Forskolin (F), PGE2, propidium iodide (PI), cycloheximide (CHX) and nicotinamide (NIA) were obtained from Sigma-Aldrich, 8-CPT-cAMP was purchased from Biolog and Trichostatin A (TSA) was obtained from Calbiochem. Antibodies were: total p53 (DO-1, FL-393, Bp53-12), HDM2 (SMP14) and actin (C2) from Santa Cruz Biotechnology; HDM2 (IF2) from Calbiochem; HDM2 (4B2) was a kind gift from Dr A. Levine (Princeton University, NJ); acetyl-p53 (K373) and acetyl-p53 (K382) were from Epitomics and Cell Signaling, respectively.

The BCP-ALL cell line Reh (18) was cultured as previously described (15). For treatment of cells with γ-radiation, cells were exposed to a 137 Cs source at a dose rate of 4.3 Gy/min using a Gammacell irradiator from MSD Nordion. To analyze cell death, cells were incubated with PI (20 μg/ml) at room temperature for 10 min before examination for PI uptake by flow cytometry.

For two-dimensional (2D) gel electrophoresis, cells were washed twice in saline and lysed in 7% trichloro-acetic acid (TCA) for 30 min on ice. After homogenization and centrifugation, the precipitated proteins were washed once in 5% TCA and three times in water-saturated ether to remove salts, each time followed by centrifugation at 13,000 rpm for 20 min at 4°C. The protein pellet was resuspended in sample buffer for 2D gel electrophoresis (7 M urea, 2 M thiourea, 100 mM dithiotreitol, 1.5% ampholyte 3–10, 0.5% ampholyte 5–6, 4% CHAPS). The protein concentration was measured by use of the Bradford method (19). Protein sample (100 μg) was diluted in rehydration buffer (7 M urea, 2 M thiourea, 100 mM dithiotreitol, 1.5% ampholyte 3–10, 0.5% ampholyte 5–6, 4% CHAPS, Bromophenol blue) to a final volume of 170 μl. Isoelectric focusing was performed using 7 cm pH 3.0–10.0 (Zoom Strip, Invitrogen Corp., Carlsbad, CA, USA) isoelectric focusing gel strips. Following rehydration of the strips in rehydration buffer, the strips were subjected to the electrophoresis program: 200 V for 40 min, 450 V for 30 min, 750 V for 30 min and 1,000 V for 1 h. Reduction and acetylation of sulfide bindings were performed according to the manufacturer’s procedure. Second dimension gel electrophoresis was run for 1 h at 200 V. Following electrophoresis, proteins were transferred to polyvinylidene fluoride membrane (Amersham Biosciences AB, Uppsala, Sweden) by standard electroblotting. p53 protein was detected using primary Bp53-12 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and secondary HRP-conjugated mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA). Visualization of the proteins was performed using the Supersignal West Pico or Femto Chemiluminescent Substrate system (Pierce Biotechnology Inc., Rockford, IL, USA) and Kodak Image Station 2000R (Eastman Kodak Company, Rochester, NY, USA).

For immunoblot analysis, cells were lysed in radioimmunoprecipitation buffer [RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 50 mM NaF, 10 mM β-glycerophosphate, 0.1% SDS, 0.5% EDTA, 1 mM Na₃VO₄, 0.2 mM PMSF, 10 μg/ml leupeptin, 0.5% aprotinin). Equal amounts of protein were separated on a 10% SDS-PAGE. After transfer to a nitrocellulose membrane (GE Healthcare, Amersham, UK), proteins were detected by use of standard immunoblotting 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 the p53 antibody FL-393 followed by 50 μl of a 1:1 slurry of protein G-agarose (Upstate, Temecula, CA, USA). Beads were washed four times in lysis buffer, eluted in boiling 1X SDS buffer, and subjected to immunoblot analysis. For densitometric analysis, blots were analyzed using Genetools analysis (SynGene).

In all figures, the histograms represent the mean values, with error bars corresponding to SEM values.

We have previously shown that stimulation of cAMP signalling inhibits DNA damage-induced accumulation of p53 by facilitating its interaction with HDM2 (17). Because the interaction between p53 and HDM2 is known to be regulated by post-translational modifications of p53, we decided to examine whether cAMP affected the post-translational modifications of p53 in IR-treated cells. To this end, we performed 2D-immunoblotting with anti-p53 antibodies on lysates of Reh cells that were treated with IR in the absence or presence of forskolin. Reh is a BCP-ALL cell line that expresses wt p53 and forskolin is a plant-derived diterpene known to induce intracellular cAMP levels by activating adenylyl cyclase. As shown in Fig. 1, IR not only led to an increase in the abundance of p53, but it also led to accumulation of more acidic forms of p53, indicating that IR leads to modification of p53 by phosphorylation or acetylation. Interestingly, forskolin was found to inhibit the IR-induced accumulation and acidification of p53, suggesting that cAMP protects p53 from IR-induced post-translational modifications.

