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. 2011 Jul;13(7):653–663. doi: 10.1593/neo.11542

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 *, Soheil Naderi *,3
PMCID: PMC3132851  PMID: 21750659

Abstract

The tumor suppressor p53 provides an important barrier to the initiation and maintenance of cancers. As a consequence, 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 lymphoblastic leukemia cells, leading to a profound reduction of their apoptotic response. In the present article, we provide 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 wild-type p53.

Introduction

The tumor suppressor p53 is normally activated in response to various types of cellular stress, such as DNA damage, oncogenic signaling, mitotic impairment, and oxidative stress [1]. 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 increased transcriptional activity [2]. 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 transcription 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 half of human cancers [5,6]. This, however, leaves approximately 3 million cases of cancer annually, which retain wild-type p53 [7], and there is mounting evidence that the p53 function must be attenuated for these cancers to develop, maintain, and progress [8–10]. Such attenuation can be achieved by viral proteins, deregulation of components of the p53 regulatory circuit, or disruption of upstream or downstream signaling pathways [11].

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 binding to the N-terminal domain of p53 and promoting its ubiquitination and subsequent proteasomal degradation. Exposure of cells toDNA 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 demonstrated by the fact that the embryonic lethality in mdm2-/- mice can be rescued by concomitant deletion of p53 [12,13]. Although additional 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 [18].

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 pathologic settings [19]. This includes control of normal T-cell activation [20,21] and contribution to T-cell dysfunction in human and murine immunodeficiency virus infection/acquired immunodeficiency syndrome [22,23]. Lymphocytes possess GPCRs for catecholamines 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 G1 phase [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 [31]. 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 treatments such as ionizing radiation (IR) and different classes of chemotherapeutics [32]. 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 abrogating 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

The BCP-ALL cell line Reh [33] was cultured as previously described [31]. Human CD4+ T cells were isolated, cultured, and stimulated as described [32]. U2OS and MCF-7 cell lines were cultured in McCoy and Dulbecco modified Eagle media, respectively. γ-Irradiation of cells was carried out using a 137Cs source at a dose rate of 4.3 Gy/min.

Cell death was detected by incubation of cells in PBS containing 20 µg/ml PI followed by analysis on a FACS instrument for PI uptake.

Small Interfering RNA Transfection

Reh cells (6 x 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 incubated 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 membrane (Amersham, Piscataway, NJ), and detected by use of standard 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 Na3 VO4, 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 1x 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

Total RNA (12 µg/sample) isolated using RNeasy (Qiagen, Germantown, MD) was fractionated on a formaldehyde/agarose gel, transferred 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 [31].

Statistical Methods and Calculation

SPSS 14.0 for Windows (Chicago, IL) was used to perform paired-samples t test. Error bars indicate SEM.

Results

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 [32]. 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 induction of p53 at 4 hours after IR. As a first step to assess the mechanisms 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 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 thereafter 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 experiment 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.

Figure 1.

Figure 1

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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.

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 expression of p53 in IR-treated normal human CD4+ T cells, the osteosarcoma U2OS, and the mammary adenocarcinoma MCF-7 cell lines. Exposure of these cells to IR led to accumulation of p53 within 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) [34], 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 [39] and T-ALL cells [40]. 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 activates 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 accumulation 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 by PKA.

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 modulation 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 [43]. Therefore, to understand the mechanism 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 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 [32]. To further substantiate 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 intervals. 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).

Figure 2.

Figure 2

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p53 mRNA steady-state levels are not affected by IR or cAMP. Reh cells were treated with forskolin 30minutes before 10 Gy IR. Cells were harvested at the indicated times after IR, RNA was isolated and subjected to Northern blot analysis with a p53 cDNA probe. RNA isolated formHCT116 p53+/+ and p53-/- cells was used as control for the specificity of the cDNA probe.

Figure 3.

Figure 3

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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 Affects p53 Half-life through Ubiquitination and Proteasome-Mediated Degradation

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 examined the effect of cAMP on p53 levels in the presence of the proteasome 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.

Figure 4.

Figure 4

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cAMP inhibits p53 accumulation in a proteasome-dependent manner and counteracts IR-induced reduction of p53 ubiquitination. (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.

