Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence

Abstract

Cell-cycle arrest, apoptosis, and senescence are widely accepted as the major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are the rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53(K117R)) or three (p53(3KR); K117R+K161R+K162R) of p53 acetylation sites. Although p53(K117R/K117R) cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p53(3KR/3KR) cells. Surprisingly, unlike p53 null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53(K117R/K117R) or p53(3KR/3KR) animals. Notably, p53(3KR) retains the ability to regulate energy metabolism and reactive oxygen species production. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.

Figure 1. p53^K117R Activates p21 Normally but Completely Abrogates Activation of Propoptotic Target Genes

(A) Western blot analysis of the thymus of p53^+/+, p53^K117R/K117R and p53^−/− mice. Mice were either untreated or exposed to 12.5 Gy of γ-irradiation, four hours later; thymocytes were isolated and analyzed for the expression of p53, phospho-p53, and p21, Puma, cleaved caspase 3 and β-actin. (B) Representative immunohistochemical staining of thymi from p53^+/+ and p53^K117R/K117R mice for cleaved caspase3. Eight-week-old mice were either untreated or exposed to 5 Gy of γ-irradiation. Four hours later, thymus from these mice were fixed overnight then processed, paraffin-embedded, sectioned and stained with anti-mouse cleaved caspase 3 antibodies according to the standard protocol. (C) In vivo apoptotic analysis of p53^+/+ and p53^K117R/K117R mice after 5 Gy of γ-irradiation. Mice were treated as described in (B), Then single-cell suspension of thymocytes from mice were prepared and stained with Annexin V-FITC for FACS analysis. (D) Quantification of apoptosis in vivo represented by Annexin-V positive thymocytes. Error bars represent averages ± SD from at least three mice for each genotype. (E) Representative cleaved caspase 3 immunohistochemical staining images showing apoptotic cells in the spleen, testis and small intestine of p53^+/+ and p53^K117R/K117R mice. Eight-week-old mice were either untreated or exposed to 12.5 Gy of γ-irradiation. Then tissues were collected 4 hr later and processed as described in (B). See also Figure S1, Figure S2 and Figure S3.

Figure 2. P53^K117R/K117R MEFs Retain Intact Cell Cycle Checkpoint and Cellular Senescence Function in Response to DNA Damage

(A) Western blot analysis of Mdm2, p53, phosphorylated p53, Puma and p21 in p53^+/+ and p53^K117R/K117R MEFs either left untreated or treated with 1.0 μM Dox (doxorubicin) for 8 hours, β-actin was used as a loading control. (B) qRT-PCR analysis of Mdm2, p21, Puma, Killer/DR5, and Noxa mRNA in p53^+/+ and p53 ^K117R/K117R MEFs either untreated or treated with 1.0 μM Dox for 8 hours. Data shown are the relative amount of specific mRNA normalized first to β-actin and then to the untreated wildtype sample values from three independent experiments with different MEF lines. Results are reported as average ± standard error of the mean (SEM). (C) Cell growth arrest analysis of p53^+/+ and p53^K117R/K117R MEFs either left untreated or exposed to 5 Gy of γ-irradiation. 23 hours after irradiation, MEFs were pulsed with 10 μM BrdU for 45 minutes, and then cells were fixed and processed for immunofluorescence analysis for BrdU (green). The nuclei were stained with DAPI (blue). (D) Quantification of BrdU labeling of p53^+/+ and p53^K117R/K117R MEFs irradiated with 5 and 10 Gy. Error bars represent ±SD from three independent experiments. (E) Western blot analysis of p53^+/+ and p53^K117R/K117R MEFs at different passages cultured according to a 3T3 protocol for the expression of p53, Arf and p21. (F) Senescence-associated β-galactosidase (SA-β-gal) staining of p53^+/+ and p53^K117R/K117R MEFs cultured according to a 3T3 protocol. MEFs at indicated passages were fixed and stained for β-galactosidase activity as described in the Extended Experimental Procedures. See also Figure S4.

