MOLECULAR AND CELLULAR BIOLOGY, Mar. 2011, p. 1201–1213
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 31, No. 6
The Codon 72 Polymorphism of p53 Regulates Interaction with NF-?B
and Transactivation of Genes Involved in Immunity
Amanda K. Frank,1Julia I-Ju Leu,2Yan Zhou,1Karthik Devarajan,1Tatiana Nedelko,3
Andres Klein-Szanto,4Monica Hollstein,3,5* and Maureen E. Murphy1*
Program in Developmental Therapeutics, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191111; Department of Genetics,
University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania2; Department of Genetic Alterations in Carcinogenesis,
Deutsches Krebsforschungszentrum, Heidelberg, Germany3; Department of Pathology, Fox Chase Cancer Center, Philadelphia,
Pennsylvania 191114; and Faculty of Medicine and Health, University of Leeds, Leeds, United Kingdom5
Received 27 September 2010/Returned for modification 11 November 2010/Accepted 6 January 2011
A common polymorphism at codon 72 in the p53 tumor suppressor gene encodes either proline (P72) or
arginine (R72). Several groups have reported that in cultured cells, this polymorphism influences p53’s
transcriptional, senescence, and apoptotic functions. However, the impact of this polymorphism within the
context of a living organism is poorly understood. We generated knock-in mice with the P72 and R72 variants
and analyzed the tissues of these mice for apoptosis and transcription. In the thymus, we find that the P72
variant induces increased apoptosis following ionizing radiation, along with increased transactivation of a
subset of p53 target genes, which includes murine Caspase 4 (also called Caspase 11), which we show is a direct
p53 target gene. Interestingly, the majority of genes in this subset have roles in inflammation, and their
promoters contain NF-?B binding sites. We show that caspase 4/11 requires both p53 and NF-?B for full
induction after DNA damage and that the P72 variant shows increased interaction with p65 RelA, a subunit
of NF-?B. Consistent with this, we show that P72 mice have a markedly enhanced response to inflammatory
challenge compared to that of R72 mice. Our data indicate that the codon 72 polymorphism impacts p53’s role
Within the p53 tumor suppressor gene exists a common
polymorphism at codon 72, encoding either proline or arginine
(P72 or R72, respectively). That these two variants might pos-
sess altered biological function was first suggested by the find-
ing of a linear relationship between geographic latitude and
the frequency of these variants, suggesting that there may be
selection for the P72 allele in environments subject to high
ultraviolet light or warmer winter temperatures (2, 40, 41).
Using genetically engineered inducible cell lines as well as
human tumor cell lines homozygous for each variant, we and
others have shown that these two forms of p53 differ in their
ability to induce growth arrest and apoptosis. Specifically, the
P72 variant possesses an increased ability to transactivate p21
and induce growth arrest (5, 36, 43, 46), while the R72 variant
demonstrates superior mitochondrial localization in tumor cell
lines (8). However, differences in the function of these variants
within an intact organism are unclear. To date, there have been
a large number of epidemiological studies investigating the
impact of this polymorphism on cancer risk. At present, the
combined studies suggest that there may be a minor associa-
tion between the P72 allele and increased cancer risk (10, 55).
The high degree of genetic variability in human populations,
combined with a dearth of information regarding potential
tissue-specific effects of the codon 72 polymorphism on p53
function, suggests that it is critical that a mouse model be
created in order to analyze these variants.
Like p53, NF-?B is a stress-inducible transcription factor
that plays a central role in proliferation and apoptosis. It
also plays a key role in the regulation of immunity and
inflammation. Given the pivotal role of both proteins in
tumorigenesis, it is not surprising that there is considerable
cross talk between them. Most of the available evidence
indicates that these two transcription factors function an-
tagonistically. For example, NF-?B transcriptionally induces
the negative regulator of p53, MDM2 (21, 45). p53 inhibits
the ability of NF-?B to transactivate NF-?B-responsive pro-
moters (15, 17), and p53 and NF-?B compete for binding to
p300 on target promoters (33, 50, 52). Restoring p53 func-
tion inhibits NF-?B (26), and loss of p53 is associated with
increased NF-?B activity (22, 23). Conversely, under certain
circumstances, p53 and NF-?B can cooperate with each
other, such as in the transactivation of genes that contain
both p53 and NF-?B response elements in their promoters,
like the Skp2 gene (1). p53 and NF-?B likewise cooperate to
transactivate certain target genes in cells exposed to hy-
droxyurea (38). Finally, in certain cells, p53 requires NF-?B
in order to efficiently induce apoptosis (35). Clearly, de-
pending upon circumstances, the impact of these proteins
on each other’s function can be either antagonistic or coop-
* Corresponding author. Mailing address for Maureen E. Murphy:
Fox Chase Cancer Center, W209, 333 Cottman Avenue, Philadelphia,
PA 19111. Phone: (215) 728-5684. Fax: (215) 728-4333. E-mail:
Maureen.Murphy@FCCC.edu. Mailing address for Monica Hollstein:
Department of Genetic Alterations in Carcinogenesis, Deutsches
Krebsforschungszentrum, Heidelberg, Germany. Phone: 49 6221 42
3303. Fax: 49 6221 42 3342. E-mail: firstname.lastname@example.org.
?Published ahead of print on 18 January 2011.
