DeltaNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors

Abstract

p73 has significant homology to p53. However, tumor-associated up-regulation of p73 and genetic data from human tumors and p73-deficient mice exclude a classical Knudson-type tumor suppressor role. We report that the human TP73 gene generates an NH(2) terminally truncated isoform. DeltaNp73 derives from an alternative promoter in intron 3 and lacks the transactivation domain of full-length TAp73. DeltaNp73 is frequently overexpressed in a variety of human cancers, but not in normal tissues. DeltaNp73 acts as a potent transdominant inhibitor of wild-type p53 and transactivation-competent TAp73. DeltaNp73 efficiently counteracts transactivation function, apoptosis, and growth suppression mediated by wild-type p53 and TAp73, and confers drug resistance to wild-type p53 harboring tumor cells. Conversely, down-regulation of endogenous DeltaNp73 levels by antisense methods alleviates its suppressive action and enhances p53- and TAp73-mediated apoptosis. DeltaNp73 is complexed with wild-type p53, as demonstrated by coimmunoprecipitation from cultured cells and primary tumors. Thus, DeltaNp73 mediates a novel inactivation mechanism of p53 and TAp73 via a dominant-negative family network. Deregulated expression of DeltaNp73 can bestow oncogenic activity upon the TP73 gene by functionally inactivating the suppressor action of p53 and TAp73. This trait might be selected for in human cancers.

Figure 3.

ΔNp73 is an efficient dominant-negative inhibitor of the transcriptional activity of wild-type p53 and TAp73. (A) ΔNp73α-mediated suppression of the wild-type p53/TAp73-responsive reporter construct PG13-luciferase in p53 null H1299 cells. Luciferase activity is normalized for renilla luciferase activity. Coexpressed ΔNp73α causes a dose-dependent complete suppression of the transcriptional activity of wild-type p53 and TAp73β. Suppression by ΔNp73α for the molar ratios of wild-type p53 or TAp73β to ΔNp73α are indicated. Results are the average ± SD of three independent experiments. Results were similar with TAp73α (unpublished data). (B) Immunoblots of H1299 cells transfected with p53 or TAp73β alone or with increasing amounts of ΔNp73α. GFP was cotransfected in all cases. Transfections were done in parallel with A. Lane loading was normalized for GFP levels. (C) ΔNp73α suppresses the wild-type p53 and TAp73β-induced transactivation of endogenous target genes. p53 null H1299 cells were transfected with expression plasmids containing wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:3 molar ratio. Lane 7 was transfected with empty vector plus ΔNp73α at a 1:3 molar ratio. Transfected crude lysates, normalized for equal protein loading by vimentin, were immunoblotted for HDM2, 14-3-3σ, and p21Waf1. Cells in the Vect lane are transfected with pcDNA3 only.

Figure 3.

ΔNp73 is an efficient dominant-negative inhibitor of the transcriptional activity of wild-type p53 and TAp73. (A) ΔNp73α-mediated suppression of the wild-type p53/TAp73-responsive reporter construct PG13-luciferase in p53 null H1299 cells. Luciferase activity is normalized for renilla luciferase activity. Coexpressed ΔNp73α causes a dose-dependent complete suppression of the transcriptional activity of wild-type p53 and TAp73β. Suppression by ΔNp73α for the molar ratios of wild-type p53 or TAp73β to ΔNp73α are indicated. Results are the average ± SD of three independent experiments. Results were similar with TAp73α (unpublished data). (B) Immunoblots of H1299 cells transfected with p53 or TAp73β alone or with increasing amounts of ΔNp73α. GFP was cotransfected in all cases. Transfections were done in parallel with A. Lane loading was normalized for GFP levels. (C) ΔNp73α suppresses the wild-type p53 and TAp73β-induced transactivation of endogenous target genes. p53 null H1299 cells were transfected with expression plasmids containing wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:3 molar ratio. Lane 7 was transfected with empty vector plus ΔNp73α at a 1:3 molar ratio. Transfected crude lysates, normalized for equal protein loading by vimentin, were immunoblotted for HDM2, 14-3-3σ, and p21Waf1. Cells in the Vect lane are transfected with pcDNA3 only.

Figure 3.

ΔNp73 is an efficient dominant-negative inhibitor of the transcriptional activity of wild-type p53 and TAp73. (A) ΔNp73α-mediated suppression of the wild-type p53/TAp73-responsive reporter construct PG13-luciferase in p53 null H1299 cells. Luciferase activity is normalized for renilla luciferase activity. Coexpressed ΔNp73α causes a dose-dependent complete suppression of the transcriptional activity of wild-type p53 and TAp73β. Suppression by ΔNp73α for the molar ratios of wild-type p53 or TAp73β to ΔNp73α are indicated. Results are the average ± SD of three independent experiments. Results were similar with TAp73α (unpublished data). (B) Immunoblots of H1299 cells transfected with p53 or TAp73β alone or with increasing amounts of ΔNp73α. GFP was cotransfected in all cases. Transfections were done in parallel with A. Lane loading was normalized for GFP levels. (C) ΔNp73α suppresses the wild-type p53 and TAp73β-induced transactivation of endogenous target genes. p53 null H1299 cells were transfected with expression plasmids containing wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:3 molar ratio. Lane 7 was transfected with empty vector plus ΔNp73α at a 1:3 molar ratio. Transfected crude lysates, normalized for equal protein loading by vimentin, were immunoblotted for HDM2, 14-3-3σ, and p21Waf1. Cells in the Vect lane are transfected with pcDNA3 only.