Figure 3. Loss of Acetylation Sites at K117, K161 and K162 of Mouse p53 Abolishes the Activation of p21 and PUMA

(A) Schematic representation of the mouse p53 protein with three mutated acetylation sites indicated and alignment of the K117, K161 and K162 flanking regions of the mouse p53 with those of other species. The conserved lysines were highlighted in red and Q165 of human p53 highlighted in blue is evolutionarily reminiscent of acetylated K. (B) Mass spectrometry analysis of tryptic mouse p53 peptides containing K161 and K162. The fragmentation spectrum of ¹⁵⁶AM_oxAIYK_acK¹⁶² and ¹⁶² K_acSQHM_oxTEVVR¹⁷¹ revealed the presence of peptides with acetylation at K161 and K162, respectively. The protein was prepared as described in the Experimental Procedures. Inset shows the high-resolution precursor ion mass. The label “Δ” designates “b” or “y” ions with water and/or ammonia loss. “K_ac” and “M_ox” designate acetyllysine and oxidized methionine, respectively. Neutral loss of sulfenic acid from oxidized methionine was indicated as “-64”. (C) Western blot analysis of p53, Mdm2, Puma and p21 in H1299 cells transfected with plasmids expressing mouse wildtype p53, p53-K117R and p53-3KR mutants. β-actin was used as a loading control. (D) ChIP assay for the binding of p53 or p53-3KR mutant to the consensus sites in the p21, Puma and Mdm2 promoters. H1299 cells transfected with plasmids DNA expressing mouse p53 wildtype (WT) and p53-3KR mutant were treated with 1% formaldehyde for 10 min and processed for ChIP analysis. The occupancy of p53 or p53-3KR of the p21, Puma and Mdm2 promoters were detected by PCR-agarose gel electrophoresis. IgG antibody serves as a negative control.

Figure 4. p53^3KR Mutation Severely Abrogates the p53-mediated Cell Cycle Arrest and Apoptotic Cell Death

(A) Analysis of apoptosis in vivo for p53^+/+ and p53^3KR/3KR mice. Mice were either untreated or exposed to 5 Gy of γ-irradiation, and then thymocytes were isolated and stained with Annexin V-FITC for FACS analysis. (B) Annexin-V positive cells representing apoptotic thymocytes are quantified in (E). Data are shown as average ± SD from at least three mice for each genotype. (C) Western blot analysis of Mdm2, p53, phosphorylated p53, Puma and p21 in p53^+/+ and p53^3KR/3KR and p53^−/− MEFs either untreated or treated with 0.2 μg/ml Dox for 2, 4 and 8 hours. β-actin serves as a loading control. (D) Cell-cycle arrest analysis of p53^+/+ and p53^3KR/3KR MEFs either left untreated or exposed to 5 or 10 Gy of γ-irradiation. 23 hours after irradiation, MEFs were pulsed with 10 μM BrdU for 45 minutes, and then cells were fixed and processed for immunofluorescence analysis for BrdU. BrdU-positive cells representing cells in S phase of cell cycle during BrdU incorporation after 5 or 10 Gy of γ-irradiation are quantified. Values shown are the averages ±SD of three independent replicates. (E) Immunoblot assays of p53, phopho-p53, Puma, p21, cleaved caspase 3 and β-actin protein in the lysates prepared from the thymus of p53^+/+, p53^3KR/3KR and p53^−/− mice 4 hours after 12.5 Gy of γ-irradiation. (F) Western blot analysis of apoptosis in the thymus of p53^+/+, p53^K117R/K117R, p53^3KR/3KR and p53^−/− mice upon DNA damage treatment. Mice were either untreated or exposed to 12.5 Gy of γ-irradiation; thymocytes were isolated at different time points after irradiation and analyzed for the level of cleaved caspase 3. β-actin was used as a loading control. See alsow Figure S5 and Figure S6.