A clear role for p53 in the control of innate immunity has
emerged in recent years. The transcription of the p53 gene is
controlled by type I interferon (IFN) signaling (44), and the
induction of p53 participates in the host defense against viral
infection (6, 28). p53 also interacts with interferon regulatory
factor 9 (IRF9) to enhance IFN signaling (6). Additionally, p53
regulates the transcription of several cytokines and chemo-
kines involved in innate immunity; this activity of p53 is be-
lieved to contribute to the ability of the immune system to
eliminate senescent cells (56). The role of p53 in the control of
innate immunity was recently found to be evolutionarily con-
served: specifically, a mutation in the nucleolar protein Nol-6
leads to ribosomal stress-induced p53 activation in Caenorhab-
ditis elegans, which results in increased innate immune function
We have focused on elucidating the impact of the codon 72
polymorphism on p53 function. While previous studies support
the premise that these two variants have altered functions, the
majority of these studies were cell line based and utilized
overexpression of p53 alleles. We recently described a human-
ized p53 knock-in (Hupki) mouse in which exons 4 to 9, en-
coding the human proline-rich and DNA binding domains of
p53, replace those of the mouse. Because codon 72 in mouse
encodes alanine, it was important to study the codon 72 poly-
morphism in a humanized version of p53. Importantly, Hupki
p53 is fully functional and tumor suppressive in the mouse (24,
34, 54). In the present study, we compare the biological func-
tions of the P72 and R72 variants in inbred mice and show that
these variants demonstrate significant differences in apoptotic
function. Moreover, these studies led us to find for the first
time that the codon 72 polymorphism markedly affects the
ability of p53 to interact and cooperate with NF-?B in the
transactivation of genes involved in immunity and inflamma-
MATERIALS AND METHODS
Mouse studies and treatment. P72 and R72 mice were generated on a mixed
C57BL/6-129 (C57/129) background. These mice were backcrossed to C57BL/6
mice (Jackson Laboratories) for seven generations. P72 and R72 mice were then
crossed to each other for three generations; single nucleotide polymorphism
(SNP) genotyping analysis of markers polymorphic between the C57BL/6 and
129 backgrounds (performed by the Jackson Laboratories) indicates that these
mice can be considered C57BL/6 in genotype. All studies with mice complied
with all federal and institutional guidelines. For irradiation experiments, mice
were exposed to a cesium-137 gamma source (Fox Chase Cancer Center Irradi-
ation Facility). For lipopolysaccharide (LPS) treatment, P72 and R72 mice were
injected intraperitoneally with 20 mg/kg LPS (Escherichia coli 0111; B4 Calbio-
chem), and survival was tracked daily. A total of 4 h after injection, the thymuses
from mice were harvested and analyzed by immunohistochemistry, or instead,
thymocytes were purified, and RNA was used for quantitative reverse transcrip-
tion-PCR (QPCR). For tumor studies, E?-myc and p53?/?mice were obtained
from Jackson Laboratories (C57BL/6 background).
Cell culture, drug treatments, and Western analysis. Primary mouse embryo
fibroblast (MEF) lines were derived from 13.5-day-old embryos as described
previously (54); only cells from passages 0 to 4 were used for these studies. H1299
cells with Tet-inducible P72 and R72 were treated with 0.75 ?g/ml doxycycline
for 6 h to induce p53. Normal human fibroblast lines 6113 (Pro/Pro) and 5386
(Arg/Arg) were obtained from the Coriell Institute for Medical Research and
cultured in Dulbecco modified Eagle medium (DMEM), 15% fetal bovine serum
(FBS), 1% Pen/Strep, and 1% L-glutamine. Adriamycin (Sigma) at a concentra-
tion of 0.5 ?g/ml was used. Etoposide (Sigma) at a concentration of 100 ?M was
used. BAY-11-7082 (Cayman Chemicals) at 1 ?M was used. Western analysis
was performed as described previously (8). Membranes were blocked and probed
with antibodies for 1:500 p53 505 (Novocastra), 1:10,000 actin AC15 (Sigma),
1:200 each Mdm2 Ab1 and Ab2 (Calbiochem), 1:100 p21 Ab 4 (Calbiochem),
1:1,000 Ras (BD Biosciences), 1:500 cleaved caspase 3 (Cell Signaling), 1:200 p53
FL393 goat (Santa Cruz), 1:1,000 cleaved lamin A (Cell Signaling), 1:200 cleaved
caspase 11 M20 (Santa Cruz), 1:1,000 tubulin (Sigma), 1:1,000 p65 ab7970
(Abcam), 1:1,000 p105/p50 ab7971 (Abcam), 1:1,000 p53 Ab 6 (Calbiochem),
and 1:500 p53 ser15 (Cell Signaling). Horseradish peroxidase-conjugated sec-
ondaries (Jackson Immunochemicals) were used at a dilution of 1:10,000. Blots
were exposed to ECL (Amersham).
RNA isolation, QPCR, and microarray. RNA was isolated from MEFs and cell
lines using RNeasy (Qiagen), and RNA from thymocytes was isolated using
Trizol (Invitrogen). For microarray analysis, RNA was amplified and labeled
using the Agilent Quick Amp labeling kit. A total of 1.65 ?g of Cy3-labeled
cRNA targets was hybridized onto Agilent 4?44k whole-genome arrays for 17 h
at 65 degrees and washed according to the Agilent protocol. Hybridized slides
were scanned at a 5-?m resolution on an Agilent scanner, and fluorescence
intensities of hybridization signals were extracted using Agilent Feature Extrac-
tion software. Raw expression data obtained from Agilent microarrays were
background corrected and quantile normalized across the experimental condi-
tions (3). The LIMMA (Linear Models for Microarray Data) methodology out-
lined in reference 42 was applied to the log2-transformed expression data to
identify differentially expressed genes in each comparison. The LIMMA module
in the Open Source R/Bioconductor package (13) was utilized in the computa-
tions. Differentially expressed genes were identified based on statistical signifi-
cance as well as biological significance. Statistical significance was measured by
the false discovery rate (FDR) to account for multiple testing; genes showing an
FDR of less than 5% were considered statistically significant. QPCR was per-
formed in the Fox Chase Cancer Center QPCR Facility, using ABI primer sets
purchased for the indicated genes.
siRNA transfection. A total of 50 ?M of small interfering RNA (siRNA)
(negative control; p65 and p50 from DharmaFECT) was transfected using
DharmaFECT 1 for 20 h. After an 8-hour recovery period, cells were treated
with etoposide (100 ?M) for 16 to 24 h.
