Dominant Effects of Δ40p53 on p53 Function and Melanoma Cell Fate

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Dominant Effects of Δ40p53 on p53 Function and Melanoma Cell Fate Rie Takahashi, Svetomir N. Markovic, Heidi J. Scrable Journal of Investigative Dermatology Volume 134, Issue 3, Pages 791-800 (March 2014) DOI: 10.1038/jid.2013.391 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Δ40p53 increases the relative number of dead cells. (a) Lentiviral constructs encoding Δ40p53 and green fluorescence protein (GFP) (Δ40p53V) or GFP alone (empty vector (EV)). cPPT, HIV central polypurine tract; LTR, long terminal repeat; RRE, Rev-responsive element; SFFVp, spleen focus-forming virus promoter; Ubp, ubiquitin promoter; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ψ, packaging signal. (b) Representative images of cells infected with EV, Δ40p53V, or no lentivirus (no infection (NI)). Bar=1 μM. (c) p53 and Δ40p53 domain structures and antibody epitopes. Δ40p53 is missing transcription factor domain 1 (TFD1), but retains transcription factor domain 2 (TFD2), proline-rich domain (PrD), DNA-binding domain, nuclear localization signal (NLS), oligomerization domain (OD), and regulatory domain (Reg). (d) Western blot analysis of A375 melanoma cells infected with EV, Δ40p53V, or NI. Δ40p53V was detected by HR231 and CM1 but not DO1. (e) Relative fold-change of dead cells in EV- and Δ40p53V-infected cells normalized to NI control. ****P<0.0001. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Investigative Dermatology 2014 134, 791-800DOI: (10.1038/jid.2013.391) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Δ40p53 causes apoptosis, not cell cycle arrest. (a) Flow cytometric analysis of apoptotic and subdiploid populations in A375 cells infected with empty vector (EV), Δ40p53V, or no infection (NI). Percentages within each quadrant represent fraction of total cells. Bottom panel percentages indicate subdiploid fraction of cells. At least 20,000 events were collected per experimental sample. Representative plots shown. (b) Western blot analysis of poly-(ADP-ribose) polymerase (PARP I) cleavage. (c) Cell cycle profiles of infected A375 cells in the presence of propidium iodide following 0 or 7 Gy of γ-irradiation (+IR). (d) Cell cycle distribution of infected versus uninfected cells with (shaded area) or without γ-irradiation (non-shaded area). Subdiploid (non-cycling) percentages are shown separately. (e) Western blot analysis of p53 expression in infected A375 cells with γ-irradiation (7 Gy). Cells were harvested at 0, 3, 6, and 9 hours after irradiation. Journal of Investigative Dermatology 2014 134, 791-800DOI: (10.1038/jid.2013.391) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Δ40p53 activates p53 under proteotoxic, but not genotoxic, conditions. (a) Serine 15 (ser15)–phosphorylated p53 in A375 cells infected with empty vector (EV), Δ40p53V, or no infection (NI) treated with ataxia telangiectasia mutated/ATM- and Rad3-related (ATM/ATR) inhibitor CGK733 (10 μM). (b) Kinases and phosphatases affecting p53 serine 15 phosphorylation in Δ40p53V, EV, and NI in A375 cells. Phosphorylated AMP-activated protein kinase α (p-AMPKα); phosphorylated mammalian target of rapamycin (p-mTOR); phosphorylated ribosomal protein S6 kinase 2 (p-RSK2); p70 ribosomal S6 kinase (p70 RSK2); cyclin-dependent kinase 5 (CDK5); and cell division cycle 25 homolog A (CDC25A). (c) Serine 15–phosphorylated p53 in Δ40p53-infected A375 cells treated with cycloheximide (CHX). Cells were infected with EV or Δ40p53V and treated with 0 or 100 μg ml-1 cycloheximide for 1, 2, 3, 6, or 12 hours. p53 and Δ40p53V detected by ser15 and CM5. Cyclin B1 levels shown to determine effectiveness of cycloheximide treatment. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Investigative Dermatology 2014 134, 791-800DOI: (10.1038/jid.2013.391) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Δ40p53 increases the expression of apoptotic gene, PIDD, and suppresses expression of cell cycle arrest gene, p21. (a) Potential p53 gene targets altered by Δ40p53V. Log-scale dot plot of gene target levels in Δ40p53V versus empty vector (EV). Eight genes (red circles) fell outside of the 3-fold dynamic range (delineated by parallel lines); see Supplementary Figure S7 online. (b) Relative expression of p53 gene targets. Quantitative PCR analysis of selected p53 targets in A375 cells infected with EV (grey bars) and Δ40p53V (black bars). p53-induced protein with a death domain (PIDD); poly(rC)-binding protein 4 (PCBP4); apoptotic protease activating factor 1 (APAF1); Bcl-2–associated X protein (Bax); cyclin-dependent kinase 1 (p21); growth arrest and DNA-damage–inducible protein α (GADD45A). *P<0.05. (c) Relative expression of p53 targets (PIDD, PCBP4, p21, and GADD45A) in Δ40p53-infected, γ-irradiated A375 cells. *P<0.05. Journal of Investigative Dermatology 2014 134, 791-800DOI: (10.1038/jid.2013.391) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Δ40p53 oligomerizes with endogenous activated p53 in the nucleus and increases promoter occupancy of PIDD and p21. (a) Nuclear and cytoplasmic fractions of A375 cells infected with Δ40p53V. Nucleoporin 62 (Np62) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as nuclear and cytoplasmic markers, respectively. (b) Higher order oligomers in A375 melanoma cells infected with Δ40p53V. Nuclear fractions of cells infected were treated with glutaraldehyde (0.1%). p53 promoter occupancy at PIDD (c) and p21 (d) promoters. p53 antibodies were used to immunoprecipitate p53, Δ40p53, and activated p53 from Δ40p53V-infected A375 cells for chromatin immunoprecipitation (ChIP) analysis (see Supplementary Figure S8 online). *P<0.05; ***P<0.001. (e) A model for how Δ40p53 alters cell fate. Endogenous p53 can be activated and directed by Δ40p53 to favor apoptosis over cell cycle arrest (even with γ-irradiation) in tumor cells. Our results are consistent with a role for Δ40p53 in the reactivation of p53-dependent tumor suppression. Journal of Investigative Dermatology 2014 134, 791-800DOI: (10.1038/jid.2013.391) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions