We describe the first detailed functional analysis of the Hupki mouse model of the p53 codon 72 polymorphism. We find that the P72 variant is associated with increased transactivation of the cyclin-dependent kinase inhibitor p21, along with an increased ability to induce growth arrest and senescence in MEFs; these data mirror the findings on human codon 72 variants of others (5
). We find that the transforming growth factor β (TGF-β) superfamily member Gdf15, which is a known p53 target gene, shows increased transactivation in P72 Hupki cells, normal human fibroblasts homozygous for P72, and inducible cell lines containing P72. We used microarray analysis to highlight the NF-κB pathway as being differentially impacted by the codon 72 polymorphism, leading us to the finding that the P72 variant shows increased interaction with p65 RelA in both mouse and human cells. That these three pieces of data are concordant between Hupki and human p53 lends credence to the premise that human polymorphisms can be effectively modeled in the mouse, with relevant biological discoveries as the outcome.
Our data indicate that the codon 72 polymorphism of p53 influences the p53-mediated inflammatory response. The role of p53 in innate immunity and the inflammatory response is now well established (6
) and, importantly, is evolutionarily conserved (11
). Whereas the impact of the codon 72 polymorphism on cancer risk appears to be minor, there are compelling examples in the literature of an effect of this polymorphism on diseases associated with inflammation. For example, studies of human ulcerative colitis (UC) indicate that there is a significant association of the P72 allele with UC (47
), with the clinical course and duration of UC (49
), and with the risk of UC-associated colorectal cancer (9
). Likewise, significant associations between this polymorphism and the incidence and severity of type II diabetes (12
) and rheumatoid arthritis (25
) have been noted, two diseases whose severity is associated with increased inflammation. Infection and chronic inflammation are known to contribute to increased cancer risk, and this may explain part of the increased cancer risk seen previously for the P72 variant. These findings suggest that when analyzing the potential impact of this polymorphism on cancer risk, it may be most informative to analyze inflammation-associated cancers.
P72 is the ancestral allele carried by the p53 gene, and it is believed that the R72 allele arose some 30,000 to 50,000 years ago (16
). It is unclear why the codon 72 polymorphism displays a geographical bias in the distribution of alleles, with P72 apparently selected for at the equator and R72 selected for in more northern latitudes. One possibility might be that the increased innate immune function associated with the P72 allele is selected for near the equator because immune challenge is greater there. In support of this notion, the P72 variant is generally associated with longevity, even following noncancerous illness (30
). The strong selection for the R72 allele in northern latitudes remains to be explained. Levine and colleagues have reported that the R72 allele demonstrates a 2-fold increased ability to transactivate LIF, a cytokine necessary for embryo implantation, thus raising the possibility that the selection for the R72 allele involves an impact on fecundity (14
). Along these lines, while the ability to fight infection is aided by a robust innate immune response, reproductive success requires a more tolerant immune response so that the fetus is not affected. In support of this, high inflammatory cytokine profiles are associated with decreased fecundity, and a reduced innate immune response is associated with increased fecundity (53
). An alternative hypothesis is that the abilities of p53 and NF-κB to play opposing roles in the control of metabolism explains the geographic distribution of these alleles (22
One reason to model the p53 polymorphism in mouse was because of the possibility that the influence of this polymorphism on apoptosis might be tissue specific. Cell line-based studies suggest that the R72 allele has superior proapoptotic function in human tumor cell lines (8
). Recently, a study by Zhu and colleagues described another mouse model for the codon 72 polymorphism; this study indicates that the R72 variant induces increased apoptosis in MEFs and in the small intestines of mice following ionizing radiation (58
). We also have found that the R72 variant is associated with increased apoptosis in these tissues (data not shown), along with decreased apoptosis in the thymus. Such tissue-specific influences of this polymorphism on apoptosis may explain why human studies have been inconclusive regarding the role of this polymorphism on cancer risk.
The present study represents the first unbiased analysis of differences in transcriptional potential between P72 and R72 variants. Somewhat surprisingly, while others have found that the R72 variant shows increased transactivation of proapoptotic genes like Perp
), and Noxa
), we find no evidence for increased transactivation of these genes in R72 thymocytes, MEFs, or normal human fibroblasts (this study; M. E. Murphy, unpublished data). The reasons for these discrepant findings are not presently clear. One explanation may be that these studies were done in cells that expressed supraphysiological levels of p53, while our Hupki mice maintain normal levels and regulation of this protein. In our model, we find that only a small subset of p53 target genes, the majority of which appear to contain binding sites for both p53 and NF-κB, show increased transactivation by P72. Interestingly, we find that NF-κB is required for the ability of p53 to bind to the promoters of caspase 4/11 and Gdf-15, suggesting that these two transcription factors may function cooperatively in the transactivation of this subset of target genes. Similar findings have been described previously for p53 and the p52 subunit of NF-κB (39
). It remains to be determined if the cooperation between these two transcription factors is stress specific or cell type specific.
There are important clinical implications for this work. The codon 72 polymorphism shows a significant ethnic bias in North American, with the P72 allele significantly more prevalent in African-Americans than in Caucasian-Americans (41
). Therefore, study of the codon 72 polymorphism of p53 has the potential to aid in our efforts to understand health disparities in African-Americans. For example, it is not understood why African-American women typically have a poorer prognosis for breast cancer or why African-American men have an increased incidence of multiple myeloma. Additionally, African-Americans show increased an incidence and severity of certain diseases associated with inflammation, including type II diabetes, heart disease, and obesity. It is anticipated that the Hupki model will be an invaluable preclinical tool to address such issues.