Cell, Vol. 119, 591–602, November 24, 2004, Copyright 2004 by Cell Press
A Single Nucleotide Polymorphism in the MDM2
Promoter Attenuates the p53 Tumor Suppressor
Pathway and Accelerates Tumor Formation in Humans
The tumor suppressor protein, p53, is activated upon
cellular stresses such as DNA damage and oncogene
activation and initiates a transcriptional program which
apoptosis (Jin and Levine, 2001). The p53 stress re-
sponse pathway has been shown to be crucial for the
prevention of tumor formation. For example, both mice
and humans harboring a germline inactivating mutation
in one allele of the p53 gene develop tumors very early
in life and at dramatically high frequencies (Donehower
et al., 1992; Garber et al., 1992; Li et al., 1990). Somatic
inactivating mutations of the p53 gene are also found
in over 50% of all human tumors (Lain and Lane, 2003).
Together, these observations and many others support
the importance of the p53 pathway in tumor suppres-
sion. It is therefore reasonable to assume that naturally
occurring polymorphic genetic variants in critical nodes
of the p53 pathway might underlie the variation seen
between individuals in their susceptibility to cancer and
the progression of their disease.
The search for genetic variation in the p53 pathway
an important negative regulator of p53. MDM2 directly
binds to and inhibits p53 by regulating its location, sta-
bility, and activity as a transcriptional activator (Michael
and Oren, 2003). MDM2 is an essential gene in murine
tation in the uterus. This lethal phenotype is rescued by
knocking out the p53 gene, clearly demonstrating an
important genetic interaction between these two genes
in murine development (Jones et al., 1995; Montes de
Oca Luna et al., 1995). Mendrysa et al. (2003) demon-
strated the importance of this interaction in the adult
mouse by genetically altering mice to express reduced
levels of Mdm2. These mice are small, lymphopenic,
and radiosensitive, with increased apoptosis in both
lymphocytes and epithelial cells. These phenotypes
were all shown to be p53 dependent, thereby further
demonstrating that Mdm2 is a key negative regulator of
p53 in both the developing and mature mouse. In hu-
mans, a subset of tumors overexpress MDM2 mRNA
and protein; this overexpression is associated with ac-
celerated cancer progression and lack of response to
therapy (Freedman and Levine, 1999). In a subset of
these tumors, overexpression of MDM2 was mutually
exclusive to p53 mutation, which could suggest that
overexpression of MDM2 can substitute for inactivating
p53 by mutation (Leach et al., 1993; Oliner et al., 1992).
As MDM2 expression levels seem to be vital to a well-
regulated p53 response, naturally occurring sequence
variations in the MDM2 promoter may result in altered
expression of the MDM2 protein, thereby impacting p53
tumor suppression and potentially cancer in humans. In
this report, data are presented which support this hy-
Gareth L. Bond,2,8Wenwei Hu,2,8
Elisabeth E. Bond,2Harlan Robins,1
Stuart G. Lutzker,2Nicoleta C. Arva,7
Jill Bargonetti,7Frank Bartel,4Helge Taubert,4
Peter Wuerl,5Kenan Onel,6Linwah Yip,3
Shih-Jen Hwang,3Louise C. Strong,3
Guillermina Lozano,3and Arnold J. Levine1,2,*
1School of Natural Sciences
Institute for Advanced Study
Princeton, New Jersey 08540
2Cancer Institute of New Jersey
University of Medicine and Dentistry of New Jersey
New Brunswick, New Jersey 08903
3Section of Cancer Genetics
M.D. Anderson Cancer Center
University of Texas
Houston, Texas 77030
4Institute of Pathology
Faculty of Medicine
University of Ulm
6Department of Medicine
Weill College of Medicine
New York, New York 10021
7Department of Biological Sciences
Hunter College and Graduate School
The City University of New York
695 Park Avenue
New York, New York 10021
The tumor suppressor p53 gene is mutated in mini-
mally half of all cancers. It is therefore reasonable to
assume that naturally occurring polymorphic genetic
variants in the p53 stress response pathway might
determine an individual’s susceptibility to cancer. A
central node in the p53 pathway is the MDM2 protein,
a direct negative regulator of p53. In this report, a
single nucleotide polymorphism (SNP309) is found in
ity of the transcriptional activator Sp1, resulting in
quent attenuation of the p53 pathway. In humans,
SNP309 is shown to associate with accelerated tumor
formation in both hereditary and sporadic cancers. A
model is proposed whereby SNP309 serves as a rate-
limiting event in carcinogenesis.
