Kang Y, Mariano J M, Angdisen J, Moody T W, Diwan B A, Wakefield L M, Jakowlew S B
Medicine Branch, National Cancer Institute, Rockville, Maryland 20850, USA.
Mol Carcinog. 2000 Oct;29(2):112-26. doi: 10.1002/1098-2744(200010)29:2<112::aid-mc8>3.0.co;2-9.
To elucidate the role of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta type II receptor (TGF-beta RII) as tumor-suppressor genes in lung carcinogenesis, we mated C57BL/6 mice heterozygous (HT) for deletion of the TGF-beta1 gene with A/J mice to produce AJBL6 TGF-beta1 HT progeny and their wild-type (WT) littermates. Immunohistochemical staining, in situ hybridization, and northern blot analyses showed lower staining and hybridization for TGF-beta1 protein and mRNA, respectively, in the lungs of normal HT mice versus WT mice. Competitive reverse transcription-polymerase chain reaction (CRT-PCR) amplification showed the level of TGF-beta1 mRNA in the lungs of HT mice to be fourfold lower than the level in WT lung. When challenged with ethyl carbamate, lung adenomas were detected in 55% of HT mice by 4 mo but only in 25% of WT littermates at this time. Whereas all HT mice had adenomas by 6 mo, it was not until 10 mo before all WT mice had adenomas. After 12 mo, the average number of adenomas was fivefold higher in HT lungs than in WT lungs. Most dramatic was the appearance of lung carcinomas in HT mice 8 mo before they were visible in WT mice. Thus, the AJBL6 TGF-beta1 HT mouse provides an excellent model system to examine carcinogen-induced lung tumorigenesis by increasing progressive lesion incidence and multiplicity relative to their WT littermates. Immunohistochemical staining showed expression of the TGF-beta type I receptor (TGF-beta RI) at moderate to strong levels in lung adenomas and carcinomas in HT and WT mice. In contrast, whereas weak immunostaining for TGF-beta RII was detected in 67% of HT carcinomas at 12 mo, only 22% of WT carcinomas showed weak staining for this protein. Individual lung carcinomas showing reduced TGF-beta RII expression and adjacent normal bronchioles were excised from HT lungs using laser capture microdissection, and CRT-PCR amplification of the extracted RNA showed 12-fold less TGF-beta RII mRNA in these carcinomas compared with bronchioles. Decreasing TGF-beta RII mRNA levels occurred with increasing tumorigenesis in lung hyperplasias, adenomas, and carcinomas, with carcinomas having fourfold and sevenfold lower levels of TGF-beta RII mRNA than adenomas and hyperplasias, respectively. These data show enhanced ethyl carbamate-induced lung tumorigenesis in AJBL6 HT mice compared with WT mice, suggesting that both TGF-beta1 alleles are necessary for tumor-suppressor activity. Reduction of TGF-beta RII mRNA expression in progressive stages of lung tumorigenesis in HT mice suggests that loss of TGF-beta RII may play an important role in the promotion of lung carcinogenesis in mice with reduced TGF-beta1 gene dosage when challenged with carcinogen.
为阐明转化生长因子-β1（TGF-β1）和TGF-βⅡ型受体（TGF-βRⅡ）作为抑癌基因在肺癌发生中的作用，我们将TGF-β1基因缺失的杂合子（HT）C57BL/6小鼠与A/J小鼠交配，以产生AJBL6 TGF-β1 HT子代及其野生型（WT）同窝小鼠。免疫组织化学染色、原位杂交和Northern印迹分析表明，正常HT小鼠肺中TGF-β1蛋白和mRNA的染色及杂交水平分别低于WT小鼠。竞争性逆转录-聚合酶链反应（CRT-PCR）扩增显示，HT小鼠肺中TGF-β1 mRNA水平比WT肺中的低四倍。用氨基甲酸乙酯攻击后，4个月时在55%的HT小鼠中检测到肺腺瘤，而此时WT同窝小鼠中只有25%出现肺腺瘤。到6个月时所有HT小鼠都有腺瘤，而直到10个月时所有WT小鼠才都有腺瘤。12个月后，HT小鼠肺中腺瘤的平均数量比WT小鼠肺中的高五倍。最显著的是，HT小鼠在WT小鼠出现肺癌前8个月就出现了肺癌。因此，AJBL6 TGF-β1 HT小鼠提供了一个极好的模型系统，通过相对于其WT同窝小鼠增加进展性病变的发生率和数量来研究致癌物诱导的肺肿瘤发生。免疫组织化学染色显示，TGF-βⅠ型受体（TGF-βRI）在HT和WT小鼠的肺腺瘤和癌中呈中度至强表达。相比之下，在12个月时，67%的HT癌中检测到TGF-βRⅡ的弱免疫染色，而只有22%的WT癌显示该蛋白的弱染色。使用激光捕获显微切割从HT小鼠肺中切除显示TGF-βRⅡ表达降低的单个肺癌及其相邻的正常细支气管，对提取的RNA进行CRT-PCR扩增显示，这些癌中的TGF-βRⅡ mRNA比细支气管中的少12倍。在肺增生、腺瘤和癌的肿瘤发生过程中，TGF-βRⅡ mRNA水平降低，癌中的TGF-βRⅡ mRNA水平分别比腺瘤和增生低四倍和七倍。这些数据表明，与WT小鼠相比，AJBL6 HT小鼠中氨基甲酸乙酯诱导的肺肿瘤发生增强，提示两个TGF-β1等位基因对于抑癌活性都是必需的。HT小鼠肺肿瘤发生进展阶段TGF-βRⅡ mRNA表达的降低表明，当受到致癌物攻击时，TGF-βRⅡ功能丧失可能在TGF-β1基因剂量降低的小鼠肺癌发生促进中起重要作用。
