ORAL CARCINOGENESIS STUDIES WITH HUMAN GINGIVIAL KEROCYTES

致癌是一个多步骤的过程，涉及到 细胞原癌基因的激活和失活 肿瘤抑制基因。上皮细胞经历了不同的 表型和基因改变的阶段，并逐渐 获得恶性生长特征。尽管有 口腔癌的发病率相对较高，人们对此知之甚少 关于导致疾病发生的分子事件和 进步。我们研究的目标是开发一种体外培养的 永生化人牙龈致口腔癌模型的建立 角质形成细胞(IHGK)的培养及生长特性 和正常人牙龈的分化特性 角质形成细胞(NHGK)、IHGK和癌基因转导的IHGK细胞。 我们还比较了几种头部和颈部的特性。 NHGK和IHGK联合作用的癌细胞系(HN细胞) 寻找口腔癌、生长和晚期的生物标志物 NHGK的分化我们制备了NHGK的原代培养物 从磨牙后垫的健康组织中分离出 上皮细胞用消解酶消化，然后用胰酶消化。在KGM 口腔菌种的最适生长和贴壁效率 角质形成细胞在0.15 mM的钙离子下可见，而角质形成细胞 人皮肤角质形成细胞的最佳钙离子浓度为 0.05毫米。NHGK细胞在KGM培养液中达到后融合的时间 含0.15-1.2 mM Ca~(++)诱导TGase I活性 (增加5-10倍)之后形成不溶细胞 被TGase I交联的包膜(CEs)。这一发现表明 培养中的NGHK细胞。就像皮肤角质形成细胞一样，可以 在体外被诱导进行终末分化。逆转录聚合酶链式反应 而Western印迹分析也显示了增加的证据 在TGase I的mRNA和蛋白中，提示TGase I是 口腔角质形成细胞终末分化的关键标志。 其他终末分化标志物的mRNA水平， 例如，总蛋白、SPR1和膜联蛋白I也在 钙离子诱导的NHGK细胞。大黄鱼的氨基酸组成 交叉链接的角化信封，显示非常高的 脯氨酸(12-18%)，说明小的富含脯氨酸 蛋白质(SPR)是角化信封的主要成分 NHGK。当这些角化的信封被消化时 蛋白水解酶、胰酶或胰凝乳酶，一些具有 与SPR1、Annexin对应的氨基酸序列 I、三氯氰菊酯、Envoplakin、Desmoplakin、八角蛋白、半胱氨酸蛋白酶抑制剂 鉴定出α和泛角蛋白。氨基酸 NHGK角化后的成分和蛋白质组成 包膜明显不同于正常的表皮 角质形成细胞(NHEK)角化的包膜表明 口腔上皮细胞的特殊屏障功能 HN鳞癌中TGase I和TGase II的表达 细胞TGase I，一种角质形成细胞特异性转谷氨酰胺酶 与末端分化密切相关。蜂窝手机 TGase II的功能没有定义得很清楚，尽管它有 与细胞凋亡、细胞与基质的相互作用有关， 转移和信号转导。我们已经比较了 细胞角蛋白1、5、8、10、14、19、TGase I和TGase I的表达 NHOK细胞和几种头颈部癌细胞中的TGase II (HN4、HN12、HN8、HN22、HN30和HN31)。的表达方式 主要细胞角蛋白(1、5、10和14)显著减少 在大多数头颈部鳞癌中。细胞角蛋白的异常表达 部分癌细胞内可见8或19个阳性细胞。的水平 在所有口腔鳞癌细胞系中，TGase I的含量都很低(约为NHGK的10% 在融合后培养中未被诱导，与 它们丧失了末端分化的能力。TGase II是 没有诱导，但观察到TGase II的急剧增加 HN12、HN3 12：00AM和HN31细胞。TGase的水平 HN细胞的II活性似乎与它们的 在软琼脂中生长的能力，表明T-Gase II的作用 在安克雷奇-独立增长。TGase II活性， 然而，与这些肿瘤的致瘤性无关 细胞。永生化和癌基因导入。自.以来 原代角质形成细胞在培养中的寿命有限(~40 世世代代)，永生是任何 长期实验。我们已经使用了包含以下内容的pLXSN载体 人乳头瘤病毒16型E6/E7基因与新霉素抗性标记pBabe 含HPV16E6/E7基因和潮霉素的载体 NHGK永生化的抗性标记。之后 用这些载体转染和选择，永垂不朽 建立了牙周角质形成细胞系。这个 永生化的人牙周角质形成细胞(IHGK)显示出 细胞角蛋白的表达模式与NHGK相似。喜欢 NHGK细胞、IHGK细胞经历终末分化 在高Ca++介质中达到融合后。然而， IHGK诱导TGase I和形成不溶物的能力 角化的信封似乎随着数量的增加而减少 文化中的段落。对潮霉素耐药IHGK细胞的作用 各种人类癌基因，包括N-ras，K-ras，c-raf，c-myc， 病毒癌基因MSV-H-ras及其激活形式MSV-H-ras RAS-V12，用pCEFL载体导入，G418- 分离耐药细胞。我们正在刻画 转癌基因细胞在体外的生长特性 液体和软琼脂中，它们对钙离子的抗性，以及它们的 在裸鼠体内形成肿瘤的能力。

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. In spite of the relatively high frequency of oral cancers, little is known about the molecular events that lead to disease initiation and progression. The goal of our study is to develop an in vitro model of oral carcinogenesis using immortalized human gingival keratinocytes (IHGK) in culture and to characterize the growth and differentiation properties of normal human gingival keratinocytes (NHGK) IHGK and oncogene-transfected IHGK cells. We also compared the properties of several head and neck cancer cell lines (HN cells) with NHGK and IHGK in an effort to find biomarkers for oral cancer.Growth and terminal differentiation of NHGKWe prepared primary cultures of NHGK from healthy tissue of the retromolar pad by separation of epithelium by dispase, followed by trypsin digestion. In KGM medium, optimum growth and plating efficiency of oral keratinocytes were observed at 0.15 mM Ca++, whereas the optimum Ca++ concentration for human skin keratinocytes is 0.05 mM. When NHGK cells reached post-confluency in KGM media containing 0.15 - 1.2 mM Ca++, induction of TGase I activity (by 5-10-fold) was followed by formation of insoluble cell envelopes (CEs) crosslinked by TGase I. This finding suggests that NGHK cells in culture. Like skin keratinocytes, can be induced to