Activation of cAMP Signaling
Interferes with Stress-Induced
p53 Accumulation in ALL-Derived
Cells by Promoting the Interaction
between p53 and HDM21,2
Elin Hallan Naderi*, Aart G. Jochemsen†,
Heidi Kiil Blomhoff*and Soheil Naderi*,3
*Department of Biochemistry, Institute of Basic Medical
Sciences, University of Oslo, Oslo, Norway;†Department
of Molecular and Cell Biology, Leiden University Medical
Center, Leiden, The Netherlands
The tumor suppressor p53 provides an important barrier to the initiation and maintenance of cancers. As a con-
sequence, p53 function must be inactivated for a tumor to develop. This is achieved by mutation in approximately
50% of cases and probably by functional inactivation in the remaining cases. We have previously shown that
the second messenger cAMP can inhibit DNA damage–induced wild-type p53 accumulation in acute lympho-
blastic leukemia cells, leading to a profound reduction of their apoptotic response. In the present article, we pro-
vide a mechanistic insight into the regulation of p53 levels by cAMP. We show that increased levels of cAMP
augment the binding of p53 to its negative regulator HDM2, overriding the DNA damage–induced dissociation
of p53 from HDM2. This results in maintained levels of p53 ubiquitination and proteasomal degradation, which
in turn counteracts the DNA damage–induced stabilization of the p53 protein. The apoptosis inhibitory effect of
cAMP is further shown to depend on this effect on p53 levels. These findings potentially implicate deregulation of
cAMP signaling as a candidate mechanism used by transformed cells to quench the p53 response while retaining
Neoplasia (2011) 13, 653–663
The tumor suppressor p53 is normally activated in response to var-
ious types of cellular stress, such as DNA damage, oncogenic signal-
ing, mitotic impairment, and oxidative stress . This activation is
brought about mainly by posttranslational modifications such as
phosphorylation, acetylation, and ubiquitination, resulting in both
quantitative and qualitative changes of p53, thus allowing for its in-
creased transcriptional activity . The result of the activation of
the p53 transcriptional program may vary depending on cell type
and the nature and intensity of cellular stress and includes cell cycle
arrest, senescence, and apoptosis. In addition to its function as a tran-
scription factor, transcription-independent effects of p53 have been
demonstrated to contribute, particularly with regard to p53-induced
apoptosis [3,4]. Evasion of the tumor-suppressive effect of p53 can be
achieved by mutational inactivation as is observed in approximately
lion cases of cancer annually, which retain wild-type p53 , and there
is mounting evidence that the p53 function must be attenuated for
these cancers to develop, maintain, and progress [8–10]. Such attenu-
ation can be achieved by viral proteins, deregulation of components of
the p53 regulatory circuit, or disruption of upstream or downstream
signaling pathways .
A central component in the p53 regulatory circuit is the HDM2 E3
ubiquitin ligase (corresponding to mouse double minute 2, Mdm2,
protein). In unstressed cells, HDM2 prevents accumulation of p53 by
Abbreviations: BCP-ALL, B-cell precursor acute lymphoblastic leukemia; IR, ionizing
radiation; Epac, exchange protein directly activated by cAMP; PKA, protein kinase A
Address all correspondence to: SoheilNaderi,PhD,FacultyofHealthSciences,OsloUni-
versity College, Pilestredet 50, 0167, Oslo, Norway. E-mail: Soheil.Naderi@hf.hio.no; or
Heidi Kiil Blomhoff, PhD, Department of Biochemistry, Institute of Basic Medical
Sciences, University of Oslo, Oslo, Norway. E-mail: firstname.lastname@example.org
1This work was supported by grants from the Norwegian Cancer Society, the Jahre
Foundation, Blix Foundation, Freia Foundation and Rachel and Otto Kr. Bruum’s
Foundation. The authors declare no conflict of interest.
2This article refers to supplementary materials, which are designated by Figures W1 to
W3 and are available online at www.neoplasia.com.
3Current address: Faculty of Health Sciences, Oslo University College, Pilestredet 50,
0167, Oslo, Norway.
Received 15 April 2011; Revised 13 May 2011; Accepted 16 May 2011
Copyright © 2011 Neoplasia Press, Inc. All rights reserved 1522-8002/11/$25.00
Volume 13 Number 7July 2011pp. 653–663
damage is thought to induce a reduction in the interaction of HDM2
with p53, thus preventing the ubiquitination of p53 and promoting its
stabilization. The essential role of HDM2 in regulation of p53 is dem-
onstrated by the fact that the embryonic lethality in mdm2−/−mice can
p53-specific E3 ubiquitin ligases such as Pirh2, COP1, and ARF-BP1/
Mule have been identified [14–17], HDM2 still retains its position as a
pivotal part of the p53 regulatory circuit .