ChIP. Chromatin immunoprecipitation (ChIP) was performed using the
SimpleChIP enzymatic chromatin IP kit, as per the protocols provided (Cell
Signaling), using the following primer sets: p21-For, 5?GGTGGGGACTAGCT
TTCTGG3?; p21-Rev, 5?TCCACCACCCTGCACTGA3?; Noxa-For, 5?GGGG
TTGAGCAGGACTCGT3?; Noxa-Rev, 5?GAGCGAAGTGGAGCAGGTCT
3?; Casp4-p53-1-For, 5?AAGTTGTATTTGTCAGCTTAGGTCCA3?; Casp4-
p53-1-Rev, 5?ATGATCAGACGCTTGTCGTTTTTA3?; Casp4-p53-2-For, 5?CC
ACCTTGCTGTCTATACCAGATACT3?; Casp4-p53-2-Rev, 5?ATTAAAAGA
CAGTGTCCCAGAGAAGA3?; Casp4-NF-?B-For, 5?ACTTTCTGAGCAGCT
CTTTCAACA3?; and Casp4-NF-?B-Rev, 5?GCCATGAGAAAAAGCCTCAG
TT3?. The IGX1A negative-control primer set was purchased from Qiagen.
Immunohistochemistry. Tissues were harvested and fixed in 10% phosphate-
buffered formaldehyde (F79-4; Fischer Scientific) for 48 h and embedded in
paraffin. Following deparaffinization, antigen retrieval was performed with
citrate buffer at pH 6 for 10 min in a microwave oven. Endogenous peroxidases
were quenched with 0.3% hydrogen peroxide in methanol. Sections were incu-
bated overnight with the primary antibody, washed the next day with phosphate-
buffered saline (PBS), incubated with biotinylated secondary antibodies (Vector
Labs), stained with the Vectastain Elite ABC kit (Vector Labs), developed with
a DAB kit (Vector Labs), and lightly counterstained with Meyer’s hematoxylin.
Negative controls were stained without primary antibody. Specimens were doc-
umented photographically using a Nikon Optiphot microscope equipped with an
Optronics charge-coupled-device (CCD) camera. Antisera used included p53
CM5 (Novocastra), cleaved caspase 11 M20 (Santa Cruz), p53 ser15 (Cell Sig-
naling), cleaved caspase 3 (Cell Signaling), and cleaved lamin A (Cell Signaling).
Immunoprecipitation and Western blotting. A total of 250 to 3,000 ?g of
whole-cell lysate was incubated with 1 ?g of antibody overnight. Protein G-
agarose beads were added for 1 h, followed by washes and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antibodies used
were the same as those described above for Western blotting, with the addition
of p300 N-15 (Santa Cruz). Equal amounts of IgG were used as negative controls.
Horseradish peroxidase-conjugated light-chain-specific secondary antibody was
used (Jackson Immunochemicals).
Statistical analysis. Data were analyzed by two-sided unpaired Student’s t test
using a GraphPad software package. For the animal cancer studies, data were
analyzed with the one-sided Wilcoxon 2 sample test. For the LPS studies, data
were analyzed using the log rank test.
Microarray data accession numbers. Microarray data have been submitted to
the GEO database and have been assigned accession no. GSE26851.
1202 FRANK ET AL.MOL. CELL. BIOL.
Increased induction of p21 in P72 Hupki MEFs. Human-
ized p53 knock-in (Hupki) mice carrying the P72 and R72
alleles were generated in a mixed C57/129 background (34),
backcrossed for multiple generations to a C57BL/6 back-
ground, and then crossed to each other to generate the mice
analyzed in this study (see Materials and Methods for details).
For simplicity, these will be referred to as P72 and R72 mice.
To initially compare the expression and function of P72 and
R72 variants, primary mouse embryo fibroblast (MEF) cul-
tures were generated for each variant and analyzed for p53
stabilization and the transcriptional response to genotoxic
stress. As depicted in Fig. 1, the levels of stabilization of p53
following treatment with adriamycin in P72, R72, and wild-type
C57BL/6 (?/?) primary MEF cultures were comparable
(Fig. 1A). Similarly, quantitative reverse transcription-PCR
(QPCR) indicated that the transactivation of two p53 target
genes, Mdm2 and Puma (Bbc3), was indistinguishable between
these two variants and murine wild-type p53 (Fig. 1B).
In response to the DNA-damaging agent etoposide, the sta-
bilization of the P72 and R72 proteins and the induction of the
p53 target gene Mdm2 were similar between these variants in
primary MEF cultures (Fig. 1C). There was, however, a mod-
erate but consistent increase in the induction of the cyclin-
dependent kinase inhibitor p21 (Cdkn1a) in P72 MEFs, as
determined by Western analysis and QPCR (Fig. 1C); this
FIG. 1. The P72 variant demonstrates an increased ability to transactivate p21 and induce senescence. (A) Western analysis of whole-cell lysates
from primary mouse embryo fibroblasts (MEFs) isolated from P72, R72, and wild-type (?/?) mice treated with 0.5 ?g/ml of adriamycin (ADR)
for 8 or 24 h and probed with antisera for p53 and actin. (B) Quantitative reverse transcription-PCR (QPCR) to obtain the levels of Mdm2 and
Puma in mouse embryo fibroblasts (MEFs) treated with 0.5 ?g/ml adriamycin. The results shown are averaged values from two independent MEF
cultures of each genotype. (C, left) Western analysis of primary MEFs treated with 100 ?M etoposide (Etop) for 8 or 24 h and probed with the
indicated antibodies. (Right) Quantitative reverse transcription-PCR (QPCR) analysis to obtain the level of p21 normalized to that of the control.
