Prasad S C, Thraves P J, Dritschilo A, Kuettel M R
Department of Radiation Medicine, Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007-2197, USA.
Electrophoresis. 1997 Mar-Apr;18(3-4):629-37. doi: 10.1002/elps.1150180348.
Carcinogenic progression in most epithelial systems is a multistep process and presents as numerous (un)stable intermediate stages prior to the development of a fully malignant phenotype. Recently, we reported the neoplastic transformation of an SV40 immortalized, neonatal human prostate epithelial cell line (267B1) by multiple exposures to X-rays [1, 2]. The parental 267B1 cells acquired anchorage-independence and exhibited morphological transformation following exposure to two consecutive doses of 2 Gy. Exposure of either the parental 267B1 cells or the anchorage-independent derivatives (F3-SAC) to a total dose of 30 Gy of X-rays yielded tumorigenic transformants (267B1-XR and 267B1-SXR, respectively). All of these radiation-treated derivatives (F3-SAC, 267B1-XR, and 267B1-SXR) were characterized by reduced cell size and poorly organized actin stress fibers [2, 3]. The present study examines the protein expression changes associated with cytoskeletal alterations during the different steps of neoplastic progression induced by X-rays in the in vitro human prostate cell system. This analysis was achieved by using the high resolving power of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in the 267B1, F3-SAC, 267B1-XR, and 267B1-SXR cells. We report changes in the expression of gelsolin in the partially transformed, anchorage-independent, nontumorigenic (F3-SAC) cells and a progressive loss of expression of tropomyosin isoforms (TM-1 and TM-3), and myosin light chain-2 (MLC-2) in the tumorigenic (267B1-XR; 267B1-SXR) cells, respectively. In contrast, our results demonstrate that the levels of the small GTP-binding protein Rho-A, an active participant in the actin stress fiber organization, are not altered during neoplastic progression of these 267B1 cells. Thus the changes in synthesis of gelsolin, tropomyosins, and MLC-2 provide a rationale for the alterations in the actin stress fiber formation and reduction in cell size during the exposure of prostate epithelial cells to multiple doses of X-rays.
在大多数上皮系统中，致癌进展是一个多步骤过程，在完全恶性表型出现之前会呈现出许多（不）稳定的中间阶段。最近，我们报道了通过多次暴露于X射线使一种SV40永生化的新生儿人前列腺上皮细胞系（267B1）发生肿瘤转化[1,2]。亲代267B1细胞获得了不依赖贴壁生长的能力，并在连续暴露于两剂2 Gy的X射线后表现出形态学转化。将亲代267B1细胞或不依赖贴壁生长的衍生物（F3-SAC）暴露于总剂量为30 Gy的X射线下，产生了致瘤性转化细胞（分别为267B1-XR和267B1-SXR）。所有这些经辐射处理的衍生物（F3-SAC、267B1-XR和267B1-SXR）的特征是细胞尺寸减小且肌动蛋白应力纤维组织紊乱[2,3]。本研究检测了在体外人前列腺细胞系统中，X射线诱导的肿瘤进展不同阶段与细胞骨架改变相关的蛋白质表达变化。该分析是通过在267B1、F3-SAC、267B1-XR和267B1-SXR细胞中使用二维聚丙烯酰胺凝胶电泳（2-D PAGE）的高分辨能力来实现的。我们报告了在部分转化的、不依赖贴壁生长的、非致瘤性（F3-SAC）细胞中凝溶胶蛋白表达的变化，以及在致瘤性（267B1-XR；267B1-SXR）细胞中原肌球蛋白异构体（TM-1和TM-3）和肌球蛋白轻链-2（MLC-2）表达的逐渐丧失。相比之下，我们的结果表明，小GTP结合蛋白Rho-A（肌动蛋白应力纤维组织的积极参与者）的水平在这些267B1细胞的肿瘤进展过程中没有改变。因此，凝溶胶蛋白、原肌球蛋白和MLC-2合成的变化为前列腺上皮细胞暴露于多剂量X射线期间肌动蛋白应力纤维形成的改变和细胞尺寸减小提供了理论依据。