cAMP is the prototypical second messenger that is generated by
adenylyl cyclase on stimulation of G protein–coupled receptors
(GPCRs). In cells of the immune system, cAMP is established as
an important signal transducer in several physiological and patho-
logic settings . This includes control of normal T-cell activa-
tion [20,21] and contribution to T-cell dysfunction in human and
murine immunodeficiency virus infection/acquired immunodefi-
ciency syndrome [22,23]. Lymphocytes possess GPCRs for cate-
cholamines and prostaglandin E2(PGE2), and engagement of these
receptors by their respective ligands has been shown to exert a
growth-inhibitory effect mediated by the elevation of cAMP levels
[24–27]. We have previously shown that elevation of intracellular
cAMP leads to accumulation of lymphoid cells in the G1phase
[28–30], and later, we demonstrated that cAMP exerts an inhibitory
effect on DNA replication, thus leading to arrest of cells in S phase
and block of apoptosis induced by S phase–specific anticancer agents
. Recently, we reported that augmentation of cAMP signaling in
primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL)
cells, as well as in BCP-ALL cell lines and primary nontransformed B
and T cells, inhibits their apoptotic response to DNA-damaging treat-
ments such as ionizing radiation (IR) and different classes of che-
motherapeutics . This effect was further shown to be mediated
through attenuation of the IR-induced accumulation of p53. Here,
we reveal the mechanism whereby activation of the cAMP signaling
pathway attenuates p53 accumulation. We show that cAMP exerts a
stimulatory effect on the interaction of p53 with HDM2, thus abrogat-
ing the IR-induced inhibition of p53 ubiquitination and degradation.
Materials and Methods
Reagents and Antibodies
Forskolin, propidium iodide (PI), and N-ethylmaleimide were from
Sigma-Aldrich (St Louis, MO). MG-132 and Nutlin-3a were obtained
from Calbiochem (La Jolla, CA). 8-CPT-cAMP and 8-pCPT-2′-O-
Me-cAMP were from BioLog (Bremen, Germany). Antibodies were
as follows: total p53 (DO-1 and FL-393), HDM2 (SMP14), and actin
(C-2) from Santa Cruz Biotechnology (Santa Cruz, CA); HDM2
(IF2) from Calbiochem; HDM2 (4B2) that was a kind gift from
Dr A. Levine; HDMX from Bethyl Laboratories (Montgomery, TX);
phospho-p53 (S15, no. 9286; T18, no. 2529) from Cell Signaling
(Danvers, MA); and phospho-p53 (S20, AF2286) and ubiquitin
(PW8810) from R&D Systems (Minneapolis, MN) and Biomol
(Plymouth Meeting, PA), respectively.
Cell Cultures, Radiation Treatment, and Cell Death Analysis
. Human CD4+T cells were isolated, cultured, and stimulated as
described . U2OS and MCF-7 cell lines were cultured in McCoy
and Dulbecco modified Eagle media, respectively. γ-Irradiation of cells
was carried out using a137Cs source at a dose rate of 4.3 Gy/min.
Cell death was detected by incubation of cells in PBS contain-
ing 20 μg/ml PI followed by analysis on a FACS instrument for
Small Interfering RNA Transfection
Reh cells (6 × 106) were transfected with 500 nmol of HDM2
small interfering RNA (siRNA) or control siRNA (L003279-00 or
D-001810-10, respectively; Dharmacon, Lafayette, CO) using the
nucleofection solution R and the O-17 program with a nucleofector
device (Amaxa Biosciences, Basel, Switzerland). Cells were then in-
cubated for 24 hours before further treatment.
Immunoblot Analysis and Immunoprecipitation
For immunoblot analysis, cells were lysed in RIPA buffer. For
detection of ubiquitinated p53, the RIPA buffer was supplemented
with 2 mM N-ethylmaleimide. Equal amounts of proteins (30 μg)
were separated on SDS-PAGE, transferred to a nitrocellulose mem-
brane (Amersham, Piscataway, NJ), and detected by use of stan-
dard immunoblot analysis procedures. For immunoprecipitation of
HDM2 in complex with p53, cells were lysed in NP-40 lysis buffer
(50 mM Tris [pH 7.5], 150 mM NaCl, 0.5% NP-40, 10 mM NaF,
1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride, 10 mg/ml
leupeptin, and 0.5% aprotinin). Lysates containing 600 μg of protein
were immunoprecipitated with FL-393 followed by 50 μl of a 1:1 slurry
of protein G-agarose (Upstate, Temecula, CA). Beads were washed four
times in lysis buffer, eluted in boiling 1× SDS buffer, and subjected to
immunoblot analysis. For densitometric analysis, blots were scanned,
and the intensity of protein bands was quantified using the Scion Image
software (Scion Corp, Frederick, MD).
Messenger RNA Purification and Northern Analysis
town, MD) was fractionated on a formaldehyde/agarose gel, trans-
ferred onto Hybond-N filter (Amersham), and then hybridized with
a complementary DNA (cDNA) probe prepared from pMEV-p53-
WT (Biomyx, San Diego, CA) as described previously .
