Oral Carcinogenesis--Human Gingival Kerocytes

正常人牙龈角质形成细胞（NHGK）细胞在高Ca++培养基中达到融合后，以与体内相似的方式进行终末分化。诱导TGase 1活性（增加5 - 10倍）后，形成不溶性细胞包膜（CEs），表明TGase 1是口腔角质形成细胞终末分化的关键标志物。末梢分化的NHGK细胞中其他末梢分化标志物如involucrin、SPR1、annexin 1 mRNA水平均升高。SPR1、膜联蛋白1、loricrin、envoplakin、desmoplakin、involucrin、胱抑素α和pancornulin是NHGK ce的组成成分。这些上皮细胞含有异常高的SPR1，提示SPR1在口腔上皮的特化屏障功能中起重要作用。我们比较了细胞角蛋白1、5、8、10、14和19、TGase I和TGase II在NHGK细胞和HN4、HN12、HN8、HN22、HN30、HN31、HN13和HN19等HNSCC（头颈鳞状细胞癌）细胞系中的表达。在大多数HNSCC细胞系中，主要细胞角蛋白（1、5、10和14）的表达显著降低。细胞角蛋白8或19在部分癌细胞中异常表达。与NHGK细胞相比，HNSCC细胞不能诱导TGase 1，也不能形成确证的细胞包膜，这与它们丧失终端分化能力一致。TGase 2不能诱导，但在HN12、HN30和HN31细胞中观察到异常高水平的TGase 2。这些HN细胞的TGase 2活性与裸鼠的致瘤性无关。永生化的人牙龈角质形成细胞（IHGK）表现出与NHGK相似的细胞角蛋白表达模式。与NHGK细胞一样，IHGK细胞用编码HPV 16e6 /E7基因的pBabe载体永活，在高Ca++培养基中达到后融合后发生终末分化。为了了解口腔癌发生的遗传和分子机制，我们使用含有6720个cDNA的cDNA微阵列（NCI-OncoChip）对NHGK、IHGK、HSG（人唾液腺系）、SGT（人唾液腺系）和11个HNSCC系进行了基因表达谱分析。分析正在进行中，以将基因表达模式与细胞表型联系起来，并确定与口腔癌发生有关的遗传变化。

Normal human gingival keratinocyte (NHGK) cells undergo terminal differentiation upon reaching confluency in high Ca++ medium in a similar fashion as in vivo. Induction of TGase 1 activity (5 to 10-fold increase) was followed by formation of insoluble cell envelopes (CEs) suggesting that TGase 1 is a key marker for terminal differentiation of oral keratinocytes. The mRNA levels of other markers of terminal differentiation, e.g. involucrin, SPR1 and annexin 1, were also increased in the terminally differentiating NHGK cells. SPR1, annexin 1, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified as components of NHGK CEs. These CEs contained an unusually high amount of SPR1, suggesting an important role of SPR1 in the specialized barrier function of oral epithelium. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHGK cells and several HNSCC (head and neck squamous cell carcinoma) lines including HN4, HN12, HN8, HN22, HN30, HN31, HN13 and HN19. The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most HNSCC lines. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. In contrast to NHGK cells, HNSCC cells failed to induce TGase 1 and failed to form cornified cell envelopes, consistent with their loss of capacity for terminal differentiation. TGase 2 was not inducible but an unusually high level of TGase 2 was observed in HN12, HN30 and HN31 cells. The TGase 2 activity of these HN cells did not correlate with tumorigenicity in nude mice. The immortalized human gingival keratinocytes (IHGK) show a similar pattern of cytokeratin expression, as do NHGK. Like NHGK cells, IHGK cells, immortalized with pBabe vector encoding HPV 16 E6/E7 genes, undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. In an effort to understand the genetic and molecular mechanisms involved in oral carcinogenesis, we have conducted gene expression profiling in NHGK, IHGK, HSG (human salivary gland line), SGT (human salivary carcinoma line) and 11 HNSCC lines using cDNA microarrays (NCI-OncoChip) containing 6720 cDNA?s. Analysis is underway to correlate the gene expression patterns with the phenotypes of the cells and to identify genetic changes involved in oral carcinogenesis.