Figure 5.

Physical interaction between ΔNp73α and wild-type p53 proteins in cancer cell lines and human tumors. (A) Crude cell lysates of p53 null SaOs2 cells transfected with the indicated expression plasmids were immunoprecipitated with the monoclonal antibody ER15 and immuno-blotted for coprecipitating wild-type p53 with polyclonal CM-1. ER15 is a p73 antibody against a COOH-terminal epitope and reacts with both ΔNp73α and TAp73α. ER15 is used here to immunoprecipitate ΔNp73α because complexes between TAp73 and wild-type p53 do not occur (lane 7 and B and C; references 18 and 30–32). Only lysate was loaded in lane 5. The shadow band in lanes 4, 6, and 7 is derived from the heavy chain of added ER15. (B) Crude cell lysates of wild-type p53 harboring U2OS cells transfected with the indicated plasmids were immunoprecipitated with ER15 or an irrelevant monoclonal antibody against GFP and immunoblotted for coprecipitating endogenous p53 with polyclonal CM-1. (C) Crude cell lysates of U2OS cells transfected with the indicated plasmids were immunoprecipitated with a monoclonal antibody against p53 (421) or irrelevant mouse IgG and immunoblotted for coprecipitating ΔNp73α with polyclonal anti-ΔNp73. This antibody is raised and immunopurified against exon 3′ and does not cross react with TAp73 isoforms or p53. Only lysate was loaded in the indicated lane. (D) Detection of tumor-specific protein complexes between ΔNp73α and wild-type p53 in an ovarian carcinoma. Immunoprecipitations of equal amounts of total protein (2 mg each) from a matched pair of homogenized tumor/normal tissues using a mixture of anti-p53 antibodies DO-1 and 1801 covalently coupled to agarose beads, followed by immunoblotting with polyclonal anti-ΔNp73. The ΔNp73α–p53 protein complex is detectable in the tumor but not in the respective normal tissue. Equal amounts of the same tumor and normal tissue lysates immunoprecipitated with protein G–coupled beads are used as an additional negative control. The last lane is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 5.

Physical interaction between ΔNp73α and wild-type p53 proteins in cancer cell lines and human tumors. (A) Crude cell lysates of p53 null SaOs2 cells transfected with the indicated expression plasmids were immunoprecipitated with the monoclonal antibody ER15 and immuno-blotted for coprecipitating wild-type p53 with polyclonal CM-1. ER15 is a p73 antibody against a COOH-terminal epitope and reacts with both ΔNp73α and TAp73α. ER15 is used here to immunoprecipitate ΔNp73α because complexes between TAp73 and wild-type p53 do not occur (lane 7 and B and C; references 18 and 30–32). Only lysate was loaded in lane 5. The shadow band in lanes 4, 6, and 7 is derived from the heavy chain of added ER15. (B) Crude cell lysates of wild-type p53 harboring U2OS cells transfected with the indicated plasmids were immunoprecipitated with ER15 or an irrelevant monoclonal antibody against GFP and immunoblotted for coprecipitating endogenous p53 with polyclonal CM-1. (C) Crude cell lysates of U2OS cells transfected with the indicated plasmids were immunoprecipitated with a monoclonal antibody against p53 (421) or irrelevant mouse IgG and immunoblotted for coprecipitating ΔNp73α with polyclonal anti-ΔNp73. This antibody is raised and immunopurified against exon 3′ and does not cross react with TAp73 isoforms or p53. Only lysate was loaded in the indicated lane. (D) Detection of tumor-specific protein complexes between ΔNp73α and wild-type p53 in an ovarian carcinoma. Immunoprecipitations of equal amounts of total protein (2 mg each) from a matched pair of homogenized tumor/normal tissues using a mixture of anti-p53 antibodies DO-1 and 1801 covalently coupled to agarose beads, followed by immunoblotting with polyclonal anti-ΔNp73. The ΔNp73α–p53 protein complex is detectable in the tumor but not in the respective normal tissue. Equal amounts of the same tumor and normal tissue lysates immunoprecipitated with protein G–coupled beads are used as an additional negative control. The last lane is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 5.

Physical interaction between ΔNp73α and wild-type p53 proteins in cancer cell lines and human tumors. (A) Crude cell lysates of p53 null SaOs2 cells transfected with the indicated expression plasmids were immunoprecipitated with the monoclonal antibody ER15 and immuno-blotted for coprecipitating wild-type p53 with polyclonal CM-1. ER15 is a p73 antibody against a COOH-terminal epitope and reacts with both ΔNp73α and TAp73α. ER15 is used here to immunoprecipitate ΔNp73α because complexes between TAp73 and wild-type p53 do not occur (lane 7 and B and C; references 18 and 30–32). Only lysate was loaded in lane 5. The shadow band in lanes 4, 6, and 7 is derived from the heavy chain of added ER15. (B) Crude cell lysates of wild-type p53 harboring U2OS cells transfected with the indicated plasmids were immunoprecipitated with ER15 or an irrelevant monoclonal antibody against GFP and immunoblotted for coprecipitating endogenous p53 with polyclonal CM-1. (C) Crude cell lysates of U2OS cells transfected with the indicated plasmids were immunoprecipitated with a monoclonal antibody against p53 (421) or irrelevant mouse IgG and immunoblotted for coprecipitating ΔNp73α with polyclonal anti-ΔNp73. This antibody is raised and immunopurified against exon 3′ and does not cross react with TAp73 isoforms or p53. Only lysate was loaded in the indicated lane. (D) Detection of tumor-specific protein complexes between ΔNp73α and wild-type p53 in an ovarian carcinoma. Immunoprecipitations of equal amounts of total protein (2 mg each) from a matched pair of homogenized tumor/normal tissues using a mixture of anti-p53 antibodies DO-1 and 1801 covalently coupled to agarose beads, followed by immunoblotting with polyclonal anti-ΔNp73. The ΔNp73α–p53 protein complex is detectable in the tumor but not in the respective normal tissue. Equal amounts of the same tumor and normal tissue lysates immunoprecipitated with protein G–coupled beads are used as an additional negative control. The last lane is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 5.

