Hendrix M J, Seftor E A, Chu Y W, Seftor R E, Nagle R B, McDaniel K M, Leong S P, Yohem K H, Leibovitz A M, Meyskens F L
Department of Anatomy, University of Arizona College of Medicine, Tucson 85724.
J Natl Cancer Inst. 1992 Feb 5;84(3):165-74. doi: 10.1093/jnci/84.3.165.
Several protein markers, including vimentin, have been used to diagnose human melanoma. Because melanoma often has metastasized by the time of diagnosis, early markers prognostic for metastatic potential need to be identified. Commonly, vimentin is found in mesenchymal cells, and keratins are present in epithelial cells, but recent studies report coexpression of vimentin and keratin(s) in epithelial and nonepithelial neoplasms, including some melanomas.
Our purpose was to determine whether coexpression of vimentin and keratin(s) is correlated with tumor cell invasion and metastatic behavior.
We evaluated nine human melanoma cell lines expressing vimentin and other markers of aggressive tumor behavior (HMB-45, S-100, HLA-ABC class I and HLA-DR class II histocompatibility antigens, and K8 and K18 keratins). Levels of K8 and K18 keratins were determined in the highly metastatic C8161 cell line, the poorly metastatic A375P line, and the moderately metastatic A375M line. To determine whether the presence of keratin affects migratory ability, we altered the conformational structure of keratin filaments in C8161 cells by transfection with a mutant K18 complementary DNA. We also determined messenger RNA levels of human type IV collagenase, an enzyme marker for invasion and metastasis.
In A375P cells, two-dimensional electrophoresis with Coomassie-stained gels, immunoblotting, and immunofluorescence staining showed no detectable levels of K8 or K18. A375M cells showed low levels of K8 and K18 by Western and Northern blotting, with a distinctive fluorescent subpopulation of cells. In comparison, K8 and K18 levels in C8161 cells were high in all cells. Type IV collagenase messenger RNA levels were lowest in A375P cells and highest in C8161 cells, correlating with invasive ability in vitro and metastatic potential in athymic nude mice. The transfectant clones C1070-10 and C1070-14 derived from the C8161 parent line showed dramatic morphological changes, disrupted keratin filaments, and decreased invasive and metastatic potential directly correlated with a reduction in migratory activity.
These findings show a correlation between the coexpression of vimentin with K8 and K18 keratins and the invasive and metastatic behavior of three representative human melanoma cell lines.
包括波形蛋白在内的几种蛋白质标志物已被用于诊断人类黑色素瘤。由于黑色素瘤在诊断时往往已经发生转移，因此需要确定能够预测转移潜能的早期标志物。通常，波形蛋白存在于间充质细胞中，角蛋白存在于上皮细胞中，但最近的研究报道波形蛋白和角蛋白在包括一些黑色素瘤在内的上皮和非上皮肿瘤中存在共表达。
我们的目的是确定波形蛋白和角蛋白的共表达是否与肿瘤细胞侵袭和转移行为相关。
我们评估了九种表达波形蛋白和其他侵袭性肿瘤行为标志物（HMB-45、S-100、I类HLA-ABC和II类HLA-DR组织相容性抗原以及K8和K18角蛋白）的人类黑色素瘤细胞系。在高转移性C8161细胞系、低转移性A375P细胞系和中度转移性A375M细胞系中测定K8和K18角蛋白的水平。为了确定角蛋白的存在是否影响迁移能力，我们通过用突变型K18互补DNA转染来改变C8161细胞中角蛋白丝的构象结构。我们还测定了人类IV型胶原酶的信使RNA水平，IV型胶原酶是侵袭和转移的一种酶标志物。
在A375P细胞中，考马斯亮蓝染色凝胶的二维电泳、免疫印迹和免疫荧光染色均未检测到K8或K18的水平。通过蛋白质免疫印迹法和Northern印迹法，A375M细胞显示出低水平的K8和K18，且有一个独特的荧光细胞亚群。相比之下，C8161细胞中的所有细胞K8和K18水平都很高。IV型胶原酶信使RNA水平在A375P细胞中最低，在C8161细胞中最高，这与体外侵袭能力和无胸腺裸鼠体内转移潜能相关。源自C8161亲代细胞系的转染克隆C1070-10和C1070-14显示出明显的形态变化、角蛋白丝破坏以及侵袭和转移潜能降低，这与迁移活性的降低直接相关。
这些发现表明波形蛋白与K8和K18角蛋白的共表达与三种代表性人类黑色素瘤细胞系的侵袭和转移行为之间存在相关性。