Figure 5. p53^3KR Loses Its Ability to Induce Cellular Senescence in p53 ^3KR/3KR MEFs

(A and B) qRT-PCR analysis of indicated mRNAs in p53^+/+, p53^3KR/3KR and p53^−/− MEFs either untreated or treated with 0.2 μg/ml Dox for 8 hours. Results shown are the relative amount of specific mRNA normalized first to β-actin and then to the untreated wildtype sample values from three independent experiments with different MEF lines. Error bars represent ± SEM. (C) Cell growth rate analysis of p53^+/+, p53^3KR/3KR and p53^−/− MEFs. 3 × 10⁴ MEFs of different genotypes were seeded into 6-well plates at day 0 and counted daily. (D) Images of SA-β-gal staining of p53^+/+ and p53^3KR/3KR MEFs at different passages cultured according to the 3T3 protocol. MEFs at indicated passages were fixed and stained for β-galactosidase activity. (E and F) Immunoblot analysis of p53, Arf and p21 in p53^3KR/3KR (E) and p53^−/− (F) MEFs at indicated passages cultured according to the 3T3 protocol. β-actin serves as a loading control. See also Figure S7.

Figure 6. p53^3KR Retains Its Tumor Suppressor Activity and the Ability to Activate Metabolic Targets

(A) Kaplan-Meier survival curves of p53^+/+, p53^K117R/K117R, p53^3KR/3KR and p53^−/− mice. (B) qRT-PCR analysis of indicated mRNAs in p53^+/+, p53^3KR/3KR and p53^−/− MEFs either untreated or treated with 0.2 μg/ml Dox for 8 hours. Data are averages ± SEM of each mRNA quantities normalized first to β-actin and then to the untreated wildtype sample values from three independent MEF lines. (C) Immunoblot assays of p53, Gls2 and Tigar protein in the whole cell lysates prepared from p53^+/+, p53^3KR/3KR and p53^−/− MEFs either untreated or treated with 0.2 μg/ml Dox for 8 hours. β-actin serves as a loading control. (D) Analysis of acetylation of p53 at K117, K161 and K162 in p53^K117R/K117R and p53^3KR/3KR MEF cells. p53^+/+ and p53^3KR/3KR and p53^K117R/K117R MEFs either untreated or treated with 0.2 μg/ml Dox, 1.0 μM TSA and 5 mM Nicotinamide for 6 hours. Cell lysates were immunoprecipitated with either anti-AcK120-p53, anti-AcK164-p53, anti- AcK CT-p53 antibodies or control IgG, and blotted with anti-p53 (CM5) antibody using ReliaBLOT systems from Bethyl Laboratories. (E) ChIP assay for the binding of p53 or p53-3KR mutant to the consensus sites of p53 metabolic targets Gls2 and Tigar in H1299 cells transfected with plasmids DNA expressing mouse p53 wildtype (WT) or p53-3KR mutant. (F) RT-PCR (upper panel) and qRT-PCR (lower panel) analysis of Glut3 expression in p53^+/+, p53^3KR/3KR and p53^−/− primary MEFs at passage 2. Data are represented as averages ± SEM from three independent experiments. See also Figure S7

Figure 7. p53^3KR Inhibits Glucose Uptake, Glycolysis, Reactive Oxygen Species Level and Colony Formation

(A) Glucose uptake measurement of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells determined by the uptake of 2-[³H]-deoxyglucose. Results shown are the averages ± SD of three different experiments. (B) Glycolysis rate analysis of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells determined by monitoring the conversion of 5-[³H] glucose to ³H₂O as described in the Experimental Procedures. Values represent averages ± SD of three different experiments. (C) Measurement of the reactive oxygen species (ROS) levels of p53^+/+, p53^3KR/3KR and p53^−/− MEF cells was performed by using mouse ROS ELISA kit as described in the Experimental Procedures. Data was reported as averages ± SD of three different experiments. (D) Western blot analysis of p53-3KR, Mdm2 and Gls2 proteins in H1299 cells transfected with FLAG-tagged expression plasmids as indicated using anti-FLAG antibody. β-actin was used as a loading control. (E) Colony formation assay of H1299 cells transfected with p53-3KR, Mdm2 and Gls2. H1299 cells were transfected with either empty vector, or FLAG-p53-3KR, FLAG-Mdm2, or FLAG-Gls2 expression plasmids for 48 hrs, and then split and subject to colony formation assay visualized by crystal violet staining. (F) Quantification of colonies formed in H1299 cells transfected with empty vector, FLAG-p53-3KR, FLAG-Mdm2, and FLAG-Gls2 expression plasmids after 12 days culture in the presence of G418. Graphs are presented as percentage of colonies of empty vector transfected cells and values are shown as average percentage ± SD of three different experiments.