Our recent result showing that cAMP has only a slight inhibitory effect on the IR-mediated phosphorylation of p53 (17), suggested that inhibition of acetylation might account for the ability of cAMP to reduce the IR-induced acidification of p53. To assess this assumption, Reh cells that were exposed to IR in the absence or presence of forskolin were harvested at 4 h post-IR and subjected to immunoblot analysis with antibodies specific for p53 acetylated at K373 and K382. Acetylation of p53 at these two residues is characteristically induced by IR (4). As shown in Fig. 2, IR led to an increase in acetylation of p53 at K373 and K382, and importantly, activation of the cAMP signalling pathway by forskolin inhibited this acetylation.

Following DNA damage, p53 is acetylated through the activity of HATs such as p300/CBP and pCAF, an event that is tightly regulated by the deacetylase activity of enzymes such as HDACs and SIRT1 (4). The cAMP signalling pathway has been linked to both SIRT activation and subcellular localization of HDACs (20–23), and we therefore hypothesized that cAMP antagonizes the IR-mediated acetylation of p53 through HDACs and/or SIRT1. To test this possibility, we examined the effects of the deacetylase inhibitors trichostatin A (TSA) and nicotineamide (NIA) on the ability of cAMP to abrogate the IR-induced acetylation of p53. TSA is an inhibitor of HDACs whereas NIA inhibits the NAD⁺-dependent deacetylases such as SIRT1. To this end, Reh cells were pretreated with TSA and/or NIA before exposure to IR in the absence or presence of forskolin. Four hours post-IR, cells were harvested and subjected to immunoblot analysis sequentially with antibodies directed against p53 acetylated at K373, K382 as well as total p53. Treatment of cells with TSA or NIA alone or the combination of the two had minor effects on p53 acetylation under unstressed conditions (Fig. 2). In accordance with previous findings (16,24), exposure of cells to IR in the presence of TSA or NIA increased the IR-mediated acetylation of p53. However, pretreatment of cells with both TSA and NIA substantially further enhanced the p53 acetylation induced by IR. Interestingly, Fig. 2A and B also show that pretreatment of IR-exposed cells with both TSA and NIA almost completely abrogated the effect of forskolin on acetylated p53 levels, suggesting that the inhibitory effect of cAMP on IR-induced acetylation of p53 depends on HDACs and SIRT1 activities.

The immunoblot shown in Fig. 2A revealed that in addition to reversal of forskolin-induced deacetylation of p53, the combination of TSA and NIA also abrogated the inhibitory effect of forskolin on IR-induced accumulation of p53. Given our previous finding that cAMP facilitated the degradation of p53, we wished to examine whether TSA and NIA abolished the inhibitory effect of cAMP on IR-mediated accumulation of p53 by increasing the stability of p53. To this end, Reh cells treated with IR in the absence or presence of forskolin were exposed to the protein synthesis inhibitor cycloheximide (CHX) and then analyzed for the level of p53 by western blot analysis.

In agreement with our previous finding, forskolin substantially decreased the half-life of p53 in IR-treated cells (Fig. 3). Interestingly, combination of TSA and NIA alleviated the inhibitory effect of forskolin on p53 stability in IR-treated cells. This result indicates that simultaneous inhibition of HDACs and SIRT1 antagonizes the destabilizing effect of cAMP on p53, and implicates the modulation of p53 acetylation as the means by which cAMP regulates the stability of p53.

We have shown that cAMP abrogates the DNA damage-induced stabilization of p53 by promoting its interaction with HDM2 (17). This, together with the finding that p53 acetylation has an inhibitory effect on its association with HDM2 (6,25) suggested deacetylation of p53 as the mechanism by which cAMP enhances the p53-HDM2 interaction in IR-treated cells. To assess this hypothesis, we examined the formation of p53-HDM2 complexes under conditions in which the inhibitory effect of cAMP on IR-induced acetylation of p53 is blocked. For this purpose, Reh cells were pretreated with TSA or NIA alone or in combination before exposure to IR in the absence or presence of forskolin. Cells were harvested at 4 h after IR, and the lysates were subjected to immunoprecipitation with anti-p53 antibodies followed by immunoblot analysis with antibodies against p53 and HDM2. In accordance with our previous results, pretreatment of cells with forskolin prevented the IR-induced dissociation of p53 from HDM2 (Fig. 4). Importantly, TSA and NIA together alleviated the facilitating effect of forskolin on the p53-HDM2 interaction and reduced the level of HDM2 in complex with p53 to a level comparable to that found in IR-only treated cells. This result suggests that cAMP counteracts the IR-induced p53-HDM2 dissociation through inhibition of p53 acetylation.

Initiation of a p53-dependent apoptotic program is dependent on acetylation of p53 (10). Given that inhibition of HDACs and SIRT1 abrogates the ability of cAMP to suppress the IR-induced acetylation of p53, we wished to examine whether blocking the HDACs and SIRT1 activities could alleviate the inhibitory effect of cAMP on IR-mediated apoptosis. To this end, Reh cells were pretreated with TSA and NIA before exposure to IR in the absence or presence of forskolin and then examined for cell death. As shown in Fig. 5, the combined inhibition of HDACs and SIRT1 opposed the inhibiting effect of forskolin on IR-induced cell death, suggesting that inhibition of p53 acetylation plays an important role in the ability of cAMP to inhibit IR-mediated cell death.