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.