The activation of the K-ras proto-oncogene by point mutations within the 61st codon appears to be an early and requisite step in the genesis of urethan-induced mouse lung tumors. We have observed that the K-ras gene is both activated by point mutation and overexpressed in lung tumors from mice that are sensitive to the carcinogenic effects of urethan. More recently, we have observed that the same two events, namely point mutation and overexpression of the K-ras gene, are both present in cell lines derived from mouse lung tumors. These cell lined provide an in vitro model to study this important gene in the lung. A major goal of the proposed studies are to determine the mechanism by which the K-ras gene is overexpressed in lung tumor-derived cells, and to determine the regulatory factors that control the expression of this gene. We will first determine whether the elevated levels of K-ras mRNA in lung tumors represents the mutated K-ras allele, the normal allele or both alleles. This will be accomplished by reverse transcription of the lung tumor cell mRNA into cDNA, followed by polymerase chain reaction and oligonucleotide, hybridization to determine the phenotype of the K-ras mRNA. Nuclear run-on transcription studies will determine whether there is increased transcription of the K-ras gene, and inhibitors of RNA and protein synthesis will be used to determine if increased mRNA stabilization might underlie the increased mRNA in lung tumors. A second goal of the proposed studies is to determine the mechanism by which glucorticoids affect K-ras expression in normal lung cells as well as determine whether lung tumor cells contain functional glucocorticoid receptors. This research goal stemmed from our observations that glucocorticoids can inhibit K-ras mRNA in nontumorigenic lung cells but not in lung tumor cell lines, and that a unique alteration in the glucocorticoid receptor gene is found in urethan- induced lung tumors. We will determine whether the lung tumor cells contain alteration in the glucocorticoid receptor that affect the responsiveness of these cells to this steroid. This will be accomplished by examining the expression of endogenous genes that are regulated by glucocorticoids as well as the expression of transfected MMTV-CAT (chloramphenicol acetyltransferase) reporter gene. The expression of CAT activity within the lung tumor cells will be dependent upon the presence of a functional glucocorticoid receptor and hormone. The identification of activating K-ras mutations in lung tumors from rodents and humans supports the use of this mouse lung cell model to understand the regulation of this gene in the lung. Further, since lung tumor development is a multistep process the elucidation of additional genetic events which may affect the expression of the K-ras gene and cell proliferation is important to our understanding of tumor development.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA046433-03
Application #
3189689
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1991-07-01
Project End
1995-02-28
Budget Start
1991-07-01
Budget End
1992-02-29
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Li, J; Beer, D G (1993) Ki-ras mRNA regulation in untransformed mouse lung cells. Mol Carcinog 8:193-201
Droms, K A; Hanson, L A; Malkinson, A M et al. (1993) Altered dexamethasone responsiveness and loss of growth control in tumorigenic mouse lung cell lines. Int J Cancer 53:1017-22
Hanson, L A; Nuzum, E O; Jones, B C et al. (1991) Expression of the glucocorticoid receptor and K-ras genes in urethan-induced mouse lung tumors and transformed cell lines. Exp Lung Res 17:371-87
Nuzum, E O; Malkinson, A M; Beer, D G (1990) Specific Ki-ras codon 61 mutations may determine the development of urethan-induced mouse lung adenomas or adenocarcinomas. Mol Carcinog 3:287-95
Pan, Y H; Nuzum, E O; Hanson, L A et al. (1990) Ki-ras activation and expression in transformed mouse lung cell lines. Mol Carcinog 3:279-86