Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway

Abstract

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and DeltaNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the DeltaNp73 isoform. Mice lacking DeltaNp73 (DeltaNp73(-/-) mice) are viable and fertile but display signs of neurodegeneration. Cells from DeltaNp73(-/-) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in DeltaNp73(-/-) cells, we discovered a completely new role for DeltaNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that DeltaNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of DeltaNp73 expression show enhanced resistance to chemotherapy.

Figure 1.

Loss of ΔNp73 reduces neuronal density in the brain. Brains from 10-mo-old wild-type (n = 5) and ΔNp73^−/− (n = 4) mice were analyzed histologically. (A) Representative Nissl-stained coronal section of a 10-mo-old ΔNp73^−/− mouse. Boxed region is the strip of cortical tissue spanning the corpus callosum to the pia that was assessed for cortical thickness. (B) Quantitation of cortical thickness. No differences in cortical thickness were detected between wild-type and ΔNp73^−/− mice at 10 mo of age. (C,D) Reduced neuronal density. Cell density in the cortical strips of the mice in A and B was assessed histologically by Nissl staining (C) and was quantified (D) (ΔNp73^−/−, 1286 ± 160.6 neurons per square millimeter, vs. wild type, 1960 ± 79.98 neurons per square millimeter). Results in D are the mean ± SEM. (E) Increased frequency of condensed cells. Condensed cells in the cortical strips of the mice in A and B were counted (ΔNp73^−/−, 687.5 ± 38.45 condensed cells per square millimeter, vs. wild type, 538.6 ± 15.14 condensed cells per square millimeter). Results shown are the mean ± SEM.

Figure 2.

Aged ΔNp73^−/− mice display signs of neurodegeneration. Brains from 26- to 27-mo-old wild-type (n = 2) and ΔNp73^−/− (n = 2) mice were analyzed histologically. (A) Representative Nissl-stained coronal sections of 26- to 27-mo-old wild-type and ΔNp73^−/− mice. A decrease in cortical thickness can be seen in the mutant compared with the wild type. (B) Quantitation of cortical thickness. A significant decrease in cortical thickness was detected in ΔNp73^−/− mice at 26–27 mo of age compared with the wild type (ΔNp73^−/−, 917 ± 6.579 mm², vs. wild type, 1082 ± 14.12 mm²). Results in B are the mean ± SEM. (C,D) Reduced neuronal density. Cell density in the cortical strips of the mice in A and B was assessed histologically by Nissl staining (C) and was quantified (D) (ΔNp73^−/−, 1243 ± 38.28 neurons per square millimeter, vs. wild type, 2025 ± 200 neurons per square millimeter). Results shown in D are the mean ± SEM. (E) Comparable frequency of condensed cells. Condensed cells in the cortical strips of the mice in A and B were counted, but no significant differences were observed.

Figure 3.

Loss of ΔNp73 increases expression of p53 target genes and enhances p53-mediated apoptosis. (A) Increased p53 target gene mRNA expression. Primary MEFs from wild-type or ΔNp73^−/− mice were left untreated or were treated with 1 μM doxorubicin (DRB) for 2 h, and mRNA expression levels of p21, Mdm2, and Puma were determined by quantitative PCR. Results shown are the fold increase relative to wild type. (B) Increased p53 target gene protein expression. Primary MEFs from wild-type or ΔNp73^−/− mice were left untreated or were treated with 1 μM DRB for 16 h, and protein levels of p21 and Mdm2 were determined by Western blotting. Actin and tubulin were used as loading controls. (C) Enhanced apoptosis. Wild-type and ΔNp73^−/− MEFs were treated with the indicated concentrations of the indicated apoptosis-inducing agents for 24 h. Cell viability was determined by Annexin V/PI staining and flow cytometry. (D) p53 dependence of enhanced apoptosis. Thymocytes from wild-type, ΔNp73^−/−, p53^−/−, or p53^−/−ΔNp73^−/− double-KO (DKO) mice were subjected to the indicated doses of γ-irradiation, and apoptosis was determined 16 h later as for C.

Figure 4.

Loss of ΔNp73 impairs tumor formation in nude mice. (A) Decreased tumor formation. E1A/Ras^V12-transformed wild-type or ΔNp73^−/− MEFs were injected subcutaneously into both flanks of athymic Balb/c nu/nu mice (n = 8 per group). The ability to form tumors was assessed at 2 wk post-injection. (Left panel) Gross presentation of representative nude mice injected with either wild-type or ΔNp73^−/− MEFs. (Right panel) Histogram showing the average weight of tumors derived in the nu/nu mice from wild-type and ΔNp73^−/− transformed MEFs (wild type, n = 15; ΔNp73^−/−, n = 11). Tumors derived from ΔNp73^−/− cells were found to be significantly smaller than those derived from wild-type cells (ΔNp73^−/−, 0.04 g ± 0.016 g, vs. wild type, 0.4 g ± 0.14 g). Results shown are the mean ± SD. P = 0.008. (B) Tumor kinetics. E1A/Ras^V12-transformed wild-type or ΔNp73^−/− MEFs were injected subcutaneously into either flank of athymic Balb/c nu/nu mice (n = 13). Tumor mass was assessed at 2-d intervals up to 20-d post-injection. Tumors derived from ΔNp73^−/− cells grow at a significantly slower rate than those derived from wild-type cells. Results are shown as the mean ± SEM. P < 0.0001. (C,D) Increased expression of senescence markers. Tumors derived from ΔNp73^−/− cells show increase in SA-β-gal (C), p16^INK4A, and DcR2 (D) compared with wild-type cells. Results are representative of four tumors from each genotype.