Statistical Methods and Calculation
SPSS 14.0 for Windows (Chicago, IL) was used to perform paired-
samples t test. Error bars indicate SEM.
cAMP Inhibits Both the Magnitude and Duration of DNA
Damage–Induced p53 Accumulation
In a recent study, we showed that an increase in cAMP levels
in primary lymphoid cells as well as cell lines, inhibited apoptosis
induced by various genotoxic agents such as IR . This effect of
cAMP was shown to depend on its ability to attenuate the DNA
damage–induced accumulation of p53. More specifically, cAMP
was found to profoundly inhibit, by approximately 70%, the induc-
tion of p53 at 4 hours after IR. As a first step to assess the mecha-
nisms that underlie the inhibitory effect of cAMP on p53 levels, we
examined the effect of cAMP on the kinetics of p53 accumulation
after IR. To this end, Reh cells were treated with IR in the absence
or presence of the adenylyl cyclase activator forskolin or the cAMP
cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al.Neoplasia Vol. 13, No. 7, 2011
analog 8-CPT-cAMP, harvested at regular intervals after IR for a
total of 24 hours, and then analyzed for the expression of p53 by
Western blot analysis. As shown in Figure 1A, p53 was induced
by 2 hours after IR, peaked within 4 to 8 hours, and declined there-
after to a level above that in untreated cells within 24 hours. In the
presence of forskolin, p53 was maximally induced by 2 hours after
IR, albeit by approximately 50% less than the level of p53 observed
in cells that were treated with IR alone. Further exposure of cells to
forskolin decreased the level of p53 slightly more so that, by 8 hours,
cells expressed p53 at a level somewhat above that seen in untreated
cells. p53 was maintained at this level by the end point of the exper-
iment at 24 hours. Exposure of cells to 8-CPT-cAMP had only a
marginal inhibitory effect on IR-induced accumulation of p53 by
2 hours after IR. By 8 hours after treatment of cells with 8-CPT-
cAMP, the expression of p53 had declined profoundly to a level
comparable to that seen in forskolin-treated cells and remained at this
level by 24 hours after IR. Taken together, these results show that
cAMP exerts an inhibitory effect on both the magnitude and the
duration of p53 response after treatment of cells with IR.
To ascertain that the ability of cAMP to inhibit DNA damage–
induced accumulation of p53 is not a cell type–specific phenomenon,
we examined the effect of forskolin and 8-CPT-cAMP on the expres-
sion of p53 in IR-treated normal human CD4+T cells, the osteo-
sarcoma U2OS, and the mammary adenocarcinoma MCF-7 cell
lines. Exposure of these cells to IR led to accumulation of p53 within
Figure 1. cAMP inhibits the IR-induced p53 accumulation in an Epac-independent fashion. (A) Reh cells were treated with forskolin or
8CPT-cAMP for 30 minutes before exposure to 10 Gy of IR. Cells were harvested at the indicated times after IR, lysed, and subjected to
immunoblot analysis with DO-1 and antiactin antibodies. p53 bands were subsequently quantified by densitometric analysis, and fold
increase of p53 band intensity was calculated relative to the unirradiated control. One representative experiment of three is shown. (B)
Human peripheral CD4+T cells, U2OS and MCF-7 were treated with forskolin or 8-CPT-cAMP for 30 minutes before exposure to IR. After
4 hours, cells were harvested, lysed, and subjected to immunoblot analysis with DO-1 and antiactin antibodies. One representative
experiment of three is shown. (C) Reh cells were treated with the indicated concentrations of 8-CPT-cAMP or 8-pCPT-2′-O-Me-cAMP
for 30 minutes before exposure to IR. After 4 hours, the cells were harvested, lysed, and subjected to immunoblot analysis with DO-1
and antiactin antibodies.
Figure 2. p53 mRNA steady-state levels are not affected by IR or
IR. Cells were harvested at the indicated times after IR, RNA was
isolated and subjected to Northern blot analysis with a p53 cDNA
as control for the specificity of the cDNA probe.
Neoplasia Vol. 13, No. 7, 2011cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al.
4 hours (Figure 1B). Importantly, pretreatment of cells with forskolin
or 8-CPT-cAMP attenuated the IR-induced p53 response in all three
cell types, indicating that inhibition of IR-mediated accumulation of
p53 represents a more general, cell type–independent action of cAMP.
cAMP Exerts Its Effect on p53 Accumulation through
Protein Kinase A
Once formed, cAMP can act on several effector proteins such as
protein kinase A (PKA) , exchange protein directly activated by
cAMP (Epac) [35,36], or cyclic nucleotide–gated (CNG) cation
channels [37,38]. PKA is generally viewed as the major cellular target
of cAMP in lymphoid cells. However, Epac has also been implicated
as a mediator of various effects, notably playing an antiapoptotic role
in B-chronic lymphocytic leukemia (B-CLL) cells  and T-ALL
cells . Therefore, to examine whether the inhibitory effect of
cAMP on IR-induced accumulation of p53 is mediated by PKA or
Epac, we treated Reh cells with IR in the absence or presence of
8-CPT-cAMP or 8-pCPT-2′-O-Me-cAMP and examined them for
the expression of p53 after 4 hours of IR. Whereas 8-CPT-cAMP ac-
tivates both PKA and Epac, 8-pCPT-2′-O-Me-cAMP functions as an
Epac-specific cAMP analog with no effect on PKA activity [41,42].