Physical interaction between ΔNp73α and wild-type p53 proteins in cancer cell lines and human tumors. (A) Crude cell lysates of p53 null SaOs2 cells transfected with the indicated expression plasmids were immunoprecipitated with the monoclonal antibody ER15 and immuno-blotted for coprecipitating wild-type p53 with polyclonal CM-1. ER15 is a p73 antibody against a COOH-terminal epitope and reacts with both ΔNp73α and TAp73α. ER15 is used here to immunoprecipitate ΔNp73α because complexes between TAp73 and wild-type p53 do not occur (lane 7 and B and C; references 18 and 30–32). Only lysate was loaded in lane 5. The shadow band in lanes 4, 6, and 7 is derived from the heavy chain of added ER15. (B) Crude cell lysates of wild-type p53 harboring U2OS cells transfected with the indicated plasmids were immunoprecipitated with ER15 or an irrelevant monoclonal antibody against GFP and immunoblotted for coprecipitating endogenous p53 with polyclonal CM-1. (C) Crude cell lysates of U2OS cells transfected with the indicated plasmids were immunoprecipitated with a monoclonal antibody against p53 (421) or irrelevant mouse IgG and immunoblotted for coprecipitating ΔNp73α with polyclonal anti-ΔNp73. This antibody is raised and immunopurified against exon 3′ and does not cross react with TAp73 isoforms or p53. Only lysate was loaded in the indicated lane. (D) Detection of tumor-specific protein complexes between ΔNp73α and wild-type p53 in an ovarian carcinoma. Immunoprecipitations of equal amounts of total protein (2 mg each) from a matched pair of homogenized tumor/normal tissues using a mixture of anti-p53 antibodies DO-1 and 1801 covalently coupled to agarose beads, followed by immunoblotting with polyclonal anti-ΔNp73. The ΔNp73α–p53 protein complex is detectable in the tumor but not in the respective normal tissue. Equal amounts of the same tumor and normal tissue lysates immunoprecipitated with protein G–coupled beads are used as an additional negative control. The last lane is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 2.

ΔNp73 is frequently overexpressed in a variety of primary human cancers. (A) Tumor-specific up-regulation of ΔNp73 (solid bars) transcripts in 35 tumor pairs, compared with their respective normal tissues of origin. In addition, Ex2Del p73 transcripts (gray bars) were measured in a subset of pairs. Ex2Del p73 is another previously described isoform of p73 that lacks most of the transactivation domain. In contrast to ΔNp73, it is generated from the same promoter as TAp73 by splicing out exon 2. It is also a transdominant inhibitor of p53 (references –24). Isoform-specific semiquantitative RT-PCR assay from total RNA extracted from human tissues (see Table I). Two tumor pairs (nos. 21 and 22) had readily detectable ΔNp73 levels in tumor tissues but failed to give detectable ΔNp73 levels in their corresponding normal tissues. Therefore, fold up-regulation could not be quantitated in those 2 cases reducing the total number plotted from 37 to 35 cases. Expression levels, standardized for their corresponding GAPDH values, were used to calculate fold induction as described in Materials and Methods. (B) Up-regulation of ΔNp73 transcripts in a series of unmatched 52 human breast cancers compared with 8 unrelated normal breast tissues. ΔNp73-specific semiquantitative RT-PCR assay from total RNA extracted from tissues. Expression levels were standardized using the corresponding GAPDH value of each sample. The relative expression of ΔNp73 in breast cancers and normal breast tissues is shown. The average normal tissue expression (gray line) is indicated. The arrow marks an arbitrary cut-off, delineating tumors with fivefold or higher ΔNp73 overexpression. 16 of 52 breast cancers (31%) overexpress ΔNp73 levels that were between 6- and 44-fold higher than that of average normal breast tissue. (C) Characterization of the polyclonal anti-ΔNp73. Anti-ΔNp73 does not recognize TAp73α, TAp73β, or p53. H1299 cells were transfected with empty vector or the indicated expression plasmids and lysates (50 μg protein for lanes 1–5 and 10 μg for lanes 6–9) were immunoblotted with either a cocktail of ER15, GC15, and DO-1 (lanes 6–9) or anti-ΔNp73 (lanes 1–5). (D) Tumor-specific up-regulation of ΔNp73α protein. Cases 9, 14, and 26 (top from left to right) and cases 10, 31, and 1 (bottom) from Table I are shown. Immunoprecipitations of equal amounts of total protein (2 mg each) from matched pairs of homogenized tumor/normal tissues with 1 μg anti-p73 antibody ER15 followed by immunoblotting with polyclonal anti-ΔNp73. Hc, heavy chain. The last lane (top) is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 2.