undergo terminal differentiation in vitro. RT-PCR and Western blot analysis also showed evidence for an increase in TGase I mRNA and protein, suggesting that TGase I 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 I, were also increased in the Ca++-induced NHGK cells. The amino acid composition of the cross-linked cornified envelope, shows a very high level of proline (12-18%), suggesting that the small proline-rich protein (SPR) is a major component of cornified envelopes of NHGK. When these cornified envelopes were digested with protease, trypsin or chymotrypsin, a number of peptides with amino acid sequences corresponding to those of SPR1, annexin I, loricrin, envoplakin, desmoplakin, involucrin, cystatin alpha and pancornulin were identified. The amino acid composition and protein composition of NHGK cornified envelopes are clearly different from those of normal epidermal keratinocytes (NHEK) cornified envelopes suggesting a specialized barrier function of oral epithelium distinct from that of the skin.Expression of TGase I and TGase II in HN SCC cellsTGase I, a keratinocyte-specific transglutaminase is intimately involved in terminal differentiation. The cellular function of TGase II is less well defined, although it has been implicated in apoptosis, cell-matrix interactions, metastasis and signal transduction. We have compared the expression of cytokeratins 1, 5, 8, 10, 14 and 19, TGase I and TGase II in NHOK cells and several head and neck cancer cells (HN4, HN12, HN8, HN22, HN30 and HN31). The expression of the major cytokeratins (1, 5, 10 and 14) was significantly reduced in most head and neck SCCs. Aberrant expression of cytokeratin 8 or 19 was observed in some carcinoma cells. The level of TGase I was very low (~10% of NHGK) in all oral SCC cell lines and was not induced in post-confluent culture, consistent with their lost ability for terminal differentiation. TGase II was not inducible but a drastic increase in TGase II was observed in HN12, HN3 12:00 AM and HN31 cells. The level of TGase II activity of the HN cells appeared to correlate with their ability to grow in soft agar, suggesting a role for T Gase II in anchorage-independent growth. TGase II activity, however, did not correlate with the tumorigenicity of these cells. Immortalization and oncogene transfection. Since primary keratinocytes have a limited life span in culture (~40 generations), immortalization is a prerequisite step to any long-term experiments. We have used a pLXSN vector containing HPV16 E6/E7 genes and a neomycin resistance marker, or a pBabe vector containing HPV16 E6/E7 genes and a hygromycin resistance marker for immortalization of NHGK. After transfection with these vectors and selection, immortalized gingival keratinocyte cell lines were derived. The immortalized human gingival keratinocytes (IHGK) cells show a similar pattern of cytokeratin expression as does 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 I and to form insoluble cornified envelopes seems decrease with the increased number of passages in culture. To the hygromycin-resistant IHGK cells various human oncogenes, including N-ras, K-ras, c-Raf, c-myc, the viral oncogene MSV-H-ras and its activated form, MSV-H- ras-V12, were introduced 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.