title: Schedule-dependent synergy between the heat shock protein 90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and doxorubicin restores apoptosis to p53-mutant lymphoma cell lines.
abstract: OBJECTIVE
  Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction.
  
  
  METHODS
  Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction.
  
  
  RESULTS
  Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G1-S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation.
  
  
  CONCLUSIONS
  Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.
authors.affiliation: Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute and Hematology Branch, National Heart, Lung and Blood Institute, NIH, Bethesda, Maryland 20892, USA. roblesa@mail.nih.gov
authors.forename: Ana I
authors.initials: AI
authors.lastname: Robles
authors.forename: Mollie H
authors.initials: MH
authors.lastname: Wright
authors.forename: Bheru
authors.initials: B
authors.lastname: Gandhi
authors.forename: Steven S
authors.initials: SS
authors.lastname: Feis
authors.forename: Christin L
authors.initials: CL
authors.lastname: Hanigan
authors.forename: Adrian
authors.initials: A
authors.lastname: Wiestner
authors.forename: Lyuba
authors.initials: L
authors.lastname: Varticovski
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D016227:Benzoquinones / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D004334:Drug Administration Schedule; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004357:Drug Synergism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D015151:Immunoblotting; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D008223:Lymphoma / Q000188:drug therapy*; D009154:Mutation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D016159:Tumor Suppressor Protein p53; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; D004317:Doxorubicin