title: Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia.
abstract: BACKGROUND
  The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy.
  
  
  RESULTS
  Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells.
  
  
  CONCLUSIONS
  Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML.
authors.forename: Ingvild
authors.initials: I
authors.lastname: Haaland
authors.forename: Jill A
authors.initials: JA
authors.lastname: Opsahl
authors.forename: Frode S
authors.initials: FS
authors.lastname: Berven
authors.forename: Håkon
authors.initials: H
authors.lastname: Reikvam
authors.forename: Hanne K
authors.initials: HK
authors.lastname: Fredly
authors.forename: Ragnhild
authors.initials: R
authors.lastname: Haugse
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
authors.forename: Emmet
authors.initials: E
authors.lastname: McCormack
authors.forename: Sonia
authors.initials: S
authors.lastname: Lain
authors.forename: Oystein
authors.initials: O
authors.lastname: Bruserud
authors.affiliation: Department of Internal Medicine, Haukeland University Hospital, Bergen N-5021, Norway. bjorn.gjertsen@med.uib.no.
authors.forename: Bjørn Tore
authors.initials: BT
authors.lastname: Gjertsen
mesh_terms: D000107:Acetylation; D000970:Antineoplastic Agents / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004357:Drug Synergism; D015973:Gene Expression Regulation, Leukemic; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*; D061251:Primary Cell Culture; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D016227:Benzoquinones; D055551:HSP27 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006657:Histones; D007093:Imidazoles; D047029:Lactams, Macrocyclic; D010879:Piperazines; D016159:Tumor Suppressor Protein p53; C482205:nutlin 3; C001277:geldanamycin