ΔNp73 is frequently overexpressed in a variety of primary human cancers. (A) Tumor-specific up-regulation of ΔNp73 (solid bars) transcripts in 35 tumor pairs, compared with their respective normal tissues of origin. In addition, Ex2Del p73 transcripts (gray bars) were measured in a subset of pairs. Ex2Del p73 is another previously described isoform of p73 that lacks most of the transactivation domain. In contrast to ΔNp73, it is generated from the same promoter as TAp73 by splicing out exon 2. It is also a transdominant inhibitor of p53 (references –24). Isoform-specific semiquantitative RT-PCR assay from total RNA extracted from human tissues (see Table I). Two tumor pairs (nos. 21 and 22) had readily detectable ΔNp73 levels in tumor tissues but failed to give detectable ΔNp73 levels in their corresponding normal tissues. Therefore, fold up-regulation could not be quantitated in those 2 cases reducing the total number plotted from 37 to 35 cases. Expression levels, standardized for their corresponding GAPDH values, were used to calculate fold induction as described in Materials and Methods. (B) Up-regulation of ΔNp73 transcripts in a series of unmatched 52 human breast cancers compared with 8 unrelated normal breast tissues. ΔNp73-specific semiquantitative RT-PCR assay from total RNA extracted from tissues. Expression levels were standardized using the corresponding GAPDH value of each sample. The relative expression of ΔNp73 in breast cancers and normal breast tissues is shown. The average normal tissue expression (gray line) is indicated. The arrow marks an arbitrary cut-off, delineating tumors with fivefold or higher ΔNp73 overexpression. 16 of 52 breast cancers (31%) overexpress ΔNp73 levels that were between 6- and 44-fold higher than that of average normal breast tissue. (C) Characterization of the polyclonal anti-ΔNp73. Anti-ΔNp73 does not recognize TAp73α, TAp73β, or p53. H1299 cells were transfected with empty vector or the indicated expression plasmids and lysates (50 μg protein for lanes 1–5 and 10 μg for lanes 6–9) were immunoblotted with either a cocktail of ER15, GC15, and DO-1 (lanes 6–9) or anti-ΔNp73 (lanes 1–5). (D) Tumor-specific up-regulation of ΔNp73α protein. Cases 9, 14, and 26 (top from left to right) and cases 10, 31, and 1 (bottom) from Table I are shown. Immunoprecipitations of equal amounts of total protein (2 mg each) from matched pairs of homogenized tumor/normal tissues with 1 μg anti-p73 antibody ER15 followed by immunoblotting with polyclonal anti-ΔNp73. Hc, heavy chain. The last lane (top) is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 2.

ΔNp73 is frequently overexpressed in a variety of primary human cancers. (A) Tumor-specific up-regulation of ΔNp73 (solid bars) transcripts in 35 tumor pairs, compared with their respective normal tissues of origin. In addition, Ex2Del p73 transcripts (gray bars) were measured in a subset of pairs. Ex2Del p73 is another previously described isoform of p73 that lacks most of the transactivation domain. In contrast to ΔNp73, it is generated from the same promoter as TAp73 by splicing out exon 2. It is also a transdominant inhibitor of p53 (references –24). Isoform-specific semiquantitative RT-PCR assay from total RNA extracted from human tissues (see Table I). Two tumor pairs (nos. 21 and 22) had readily detectable ΔNp73 levels in tumor tissues but failed to give detectable ΔNp73 levels in their corresponding normal tissues. Therefore, fold up-regulation could not be quantitated in those 2 cases reducing the total number plotted from 37 to 35 cases. Expression levels, standardized for their corresponding GAPDH values, were used to calculate fold induction as described in Materials and Methods. (B) Up-regulation of ΔNp73 transcripts in a series of unmatched 52 human breast cancers compared with 8 unrelated normal breast tissues. ΔNp73-specific semiquantitative RT-PCR assay from total RNA extracted from tissues. Expression levels were standardized using the corresponding GAPDH value of each sample. The relative expression of ΔNp73 in breast cancers and normal breast tissues is shown. The average normal tissue expression (gray line) is indicated. The arrow marks an arbitrary cut-off, delineating tumors with fivefold or higher ΔNp73 overexpression. 16 of 52 breast cancers (31%) overexpress ΔNp73 levels that were between 6- and 44-fold higher than that of average normal breast tissue. (C) Characterization of the polyclonal anti-ΔNp73. Anti-ΔNp73 does not recognize TAp73α, TAp73β, or p53. H1299 cells were transfected with empty vector or the indicated expression plasmids and lysates (50 μg protein for lanes 1–5 and 10 μg for lanes 6–9) were immunoblotted with either a cocktail of ER15, GC15, and DO-1 (lanes 6–9) or anti-ΔNp73 (lanes 1–5). (D) Tumor-specific up-regulation of ΔNp73α protein. Cases 9, 14, and 26 (top from left to right) and cases 10, 31, and 1 (bottom) from Table I are shown. Immunoprecipitations of equal amounts of total protein (2 mg each) from matched pairs of homogenized tumor/normal tissues with 1 μg anti-p73 antibody ER15 followed by immunoblotting with polyclonal anti-ΔNp73. Hc, heavy chain. The last lane (top) is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 2.

