EXPRESSION OF CELL-CYCLE PROTEINS IN ETHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE-INDUCED MOUSE LUNG-TUMORS

Cyclin D1 dysregulation and differential inactivation of p16(INK4a) an d Rb have been observed in human lung cancer. In chemically induced mo use lung tumors, the p16(INK4a) gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at th e protein level (Western blot) when compared to normal lung tissues. T he expression of Cyclin D1, cdk4, p16(INK4a), and Rb protein was exami ned by immunohistochemistry in 4-( methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-induced mouse lung tumors. Immunohistochemical staining revealed exclusive nuclear staining of both cyclin D1 and cdk4 that w as light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the inc reased expression of cyclin D1 and cdk4 in lung tumors compared to nor mal lung. Immunohistochemical analyses of lung tumors showed focal are as which lacked p16(INK4a) staining. Expression of p16(INK4a), as dete rmined by RT-PCR, was variable in lung turners. Mutations in p16(INK4a ) were not found by SSCP analysis. Immunohistochemical analyses of nor mal lung tissues showed intense staining for Rb protein in alveolar ep ithelial cells and in other lung cell types; however, in the lung tumo rs the staining intensity was reduced and the distribution was altered . Expression of Rb was detected in normal lung tissues but was barely detectable by Northern blot hybridization in lung tumors. Western blot analysis indicated the presence of both hypophosphorylated and hyperp hosphorylated Rb protein in lung tumors and in normal lung tissues. Th ese results suggest that alterations in the cell cycle proteins, cycli n D1, cdk4, p16(INK4a), and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the impor tance of immunohistochemical studies to examine expression in tissues that contain multiple cell types, such as the lung, and in tumors that by nature are heterogeneous.