Error bars depict standard errors. Statistical significance was calculated using the Student’s t test. (D) Percentage of bromodeoxyuridine
(BrdU)-positive cells from primary P72, R72, ?/?, and ?/? MEFs treated with etoposide (Etop; 100 ?M) after 24 h. Error bars depict standard
errors. (E, left) Senescence-associated ?-galactosidase (SA ?-gal) staining of primary P72 and R72 MEF cultures infected with oncogenic Ha-Ras
retrovirus, selected for 4 days for Ha-Ras expression, and assayed after the times indicated; the data depicted are representative of 4 days
postselection. (Right) Western analysis of the levels of p53, p21, Ras, and control (actin) in Ha-Ras-infected MEFs of the genotypes shown.
(F) Western analysis of lysates from normal human fibroblasts (NHFs) that are homozygous P72 or R72 treated with 100 ?M etoposide for 8 and
24 h and probed for Mdm2, p53, p21, and actin.
VOL. 31, 2011MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p531203
difference was by approximately 1.5-fold and was evident in
multiple P72 and R72 MEF lines (A. K. Frank, unpublished
results). These data were notable because in human cell lines,
a similar increased ability of the P72 variant to transactivate
p21 has been noted (5, 36, 46). To investigate further, we
compared the abilities of the P72 and R72 variants to induce
cell cycle arrest and Ras-mediated senescence, both of which
require p21. P72 MEFs showed a moderate but consistent
increase in cell cycle arrest following etoposide treatment (Fig.
1D) and gamma irradiation (data not shown), as assessed by
the percentage of cells positive for bromodeoxyuridine
(BrdU). We also noted increased p21 levels in P72 MEFs
following infection with oncogenic Ha-ras, along with in-
creased numbers of cells positive for senescence-associated
?-galactosidase (Fig. 1E). Similarly, we noted a consistently
increased ability of P72 to induce p21 in normal human fibro-
blasts homozygous for P72 and R72 (Fig. 1F). Overall, these
data were notable because they recapitulate what has been
found for the human codon 72 variants in normal fibroblasts
(5, 36) and therefore support the use of Hupki mice as a valid
model in which to investigate functional differences between
codon 72 variants.
Increased apoptosis in the P72 thymus. P72 and R72 mice
were next subjected to gamma irradiation, and the thymuses of
these mice were analyzed by immunohistochemistry using an-
tisera for p53, cleaved caspase 3, and caspase-cleaved lamin A
to measure apoptosis. Immunostaining for p53 in the thymuses
of P72, R72, and wild-type (?/?) mice showed no difference in
p53 accumulation at 2 h following 10 Gy of gamma radiation
(Fig. 2A, left). There were, however, marked differences in
apoptosis, with approximately a 2-fold increased number of
apoptotic cells in the P72 thymus compared to that in the R72
thymus (Fig. 2A, right); this difference was also evident by
Western analysis of cleaved caspase 3 (Fig. 2B). Interestingly,
this was not the case in other tissues, as there was consistently
greater apoptosis in the R72 small intestine and equal apop-
tosis seen between these variants in the spleen (data not
shown). To account for possible differences due to biological
variation, we next analyzed three P72 and R72 sibling litter-
mates side by side for their levels of apoptosis after gamma
irradiation using Western analysis of cleaved caspase 3 and
cleaved lamin A. Again these data indicated between 2- and
2.5-fold increased apoptosis levels in the thymuses of P72 mice
(Fig. 2C and D). These data could not be explained by differ-
ences in the differentiation of the thymocytes in these mice, as
P72 and R72 mice showed identical profiles of thymocyte de-
velopment (Fig. 2E). We next performed a time course exper-
iment following a lower dose of gamma irradiation (5 Gy). This
analysis also showed increased apoptosis in the P72 thymus at
all time points, as assessed by immunohistochemical (IHC)
analysis of cleaved lamin A (Fig. 2F), with the most marked
differences between P72 and R72 mice at 4 and 8 h postradia-
Increased induction of a subset of p53 target genes in the
P72 thymus. The p53-dependent apoptotic pathway possesses
both transcription-dependent and -independent arms (4). To
determine the underlying basis for increased apoptosis in the
P72 thymus, we analyzed thymocytes from gamma-irradiated
P72 and R72 mice for mitochondrial localization of p53 and for
the transactivation of p53 target genes with known roles in cell
death (Puma [Bbc3], Noxa [Pmaip1], Bax, and Pidd [Lrdd]).
Surprisingly, no differences in the localization of these variants
to mitochondria, or in the transactivation of these target genes,
were detected (data not shown). These findings prompted us to
consider the possibility that a previously unidentified p53 tar-
get gene(s) might play a role in the increased apoptosis found
in P72 thymocytes. With this hypothesis in mind, we performed
microarray analysis on thymocytes isolated from P72 and R72
mice that were subjected to 5 Gy of gamma radiation and
isolated after 0, 2, and 4 h. Four independent biological rep-
licates of each experiment were conducted, using two mice per
time point along with p53?/?and p53?/?mice as controls. A
heat map depicting known p53 target genes, including Apaf1,
Bax, Puma (Bbc3), and Pidd (Lrdd), is depicted in Fig. 3A; this
heat map indicates no differences in the transactivation of
proapoptotic p53 target genes following gamma radiation in
P72, R72, and ?/? mice (Fig. 3A). Indeed, of the more than
100 genes that were upregulated in a p53-dependent manner
following gamma radiation, the overwhelming majority dis-
played no differences between the P72 and R72 thymocytes
(data not shown). There was, however, a small subset of genes
that were upregulated in a p53-dependent manner and which
were transactivated to higher levels in P72 thymocytes. These
genes included the known p53 target genes p21 (Cdkn1a),
Gdf15 (31), Csf-1, and Cxcl1 (56). Other preferential P72 tar-
gets included Ccl4, Caspase 4 (originally called Caspase 11 in
mouse and most likely the orthologue of human Caspase 5),
Gpr77, Aire, Slpi, and ThyN1 (Fig. 3A). Quantitation of the
data from the microarray analysis of the genes Gdf15, ThyN1,
and Caspase 4/11, as well as confirmatory QPCR analysis of
thymocytes and primary MEFs, is depicted in Fig. 3B. Overall,
we noted a 2- to 4-fold increase in the levels of these genes in
P72 cells relative to those in R72 cells (Fig. 3B). Of these three
genes, only Gdf15 was expressed in normal human fibroblasts;
QPCR analysis confirmed that there is increased expression of
Gdf15 in P72 normal human fibroblasts (NHFs) and in induc-
ible P72 cells compared to that in R72 NHFs (Fig. 3C). West-
ern analysis of caspase 4/11 confirmed that this protein is
induced by gamma radiation to higher levels in P72 thymocytes
than in R72 thymocytes (Fig. 3D). Caspase 4/11 functions in
innate immunity by processing caspase 1, which processes and
activates interleukin-1?; alternatively, caspase 4/11 can behave
as an initiator caspase by directly activating caspase 3 (19, 20).
