Oral Carcinogenesis: Human Gingival Keratinoocytes

口腔癌发生：人类牙龈角化细胞

• 批准号: 6432049
• 负责人: MYUNG HEE PARK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432049
• 关键词: biomarker; carcinogenesis; cell differentiation; cell line; cellular oncology; gingiva; keratin; keratinocyte; laboratory mouse; oncogenes; oral pharyngeal neoplasm; protein glutamine gamma glutamyltransferase

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.

我们已经证明，正常人牙龈角质形成细胞（NHGK）细胞在高 Ca++ 达到汇合后会以与体内类似的方式进行终末分化。诱导 TGase 1 活性（增加 5 至 10 倍）后形成不溶性细胞包膜 (CE)，表明 TGase 1 是口腔角质形成细胞终末分化的关键标志物。其他终末分化标志物的 mRNA 水平，例如外皮蛋白、SPR1 和膜联蛋白 1 在终末分化的 NHGK 细胞中也有所增加。 SPR1、annexin 1、loricrin、envoplakin、desmoplakin、involucrin、cystatin α 和 pancornulin 被确定为 NHGK CE 的组成部分。这些 CE 含有异常高含量的 SPR1，表明 SPR1 在口腔上皮的特殊屏障功能中发挥着重要作用。 我们比较了 NHGK 细胞和几种头颈癌细胞（HN4、HN12、HN8、HN22、HN30、HN31、HN13 和 HN19）中细胞角蛋白 1、5、8、10、14 和 19、TGase I 和 TGase II 的表达。在大多数 HNSCC 系中，主要细胞角蛋白（1、5、10 和 14）的表达显着降低。在一些癌细胞中观察到细胞角蛋白8或19的异常表达。在所有口腔 SCC 细胞系中，TGase 1 活性水平非常低（约 NHGK 的 10%）。与 NHGK 细胞相反，HNSCC 细胞无法诱导 TGase 1，也无法形成角化细胞包膜，这与它们丧失终末分化能力相一致。 TGase 2 不可诱导，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 活性似乎与它们在软琼脂中生长的能力相关，但与裸鼠的致瘤性无关。我们使用 HPV16 E6/E7 基因使 NHEK 细胞永生化。永生化人牙龈角化细胞 (IHGK) 显示出与 NHGK 类似的细胞角蛋白表达模式。与 NHGK 细胞一样，IHGK 细胞在高 Ca++ 培养基中达到汇合后会经历终末分化。 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。使用pCEFL载体转染各种人类原癌基因（包括H-Ras、N-Ras、K-Ras、c-Raf和c-Myc），并分离激活的癌基因H-ras-V12、H-ras-D12、K-ras-V12，并分离G418耐药细胞。我们正在表征这些致癌基因转染细胞在液体和软琼脂中的生长特性、它们的 Ca++ 抗性以及它们在裸鼠中形成肿瘤的能力。