ΔNp73 is frequently overexpressed in a variety of primary human cancers. (A) Tumor-specific up-regulation of ΔNp73 (solid bars) transcripts in 35 tumor pairs, compared with their respective normal tissues of origin. In addition, Ex2Del p73 transcripts (gray bars) were measured in a subset of pairs. Ex2Del p73 is another previously described isoform of p73 that lacks most of the transactivation domain. In contrast to ΔNp73, it is generated from the same promoter as TAp73 by splicing out exon 2. It is also a transdominant inhibitor of p53 (references –24). Isoform-specific semiquantitative RT-PCR assay from total RNA extracted from human tissues (see Table I). Two tumor pairs (nos. 21 and 22) had readily detectable ΔNp73 levels in tumor tissues but failed to give detectable ΔNp73 levels in their corresponding normal tissues. Therefore, fold up-regulation could not be quantitated in those 2 cases reducing the total number plotted from 37 to 35 cases. Expression levels, standardized for their corresponding GAPDH values, were used to calculate fold induction as described in Materials and Methods. (B) Up-regulation of ΔNp73 transcripts in a series of unmatched 52 human breast cancers compared with 8 unrelated normal breast tissues. ΔNp73-specific semiquantitative RT-PCR assay from total RNA extracted from tissues. Expression levels were standardized using the corresponding GAPDH value of each sample. The relative expression of ΔNp73 in breast cancers and normal breast tissues is shown. The average normal tissue expression (gray line) is indicated. The arrow marks an arbitrary cut-off, delineating tumors with fivefold or higher ΔNp73 overexpression. 16 of 52 breast cancers (31%) overexpress ΔNp73 levels that were between 6- and 44-fold higher than that of average normal breast tissue. (C) Characterization of the polyclonal anti-ΔNp73. Anti-ΔNp73 does not recognize TAp73α, TAp73β, or p53. H1299 cells were transfected with empty vector or the indicated expression plasmids and lysates (50 μg protein for lanes 1–5 and 10 μg for lanes 6–9) were immunoblotted with either a cocktail of ER15, GC15, and DO-1 (lanes 6–9) or anti-ΔNp73 (lanes 1–5). (D) Tumor-specific up-regulation of ΔNp73α protein. Cases 9, 14, and 26 (top from left to right) and cases 10, 31, and 1 (bottom) from Table I are shown. Immunoprecipitations of equal amounts of total protein (2 mg each) from matched pairs of homogenized tumor/normal tissues with 1 μg anti-p73 antibody ER15 followed by immunoblotting with polyclonal anti-ΔNp73. Hc, heavy chain. The last lane (top) is a positive control of H1299 lysate transfected with a ΔNp73α expression vector.

Figure 4.

ΔNp73α counteracts apoptosis and suppression of tumor cell growth induced by wild-type p53 and TAp73. (A) Annexin V analysis of apoptosis. HeLa cells were transfected with expression plasmids encoding wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:1 molar ratio and analyzed after 16 h. As control, ΔNp73α plus empty vector was used. For each construct, the number of expressing cells was determined by immunofluorescence and the apoptotic index of expressing cells is indicated. Results are the average ± SD of four independent experiments. Similar results were obtained with SaOs2 cells and with TUNEL analysis. (B) SaOs2 colony suppression induced by wild-type p53 and TAp73α is inhibited by ΔNp73α. Number of colonies are shown in Table II. (C) p53 expression analysis of random SaOs2 clones derived from surviving colonies of a parallel experiment to the one shown in B. Immunoblots with anti-p53 antibody DO-1 using 30 μg total cell lysate per lane. Clones from cells originally transfected with expression plasmids for wild-type p53 alone (left) or wild-type p53 plus ΔNp73α (right). Except for clone 6, all other wild-type p53-transfected clones have lost full-length p53 protein expression. Clones 1, 3, and 5 show no detectable p53 protein at all, whereas clones 2, 4, and 6 express truncated p53 polypeptides (left). In contrast, all six colonies derived from cotransfection with wild-type p53 and ΔNp73α show detectable levels of full-length p53 protein with sequence-confirmed wild-type status of the DNA binding domain in three of three tested clones (right, five clones are shown). Immunoblot to show ΔNp73α expression in SaOs2 clones 8–12 compared with a SaOs2 clone transfected with empty vector (bottom). (D) ΔNp73α confers drug resistance to wild-type p53/TAp73 harboring tumor cells. RKO cells were transfected with empty vector (left), the irrelevant expression plasmid LcRel (center), or with ΔNp73α expression plasmid (right). 5 h after transfection, cells were treated with 5 μM camptothecin overnight or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNEL staining in green and immunofluorescence with ΔNp73-specific polyclonal antibody (left and right) or Flag antibody (center) in red to assess apoptosis and expression. In contrast to vector-only or LcRel-transfected cells, ΔNp73α-expressing cells (right white arrows) are virtually protected from camptothecin-induced apoptosis. The p73 antibody produces a slight background staining in empty vector–transfected RKO cells, which allows the counting of all cells in the well (left). The percentage of apoptosis of ΔNp73α-expressing and control-transfected cells after 24 h of treatment with 5 μM camptothecin is shown.

Figure 4.