As shown in Figure 1C, pretreatment of cells with 200 or 400 μM
8-CPT-cAMP led to a progressive inhibition of IR-induced p53 ac-
cumulation in a dose-responsive manner. In contrast, treatment of
cells with as high as 400 μM pCPT-2′-O-Me-cAMP had no effect
on the expression of p53 in IR-treated cells. These results suggest
that the inhibitory effect of cAMP on p53 expression is mediated
cAMP Affects p53 Levels by Alteration of Protein Stability
p53 is a labile protein expressed at low levels under unperturbed
conditions but is rapidly stabilized and accumulates in response to
cellular stress such as DNA damage. However, apart from modula-
tion of its protein stability, other mechanisms have been suggested to
contribute to regulation of p53 protein levels. For instance, it was
recently shown that modulation of p53 messenger RNA (mRNA)
levels by Wrap53, a natural p53 antisense transcript, contributes to
the regulation of p53 levels . Therefore, to understand the mech-
anism by which cAMP inhibits DNA damage–induced accumulation
of p53, we first examined the effect of cAMP on p53 mRNA steady-
state levels. Northern blot analysis of Reh cells that were treated with
IR in the presence or absence of forskolin showed that the steady-
state levels of p53 mRNA remained unaffected by IR alone or IR in
the presence of forskolin (Figure 2). This result points toward our
Figure 3. cAMP inhibits the IR-induced stabilization of the p53 protein. Reh cells were treated with forskolin (60 μM) or 8-CPT-cAMP
(200 μM) for 30 minutes before IR. Four hours after IR, cells were treated with cycloheximide (CHX; 25 μg/ml) and then harvested at the
indicated times. Whole-cell lysates were prepared and analyzed by immunoblot analysis with DO-1 and antiactin antibodies. Upper panel
shows one representative experiment of four. Lower panel: the immunoblots represented in the upper panel were scanned, and the
intensity of the p53 protein bands was quantitated and plotted with the value obtained for cells not treated with CHX set as 1. Values
were normalized with those of actin (n = 4).
cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al. Neoplasia Vol. 13, No. 7, 2011
previous finding demonstrating an inhibitory effect of forskolin on
p53 protein stability as the primary mechanism by which forskolin
attenuates the IR-induced accumulation of p53 . To further sub-
stantiate that the effect of forskolin on IR-induced p53 stability indeed
depends on cAMP, we treated Reh cells with forskolin or 8-CPT-
cAMP before IR. After 4 hours, cycloheximide was added, and the
levels of p53 were assessed by immunoblot analysis at 30-minute in-
tervals. Whereas IR led to stabilization of p53 protein, both 8-CPT-
cAMP and forskolin profoundly reduced the IR-induced stabilization
of p53 protein (Figure 3).
cAMP Affects p53 Half-life through Ubiquitination and
The half-life of the p53 protein is predominantly regulated through
the proteasomal degradation pathway [1,44]. Therefore, to unravel the
mechanism whereby cAMP reduces the stability of p53, we first exam-
ined the effect of cAMP on p53 levels in the presence of the protea-
some inhibitor MG-132. As shown in Figure 4A, exposure of cells to
MG-132 abrogated the inhibitory effect of forskolin or 8-CPT-cAMP
on IR-induced accumulation of p53, indicating that cAMP negatively
regulates the p53 levels through the proteasomal degradation pathway.
Ubiquitination of p53 is a tightly regulated event required for p53
degradation by proteasomes. Therefore, we wished to examine whether
cAMP affected the ubiquitination of p53. To do so, Reh cells were first
treated with MG-132 to inhibit the proteasomal degradation p53.
Cells were then exposed to IR in the absence or presence of forskolin
and harvested 4 hours after IR treatment for examination of p53 pro-
tein by Western blot analysis. IR reduced the level of p53 ubiquitina-
tion as visualized by the appearance of high-molecular weight bands of
p53 (Figure 4B, upper panel). Importantly, pretreatment of cells with
forskolin restored the ubiquitination of p53 to levels comparable to
those seen in untreated cells. This effect of forskolin on p53 ubiqui-
tination in IR-treated cells was not due to the presence of MG-132 be-
cause forskolin was found to exert a similar effect in the absence of
MG-132 (Figure W1). To further substantiate that the changes in in-
tensity of high-molecular weight bands of p53 represent changes in the
degree of ubiquitination, p53 was immunoprecipitated from the cell
lysates used in the experiment shown in the upper panel of Figure 4B
Figure 4. cAMP inhibits p53 accumulation in a proteasome-dependent manner and counteracts IR-induced reduction of p53 ubiquitina-
tion. (A) Reh cells were preincubated with (upper panel) or without (lower panel) MG-132 for 2 hours before treatment with forskolin or
8-CPT-cAMP for 30 minutes. Cells were then exposed to IR, harvested at the indicated times post-IR, and subjected to immunoblot
analysis with DO-1 and antiactin antibodies. Vertical lines have been inserted to indicate repositioned gel lanes. Lower panel is shown
as comparative data on the effect of MG-132 on p53 levels. (B) Upper panel: Reh cells were pretreated with MG-132 for 2 hours before
addition of forskolin. After 30 minutes, cells were exposed to IR, harvested after 4 hours, and then subjected to immunoblot analysis
with DO-1 and antiactin antibodies. Lower panel: Reh cells were treated as described for the upper panel. Whole-cell extracts were
prepared and immunoprecipitated (IP) with FL-393 antibody. The recovered proteins were resolved on SDS-PAGE and then subjected
to immunoblot analysis with antibodies against ubiquitin. Subsequently, the blot was stripped and then reprobed with DO-1.
