The long-term goal of this proposal is to identify the Papg1 gene, which is responsible for lung tumor progression in mice. A quantitative trait loci (QTL) responsible for lung tumor progression has been mapped to chromosome 4. Increasing evidence suggests that the Cdkn2a gene is the primary candidate for the Papg1 locus. The Cdkn2a gene encodes two distinct proteins: p16 (INK4a) and ARF. p16 (INK4a) is a member of the INK4 (Inhibitors of CDK4) family of cyclin-dependent kinase inhibitors that controls cellular transit through the G1 phase of the cell cycle by inhibiting the D-type cyclin-dependent kinases (CDK4 and CDK6). ARF is encoded by the Cdkn2a gene using an alternative first exon (Alternative Reading Frame or ARF) and is distinct from p16(INK4a). ARF arrests cells in either G1 or G2 phase through binding with p53 and mdm2. ARF has been excluded as a candidate for the Papg1 gene because both ARF variants suppressed cell growth with similar potencies. In contrast, allelic forms of the p16(INK4a) gene exhibit characteristics of a strong candidate for the Papg 1 gene. The supporting evidence includes: 1) The Cdkn2a gene maps to the same region that contains Papg1 and displays 100% concordance between allele type and susceptibility to lung tumor progression; 2) There are sequence variations in the p16(INK4a) gene resulting in amino acid differences among mouse strains; 3) Functional analysis showed that the BALB/cByJ p16(INK4a) variant was diminished in NIH/3T3 fibroblast growth suppression, CDK6 binding, and CDK6 inhibition compared with the SWR/J variant and it segregates with susceptibility to lung tumor progression; And 4) LOH of the Papg1 region is seen exclusively in lung adenocarcinomas of intervariant F1 hybrids. In the first phase of this proposal, we will evaluate the BALB/cByJ p16(INK4a) allele as the Papg1 gene by both fine-structure mapping using congenic mice and the construction of knock-in mice. If p 16(INK4a) is confirmed to be the Papg1 gene, further studies of p 16(INK4a) and ARF interactions in promoting lung tumor progression will be determined. If the BALB/cByJ p 16(INK4a) allele is excluded as the Papg1 gene, we will clone the Papg1 locus by fine mapping of the Papgl QTL to an approximate 0.5 cM subregion of the chromosome by congenic mice construction and generation of congenic substrains followed by positional cloning of the Papg1 QTL. The completion of this proposal will significantly further our understanding of tumor suppressor role of p 16(INK4a) in lung tumor progression, and of the genetic basis of lung tumor progression in mice.
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