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 protein by Western blot analysis. IR reduced the level of p53 ubiquitination 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 ubiquitination in IR-treated cells was not due to the presence of MG-132 because forskolin was found to exert a similar effect in the absence of MG-132 (Figure W1). To further substantiate that the changes in intensity 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 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 [45]. Therefore, our observation that cAMP-mediated inhibition of IR-induced accumulation of p53 was associated with an increase in ubiquitination of p53 suggested that HDM2 might be involved in mediating this inhibitory effect of cAMP on p53. To examine this possibility, we wished to assess 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 disrupting 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 mediates the ability of cAMP to induce the degradation of p53.

Figure 5.

Figure 5

Figure 5

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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 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 (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.

To further characterize the mechanism by which cAMP uses 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 HDM2 protein levels. On exposure of cells to IR, HDM2 was induced, 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 followed 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 exposed to IR alone. However, this difference was statistically insignificant 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 activity, 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 antagonizing 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 immunoprecipitation with anti-p53 antibodies followed by immunoblot analysis with antibodies against p53 and HDM2. p53 immunocomplexes 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 dissociation 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 (Figure 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 stabilization 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 notion 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 immunoblotted 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%.

Figure 6.

Figure 6

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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 immunoprecipitated 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 densitometrically. 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).

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 inhibitory effect on apoptosis induced by DNA damage [32]. This observation, together with our present finding that cAMP inhibits the IR-induced accumulation of p53 in an HDM2-dependent manner, 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). Unfortunately, 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.

Figure 7.

Figure 7

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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 representative 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).

Discussion

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).

Figure 8.

Figure 8

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Model depicting how cAMP regulates the DNA damage-induced accumulation of p53.

HDM2 plays an essential role in the regulation of p53 function. Under normal conditions, HDM2 binds p53 and primes it for ubiquitin-dependent degradation by nuclear and cytoplasmic proteasomes [44]. 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 [45]. 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]. Furthermore, overexpression of HDM2 has been observed in soft tissue tumors, osteosarcomas, and esophageal carcinomas [50]. Specifically, in pediatric ALLs, most of which retain wild-type p53, overexpression of HDM2 is a rather common event [51]. These results suggest that p53-HDM2 interaction plays a major role in inhibition of p53 function in tumors that express wild-type p53. Therefore, modulation of the interaction between p53 and HDM2 in these tumors would impact 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 IR-induced stabilization of p53 but also suggest that inhibition of this dissociation by activating the cAMP signaling pathway suffices to attenuate 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 dissociation of p53-HDM2 complex after DNA damage. However, conflicting 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 studies 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 accumulation 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 studies 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 inhibitory 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 contrast 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.

Because the inhibitory effect of cAMP on p53 accumulation is mediated by PKA, a question emerging is whether PKA exerts its stimulating effect on the interaction of p53 with HDM2 directly by phosphorylating p53 or HDM2 or indirectly through modification of other proteins that regulate the binding of p53 to HDM2. In vitro, PKA has been shown to phosphorylate both virally and bacterially produced p53 [62]. However, this reaction was shown to depend on p53 conformation and high concentrations of PKA, and its occurrence in vivo has not been established. Furthermore, database searches did not identify 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 targets is currently under way.

Regardless of the mechanism by which cAMP regulates p53-HDM2 interaction, the physiological significance of the inhibitory effect of cAMP on p53 accumulation and apoptosis should be addressed. In normal, unstressed cells, the driving force for p53 accumulation and signaling is assumed to be minimal, thus variations in cAMP levels would not be expected to affect p53 expression in a physiologically relevant manner in these cells. Conversely, in transformed cells as well as in fully 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 malignant cells, which do not shield themselves from the detrimental effects 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 enhanced cAMP levels in tumor tissues. However, these estimations of the cAMP content have been considered inaccurate because of the inefficient separation of malignant cells of solid tumors from the surrounding stroma cells [66,67]. In contrast, ALL cells can efficiently be separated from the surrounding bone marrow stroma cells. This, together 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 [32] make this disease a suitable model system to investigate the possible in vivo role of augmented cAMP signaling as a p53-inactivating mechanism. We are currently in the process of collecting clinical material to examine cAMP levels in primary BCP-ALL cells.

Supplementary Material

Supplementary Figures and Tables
neo1307_0653SD1.pdf (137.6KB, pdf)

Abbreviations

BCP-ALL

B-cell precursor acute lymphoblastic leukemia

IR

ionizing radiation

Epac

exchange protein directly activated by cAMP

PKA

protein kinase A

Footnotes

1

This 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.

2

This article refers to supplementary materials, which are designated by Figures W1 to W3 and are available online at www.neoplasia.com.

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Supplementary Materials

Supplementary Figures and Tables
neo1307_0653SD1.pdf (137.6KB, pdf)

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