8These authors contributed equally to this work.
Figure 1. The MDM2 Promoter Contains a Single Nucleotide Polymorphism Which Alters the Affinity of the Transcriptional Activator Sp1
(A) A schematic diagram depicts the intronic promoter of the MDM2 gene. The position of SNP309 is indicated in relation to the exon/intron
boundaries and the transcription factor binding sites for p53 and Ets/AP-1. The region analyzed for sequence variation is marked by the bar
drawn below the diagram.
(B) The analysis of transcription factor binding sites in the region containing SNP309 is depicted. Potential Sp1 sites are underlined.
(C)An autoradiographof anelectrophoretic mobilityshift assayis depictedwhere varyingconcentrations ofpurifiedSp1 proteinwere incubated
with biotin-labeled oligonucleotides containing either SNP309 (G/G; lanes 6–10), wild-type sequence (T/T; lanes 1–5), or an Sp1 optimal binding
site (lane 11, oligo alone, and lane 12, oligo and Sp1). The arrow indicates the specific Sp1-DNA complex.
(D) Chromatin immunoprecipitations (ChIP) were preformed using antibodies against either Sp1 or lamin A. The presence of the MDM2
promoter was assayed for using PCR. The PCR products are depicted here after electrophoreses on an 8% nondenaturing polyacrylamide
gel and subsequent staining with EtBr.
(E) The relative measured luciferase levels are depicted in a bar graph, whereby pGL2 luciferase reporter plasmids containing either SNP309
sequence or wild-type sequence were cotransfected with pPac (control) or pPac-Sp1 expression plasmids. Each value is the average of at
least three independent experiments and the error bars represent the standard deviations.
Analysis of this region of the MDM2 promoter using a
computer algorithm (AliBaba) revealed several putative
binding sites for the transcription factor Sp1 (Figure 1B).
Interestingly, the presence of SNP309, a T to G change,
extended the length of one of the putative Sp1 DNA
binding sites, suggesting that the presence of SNP309
could increase the affinity of Sp1 to this region of the
identified by computer analysis and to investigate the
functional consequences of SNP309, electro-mobility
shift assays (EMSAs) were performed. EMSAs were car-
ried out with purified recombinant human Sp1 protein
and labeled double-stranded oligonucleotides contain-
ing either the wild-type sequence (T/T) or the SNP309
sequence (G/G). Interestingly, as predicted by the com-
puter analysis, the binding affinity of oligonucleotides
containing SNP309 to a range of concentrations (150
ng–450ng) ofpurifiedSp1 ismuchhigher (2–4-fold)than
The search for genetic variation in the MDM2 promoter
was focused to a well-characterized region in the first
intron of the intronic promoter, which is utilized by both
the p53 and ras pathways to activate MDM2 transcrip-
tion (Ries et al., 2000; Zauberman et al., 1995). To look
for sequence variation in this region, 300 base pairs
from genomic DNAs isolated from 50 healthy volunteers
were amplified by PCR and sequenced (Figure 1A). Two
single nucleotide polymorphisms (SNPs) were found in
this region. One of these, SNP309 (a T to G change at
the 309thnucleotide in the first intron), was found at
relatively high frequency both in the heterozygous state
(T/G, 40%) and in the homozygous state (G/G, 12%).
