title: Reversal of cytosine arabinoside (ara-C) resistance by the synergistic combination of 6-thioguanine plus ara-C plus PEG-asparaginase (TGAP) in human leukemia lines lacking or expressing p53 protein.
abstract: BACKGROUND
  Sequence-specific combinations of purine analogs, such as fludarabine or 6-mercaptopurine (6-MP), administered prior to cytosine arabinoside (ara-C) have been shown to abrogate ara-C resistance in human leukemia cells in vitro and in patients with relapsed acute myeloid or lymphoblastic leukemias. The two-drug combination of 6-MP plus ara-C results in greater cytotoxicity than that achieved with either ara-C or 6-MP alone. Further preclinical investigations have shown that the addition of PEG-asparaginase (PEG-ASNase) to the combination of 6-MP plus ara-C (6-MP + ara-C + PEG-ASNase) results in 15.6-fold synergism over that achieved with the two-drug regimen. This is due to increased DNA damage leading to apoptotic cell death.
  
  
  OBJECTIVE
  Since the intravenous preparation of 6-MP is no longer available and since oral 6-thioguanine (6-TG) provides higher levels of intracellular thioguanine nucleotides than an isotoxic dose of oral 6-MP, we investigated the potential drug synergism of 6-TG plus ara-C plus PEG-ASNase (TGAP) in myeloid (HL60/S, HL60/SN3, U937) and lymphoblastic (CEM/0, CEM/ ara-C/B, CEM/ara-C/I, MOLT-4) leukemia cell lines. The CEM clones, MOLT-4 and HL60/SN3 cell lines expressed functional or measurable p53 protein, while the other cell lines did not.
  
  
  METHODS
  The MTT and trypan blue dye exclusion assays were used to determine drug cytotoxicity. In addition, cellular apoptosis and cellular p53, p21/waf-1 and bcl-2 protein concentrations were determined by FACS analysis and ELISA assays.
  
  
  RESULTS
  Sequential exposure to 6-TG (24 h) plus ara-C (24 h) plus PEG-ASNase (24 h) produced 1.3- to 18.3-fold drug synergism over the two-drug combination of 6-TG plus ara-C. The molecular mechanism of synergism was due to the fact that the three-drug combination was capable of downregulating bcl-2 oncoprotein levels in these cell lines even when p53 was absent.
  
  
  CONCLUSIONS
  These studies strongly demonstrate that the TGAP regimen is highly synergistic in p53-null and p53-expressing leukemia cell lines. We conclude that this combination regimen is collaterally sensitive with ara-C and further evaluation in an investigational phase I trial in relapsed leukemia patients is warranted.
authors.affiliation: Department of Pediatrics, USC School of Medicine, Childrens Hospital Los Angeles, CA 90027, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Fu
authors.forename: S
authors.initials: S
authors.lastname: Martin-Aragon
authors.forename: K I
authors.initials: KI
authors.lastname: Weinberg
authors.forename: V C
authors.initials: VC
authors.lastname: Ardi
authors.forename: P V
authors.initials: PV
authors.lastname: Danenberg
authors.forename: V I
authors.initials: VI
authors.lastname: Avramis
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001215:Asparaginase / Q000008:administration & dosage / Q000494:pharmacology; D003561:Cytarabine / Q000008:administration & dosage / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D007945:Leukemia, Lymphoid / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D015458:Leukemia, T-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D013866:Thioguanine / Q000008:administration & dosage / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000172:deficiency / Q000502:physiology*; D020298:U937 Cells
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D003561:Cytarabine; D011092:Polyethylene Glycols; C042705:pegaspargase; D001215:Asparaginase; D013866:Thioguanine