Lesley Jarvis MD, PhD, Presenter: Nothing to Disclose

Tom Johnson, Abstract Co-Author: Nothing to Disclose

Laura Attardi, Abstract Co-Author: Nothing to Disclose

Greater than fifty percent of human tumors harbor mutations in the tumor suppressor gene p53. The p53 protein, in its wild type form, prevents tumor formation by regulating apoptosis, G1 cell cycle arrest and cellular senescence. p53 is proposed to function through both transactivation-independent and dependent pathways. The transactivation function has been mapped to residues in the amino terminus of the protein. Attardi et al. developed a hypermorphic allele of p53, in which the transactivation domain is replaced by the herpes simplex virus VP16 transactivator protein. This form of p53 was found to induce senescence. We have designed a series of genetic experiments to study the importance of p53 transactivation and senescence in tumor suppression.

Knock-in mice were used to be the most physiologically relevant. The p53 mutant mouse strain was developed by replacing the amino-terminal 80 amino acids of p53 with the VP16 transactivation domain. The targeting vector contained this p53VP16 mutant with an upstream transcriptional stop site flanked by LoxP recombination sites. The resulting LSL-p53 VP16 mice were used. To study the effect of this p53 VP16 mutant on lung cancer induced by K-ras, a mouse strain with a conditional and active form of the protein was utilized, termed LSL-K-rasG12D. To study the effect of this p53 VP16 mutant on lymphoma induction and maintenance, the Eu-myc transgenic mouse was utilized. This mouse strain expresses c-Myc in B-cells and develop B-cell lymphomas with short latency in a p53 heterozygous background.

For the lung cancer model studies, a cohort of compound conditional mutant mice were generated by the following crosses:LSL- p53 VP16 /+ X p53+/-; LSL-K-ras -> LSL- p53 VP16 /-; LSL-K-ras and LSL- p53 VP16 /+; LSL-K-rasLSL-p53/+ X p53+/-; LSL-K-ras -> LSL-p53/-; LSL-K-ras and LSL-p53/+; LSL-K-rasp53+/- X p53+/-; LSL-K-ras -> p53-/-; LSL-K-rasAll genotypes were confirmed by PCR. Mice were infected with an inhaled form of adeno-cre to excise the stop elements and activate the stopped alleles in lung tissue only. Tumors were allowed to initiate and progress for 3 months. Tissue sections from harvested lungs stained with H&E showed the development of adenocarcinoma.For the lymphoma model, a cohort of mice are being generated that expess Eu-myc and the conditional p53 VP16 allele by the following crosses:LSL- p53 VP16 /+ X Eu-myc/+ -> LSL- p53 VP16 /+; Eu-mycP53+/- X Eu-myc/+ -> p53+/-; Eu-mycAll genotypes were confirmed by PCR. Mice are being observed for the development of lymphoma. Lymphoma cells will be harvested and retroviral cre will be utilized to activate of the LSL-p53 VP16 allele. The effect of p53 VP16 will be determined by transplantation studies.

This work shows a strategy to better understand the in vivo tumor suppression activity of the p53 transactivation domain by use of a conditional knock-in mutation of p53 VP16. Continued studies using this approach in both a lung cancer and lymphoma model will yield valuable information on the importance of senescence in tumor suppression.

Jarvis, L, Johnson, T, Attardi, L, Dissecting the Mechanism of P53 Tumor Suppression in Vivo Using a Hyperactive Transactivation Mutant of P53.  Radiological Society of North America 2007 Scientific Assembly and Annual Meeting, November 25 - November 30, 2007 ,Chicago IL. http://archive.rsna.org/2007/6001093.html