Neoplasia Vol. 13, No. 7, 2011 cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al.
and then immunoblotted with antiubiquitin antibody. In accordance
with results obtained with whole-cell lysates, exposure of cells to IR led
to reduction of ubiquitinated proteins that precipitated with anti-p53
antibody, whereas cotreatment of cells with forskolin increased the
amount of ubiquitin-conjugated p53 compared with cells exposed to
IR alone (Figure 4B, lower panel). Taken together, these results show
that cAMP inhibits the accumulation of p53 after IR by antagonizing
the IR-induced loss of p53 ubiquitination.
Effect of cAMP on IR-Induced p53 Accumulation and
Apoptosis Depends on HDM2 and Involves Enhanced
Interaction between HDM2 and p53
The degradation of p53 is primarily mediated by the E3 ubiquitin
ligase, HDM2, which, on binding to p53, induces the ubiquitination
of p53, priming it for proteasomal recognition . Therefore, our
observation that cAMP-mediated inhibition of IR-induced accumu-
lation of p53 was associated with an increase in ubiquitination of p53
Figure 5. The inhibitory effect of cAMP on p53 accumulation requires functional HDM2 and involves inhibition of IR-induced dissociation
of the p53-HDM2 complex. (A) Reh cells were treated with Nutlin-3a for 10 minutes before the addition of 8-CPT-cAMP, forskolin, or
corresponding volumes of their solvents dH2O or DMSO, respectively. After 30 minutes, cells were exposed to IR and incubated for an
additional 4 hours. Whole-cell lysates were then prepared and analyzed by immunoblot analysis with the DO-1 and antiactin antibodies.
The immunoblot shows one representative experiment of three. The histogram depicts the average densitometric value of the p53
protein bands. Values from cAMP-treated samples have been normalized to their relevant solvent controls, whose values were set
to 100% (n = 3). (B) Reh cells were transfected with control siRNA or siRNA against HDM2. After 24 hours, cells were cultured in
the presence or absence of forskolin for 30 minutes before exposure to IR and incubated for an additional 4 hours. Whole-cell lysates
were then prepared and analyzed by immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2), DO-1, and antiactin
antibodies. (C) Reh cells were treated with forskolin for 30 minutes before exposure to IR. Cells were then harvested at the indicated
times after IR and subjected to immunoblot analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2) and antiactin antibodies. The
immunoblot shows one representative experiment of seven. The immunoblots represented above were scanned, and the intensity of
the p53 protein bands was quantitated and plotted with the value obtained for untreated cells set as 1 (n = 7). (D) Reh cells were treated
as in C and analyzed by immunoblot analysis with the indicated antibodies. One representative experiment of three is shown. (E) Reh
cells were pretreated with MG-132 for 2 hours before addition of forskolin. After 30 minutes of forskolin treatment, cells were exposed
to IR and then harvested at the indicated times. Whole-cell extracts were prepared and immunoprecipitated with FL-393. The recovered
proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with the anti-HDM2 (a mixture of SMP14, IF2, and
4B2) and DO-1 antibodies. The immunoblot shows one representative experiment of seven. The intensity of protein bands was quan-
tified densitometrically, and the ratio of signal intensity for HDM2 relative to p53 was calculated and plotted with the value obtained for
untreated cells set as 1 (n = 7; at all three time points, there is a significant difference between cells treated with IR alone and those
treated with IR + forskolin, P < .05). Panel marked input represents Western blots for HDM2, p53, and actin in cell extracts before
immunoprecipitation. Vertical lines have been inserted to indicate repositioned gel lanes.
cAMP Signaling Promotes p53-HDM2 Association Naderi et al.Neoplasia Vol. 13, No. 7, 2011
suggested that HDM2 might be involved in mediating this inhibitory
effect of cAMP on p53. To examine this possibility, we wished to as-
sess the regulation of p53 by cAMP under conditions in which the
effect of HDM2 on p53 is blocked. To this end, we examined the
effect of cAMP on p53 levels in the presence of Nutlin-3a, a small
molecule that alleviates the inhibitory effect of HDM2 on p53 by dis-
rupting the p53-HDM2 interaction. As shown in Figure 5A, forskolin
or 8-CPT-cAMP was unable to inhibit the IR-induced accumulation
of p53 in the presence of Nutlin-3a. Similarly, knockdown of HDM2
strongly attenuated the effect of forskolin on the accumulation of p53
in IR-treated cells (Figure 5B). These results show that HDM2 me-
diates the ability of cAMP to induce the degradation of p53.
Tofurther characterizethemechanismbywhichcAMPuses HDM2
to exert its inhibitory effect on p53, we investigated in more detail
whether cAMP modulates the levels of HDM2 protein. As shown in
Figure 5C, forskolin alone led to a slight and transient reduction in
peaked by 4 hours, and remained essentially at this level by the end
point of the experiment at 8 hours. In the presence of forskolin,
HDM2 was also maximally induced by 4 hours after IR. By 6 hours
after IR, forskolin had inhibited the expression of HDM2 to a level
below that seen in cells that were treated with IR alone. This was fol-
lowed by an increase in HDM2 levels so that, by 8 hours after IR,
HDM2 was expressed at a level similar to that in IR-exposed cells in
the absence of forskolin. By 2 and 4 hours after IR, forskolin-treated
cells expressed HDM2 to slightly higher levels than cells that were ex-
posed to IR alone. However, this difference was statistically insignifi-
cant and thus could not convincingly explain the inhibitory effect of
cAMP on IR-induced accumulation of p53.
