Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice

We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung carcinogenesis, suggesting that components of this pathway may be potential targets for gene therapy.