ΔNp73α counteracts apoptosis and suppression of tumor cell growth induced by wild-type p53 and TAp73. (A) Annexin V analysis of apoptosis. HeLa cells were transfected with expression plasmids encoding wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:1 molar ratio and analyzed after 16 h. As control, ΔNp73α plus empty vector was used. For each construct, the number of expressing cells was determined by immunofluorescence and the apoptotic index of expressing cells is indicated. Results are the average ± SD of four independent experiments. Similar results were obtained with SaOs2 cells and with TUNEL analysis. (B) SaOs2 colony suppression induced by wild-type p53 and TAp73α is inhibited by ΔNp73α. Number of colonies are shown in Table II. (C) p53 expression analysis of random SaOs2 clones derived from surviving colonies of a parallel experiment to the one shown in B. Immunoblots with anti-p53 antibody DO-1 using 30 μg total cell lysate per lane. Clones from cells originally transfected with expression plasmids for wild-type p53 alone (left) or wild-type p53 plus ΔNp73α (right). Except for clone 6, all other wild-type p53-transfected clones have lost full-length p53 protein expression. Clones 1, 3, and 5 show no detectable p53 protein at all, whereas clones 2, 4, and 6 express truncated p53 polypeptides (left). In contrast, all six colonies derived from cotransfection with wild-type p53 and ΔNp73α show detectable levels of full-length p53 protein with sequence-confirmed wild-type status of the DNA binding domain in three of three tested clones (right, five clones are shown). Immunoblot to show ΔNp73α expression in SaOs2 clones 8–12 compared with a SaOs2 clone transfected with empty vector (bottom). (D) ΔNp73α confers drug resistance to wild-type p53/TAp73 harboring tumor cells. RKO cells were transfected with empty vector (left), the irrelevant expression plasmid LcRel (center), or with ΔNp73α expression plasmid (right). 5 h after transfection, cells were treated with 5 μM camptothecin overnight or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNEL staining in green and immunofluorescence with ΔNp73-specific polyclonal antibody (left and right) or Flag antibody (center) in red to assess apoptosis and expression. In contrast to vector-only or LcRel-transfected cells, ΔNp73α-expressing cells (right white arrows) are virtually protected from camptothecin-induced apoptosis. The p73 antibody produces a slight background staining in empty vector–transfected RKO cells, which allows the counting of all cells in the well (left). The percentage of apoptosis of ΔNp73α-expressing and control-transfected cells after 24 h of treatment with 5 μM camptothecin is shown.

Figure 4.

ΔNp73α counteracts apoptosis and suppression of tumor cell growth induced by wild-type p53 and TAp73. (A) Annexin V analysis of apoptosis. HeLa cells were transfected with expression plasmids encoding wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:1 molar ratio and analyzed after 16 h. As control, ΔNp73α plus empty vector was used. For each construct, the number of expressing cells was determined by immunofluorescence and the apoptotic index of expressing cells is indicated. Results are the average ± SD of four independent experiments. Similar results were obtained with SaOs2 cells and with TUNEL analysis. (B) SaOs2 colony suppression induced by wild-type p53 and TAp73α is inhibited by ΔNp73α. Number of colonies are shown in Table II. (C) p53 expression analysis of random SaOs2 clones derived from surviving colonies of a parallel experiment to the one shown in B. Immunoblots with anti-p53 antibody DO-1 using 30 μg total cell lysate per lane. Clones from cells originally transfected with expression plasmids for wild-type p53 alone (left) or wild-type p53 plus ΔNp73α (right). Except for clone 6, all other wild-type p53-transfected clones have lost full-length p53 protein expression. Clones 1, 3, and 5 show no detectable p53 protein at all, whereas clones 2, 4, and 6 express truncated p53 polypeptides (left). In contrast, all six colonies derived from cotransfection with wild-type p53 and ΔNp73α show detectable levels of full-length p53 protein with sequence-confirmed wild-type status of the DNA binding domain in three of three tested clones (right, five clones are shown). Immunoblot to show ΔNp73α expression in SaOs2 clones 8–12 compared with a SaOs2 clone transfected with empty vector (bottom). (D) ΔNp73α confers drug resistance to wild-type p53/TAp73 harboring tumor cells. RKO cells were transfected with empty vector (left), the irrelevant expression plasmid LcRel (center), or with ΔNp73α expression plasmid (right). 5 h after transfection, cells were treated with 5 μM camptothecin overnight or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNEL staining in green and immunofluorescence with ΔNp73-specific polyclonal antibody (left and right) or Flag antibody (center) in red to assess apoptosis and expression. In contrast to vector-only or LcRel-transfected cells, ΔNp73α-expressing cells (right white arrows) are virtually protected from camptothecin-induced apoptosis. The p73 antibody produces a slight background staining in empty vector–transfected RKO cells, which allows the counting of all cells in the well (left). The percentage of apoptosis of ΔNp73α-expressing and control-transfected cells after 24 h of treatment with 5 μM camptothecin is shown.

Figure 4.

ΔNp73α counteracts apoptosis and suppression of tumor cell growth induced by wild-type p53 and TAp73. (A) Annexin V analysis of apoptosis. HeLa cells were transfected with expression plasmids encoding wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:1 molar ratio and analyzed after 16 h. As control, ΔNp73α plus empty vector was used. For each construct, the number of expressing cells was determined by immunofluorescence and the apoptotic index of expressing cells is indicated. Results are the average ± SD of four independent experiments. Similar results were obtained with SaOs2 cells and with TUNEL analysis. (B) SaOs2 colony suppression induced by wild-type p53 and TAp73α is inhibited by ΔNp73α. Number of colonies are shown in Table II. (C) p53 expression analysis of random SaOs2 clones derived from surviving colonies of a parallel experiment to the one shown in B. Immunoblots with anti-p53 antibody DO-1 using 30 μg total cell lysate per lane. Clones from cells originally transfected with expression plasmids for wild-type p53 alone (left) or wild-type p53 plus ΔNp73α (right). Except for clone 6, all other wild-type p53-transfected clones have lost full-length p53 protein expression. Clones 1, 3, and 5 show no detectable p53 protein at all, whereas clones 2, 4, and 6 express truncated p53 polypeptides (left). In contrast, all six colonies derived from cotransfection with wild-type p53 and ΔNp73α show detectable levels of full-length p53 protein with sequence-confirmed wild-type status of the DNA binding domain in three of three tested clones (right, five clones are shown). Immunoblot to show ΔNp73α expression in SaOs2 clones 8–12 compared with a SaOs2 clone transfected with empty vector (bottom). (D) ΔNp73α confers drug resistance to wild-type p53/TAp73 harboring tumor cells. RKO cells were transfected with empty vector (left), the irrelevant expression plasmid LcRel (center), or with ΔNp73α expression plasmid (right). 5 h after transfection, cells were treated with 5 μM camptothecin overnight or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNEL staining in green and immunofluorescence with ΔNp73-specific polyclonal antibody (left and right) or Flag antibody (center) in red to assess apoptosis and expression. In contrast to vector-only or LcRel-transfected cells, ΔNp73α-expressing cells (right white arrows) are virtually protected from camptothecin-induced apoptosis. The p73 antibody produces a slight background staining in empty vector–transfected RKO cells, which allows the counting of all cells in the well (left). The percentage of apoptosis of ΔNp73α-expressing and control-transfected cells after 24 h of treatment with 5 μM camptothecin is shown.

