Table of Contents

1. Mutagenesis in a mammalian test system with a cancer-relevant gene as mutagen target.

1. A comparison of the types of base substitutions found in cell lines derived from benzo(a)pyrene-treated Hupki cells with the types of mutations in human lung and colon tumors in the IARC TP53 Database. The electropherogram shows the G to T transversions in codon 157/ 158 of the p53 gene in 2 B(a)P Hupki cell lines. | © dkfz.de

To test hypotheses on the origins of p53 tumor suppressor gene mutations in human tumors, novel strategies are needed that allow the researcher to generate and select mutations experimentally in the human p53 gene sequence. Towards this aim, we have combined the advantages of working with mouse embryonic fibroblasts, which readily undergo immortalization in vitro by acquiring p53 mutations, with the strategies developed by molecular biologists allowing targeted insertion of specific sequences in the mouse genome. First, we generated a unique gene-targeted mouse strain harboring normal human p53 sequences (the Hupki mouse), and then we developed an embryonic fibroblast immortalization assay (the HUF assay) with cells from these mice, which can be used as an in vitro approximation of p53 gene mutagenesis in human cancer development.
With this approach we showed that immortalized cultures derived from Hupki embryonic primary cells exposed to the tobacco smoke pro-carcinogen benzo(a)pyrene harbor p53 mutations in their human p53 gene sequences that are characteristic of smokers' lung tumors, strengthening the link between exposure to tobacco smoke and critical cancer mutations in people. We are currently employing the HUF assay to examine the mutation signatures in the human p53 gene of other putative exogenous and endogenous human cancer risk factors.

2. Development of mouse models to investigate the potential biological relevance of critical human polymorphisms in cancer-related genes. Impact on cancer risk, apoptosis, and response to chemotherapy.

2. Functional domains of the human and murine p53 proteins. The Hupki mouse p53 protein (center) is encoded by a chimeric allele generated by homologous recombination with a vector harbouring sequences from wild-type human p53 polyproline and DNA binding domains. | © dkfz.de

It has been difficult to assess the in vivo biological impact, if any, of certain common single nucleotide polymorphisms in human cancer development. One controversial example of a common human polymorphism of possible significance in cancer is at codon 72 of the human p53 gene. In humans, this codon encodes either arginine or proline. The 2 common allelic variants have been the subject of numerous epidemiological, clinical and molecular studies, but results have been mixed, or often conflicting. An inbred mouse model for studying the variants in vivo would shed new light on the potential impact of the polymorphism on cancer risk, response to therapy, and aging. We have generated mice with human p53 gene sequences that are homozygous for one or the other allelic variant, and crossed these to generate mice that are heterozygous at the polymorphic site. These mice, and primary cells derived from them, are currently being tested for possible phenotypic differences related to p53 genotype.