The HDM2-related protein HDMX is another negative regulator of
p53. In addition to its ability to suppress the p53 transcriptional activ-
ity, HDMX inhibits the accumulation of p53 by augmenting HDM2-
mediated ubiquitination of p53 [46–48]. Indeed, down-regulation of
HDMX protein levels in response to DNA damage is critical for p53
accumulation [46,49]. This property of HDMX raised the possibility
that cAMP might inhibit IR-mediated degradation of p53 by antago-
nizing the IR-induced down-regulation of HDMX. Therefore, we
examined the expression of HDMX in IR-exposed Reh cells in the
absence or presence of forskolin. As shown in Figure 5D, IR led to a
pronounced reduction in HDMX levels, but forskolin failed to prevent
the degradation of HDMX in IR-treated cells.
We then examined whether cAMP affected the interaction of
HDM2 with p53. Reh cells were treated with MG-132 to maintain
p53 at a relatively constant level before exposure to IR in the absence
or presence of forskolin. Cells were harvested at regular intervals after
IR for a total of 6 hours, and the lysates were then subjected to immu-
noprecipitation with anti-p53 antibodies followed by immunoblot
analysis with antibodies against p53 and HDM2. p53 immunocom-
plexes recovered from IR-treated cells contained significantly lower
levels of HDM2 than those of untreated cells (Figure 5E). Importantly,
pretreatment of cells with forskolin antagonized the IR-induced dis-
sociation of HDM2 from p53, so that, by 4 and 6 hours after IR,
forskolin had restored the level of HDM2 in association with p53 to
that found in untreated cells. The positive effect of forskolin on p53-
HDM2 association was not due to the presence of MG-132 because
forskolin exerted a similar effect in the absence of MG-132 (Fig-
ure W2). Importantly, similar results were obtained with cells that were
exposed to PGE2, a physiological inducer of intracellular cAMP levels
(Figure W3). These results suggest that cAMP inhibits IR-induced sta-
bilization of p53 by increasing the association between HDM2 and
p53. Interestingly, Figure 5E also shows that forskolin augments the
association of HDM2 with p53 in the absence of IR, indicating that
the IR-induced dissociation of HDM2 from p53 is not a prerequisite
for the positive effect of cAMP on p53-HDM2 interaction. This no-
tion is in accordance with the observation that cAMP also inhibits the
expression of p53 protein in nonirradiated cells (Figures 1B and 3).
Effect of cAMP on p53 Phosphorylation
The DNA damage–induced phosphorylation of p53 at sites within
its N-terminus has been proposed to contribute to p53 stabilization
by attenuating its binding to HDM2. Therefore, we wished to assess
whether cAMP antagonizes the IR-induced dissociation of p53 from
HDM2 by inhibiting the phosphorylation of p53 after IR. To do
so, Reh cells were first treated with MG-132 to minimize variations
in p53 levels, before exposure to IR in the absence or presence of
forskolin. Cells were harvested at different times after IR and immuno-
blotted with antibodies against p53 phosphorylated at S15, T18, or
S20 as phosphorylation of these sites has been suggested to disrupt
the p53-HDM2 complex. IR led to a rapid and pronounced increase
in phosphorylation of p53 at S15 and S20 and, to a lesser extent, at
T18 (Figure 6). Whereas forskolin had only a slight or no effect on
IR-induced phosphorylation of p53 at S15 and T18, respectively, it
reduced the level of phosphorylation at S20 by approximately 20%.
Attenuation of p53 Accumulation Is Required for
cAMP-Induced Inhibition of p53-Dependent Apoptosis
Recently, we showed that cAMP depends on p53 to exert its inhib-
itory effect on apoptosis induced by DNA damage . This ob-
servation, together with our present finding that cAMP inhibits the
IR-induced accumulation of p53 in an HDM2-dependent manner,
Figure 5. (continued)
Neoplasia Vol. 13, No. 7, 2011 cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al.
Figure 6. Effects of cAMP on p53 phosphorylation. Upper panel: Reh cells were treated with MG-132 (20 μM) for 2 hours before addition
of forskolin (60 μM). After 30 minutes, cells were exposed to 10 Gy of IR and harvested at the indicated times. For the examination of
p53 phosphorylation at S15 and S20, whole-cell lysates were subjected to immunoblot analysis with phospho-specific antibodies
against p53 phosphorylated at S15 or S20. Subsequently, the membranes were stripped and reprobed with DO-1 to detect total p53
protein. One representative experiment of five is shown. For detection of p53 phosphorylated at T18, whole-cell lysates were immuno-
precipitated with DO-1, and the recovered proteins were immunoblotted with phospho-specific antibodies against p53 phosphorylated
at T18. The blot was then stripped and reprobed with total p53 (FL-393) antibody. One representative experiment of four is shown.