Figure 4.

ΔNp73α counteracts apoptosis and suppression of tumor cell growth induced by wild-type p53 and TAp73. (A) Annexin V analysis of apoptosis. HeLa cells were transfected with expression plasmids encoding wild-type p53 or TAp73β with either empty vector or ΔNp73α at a 1:1 molar ratio and analyzed after 16 h. As control, ΔNp73α plus empty vector was used. For each construct, the number of expressing cells was determined by immunofluorescence and the apoptotic index of expressing cells is indicated. Results are the average ± SD of four independent experiments. Similar results were obtained with SaOs2 cells and with TUNEL analysis. (B) SaOs2 colony suppression induced by wild-type p53 and TAp73α is inhibited by ΔNp73α. Number of colonies are shown in Table II. (C) p53 expression analysis of random SaOs2 clones derived from surviving colonies of a parallel experiment to the one shown in B. Immunoblots with anti-p53 antibody DO-1 using 30 μg total cell lysate per lane. Clones from cells originally transfected with expression plasmids for wild-type p53 alone (left) or wild-type p53 plus ΔNp73α (right). Except for clone 6, all other wild-type p53-transfected clones have lost full-length p53 protein expression. Clones 1, 3, and 5 show no detectable p53 protein at all, whereas clones 2, 4, and 6 express truncated p53 polypeptides (left). In contrast, all six colonies derived from cotransfection with wild-type p53 and ΔNp73α show detectable levels of full-length p53 protein with sequence-confirmed wild-type status of the DNA binding domain in three of three tested clones (right, five clones are shown). Immunoblot to show ΔNp73α expression in SaOs2 clones 8–12 compared with a SaOs2 clone transfected with empty vector (bottom). (D) ΔNp73α confers drug resistance to wild-type p53/TAp73 harboring tumor cells. RKO cells were transfected with empty vector (left), the irrelevant expression plasmid LcRel (center), or with ΔNp73α expression plasmid (right). 5 h after transfection, cells were treated with 5 μM camptothecin overnight or left untreated (not depicted). After fixation with paraformaldehyde, each well underwent both TUNEL staining in green and immunofluorescence with ΔNp73-specific polyclonal antibody (left and right) or Flag antibody (center) in red to assess apoptosis and expression. In contrast to vector-only or LcRel-transfected cells, ΔNp73α-expressing cells (right white arrows) are virtually protected from camptothecin-induced apoptosis. The p73 antibody produces a slight background staining in empty vector–transfected RKO cells, which allows the counting of all cells in the well (left). The percentage of apoptosis of ΔNp73α-expressing and control-transfected cells after 24 h of treatment with 5 μM camptothecin is shown.

Figure 1.

Gene architecture of human TP73. (A) In contrast to TP53, which harbors a single promoter generating a single protein composed of the transactivation domain (TAD), DNA-binding domain (DBD), and tetramerization domain (TD), the TP73 gene is complex and contains two promoters and an additional sterile α motif domain (SAM). The P1 promoter in the 5′ UTR region produces transactivation-competent full-length proteins containing the TA domain (TAp73). The P2 promoter in intron 3 produces TA-deficient protein(s) (ΔNp73) with dominant-negative function toward TAp73 and wild-type p53. ΔNp73 starts with exon 3′, which encodes 13 unique amino acids that are highly conserved between human and mouse. Another NH₂ terminally truncated p73, Ex2Del, also lacks the TA domain but is created by splicing out exon 2 from the P1 transcript (reference 1). The COOH-terminal of TAp73 undergoes additional exon splicing, which generates β-φ isoforms. (B) Positions of the P1 and P2 promoters. Positions of the RT-PCR primers are indicated: TAp73 (bottom), ΔNp73 (center), and Ex2Delp73 (top). (C) Sequence of the 5′ UTR region of ΔNp73. The putative TATA box is indicated.

Figure 1.

Gene architecture of human TP73. (A) In contrast to TP53, which harbors a single promoter generating a single protein composed of the transactivation domain (TAD), DNA-binding domain (DBD), and tetramerization domain (TD), the TP73 gene is complex and contains two promoters and an additional sterile α motif domain (SAM). The P1 promoter in the 5′ UTR region produces transactivation-competent full-length proteins containing the TA domain (TAp73). The P2 promoter in intron 3 produces TA-deficient protein(s) (ΔNp73) with dominant-negative function toward TAp73 and wild-type p53. ΔNp73 starts with exon 3′, which encodes 13 unique amino acids that are highly conserved between human and mouse. Another NH₂ terminally truncated p73, Ex2Del, also lacks the TA domain but is created by splicing out exon 2 from the P1 transcript (reference 1). The COOH-terminal of TAp73 undergoes additional exon splicing, which generates β-φ isoforms. (B) Positions of the P1 and P2 promoters. Positions of the RT-PCR primers are indicated: TAp73 (bottom), ΔNp73 (center), and Ex2Delp73 (top). (C) Sequence of the 5′ UTR region of ΔNp73. The putative TATA box is indicated.

