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

Zaika AI, Slade N, Erster SH, Sansome C, Joseph TW, Pearl M, Chalas E, Moll UM - J. Exp. Med. (2002)

Bottom Line: p73 has significant homology to p53.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.

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.

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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.
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fig4: 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.

Mentions: Hela and SaOs2 cells were seeded into 8-well chamber slides and cotransfected with 300 ng of the indicated pcDNA3-based expression plasmids per well using Fugene (see Fig. 4 A). Control wells (vector alone) received 600 ng. After 16 or 24 h, cells were stained with Annexin V or Tdt-mediated dUTP-X nick-end labeling (TUNEL), respectively, according to the manufacturer's instructions (Roche). Expression was determined by immunofluorescence in duplicate wells. Transfection efficiency was reproducibly ∼30% of cells, similar among all constructs and evenly distributed throughout the wells. Annexin V or TUNEL positive cells (494 fields at 40×) and plasmid-expressing cells (15 random fields, >500 cells) were counted and the percentage of apoptosis in transfected cells was determined after correction for background with vector alone.


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

Zaika AI, Slade N, Erster SH, Sansome C, Joseph TW, Pearl M, Chalas E, Moll UM - J. Exp. Med. (2002)

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.
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Related In: Results  -  Collection

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fig4: 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.
Mentions: Hela and SaOs2 cells were seeded into 8-well chamber slides and cotransfected with 300 ng of the indicated pcDNA3-based expression plasmids per well using Fugene (see Fig. 4 A). Control wells (vector alone) received 600 ng. After 16 or 24 h, cells were stained with Annexin V or Tdt-mediated dUTP-X nick-end labeling (TUNEL), respectively, according to the manufacturer's instructions (Roche). Expression was determined by immunofluorescence in duplicate wells. Transfection efficiency was reproducibly ∼30% of cells, similar among all constructs and evenly distributed throughout the wells. Annexin V or TUNEL positive cells (494 fields at 40×) and plasmid-expressing cells (15 random fields, >500 cells) were counted and the percentage of apoptosis in transfected cells was determined after correction for background with vector alone.

Bottom Line: p73 has significant homology to p53.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.

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.

Show MeSH
Related in: MedlinePlus