free PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al., Cancer Res., 51: 222-226, 1991; H. Lilja et al., Clin. Chem., 37: 1618-1625, 1991; D. L. Woodrum et al., J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 1542-1547, 1998). The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT). Two major mechanistic questions have previously been unknown: (a) Does PSA in human prostate cancer cells in tissue exist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populations of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al., Science, 278: 1481-1483, 1997). This technology provides a unique opportunity to study intracellular protein composition and structure from human cells. In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and malignant epithelium procured from prostate tissue. One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tissue specimens. Western blotting analysis of one-dimensional PAGE gels revealed a strong band at M(r) 30,000 (expected molecular weight of unbound PSA) in all cases demonstrating that the vast majority of intracellular tumor and normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bind ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA/ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.
据报道，与良性前列腺疾病患者相比，前列腺癌患者血清中未结合的前列腺特异性抗原（PSA；游离PSA百分比）比例较低（U. H. 斯滕曼等人，《癌症研究》，51: 222 - 226，1991；H. 利尔雅等人，《临床化学》，37: 1618 - 1625，1991；D. L. 伍德拉姆等人，《泌尿外科杂志》，159: 5 - 12，1998；W. J. 卡塔拉诺等人，《美国医学会杂志》，279: 1542 - 1547，1998）。血清中大多数具有免疫反应性的PSA与α-1-抗糜蛋白酶（ACT）结合。此前有两个主要的机制问题尚不清楚：（a）组织中人类前列腺癌细胞内的PSA是以游离形式还是结合形式存在？（b）恶性细胞产生的PSA是否以游离形式存在，因为它已经失去了与ACT形成复合物的能力？激光捕获显微切割（LCM）能够从组织中获取特定细胞类型的纯细胞群体（M. R. 埃默特 - 巴克等人，《科学》，274: 998 - 1001，1996；R. F. 邦纳等人，《科学》，278: 1481 - 1483，1997）。这项技术为研究人类细胞内的蛋白质组成和结构提供了独特的机会。在本研究中，我们使用LCM来评估从前列腺组织获取的良性和恶性上皮细胞内PSA的结合形式与游离形式。对从冷冻组织标本中通过LCM获取的良性和恶性前列腺上皮细胞裂解物进行了一维和二维聚丙烯酰胺凝胶电泳（PAGE）。一维PAGE凝胶的蛋白质印迹分析显示，在所有情况下，分子量（M(r)）为30,000处（游离PSA的预期分子量）都有一条强带，这表明绝大多数细胞内肿瘤和正常PSA以“游离”形式存在于细胞内。结合研究表明，从通过LCM获取的细胞中回收的PSA保留了与ACT结合的全部能力，二维PAGE蛋白质印迹分析表明，PSA/ACT复合物在强还原条件下是稳定的。我们得出结论，细胞内PSA以“游离”形式存在，并且与ACT的结合仅在细胞外发生。