Figure 1.

Gene architecture of human TP73. (A) In contrast to TP53, which harbors a single promoter generating a single protein composed of the transactivation domain (TAD), DNA-binding domain (DBD), and tetramerization domain (TD), the TP73 gene is complex and contains two promoters and an additional sterile α motif domain (SAM). The P1 promoter in the 5′ UTR region produces transactivation-competent full-length proteins containing the TA domain (TAp73). The P2 promoter in intron 3 produces TA-deficient protein(s) (ΔNp73) with dominant-negative function toward TAp73 and wild-type p53. ΔNp73 starts with exon 3′, which encodes 13 unique amino acids that are highly conserved between human and mouse. Another NH₂ terminally truncated p73, Ex2Del, also lacks the TA domain but is created by splicing out exon 2 from the P1 transcript (reference 1). The COOH-terminal of TAp73 undergoes additional exon splicing, which generates β-φ isoforms. (B) Positions of the P1 and P2 promoters. Positions of the RT-PCR primers are indicated: TAp73 (bottom), ΔNp73 (center), and Ex2Delp73 (top). (C) Sequence of the 5′ UTR region of ΔNp73. The putative TATA box is indicated.

Figure 6.

Mechanism of the dominant-negative effect of ΔNp73. (A) EMSA using nuclear extracts of H1299 cells transfected with expression plasmids for p53 or ΔNp73α alone or in combination. Competition of sequence-specific binding of p53 by ΔNp73α is shown (compare lanes 6 and 7). ΔNp73α alone does not bind to the p53 cognate site, p53CON (lane 8). Anti-p53 antibody (421) was added as indicated. (B) p53 reporter assay using the p53-responsive reporter sequence PG13. A tetramerization-incompetent but DNA binding–competent mutant of ΔNp73α, ΔNp73 L(322)P (corresponding to L371P in TAp73α; reference 4), shows a significant but incomplete reversal of the dominant-negative inhibition of p53 transactivation by ΔNp73α.

Figure 7.

Down-regulation of endogenous ΔNp73 levels alleviates its suppressive action and thereby enhances apoptosis mediated by p53 and TAp73. (A) Wild-type p53 harboring RKO cells were transfected with antisense (as) or sense (s) oligonucleotides (200 nM each) directed against exon 3′ of DNp73. After 8 h, cells were subjected to DNA damage by 1 mM camptothecin for an additional 16 h before TUNEL staining. Apoptosis was determined by measuring green fluorescence as described in Materials and Methods. Camp, cells treated with camptothecin alone. (B) RKO cells were transfected with 360 ng wild-type p53 expression plasmid with 200 nM of either ΔNp73 antisense (as) or sense (s) oligonucleotides. 20 h after transfection, TUNEL staining was performed. Apoptosis was quantitated as described above. (C) Cells were transfected with 200 nM each of antisense (as) or sense (s) oligonucleotides. 36 h later, equal amounts of lysates (2 mg total protein) were immunoprecipitated with ER15 and blotted with the polyclonal anti-ΔNp73.

Figure 7.

Down-regulation of endogenous ΔNp73 levels alleviates its suppressive action and thereby enhances apoptosis mediated by p53 and TAp73. (A) Wild-type p53 harboring RKO cells were transfected with antisense (as) or sense (s) oligonucleotides (200 nM each) directed against exon 3′ of DNp73. After 8 h, cells were subjected to DNA damage by 1 mM camptothecin for an additional 16 h before TUNEL staining. Apoptosis was determined by measuring green fluorescence as described in Materials and Methods. Camp, cells treated with camptothecin alone. (B) RKO cells were transfected with 360 ng wild-type p53 expression plasmid with 200 nM of either ΔNp73 antisense (as) or sense (s) oligonucleotides. 20 h after transfection, TUNEL staining was performed. Apoptosis was quantitated as described above. (C) Cells were transfected with 200 nM each of antisense (as) or sense (s) oligonucleotides. 36 h later, equal amounts of lysates (2 mg total protein) were immunoprecipitated with ER15 and blotted with the polyclonal anti-ΔNp73.

Figure 7.

Down-regulation of endogenous ΔNp73 levels alleviates its suppressive action and thereby enhances apoptosis mediated by p53 and TAp73. (A) Wild-type p53 harboring RKO cells were transfected with antisense (as) or sense (s) oligonucleotides (200 nM each) directed against exon 3′ of DNp73. After 8 h, cells were subjected to DNA damage by 1 mM camptothecin for an additional 16 h before TUNEL staining. Apoptosis was determined by measuring green fluorescence as described in Materials and Methods. Camp, cells treated with camptothecin alone. (B) RKO cells were transfected with 360 ng wild-type p53 expression plasmid with 200 nM of either ΔNp73 antisense (as) or sense (s) oligonucleotides. 20 h after transfection, TUNEL staining was performed. Apoptosis was quantitated as described above. (C) Cells were transfected with 200 nM each of antisense (as) or sense (s) oligonucleotides. 36 h later, equal amounts of lysates (2 mg total protein) were immunoprecipitated with ER15 and blotted with the polyclonal anti-ΔNp73.