Meade-Tollin L C, Boukamp P, Fusenig N E, Bowen C P, Tsang T C, Bowden G T
University of Arizona, Arizona Cancer Center, Department of Radiation Oncology, Tucson 85724, USA.
Br J Cancer. 1998 Mar;77(5):724-30. doi: 10.1038/bjc.1998.119.
Elevated expression of matrix metalloproteinases (MMPs), a family of secreted proteinases that degrade matrix components of basement membranes and connective tissues, is strongly correlated with malignant expression in various human epithelial cancers and epithelial cancer cell lines. We have tested whether elevated levels of MMP expression are also associated with malignant progression in human cutaneous squamous cell carcinoma. Constitutive levels of expression of steady-state mRNA and of secreted protein encoded by three MMP genes (matrilysin, gelatinases A and B) were compared in a unique in vitro model of human skin carcinogenesis. This model is composed of the parental immortalized non-tumorigenic human keratinocyte line (HaCaT), and three activated c-Harvey-ras-oncogene transfected variants (A-4, I-7 and II-4). Although clone A-4 is non-tumorigenic, clones I-7 and II-4 exhibit benign and malignant tumorigenic phenotypes, respectively, after subcutaneous injection into athymic nude mice. Northern blot, Western blot, and zymogram analyses revealed three MMP-specific patterns of expression. Constitutive matrilysin mRNA expression was markedly increased in the I-7 cells compared with HaCaT, A-4 or II-4 cells. Secreted promatrilysin was distinctly increased in the tumorigenic I-7 and II-4 cells compared with the non-tumorigenic HaCaT and A-4 cells. Gelatinase A mRNA and secreted gelatinase A protein levels were increased in each transfectant compared with HaCaT. Both active and inactive forms of gelatinase A were detected. Gelatinase B transcripts were not detected, but an EDTA-inhibitable gelatinase activity comigrating with gelatinase B was moderately enhanced in both tumorigenic variants compared with the non-tumorigenic cells. Because promatrilysin and 92-kDa gelatinase secretion were increased in both benign and malignant tumorigenic cells, and not related to invasiveness in this model, it is concluded that enhanced constitutive expression of these two MMPs is associated with acquisition of the tumorigenic phenotype, before acquisition of the malignant phenotype.
基质金属蛋白酶（MMPs）是一类可降解基底膜和结缔组织基质成分的分泌蛋白酶家族，其表达升高与多种人类上皮癌及上皮癌细胞系的恶性表型密切相关。我们检测了MMP表达水平升高是否也与人类皮肤鳞状细胞癌的恶性进展有关。在一种独特的人类皮肤癌发生体外模型中，比较了三种MMP基因（基质溶素、明胶酶A和B）编码的稳态mRNA及分泌蛋白的组成性表达水平。该模型由亲代永生化非致瘤性人类角质形成细胞系（HaCaT）以及三种转染了激活型c-Harvey-ras癌基因的变体（A-4、I-7和II-4）组成。虽然克隆A-4无致瘤性，但将克隆I-7和II-4皮下注射到无胸腺裸鼠后，它们分别表现出良性和恶性致瘤表型。Northern印迹、Western印迹和酶谱分析揭示了三种MMP特异性表达模式。与HaCaT、A-4或II-4细胞相比，I-7细胞中基质溶素mRNA的组成性表达明显增加。与非致瘤性的HaCaT和A-4细胞相比，致瘤性的I-7和II-4细胞中分泌的前基质溶素明显增加。与HaCaT相比，每个转染子中明胶酶A mRNA和分泌的明胶酶A蛋白水平均升高。同时检测到了明胶酶A的活性和非活性形式。未检测到明胶酶B转录本，但与非致瘤性细胞相比，两种致瘤变体中与明胶酶B共迁移的EDTA抑制性明胶酶活性均适度增强。由于前基质溶素和92-kDa明胶酶的分泌在良性和恶性致瘤细胞中均增加，且在该模型中与侵袭性无关，因此得出结论，在获得恶性表型之前，这两种MMP的组成性表达增强与致瘤表型的获得有关。