We confirmed that the increased apoptosis in P72 thymocytes
may be caused by enhanced induction of caspase 4/11, as we
found that expression of full-length caspase 4/11 in transfected
cells is sufficient to induce apoptosis (data not shown) (19, 20).
A list of all genes identified by microarray analysis as being
induced to higher levels in P72 thymocytes is presented in
Table 1. In this table, the ratio of P72/R72 induction of each
gene is presented at 2 and 4 h after gamma radiation and
ranges from 1.4- to 2.9-fold. Several of these genes (Gdf15,
Rnd3, Csf-1, and Cxcl1) are known to be upregulated by p53
(29, 31, 56, 57). Interestingly, 10 out of 12 of these genes are
known or postulated NF-?B target genes (see, for example,
http://bioinfo.lifl.fr/NF-KB/), suggesting the involvement of
this transcription factor. Consistent with this, IPA pathway
analysis of this subset of genes indicated that NF-?B repre-
sented a common node of regulation (data not shown). We
cloned the 5? regulatory sequences of the murine Caspase 4/11
1204 FRANK ET AL.MOL. CELL. BIOL.
FIG. 2. Increased apoptosis in P72 thymocytes following ionizing radiation. (A) Wild-type (?/?), p53-null (?/?), P72, and R72 mice were
irradiated with 10 Gy, and after 2 h, thymuses were collected and subjected to immunohistochemical (IHC) analysis of p53 (left) and cleaved
(activated) caspase 3 (right). UNT, untreated. (B) Western analysis of thymic extracts from P72 and R72 mice and p53-null mice (?/?) that were
irradiated with 10 Gy, harvested after 2 h, and probed for p53, actin, and cleaved caspase 3 (CC3). The lower-molecular-weight band is the fully
active form of caspase 3. WCL, whole-cell lysate. (C) As in panel B, except that sibling littermates were analyzed. (D) The averaged densitometry
results for Western analyses for cleaved caspase 3 shown in panel B and cleaved lamin A in irradiated thymocytes normalized to actin. The data
depicted are averaged values from 3 mice per genotype. Statistical significance was calculated using the Student’s t test. (E) Thymocytes were
isolated from P72, R72, wild-type, and p53-null mice and subjected to fluorescence-activated cell sorter (FACS) analysis of the cell surface markers
CD8 and CD4. The percentages of cells that were double negative (DN), double positive (DP), or singly positive (CD8?and CD4?) were graphed.
Three mice with each genotype were analyzed. (F) Immunohistochemical analysis of cleaved lamin A obtained from thymuses from P72 and R72
mice that were irradiated with 5 Gy and harvested after the times indicated.
VOL. 31, 2011 MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p531205
promoter and identified three consensus NF-?B binding sites
in the upstream regulatory sequences and two closely linked
consensus binding sites for p53 in the first intron (Fig. 4A,
diagram). In a reporter construct controlled by this promoter,
we found that both p65 RelA and p53 were able to substan-
tially activate reporter activity (Fig. 4A and B), suggesting that
this promoter is positively regulated by both NF-?B and p53.
We next tested the possibility that p53 cooperates with NF-?B
FIG. 3. Increased expression of Gdf15, Casp4, and ThyN1 in the P72 thymus after irradiation. (A) Portions of the heat map generated from
microarray analysis of thymocytes purified from P72, R72, wild-type (?/?), and p53-null (?/?) mice following treatment with 5 Gy and harvested
after 2 or 4 h. Each square represents one of four independent biological replicates, using 2 mice per genotype. (B, left) Graphic representation
of the generated microarray data shown in panel A, showing values for Gdf-15, caspase 4/11, and ThyN1. (Middle) QPCR analysis of a time course
experiment with an independent set of thymocyte RNA samples for P72 and R72 thymocytes following treatment with 5 Gy, depicting Gdf-15,
caspase 4/11, and ThyN1. IR, ionizing radiation. (Right) QPCR of RNA isolated from P72 and R72 mouse embryo fibroblasts (MEFs) treated with
100 ?M etoposide for 0, 8, or 24 h. The results depicted are averaged values from four independent QPCR samples performed in duplicate; the
error bars depict standard errors. (C, top) QPCR of the Gdf15 level in normal human fibroblasts (NHFs) homozygous for either P72 or R72 that
were irradiated with 5 Gy and harvested after 2 and 4 h. (Bottom) QPCR of the Gdf15 level in Saos-2 cells containing a temperature-sensitive
inducible form of P72 or R72 shifted to the permissive temperature (wild-type p53) for 8 and 24 h. (D) Western analysis of thymocytes purified
from P72 and R72 mice following irradiation with 5 Gy and harvested after the time points indicated for p53, caspase 4/11 (Casp4 p20), and tubulin;
the loss of tubulin at 8 h likely represents substantial cell death at this time point.
