Oral Carcinogenesis: Human Gingival Keratinocytes

癌的发生是一个多步骤的过程，涉及细胞原癌基因的激活和肿瘤抑制基因的失活。上皮细胞经历不同阶段的表型和基因型改变，并逐渐获得恶性生长特征。本研究的目的是建立一种永生化人牙龈角质形成细胞（IHGK）体外口腔癌发生模型，并研究正常人牙龈角质形成细胞（NHGK）、IHGK和一些头颈部鳞状细胞癌（HNSCC）细胞系的生长和分化特性，以及表型变化与基因表达谱的关系。 我们利用含有10 K克隆的人cDNA微阵列，对25个HNSCC细胞系和1个HPV 16 E6/E7基因转化的IHOK进行了基因表达谱的系统表征。NHOK被用作参考。我们的研究表明，主要参与细胞周期调控，肿瘤发生，细胞增殖，分化，凋亡和细胞粘附的基因被广泛改变，在所有26个细胞系研究。上调的基因包括已知的癌基因、蛋白激酶、DNA结合蛋白和细胞周期调节因子。而那些通常下调的包括分化标志物、细胞粘附蛋白、细胞外基质蛋白、结构蛋白（角蛋白）和蛋白酶抑制剂蛋白。与NHOK相比，在HNSCC中观察到参与终末分化和细胞粘附的基因的表达显著降低，表明该过程中的损失是HNSCC的重要特征。此外，数据的分层聚类分析揭示了26个细胞系中两种不同的基因表达模式亚型，反映了HNSCC的异质性。应用基因芯片显著性分析，筛选出136个基因在两组间差异表达。在两个亚组中差异表达的基因包括细胞增殖相关基因IGFBP 6、EGFR和VEGFC;肿瘤抑制和凋亡相关基因如CASP 4（半胱天冬酶4）、Tp 53、Tp 63;以及细胞周期调节因子如CCND 1和CCND 2（细胞周期蛋白D2），这表明两个亚组可能经历了不同的致癌途径。

Carcinogenesis is a multi-step process involving the activation of cellular proto-oncogenes and inactivation of tumor suppressor genes. Epithelial cells undergo different stages of phenotypic and genotypic alterations and gradually acquire malignant growth characteristics. The goals of our study are to develop an in vitro model of oral carcinogenesis using immortalized human gingival keratinocytes (IHGK) and to characterize the growth and differentiation properties of normal human gingival keratinocytes (NHGK), IHGK and a number of head and neck squamous cell carcinoma (HNSCC) lines and to relate the changes in phenotype with the gene expression profiles. We have conducted a systematic characterization of gene expression profiles in 25 HNSCC cell lines, and 1 IHOK transformed by HPV16 E6/E7 genes, using human cDNA microarrays containing 10K clones. NHOK were used as a reference. Our study shows that genes primarily involved in cell cycle regulation, oncogenesis, cell proliferation, differentiation, apoptosis and cell adhesion were widely altered in all 26 cell lines studied. Up-regulated genes included known oncogenes, protein kinases, DNA binding proteins and cell cycle regulators. While those commonly down-regulated included differentiation markers, cell adhesion proteins, extracellular matrix proteins, structural proteins (keratins) and protease inhibitor proteins. Compared to NHOK, a notable reduction in the expression of genes involved in terminal differentiation and cell adhesions was observed in HNSCC, suggesting that a loss in this process is an important signature of HNSCC. In addition, hierarchical clustering analysis of the data revealed two distinctive subtypes of gene expression patterns among the 26 cell lines, reflecting a degree of heterogeneity in HNSCC. By applying Significance Analysis of Microarrays, 136 genes were selected for being distinctively expressed between the two groups. Genes differentially expressed in the two subgroups include cell proliferation related genes, IGFBP6, EGFR, and VEGFC; tumor suppression and apoptosis related genes such as CASP4 (caspase 4), Tp53, Tp63; as well as cell cycle regulators such as CCND1 and CCND2 (cyclin D2) suggesting that the two subgroups might have undergone different pathways of carcinogenesis.