The other SNP, SNP344 (a T to A change at the 344th
nucleotide in the first intron), is rare and is not further
studied (found only in the heterozygous state in 8%
A SNP in Mdm2, the p53 Pathway, and Tumor Formation
by tandemly inserting two copies of double-stranded oligonucleo-
tides containing the same sequence as used for the EMSAs into
pGL2 luciferase reporter plasmid (Promega). The clones were con-
firmed by DNA sequencing.
Drosophila Schneider’s SL2 cells were seeded at 1.5 ? 106cells
per well in a six-well plate 24 hr before transfection. Transient trans-
to the manufacturer’s instructions. The DNA transfection mixture
contained 250 ng reporter plasmid, varying amounts of pPac-Sp1
plasmid, and empty pPac vector to normalize DNA concentrations.
The expression plasmids pPac and pPac-Sp1 were kindly provided
by Dr. Richard D. Kolodoner (La Jolla, California). Cells were har-
vested 48 hr after transfection in reporter lysis buffer (Promega)
and assayed for luciferase activity. Each extract was analyzed in
duplicate, and at least three independent experiments were per-
formed. Luciferase activities were normalized to cellular protein,
measured by the Bio-Rad protein assay system.
that the G allele can serve as a rate-limiting event in the
formation of a sarcoma.
It is not difficult to propose a model to explain why
Over the past 50 years, it has become clear that age-
specific incidence of cancer is dependent minimally on
three factors: the number of rate-limiting mutations re-
quired for a given cancer, the mutation rate per mitosis,
and the net proliferation rate of the effected cells (cell
division rate minus cell death rate; Knudson, 2001). Inhi-
bition of the p53 pathway by SNP309 could potentially
affect all three of these factors (Jin and Levine, 2001;
Lain and Lane, 2003). The p53 gene itself is thought of
as a rate-limiting mutation in many cancer types, as it
is found mutated in over 50% of all human tumors, and
humans who carry a germline p53 mutation develop
cancer with increased incidence and on average early
in life. The wild-type p53 pathway is also thought to
reduce mutation rates per mitosis, as loss of p53 leads
mosomal abnormalities. Finally, the p53 pathway also
impacts the net proliferation rate of cells, as it functions
signals like DNA damage and oncogene activation.
Therefore, inhibition of the p53 pathway by SNP309
could affect all three factors, which have been shown
to influence the age-specific incidence of cancer to ac-
celerate carcinogenesis in an individual.
To analyze MDM2 RNA levels, total RNA was isolated from cell
pellets using RNeasy (Qiagen). cDNAs were made using TaqMan
reverse transcription reagents from Applied Biosystems. Real-time
PCR was carried out using the ABI Prism 7000 sequence detection
system. Probe and primer sets for MDM2 and Gapdh were pur-
chased as predeveloped assays from Applied Biosystems.
To analyze protein levels, total cell extracts were made by using a
detergent lysis buffer (50 mM Tris [pH 7.5] 150 mM NaCl, 1% NP40,
0.1% SDS, 0.5% deoxycholic acid, 5mM EDTA, and a protease
protein was run on a 4%–20% tris-glycine gel (Invitrogen) and trans-
ferred to a PVDF membrane. MDM2 was detected using the mouse
monoclonal antibody SMP-14; Sp1 and Sp3 were detected using
the rabbit polyclonal antibody PEP2 and D-20, respectively. Lamin
clonal antibody 346, DCS-6, Do-1, and C-2, respectively. Gapdh
were purchased from Santa Cruz.
The MDM2 promoter was analyzed for sequence variation by PCR
amplification and subsequent sequencing, primer 1: CGGGAGTT
CAGGGTAAAGGT and primer 2: AGCAAGTCGGTGCTTACCTG.
Gene Silencing with siRNA
Sp1 siRNA targeted to AATGAGAACAGCAACAACTCC was used to
lower Sp1 expression. Two hundred picomoles siRNA duplex was
reagent (Invitrogen) according to the manufacturer’s instructions.
Control siRNA duplex has no known target in mammalian genomes
and was usedas follows: sense UUCUCCGAACGUGUCACGUdTdT,
antisense ACGUGACACGUUCGGAGAAdTdT. Lamin A/C siRNAs
were purchased from Qiagen and MDM2 siRNAs from Dharmacom.