1206 FRANK ET AL.MOL. CELL. BIOL.
in the DNA damage-mediated induction of caspase 4/11. No-
tably, we found that transfection of primary MEFs with siRNA
of the NF-?B subunit p65 RelA or p50 markedly inhibited the
ability of p53 to induce caspase 4/11, but not p21, following
etoposide treatment (Fig. 4C). Similarly, we found that the
NF-?B inhibitor BAY-11-7082 could effectively inhibit the
ability of p53 in thymocytes to transactivate Caspase 4/11 but
not p21 or Perp (Fig. 4D). The combined data support the
premise that p53 and NF-?B cooperate in the regulation of
Caspase 4/11 in MEFs and thymocytes.
p53 and p65 RelA bind to the Caspase 4 promoter. We next
performed chromatin immunoprecipitation (ChIP) to deter-
mine whether p53 and NF-?B bind to the Caspase 4/11 pro-
moter. In P72 thymocytes treated with gamma radiation, we
were able to reproducibly detect a fragment of the Caspase
4/11 promoter containing the closely linked p53 binding site(s)
immunoprecipitating with antisera to p53 but not the negative-
control gene IGX1A (Fig. 5A). Similarly, NF-?B binding was
readily detectable at the closely linked consensus NF-?B sites
(Fig. 5B). In MEFs treated with etoposide, we were likewise
able to perform ChIP of the p53 binding site of Caspase 4/11
with p53 antisera (Fig. 5C). These data prompted us to eval-
uate the impact of the silencing of p65 RelA on the ability of
p53 to bind to this promoter. Notably, we found that treatment
of MEFs with siRNA to p65 RelA markedly inhibited the
ability of p53 to chromatin immunoprecipitate the promoters
of caspase 4/11 and Gdf-15 but not p21 (Fig. 5D). These data
indicate that NF-?B is required for p53 binding to these pro-
moters. We noted in these experiments that both the P72 and
R72 proteins had comparable abilities in chromatin immuno-
precipitating the Caspase 4/11 promoter in thymocytes and
MEFs (A. K. Frank, unpublished results). These data
prompted us to analyze the interaction between the codon 72
variants of p53 with NF-?B.
Increased association of P72 with the p65 subunit of NF-?B.
p53 and the p65 RelA subunit of NF-?B directly interact (15,
17). We isolated thymocytes from irradiated P72 and R72 mice
and immunoprecipitated p65 RelA, followed by immunoblot-
ting for associated p53. As shown in Fig. 6A, p53 was clearly
immunoprecipitated with p65 RelA in P72 thymocytes; how-
ever, there was evidence showing minimal R72 protein in these
p65 immunoprecipitates, despite comparable total levels of
p53. Similarly, in a time course experiment of etoposide-
treated P72 MEFs, a p65 RelA/p53 complex was detectable at
8 h posttreatment and was enriched at 24 h (Fig. 6B). Again,
however, there was minimal interaction between the R72 vari-
ant and p65 RelA. As both p53 and p65 can “shuttle” between
the nucleus and the cytoplasm, it was formally possible that the
impaired interaction between p65 and R72 was the result of
differences in the nuclear/cytosolic localization of these p53
variants. However, cell fractionation experiments demon-
strated no differences in the nuclear/cytosolic localization of
P72 and R72 variants in etoposide-treated MEFs (data not
shown). In transfected cells, we found that the R72 variant
possessed a decreased ability to interact with p65 RelA and
that deletion of the proline-rich domain of p53 likewise im-
paired the ability of p53 to interact with p65 RelA (Fig. 6C).
We next tested the influence of the codon 72 polymorphism on
the ability of human p53 to interact with p65. As depicted in
Fig. 6D, we consistently found that the R72 variant had an
impaired ability to interact with p65 RelA (Fig. 6D, left); in
contrast, both variants showed identical abilities to immuno-
precipitate with p300 (Fig. 6D, right). The combined data
support the premise that the codon 72 polymorphism of p53
influences its ability to interact and cooperate with NF-?B.
In order to assess the potential impact of the codon 72
polymorphism on cancer incidence, we crossed P72 and R72
mice with the E?-myc mouse, which develops B cell lym-
phoma; the development of this tumor is known to be lim-
ited by p53 (32, 37). We also crossed P72 and R72 mice with
p53?/?mice to generate P72/? and R72/? mice; these
would be expected to develop T cell lymphoma and sarcoma,
predominantly (7). As depicted in Fig. 7A, in the E?-myc
background there was a modest increase in survival im-
parted by the P72 allele; this may be due to the increased
ability of P72 to induce senescence, which is known to con-
trol the development of tumors in E?-myc mice (32). We
noted, however, no difference in the survival (Fig. 7B) or
tumor spectrum (Table 2) between P72/?, R72/?, and ?/?
mice. Therefore, consistent with the current literature on
human populations and the findings of others (55, 58), we
noted only the minor impact of the codon 72 polymorphism
on cancer risk, at least in these models.
TABLE 1. List of genes demonstrating increased transactivation in P72 thymocytes following gamma radiation
2 h4 hNF-?B target p53
Secretory leukocyte peptidase inhibitor
Macrophage inhibitory cytokine-1
Thymocyte nuclear protein 1
G protein coupled receptor 77
Chemokine ligand 4
Rho family GTPase 3
Macrophage colony-stimulating factor 1
Chemokine ligand 1
aThe fold increase in transactivation in P72 cells compared to that detected in R72 cells is depicted after 2 and 4 h.