Lower panel: The immunoblots represented in the upper panel were scanned, and the intensity of protein bands was quantified densi-
tometrically. The ratio of signal intensity for phosphorylated p53 at S15, S20, or T18 relative to total p53 was then calculated, and the
obtained values were normalized to the value obtained for cells at time 0 (S15 and S20, n = 5; T18, n = 4).
cAMP Signaling Promotes p53-HDM2 Association Naderi et al.Neoplasia Vol. 13, No. 7, 2011
suggests that the inhibitory effect of cAMP on IR-induced cell death is
mediated through its ability to attenuate the stabilization of p53. To
verify this notion, we examined the effect of cAMP on IR-induced
apoptosis in the presence of Nutlin-3a, a condition under which
cAMP is unable to modulate the stability of p53 (Figure 5A). Unfor-
tunately, the combination of Nutlin-3a and IR was extremely toxic to
the cells (data not shown), complicating the interpretation of results.
To circumvent this problem, we examined the effect of cAMP on
Nutlin-3a–induced cell death, an event shown to depend on p53.
As shown in Figure 7, treatment of cells with Nutlin-3a alone led to
accumulation of p53 to a level comparable to that observed 4 hours
after exposure of cells to 10 Gy of IR and resulted in cell death within
20 hours. Whereas forskolin markedly inhibited the IR-mediated p53
accumulation and cell death, it had little effect on Nutlin-3a–induced
p53 accumulation and no inhibitory effect on Nutlin-3a–induced cell
death. This result further supports the notion that the cAMP inhibits
IR-induced cell death through its ability to abrogate p53 accumulation
and indicates that this property of cAMP depends on the presence of
functionally intact HDM2.
The study outlined here shows that activation of cAMP signaling
attenuates IR-induced p53 stabilization in an HDM2-dependent
manner by counteracting the IR-induced p53-HDM2 dissociation,
thereby restoring p53 ubiquitination and degradation (Figure 8).
HDM2 plays an essential role in the regulation of p53 func-
tion. Under normal conditions, HDM2 binds p53 and primes it for
ubiquitin-dependent degradation by nuclear and cytoplasmic pro-
teasomes . In response to stress signals such as DNA damage,
the p53-HDM2 interaction is disrupted, leading to accumulation of
p53 and induction of a p53 response . The role of HDM2 as a
critical regulator of p53 activity is demonstrated by genetic studies
showing that the embryonic lethality in mdm2-knockout mice can
be rescued by additional deletion of the Trp53 gene [12,13]. Further-
more, overexpression of HDM2 has been observed in soft tissue tu-
mors, osteosarcomas, and esophageal carcinomas . Specifically,
in pediatric ALLs, most of which retain wild-type p53, overexpression
of HDM2 is a rather common event . These results suggest that
p53-HDM2 interaction plays a major role in inhibition of p53 func-
tion in tumors that express wild-type p53. Therefore, modulation of
the interaction between p53 and HDM2 in these tumors would im-
pact their response to the presence of wild-type p53. On the basis of
the results of the present study, we propose that one such regulatory
mechanism might be the activity of the cAMP signaling pathway.
Our results not only support a role for p53-HDM2 dissociation in
Figure 7. Inhibition of p53-induced cell death requires binding of
HDM2 to p53. Reh cells were treated with forskolin for 30 minutes
before addition of Nutlin-3a or exposure to 10 Gy IR. After 4 hours,
a portion of cells were harvested and subjected to immunoblot
analysis with anti-HDM2 (a mixture of SMP14, IF2, and 4B2),
DO-1, and antiactin antibodies. The immunoblot shows one repre-
sentative experiment of four. The remaining cells were analyzed
for cell death by PI staining at 20 hours after IR (n = 4, *P <
.01, relative to cells treated with IR only).
Figure 8. Model depicting how cAMP regulates the DNA damage–induced accumulation of p53.
Neoplasia Vol. 13, No. 7, 2011cAMP Signaling Promotes p53-HDM2 Association Naderi et al.
IR-induced stabilization of p53 but also suggest that inhibition of this
dissociation by activating the cAMP signaling pathway suffices to at-
tenuate p53 stabilization, accumulation, and downstream signaling.
DNA damage–induced activation of stress kinases such as ATM
and Chk2 leads to phosphorylation of S15, T18, and S20 on p53.
Because of their localization within the HDM2-binding region of
p53, phosphorylation of these residues has been proposed to reduce
the affinity of p53-HDM2 complexes and thus contribute to dissoci-
ation of p53-HDM2 complex after DNA damage. However, conflict-
ing data have been published regarding the effect of phosphorylation
of S15 or S20 on the interaction of p53 with HDM2. Whereas data
obtained from in vitro p53-HDM2 binding assays or transfection stud-
ies suggest that phosphorylation of S15 or S20 interferes with p53
binding to HDM2, germ line mutations of the murine equivalents
of S15 or S20 to alanine have failed to significantly alter the accumu-
lation of p53 after DNA damage, indicating that phosphorylation of
S15 or S20 does not play a key regulatory role in affecting the p53-
HDM2 interaction [52–57]. In support of this notion, in vitro stud-
ies with peptides that encompass S15 and S20 residues in p53 show
that phosphorylation of S15 or S20 has no effect on the interaction of
p53 with HDM2 [58–61]. Therefore, we believe that the slight inhib-
itory effect of cAMP on DNA damage–induced phosphorylation of
S15 and S20 cannot account for the ability of cAMP to antagonize
the DNA damage–mediated dissociation of p53 from HDM2. In con-
trast to S15 and S20, several studies have shown that phosphorylation
of T18 significantly attenuates the interaction of p53 with HDM2
[58–61]. However, our finding that cAMP does not affect the DNA
damage–induced phosphorylation of T18 excludes a role for regulation
of this modification in mediating the effect of cAMP on p53-HDM2
interaction. It thus seems unlikely that cAMP exerts its effect on IR-
induced accumulation of p53 through modulation of its N-terminus
phosphorylation. This view is further supported by our observation
that cAMP can also stimulate p53-HDM2 binding and decrease
p53 levels in nonirradiated cells (Figures 5E and 3, respectively), which
do not express detectable phosphorylated p53.