Cells were lysed 48 hr after transfection, and protein levels of Sp1,
Sp3, and MDM2 were analyzed as described above.
Electrophoretic Mobility Shift Assays (EMSA)
EMSAs were performed with a LightShiftTM Chemiluminescent
EMSA Kit (PIERCE, Rockford, Illinois). The binding reactions were
performed for 20 min at room temperature in 10 mM Tris-HCl ([pH
7.5] at 25?C), 1 mM MgCl2, 50 mM NaCl, and 0.5 mM DTT, 4%
glycerol, 50 ?g/ml poly (dI-dC) (dI-dC), 10 fmol biotin 3?-end-labeled
double-stranded oligonucleotides, and purified recombinant Sp1
protein. After incubation, samples were separated on a native 4%
polyacrylamide gel and then transferred to a nylon membrane. The
positions of the biotin end-labeled oligonucleotides were detected
by a chemiluminescent reaction with streptavidin-horseradish per-
oxidase according to the manufacturer’s instruction and visualized
by autoradiography. The nucleotide sequence of the double-
stranded oligonucleotides with either wild-type sequence or the
SNP is as follows: 5?-CCGGGGGCTGCGGGGCCGCTT/GCGGCGC
Mithramycin A Treatment
Logarithmically growing cells were treated with various concentra-
tions of mithramycin A (Sigma) for 24 hr. After treatment, cells were
lysed and protein levels of MDM2 were analyzed.
Cell Viability Analysis
cells were treated with either 1 ?M or 5 ?M etoposide for 24 hr. To
analyze etoposide-induced cell death after inhibition of Sp1, grow-
ing cells were treated with various concentrations of mithramycin
A and etoposide for 48 hr. After treatment, cells were harvested and
viability was measured using the Guava ViaCount assay (Guava).
Proteins were crosslinked to DNA in 1% formaldehyde. After wash-
ing, cells were lysed in detergent lysis buffer. Lysates were washed
andsonicated.Two microgramsofantibodieswere addedandincu-
bated overnight. Protein A/G Plus beads (Santa Cruz) were used,
and after extensive washing, crosslinks were removed at 65?C over-
using the QIAquick PCR purification kit (Qiagen). Ten percent of
purified DNA was analyzed by PCR. The entire PCR reaction was
mide gel and subsequent ethidium bromide staining.
Cell Cycle Analysis
To analyze the DNA damage response in fibroblasts, all cells were
treated with two concentrations of etoposide (1 ?M and 5 ?M) for
24 hr. Cells were harvested and fixed with methanol and kept
at ?20?C for 40 min or overnight. After washing and equlibration in
PBS, the cell pellet was resuspended in 1 ml of staining solution
containing 50 ?g of RNase A and 0.5 ?g of propidium iodide per
ml in PBS and kept at room temperature for over 30 min. The cells
Luciferase Reporter Assays
The MDM2 promoter-luciferase reporter plasmids containing either
the wild-type sequence or the SNP309 sequence were constructed
were then applied to the fluorescence-activated cell sorter (FACS-
Calibur; Becton Dickson). The FACSCalibur program was used to
sort and count the cells.
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without SNP309. The two groups are compared pair-wise, and each
instance of an element from the second group greater than an ele-
ment of the first group adds one to the distance. This total distance
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the number of elements of each list. The calculated p value is the
percent of randomized groups that have a distance less than the
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We would like to thank A. Teresky, H. Jaramillo, B. Wypior, and Z.
Feng for their expert technical assistance. We thank W. Liu and D.
Notterman in the CINJ Core Expression Array Facility. T. Gopen
kindly provided us with the status of the p53 gene in the Manca cell
line. We thank A.G. Knudson and S. Friend for helpful discussions.
We thank S. Harris for help with the preparation of this manuscript.
Received: April 14, 2004
Revised: September 23, 2004
Accepted: October 13, 2004
Published: November 23, 2004
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