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Expression of cell cycle proteins in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced mouse lung tumors
C L Sabourin1, Q S Wang, S L Ralston
1Division of Environmental Health Sciences, School of Public Health, Ohio State University, Columbus, USA.
Abstract:
Cyclin D1 dysregulation and differential inactivation of p16INK4a and Rb have been observed in human lung cancer. In chemically induced mouse lung tumors, the p16INK4a gene is a target of inactivation, and Rb is reduced at the mRNA level (Northern blot) although similar at the protein level (Western blot) when compared to normal lung tissues. The expression of cyclin D1, cdk4, p16INK4a, and Rb protein was examined 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 was light to moderate in normal mouse lung tissues, but intense in lung adenomas and adenocarcinomas. Western blot analysis confirmed the increased expression of cyclin D1 and cdk4 in lung tumors compared to normal lung. Immunohistochemical analyses of lung tumors showed focal areas which lacked p16INK4a staining. Expression of p16INK4a, as determined by RT-PCR, was variable in lung tumors. Mutations in p16INK4a were not found by SSCP analysis. Immunohistochemical analyses of normal lung tissues showed intense staining for Rb protein in alveolar epithelial cells and in other lung cell types; however, in the lung tumors 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 hyperphosphorylated Rb protein in lung tumors and in normal lung tissues. These results suggest that alterations in the cell cycle proteins, cyclin D1, cdk4, p16INK4a, and Rb, may play a role in the acquisition of autonomous growth by adenomas. Furthermore, they demonstrate the importance 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.
Insights
Altered cell cycle proteins, including cyclin D1, cdk4, p16INK4a, and Rb, are implicated in mouse lung tumor development. Immunohistochemistry is crucial for studying these proteins in heterogeneous lung tissues.
Area of Science:
- Oncology
- Molecular Biology
- Cell Cycle Regulation
Background:
- Cyclin D1 dysregulation and p16INK4a/Rb inactivation are noted in human lung cancer.
- Mouse lung tumors show p16INK4a inactivation and reduced Rb mRNA, with similar Rb protein levels compared to normal lung.
- Cell cycle proteins are critical in cancer development and progression.
Purpose of the Study:
- To investigate the expression and potential role of cell cycle proteins (cyclin D1, cdk4, p16INK4a, Rb) in NNK-induced mouse lung tumors.
- To evaluate the utility of immunohistochemistry for analyzing protein expression in complex lung tissues.
Main Methods:
- Immunohistochemistry was used to examine cyclin D1, cdk4, p16INK4a, and Rb protein expression in normal and tumor mouse lung tissues.
- Western blot analysis confirmed protein expression levels.
- RT-PCR and SSCP analysis were employed to assess p16INK4a gene expression and mutations.
Main Results:
- Cyclin D1 and cdk4 showed increased nuclear expression in lung tumors compared to normal lung tissue.
- p16INK4a staining was focally absent in tumors, with variable expression detected by RT-PCR; no mutations were found.
- Rb protein staining intensity and distribution were reduced in lung tumors, with altered phosphorylation states observed via Western blot.
Conclusions:
- Alterations in cyclin D1, cdk4, p16INK4a, and Rb expression suggest their involvement in the autonomous growth of lung adenomas.
- Immunohistochemistry is vital for assessing protein expression in heterogeneous tissues like the lung and in complex tumor samples.