Owens D M, Wei S, Smart R C
Molecular and Cellular Toxicology, Department of Toxicology, North Carolina State University, Raleigh, NC 27695-7633, USA.
Carcinogenesis. 1999 Sep;20(9):1837-44. doi: 10.1093/carcin/20.9.1837.
Carcinogenesis involves the accumulation of genetic changes within a single cell. Tumor promotion functions in the initial clonal expansion of an initiated cell but is generally not considered to influence later stages. To investigate whether tumor promotion can influence later stages of carcinogenesis we developed a two-hit 7, 12-dimethylbenz[a]anthracene (D) protocol designed to enrich for keratinocytes that contain at least two D-induced genetic alterations. FVB/N mice were initiated with D and promoted with 12-O-tetradecanoylphorbol-13-acetate (T) or treated with acetone (A) vehicle for 6 weeks. At 7 weeks after the start of promotion, but before visible papilloma development, groups of mice were treated with a second dose of D or A and 1 week later T promotion was resumed. D/T/A/T mice developed 2.8 papillomas/mouse and D/A/D/T mice demonstrated an additive tumor response and developed 5.8 papillomas/mouse. Importantly, D/T/D/T mice developed 12.4 papillomas/mouse, thereby demonstrating a synergistic tumor response compared with D/A/D/T and D/T/A/T mice. D/T/D/T papillomas exhibited increases in suprabasal S phase cells and keratin 13 expression when compared with D/T/A/T papillomas. D/T/D/T mice developed squamous cell carcinomas (SCCs) 10 weeks earlier than D/T/A/T mice and demonstrated a 96% malignancy incidence and 1.71 SCC/mouse compared with D/T/A/T mice, which demonstrated a 28% malignancy incidence and 0.32 SCC/mouse. Greater than 90% of D/T/A/T and D/T/D/T papillomas and SCCs contained mutant Ha-ras, while a normal Ha-ras allele persisted in all cases, indicating that a gene other than the remaining normal allele of Ha-ras was a target gene for the second D hit. These data demonstrate that: (i) promotion between the first and second hits has a profound outcome on carcinogenesis, presumably by increasing the probability that a second hit will occur in a previously initiated cell; (ii) continued promotion after the second hit is required for full expression of malignancy; (iii) the classic initiation-promotion protocol can be extended to a multihit, multistage model.
致癌作用涉及单个细胞内遗传变化的积累。肿瘤促进作用在起始细胞的初始克隆扩增中发挥作用，但一般认为不影响后期阶段。为了研究肿瘤促进作用是否会影响致癌作用的后期阶段，我们开发了一种双打击7,12 - 二甲基苯并[a]蒽（D）方案，旨在富集至少含有两个D诱导的遗传改变的角质形成细胞。FVB/N小鼠先用D启动，再用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（T）促进，或用丙酮（A）载体处理6周。在促进开始后7周，但在可见乳头瘤形成之前，给小鼠组施用第二剂D或A，1周后恢复T促进。D/T/A/T小鼠每只小鼠发生2.8个乳头瘤，D/A/D/T小鼠表现出累加的肿瘤反应，每只小鼠发生5.8个乳头瘤。重要的是，D/T/D/T小鼠每只小鼠发生12.4个乳头瘤，从而与D/A/D/T和D/T/A/T小鼠相比表现出协同的肿瘤反应。与D/T/A/T乳头瘤相比，D/T/D/T乳头瘤的基底上层S期细胞和角蛋白13表达增加。D/T/D/T小鼠比D/T/A/T小鼠早10周发生鳞状细胞癌（SCC），与D/T/A/T小鼠相比，恶性发生率为96%，每只小鼠有1.71个SCC，而D/T/A/T小鼠的恶性发生率为28%，每只小鼠有0.32个SCC。超过90%的D/T/A/T和D/T/D/T乳头瘤和SCC含有突变的Ha - ras，而在所有情况下正常的Ha - ras等位基因持续存在，表明除了Ha - ras的其余正常等位基因之外的一个基因是第二次D打击的靶基因。这些数据表明：（i）第一次和第二次打击之间的促进作用对致癌作用有深远影响，大概是通过增加第二次打击发生在先前起始细胞中的概率；（ii）第二次打击后持续的促进作用是恶性肿瘤充分表达所必需的；（iii）经典的起始 - 促进方案可以扩展为多打击、多阶段模型。