Figure 5.

Increased p53 and ATM phosphorylation and activation in ΔNp73-deficient cells and tissues upon DNA damage. (A) Increased p53 protein levels. Wild-type and ΔNp73^−/− MEFs were treated with 10 μM cisplatin for 16 h, and p53 protein was analyzed by Western blot. (B,C) Increased p53Ser18 and ATMSer1987 phosphorylation. Wild-type and ΔNp73^−/− MEFs were treated with 1 μM DRB for a long (B) or short (C) time course as indicated, and total and phosphorylated p53 and ATM were analyzed by Western blot. (D) Increased ATMSer1987 phosphorylation in thymocytes. Wild-type and ΔNp73^−/− thymocytes were γ-irradiated as indicated, and total and phosphorylated ATM levels were assessed by Western blot. (E) Increased ATMSer1987 phosphorylation and γ-H2AX induction in skin tissue. Skin tissue from wild-type and ΔNp73^−/− mice were γ-irradiated as indicated, and total ATM, phosphorylated ATM, and γ-H2AX protein levels were assessed by Western blot. (F) The increased sensitivity to DNA damage in ΔNp73^−/− thymocytes is ATM-dependent. Thymocytes from mice of the indicated genotypes were γ-irradiated as indicated, and apoptosis was determined 16 h later by Annexin V/PI staining and flow cytometry. DKO, ΔNp73^−/−ATM^−/−. mice.

Figure 6.

ΔNp73 interferes with ATM and p53 activation in human tumor cells. (A) Sensitization to apoptosis in the absence of ΔNp73. Human osteosarcoma U2OS cells were treated with control siRNA (black bars) or siRNA against ΔNp73 (gray bars) for 24 h, and then exposed for an additional 24 h to the indicated doses of cisplatin. Apoptosis was determined by Annexin V/PI staining and flow cytometry. (B) Increased ATM activation in the absence of ΔNp73. U2OS cells were treated with control siRNA or siRNA against ΔNp73 for 48 h, and phosphorylation levels of ATMSer1981 and ATM/ATR substrates were determined at 1 h after exposure to the indicated levels of γ-irradiation by Western blotting. (C,D) Overexpression of ΔNp73β impairs ATMSer1981 phosphorylation, p53 protein accumulation, and Puma expression. U2OS cells engineered to ectopically express ΔNp73β or a mock vector were exposed to 5 Gy γ-irradiation for the indicated times, and protein levels of phospho-ATMSer1987 (C), or p53 and Puma (D) were determined by Western blotting.

Figure 7.

ΔNp73 interacts with 53BP1 and localizes to sites of DNA damage. (A) Overexpressed ΔNp73β, but not ΔNp73α, binds to 53BP1. U2OS cells were transiently transfected 24 h with constructs expressing HA-tagged ΔNp73α or ΔNp73β, and then were left untreated or were treated for 1 h with 5 Gy γ-irradiation. Cell extracts were immunoprecipitated with anti-53BP1 antibody to detect the binding of each ΔNp73 isoform to endogenous 53BP1. (B) Endogenous ΔNp73 binds to endogenous 53BP1. SH-SY5Y neuroblastoma cells were left untreated or were treated with 5 Gy γ-irradiation for the indicated times. Extracts were immunoprecipitated with anti-53BP1 and subjected to Western blotting. The copurification of ΔNp73 and 53BP1 decreases after γ-irradiation. (C) PLA confirming that an interaction between 53BP1 and ΔNp73β occurs in response to DNA damage. U2OS cells grown on coverslips were transfected with a plasmid expressing ΔNp73β. After 24 h, transfected cells were left untreated or were subjected to 5 Gy γ-irradiation, incubated for 10 min, and fixed and stained to detect 53BP1:ΔNp73 interaction. Red dots in the confocal microscopic images indicate positivity for 53BP1:ΔNp73 interaction. Nuclei were stained with DAPI. (D) Detection of endogenous p73 in DNA damage foci. H1299 cells were grown on coverslips, treated with 2 μM doxorubicin, and fixed after 10 min. Cells were stained with anti-p73 antibody (green) and with antibody to detect the DNA damage focus marker γ-H2AX (red). Merged fluorescence shows that p73 colocalizes with γ-H2AX and DNA damage foci upon DNA damage. (E) Detection of endogenous ΔNp73 in DNA damage foci. SH-SY5Y cells were grown on coverslips, treated with 5 Gy γ-irradiation, and fixed after 10 min. Cells were stained with anti-ΔNp73 antibody (red) and with antibody to detect the DNA damage focus marker γ-H2AX (green). Merged fluorescence shows that ΔNp73 colocalizes with γ-H2AX and DNA damage foci upon γ-irradiation. (F) siRNA knockdown confirmation of ΔNp73 colocalization with DNA damage foci. U2OS cells expressing either control siRNA or siRNA against ΔNp73 were cultured for 48 h. Cells were then either left untreated or treated with 5 Gy γ-irradiation, fixed after 10 min, and immunostained to detect 53BP1 and p53. Confocal microscopy shows that depletion of ΔNp73 increases the recruitment and colocalization of 53BP1 (green) and p53 (red) to sites of DNA damage.