Becausetheinhibitory effect of cAMP onp53 accumulation ismedi-
ated by PKA, a question emerging is whether PKA exerts its stimulating
effect on the interaction of p53 with HDM2 directly by phosphory-
lating p53 or HDM2 or indirectly through modification of other pro-
teins that regulate the binding of p53 to HDM2. In vitro, PKA has
been shown to phosphorylate both virally and bacterially produced
p53 . However, this reaction was shown to depend on p53 con-
formation and high concentrations of PKA, and its occurrence in vivo
has not been established. Furthermore, database searches did not iden-
tify canonical PKA phosphorylation consensus sequences in p53 or
HDM2. Therefore, we consider it quite possible that PKA regulates
the p53-HDM2 association through phosphorylation of other proteins
than p53 and HDM2. Investigation aiming to identify such PKA tar-
gets is currently under way.
Regardless of the mechanism by which cAMP regulates p53-HDM2
interaction, the physiological significance of the inhibitory effect of
mal, unstressed cells, the driving force for p53 accumulation and sig-
naling is assumed to be minimal, thus variations in cAMP levels would
not be expected to affect p53 expression in a physiologically relevant
manner inthese cells. Conversely, intransformedcells aswellas infully
developed tumor cells, the p53 pathway is assumed to be constitutively
activated, presumably because of the activation of the DNA damage
pathway or ARF signaling [63–65]. Our findings suggest that, in ma-
lignant cells, which do not shield themselves from the detrimental ef-
fects of p53 by mutational inactivation, high levels of cAMP might be
of advantage to quench the p53 response. In this respect, cAMP would
protect the insipient cancer cell from the tumor-suppressive effects of
p53. When later faced with the challenge of DNA-damaging cancer
treatments, cAMP could again prove protective through the inhibition
of further p53 accumulation, thus contributing to treatment resistance.
Therefore, one would expect a selection for high cAMP levels in cancer
cells that retain wild-type p53. There have been a few reports on en-
hanced cAMP levels in tumor tissues. However, these estimations of
the cAMP content have been considered inaccurate because of the in-
efficient separation of malignant cells of solid tumors from the sur-
rounding stroma cells [66,67]. In contrast, ALL cells can efficiently
be separated from the surrounding bone marrow stroma cells. This, to-
gether with the expression of wild-type p53 by almost all pediatric
BCP-ALLs, and our finding that cAMP negatively affects p53 levels
and apoptosis in these cells  make this disease a suitable model sys-
tem to investigate the possible in vivo role of augmented cAMP signal-
ing as a p53-inactivating mechanism. We are currently inthe processof
collecting clinical material to examine cAMP levels in primary BCP-
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Neoplasia Vol. 13, No. 7, 2011cAMP Signaling Promotes p53-HDM2 AssociationNaderi et al.
Figure W1. Inhibition of IR-induced reduction of p53 ubiquitination
by cAMP. Reh cells were treated with forskolin for 30 minutes be-
fore exposure to IR and harvested at 4 hours after IR. First, equal
amounts of protein were subjected to immunoblot analysis with
DO-1 followed by densitometric analysis to quantify the intensity
of p53 protein bands. Based on the fold variations of the intensity
the amount of each sample was adjusted so that all samples
contained approximately equal amounts of p53 and then subjected
to Western blot analysis with DO-1 and antiactin antibodies.
Figure W2. Inhibition of IR-induced dissociation of the p53-HDM2 complex by cAMP. Reh cells were treated with forskolin for 30 min-
utes before exposure to IR and then harvested at the indicated times. Whole-cell extracts were prepared and immunoprecipitated with
FL-393. The recovered proteins were resolved on SDS-PAGE and then subjected to immunoblot analysis with the anti-HDM2 (a mixture
of SMP14, IF2, and 4B2) and DO-1 antibodies. The immunoblot shows one representative experiment of three. The intensity of protein
bands was quantified densitometrically, and the ratio of signal intensity for HDM2 relative to p53 was calculated and plotted with the
value obtained for untreated cells set as 1. Panel marked input represents Western blots for HDM2, p53, and actin in cell extracts be-
Figure W3. PGE2inhibits IR-induced dissociation of the p53-
HDM2 complex. Reh cells were treated with PGE2for 30 minutes
before exposure to IR and then harvested at 2 hours after IR.
Whole-cell extracts were prepared and immunoprecipitated with
FL-393. The recovered proteins were resolved on SDS-PAGE and
then subjected to immunoblot analysis with the anti-HDM2 (a mix-
ture of SMP14, IF2, and 4B2) and DO-1 antibodies. The immuno-
blot shows one representative experiment of two. Panel marked
input represents Western blots for HDM2, p53, and actin in cell
extracts before immunoprecipitation.