VOL. 31, 2011 MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p531207
Enhanced response of P72 mice to LPS challenge. Our find-
ing that the codon 72 polymorphism influences the ability of p53
to cooperate with NF-?B in the transactivation of a subset of
proinflammatory p53 target genes prompted us to test the hy-
pothesis that this polymorphism might have an impact on the
inflammatory response. To test this hypothesis, we treated P72
stimulates a strong inflammatory response. Importantly, suscep-
FIG. 4. Induction of caspase 4/11 after DNA damage is impaired following silencing or inactivation NF-?B. (A, top) Schematic of the Caspase
4/11 promoter, depicting consensus NF-?B binding sites (filled ovals) and p53 binding sites (filled rectangles). The corresponding sequences of
these sites and their locations relative to the start site of transcription are shown. (Bottom) Luciferase assays on lysates from H1299 cells
transfected with a luciferase construct containing the full-length caspase 4 promoter (nucleotides ?167 to ?1083) along with increasing amounts
of p65 RelA. The graph represents two independent transfections, each analyzed in triplicate. (B) Luciferase assay of lysates from H1299 cells
transfected with a luciferase construct containing the p53 response element from caspase 4 (nucleotides ?998 to 1083) and increasing amounts
of p53. (C, top) Western analysis of primary mouse embryo fibroblasts (MEFs) transfected with a nontargeting control siRNA (siControl) or
siRNA from p65 RelA (p65), p50, or left untreated (no siRNA). Following transfection, cells were treated with 100 ?M etoposide (Etop) for 20 h
or left untreated (Un). (Bottom) QPCR determination of the levels of the p53 target genes p21 and Caspase 4/11 in the siRNA-treated cells. The
results shown were obtained from 2 independent experiments each done in duplicate; error bars depict standard errors. Statistical significance was
calculated using Student’s t test. (D) QPCR for caspase 4/11, normalized to the Hprt control. Thymocytes isolated from P72 mice were pretreated
with 1 ?M Bay-11-7082 for 1 h or left untreated, subjected to 5 Gy, and harvested after 2 h. QPCR was performed for Hprt, Casp4, ThyN1, Perp,
and p21. The results depicted represent three independent experiments analyzed in duplicate. Error bars show standard errors of measurement.
Statistical significance was calculated using Student’s t test.
1208 FRANK ET AL.MOL. CELL. BIOL.
tibility to LPS challenge is known to be mediated by caspase 4/11,
and knockout mice for this caspase show resistance to toxicity by
LPS (51). We found that LPS treatment induces an accumulation
of p53 protein that is phosphorylated on serine 15 (a common
marker for activated p53) (Fig. 8A). Additionally, a p65 RelA/p53
complex was detectable in LPS-treated P72 thymocytes (Fig. 8B).
Analysis of RNA isolated from the thymocytes of LPS-treated
mice indicated that Caspase 4/11 and Gdf-15 demonstrated mark-
edly increased transactivation in P72 mice. In contrast, there was
no difference in the transactivation of NF-?B target genes that
lack p53 binding sites, including Birc3 and Tlr2 (Fig. 8C). Notably,
the response to LPS challenge differed significantly for the two
p53 genotypes, and whereas the majority of P72 mice succumbed
to the septic shock induced by LPS within 2 days, the majority of
R72 mice survived this treatment (Fig. 7D). This difference was
highly significant (P ? 0.0155; log rank test). These data indicate
that the codon 72 polymorphism of p53 significantly affects the
inflammatory response to LPS challenge in mice.
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 transac-
tivation of the cyclin-dependent kinase inhibitor p21, along
with an increased ability to induce growth arrest and senes-
cence in MEFs; these data mirror the findings on human codon
72 variants of others (5, 36). We find that the transforming
growth factor ? (TGF-?) 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 microar-
ray analysis to highlight the NF-?B pathway as being differen-
tially 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 biolog-
ical 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, 28, 44) and, importantly, is evolution-
arily 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 poly-
morphism on diseases associated with inflammation. For ex-
ample, studies of human ulcerative colitis (UC) indicate that
there is a significant association of the P72 allele with UC (47),
FIG. 5. p53 binds to the Caspase 4/11 promoter in an NF-?B-dependent manner. (A) Chromatin immunoprecipitation using p53 antisera
(antibody fl-393G) of P72 thymocytes treated with 5 Gy and harvested after 4 h. Immunoprecipitated DNA was analyzed by QPCR for the
consensus p53 binding sites in Caspase 4/11 as well as for known p53 binding sites in p21 and Noxa and in the negative control IGX1A. All
ChIP data were obtained from two independent experiments performed in duplicate, normalized to IgG control. The error bars depict
standard errors. (B) Chromatin immunoprecipitation using antisera to p65 RelA (p65) for the p53 binding site in p21 as well as for the set
of three closely linked NF-?B binding sites in Caspase 4/11. (C) Chromatin immunoprecipitation using p53 antisera of P72 MEFs treated
with 100 ?M etoposide (Etop) for 16 h or left untreated (UNT). Immunoprecipitated DNA was analyzed by QPCR for the consensus p53
binding sites in Caspase 4/11 as well as for known p53 binding sites in p21 and Noxa. (D) Chromatin immunoprecipitation using p53 and p65
RelA antisera of P72 MEFs treated with 100 ?M etoposide for 16 h or left untreated (UNT), following 24 h transfection with siControl or
siRNA to p65 RelA. Immunoprecipitated DNA was analyzed by QPCR for the consensus p53 and NF-?B binding sites in Caspase 4/11 and
Gdf-15 as well as for the known p53 binding site in p21.
