Park N H, Gujuluva C N, Baek J H, Cherrick H M, Shin K H, Min B M
Dental Research Institute, University of California, Los Angeles 90024, USA.
Oncogene. 1995 Jun 1;10(11):2145-53.
We previously immortalized normal human oral keratinocytes by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming growth factor-beta 1 and (7) secreted higher level of vascular endothelial growth factor with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral keratinocytes can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to transforming growth factor-beta 1 and (3) the high secretion of 56 kd vascular endothelial growth factor from the cells.
我们之前通过用重组人乳头瘤病毒16型（HPV-16）DNA转染使正常人口腔角质形成细胞永生化，随后将这些细胞暴露于苯并(a)芘中7天。苯并(a)芘的暴露改变了永生化细胞：这些改变后的细胞（HOK-16B-BaP）在含有生理钙水平（1.5 mM）的普通培养基中增殖，但在裸鼠中仅表现出增殖能力增强，未形成肿瘤，且在体外未表现出不依赖贴壁生长的特性。在本研究中，我们通过将细胞在含有苯并(a)芘的培养基中传代培养6个月，进一步对HOK-16B-BaP细胞进行了改造。经长期苯并(a)芘暴露后，这些细胞被称为HOK-16B-BaP-T细胞，（1）在器官型“筏”培养中表现出恶性表型，（2）在体外表现出不依赖贴壁生长的特性，（3）皮下注射到裸鼠中会形成肿瘤，（4）含有显著更高拷贝数的完整和整合的HPV-16 DNA；（5）含有更高水平的HPV-16 E6/E7信使核糖核酸和E7蛋白，（6）对转化生长因子-β1更具抗性，（7）与亲本HOK-16B-BaP细胞相比，分泌更高水平的分子量为56 kd的血管内皮生长因子。然而，HOK-16B-BaP-T细胞中p53和ras蛋白的水平以及p53、c-myc和c-fos转录本的水平与HOK-16B-BaP细胞中的水平没有差异。肿瘤细胞中p53基因、c-Ha-ras1基因和c-Ki-ras2基因的高度保守编码区未发生突变。这些数据表明，HPV永生化的人口腔角质形成细胞通过长期暴露于苯并(a)芘可转化为致瘤细胞。致瘤性转化似乎与（1）病毒癌基因如E6和E7基因的过表达、（2）细胞对转化生长因子-β1的更高抗性以及（3）细胞中56 kd血管内皮生长因子的高分泌有关。
