Oral Carcinogenesis: Human Gingival Keratinoocytes

我们已经证明，正常的人牙龈角质形成细胞（NHGK）细胞在高Ca++达到合流后，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。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以及TGase I和TGase II在NHGK细胞和几种头颈癌细胞（HN4、HN12、HN8、HN22、HN30、HN31、HN13和HN19）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。TGase - 1活性水平在所有口腔SCC细胞系中都很低（约为NHGK的10%）。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HNSCC细胞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活性似乎与其在软琼脂中的生长能力相关，但与裸鼠的致瘤性无关。我们使用HPV16 E6/E7基因永生化NHEK细胞。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细胞在高Ca++培养基中达到后融合后发生终末分化。然而，IHGK诱导TGase 1和形成ce的能力似乎随着培养传代次数的增加而降低。将H-Ras、N-Ras、K-Ras、c-Raf、c-Myc等多种人原癌基因转染IHGK细胞，利用pCEFL载体将活化的癌基因H-Ras - v12、H-Ras - d12、K-Ras - v12分离得到g418耐药细胞。我们正在描述这些癌基因转染细胞在液体和软琼脂中的生长特性，它们对Ca++的抗性，以及它们在裸鼠体内形成肿瘤的能力。

We have shown that normal human gingival keratinocyte (NHGK) cells undergo terminal differentiation upon reaching confluency by high Ca++ 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 the 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 head and neck cancer cells (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. The level of TGase 1 activity was very low (~10% of NHGK) in all oral SCC cell lines. In contrast to NHGK cells, HNSCC cells failed to induce TGase 1 and failed to form cornified cell envelopes, consistent with their lost ability 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 appeared to correlate with their ability to grow in soft agar, but not with the tumorigenicity in nude mice. We have immortalized NHEK cells using HPV16 E6/E7 genes. The immortalized human gingival keratinocytes (IHGK) show a similar pattern of cytokeratin expression, as do NHGK. Like NHGK cells, IHGK cells undergo terminal differentiation upon reaching post-confluency in a high Ca++ medium. However, the capacity of IHGK to induce TGase 1 and to form CEs seemed to decrease with the increased number of passages in culture. IHGK cells were transfected with various human proto-oncogenes, including H-Ras, N-Ras, K-Ras, c-Raf, and c-Myc, and the activated oncogenes, H-ras-V12, H-ras-D12, K-ras-V12, using the pCEFL vector, and G418-resistant cells were isolated. We are characterizing the growth properties of these oncogene-transfected cells in liquid and in soft agar, their Ca++ resistance, and their ability to form tumors in nude mice.