K-ras is an important oncogene found to be mutated in almost 30% in the lung adenocarcinomas which has high incidence in countries like India and United States. The ras encodes for a 21kD protein, which is a crucial growth regulator of cells. Mutations in the hot spot codon of ras viz; codon 12, 13 and 61 have been frequently reported in many human and animal lung cancer. The knowledge is pretty limiting regarding functioning of K-ras p21 in lung epithelium. Therefore the goal of this study is to understand the mechanism of action of K-ras p21 functioning in normal growth and to see how its functioning differs following mutation leading to tumor development. We hypothesize that specific types of activating point mutations in K-ras oncogene can confer different growth advantages to the cells harboring it by turning on different downstream effector proteins (both quantitative and qualitative) involved in signaling. In this regard we plan to use both mouse (E10) and human (HPL1D) lung type II epithelial cells which are the target cells giving arise to adenocarcinomas. To eliminate ras independent gene expression between cell lines we will prepare tetracycline K-ras inducible constructs and transfect them into El0 and HPL1D cells. Following are the main objectives: (1) to analyze growth, cell cycle distribution and transforming efficiencies of the mouse and human lung epithelial cell lines containing different types of transfected inducible K-ras viz., wild type, mutants in codon 12,13 & 61, (2) to check for differential/preferential activation or crosstalks of Raf-MAPK and PI3-K-AKT pathway in the K-ras inducible cells (3) to identify targets of wild type and mutant K-ras by comparing the global gene expression profiles using membrane arrays. This kind of an approach will help delineate functioning of normal K-ras and also provide insights into functional consequences of specific mutations in ras and hence efforts can be directed towards molecular targeting for prevention and therapy.
| Singh, Arvind; Sowjanya, A Pavani; Ramakrishna, Gayatri (2005) The wild-type Ras: road ahead. FASEB J 19:161-9 |