VOL. 31, 2011 MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p531209
with the clinical course and duration of UC (49), and with the
risk of UC-associated colorectal cancer (9). Likewise, signifi-
cant 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 inflamma-
tion 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
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 chal-
lenge is greater there. In support of this notion, the P72 variant
is generally associated with longevity, even following noncan-
cerous illness (30, 48). The strong selection for the R72 allele
in northern latitudes remains to be explained. Levine and col-
leagues have reported that the R72 allele demonstrates a
2-fold increased ability to transactivate LIF, a cytokine neces-
FIG. 6. Impaired interaction of the R72 variant of p53 with p65 RelA. (A, left) Immunoprecipitation-Western analysis of thymocytes from P72
and R72 mice harvested 4 h after 5 Gy; the immunoprecipitating (IP) antibody is denoted, and the blot was probed for p53 and p65 RelA. Equal
exposures of p65 RelA and p53 are shown. (Right) Western analysis of thymocyte lysates for p53, the NF-?B subunits p105, p65, and p50, and the
actin control. The data depicted are representative of three independent experiments. (B, top) Immunoprecipitation-Western analysis of P72 and
R72 MEFs treated with 100 ?M etoposide for 8 and 24 h or untreated (UNT); the immunoprecipitating (IP) antibody is denoted, and the blot
was probed for p53. (Bottom) Western analysis of MEF lysates for p53, the NF-?B subunits p65 and p50, and the actin control. (C, left) A total
of 250 ?g of lysates from H1299 cells transfected for 24 h with plasmids containing P72, R72, ?61-75TZ (tetramerization zipper [TZ] with deletion
of positions 61 to 75), TZ, and ?81-96TZ p53 variants were immunoprecipitated (IP) with an antibody (Ab) to p65 RelA and IgG. The TZ mutant
lacks the oligomerization domain of p53, which reduces spurious interactions. IPW, immunoprecipitation-Western analysis. (Right) Western
analysis of whole-cell lysates from H1299 (p53-null) cells transfected with the p53 constructs denoted and probed using antisera specific for p53,
p65, and actin. (D) Immunoprecipitation-Western analysis of H1299 cells containing tetracycline-inducible versions of P72 and R72 following
treatment with doxycycline for 6 h; the immunoprecipitating antibody is denoted, and the blot was probed for p53 and p65 RelA. Equal exposures
of p65 RelA and p53 are shown. WCL, whole-cell lysate.
1210 FRANK ET AL.MOL. CELL. BIOL.
sary for embryo implantation, thus raising the possibility that
the selection for the R72 allele involves an impact on fecundity
(14, 18). Along these lines, while the ability to fight infection is
aided by a robust innate immune response, reproductive suc-
cess 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-?B to play opposing roles in the control of me-
tabolism explains the geographic distribution of these alleles
One reason to model the p53 polymorphism in mouse was
because of the possibility that the influence of this polymor-
phism 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 de-
creased 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 poly-
morphism 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 proapop-
totic genes like Perp, Puma (Bbc3), and Noxa (Pmaip1) (16,
58), 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 explana-
tion may be that these studies were done in cells that expressed
supraphysiological levels of p53, while our Hupki mice main-
tain normal levels and regulation of this protein. In our model,
we find that only a small subset of p53 target genes, the ma-
jority of which appear to contain binding sites for both p53 and
NF-?B, show increased transactivation by P72. Interestingly,
we find that NF-?B 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 find-
ings have been described previously for p53 and the p52 sub-
unit of NF-?B (39). It remains to be determined if the coop-
eration 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 preva-
lent 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-Ameri-
cans show increased an incidence and severity of certain dis-
eases associated with inflammation, including type II diabetes,
heart disease, and obesity. It is anticipated that the Hupki
FIG. 7. Limited impact of the codon 72 polymorphism of p53 on cancer incidence. (A) Kaplan-Meier analysis of survival data obtained from
P72 and R72 mice crossed with E?-myc transgenic mice (C57BL/6 background). Statistical significance was assessed using the log rank test.
(B) Kaplan-Meier analysis of survival data from P72 and R72 mice crossed with p53?/?mice to generate P/?, R/?, and ?/? mice (C57BL/6
background). Statistical significance was assessed using the log rank test.
TABLE 2. Tumor spectrum in P/?, R/?, and ?/? mice
Type of tumor
No. of tumors in indicated mice
(n ? 23)
(n ? 23)
(n ? 16)
Spindle cell sarcoma
Sarcoma (undetermined origin)
Squamous cell carcinoma
aOne mouse had multiple tumors.
bTwo mice had multiple tumors.
VOL. 31, 2011MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p53 1211
model will be an invaluable preclinical tool to address such
We thank Donna George and Steven McMahon for critical reading
of the manuscript and members of the Murphy lab for valuable input.
We thank Glenn Rall, Dave Wiest, and Rugang Zhang for guidance
with the MEF, thymocyte, and senescence assays, respectively. We
thank Steven McMahon (Kimmel Cancer Center, Thomas Jefferson
University) for providing Tet-inducible P72 and R72 H1299 cells. We
thank David Johnson for communication of results prior to publica-
tion. We are grateful to Emmanuelle Nicolas for QPCR expertise, and
we acknowledge the support of the Laboratory Animal Facility, DNA
Microarray, Biostatistics, Immunohistochemistry, and Genotyping Fa-
cilities at Fox Chase Cancer Center.
We acknowledge funding received from the National Institutes of
Health. We acknowledge core budget support received from the Ger-
man Cancer Research Center. This project was funded in part by a
grant from the Pennsylvania Department of Health. We declare that
no conflicts of interest exist.
The Pennsylvania Department of Health specifically disclaims re-
sponsibility for any analyses, interpretations, or conclusions found in
A.K.F designed the research, performed experiments, analyzed data,
and cowrote the paper. J.I.-J.L., T.N., and M.H. designed the research,
performed experiments, and analyzed data. K.D. and Y.Z. provided
analysis of microarray data. A.K.-S. provided analysis of immunohis-
tochemistry. M.E.M. designed the research, analyzed data, and cow-
rote the paper.
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VOL. 31, 2011 MOUSE MODEL FOR EXON 4 POLYMORPHISM IN p531213