title: Selective enhancement of ifosfamide-induced toxicity in Chinese hamster ovary cells.
abstract: OBJECTIVE
  O6-benzylguanine (BG) is a unique purine analog that has been shown to influence nitrogen mustard activity and increase cytotoxicity. Ifosfamide is a nitrogen mustard with growing clinical applications; effective modulation may lead to improved efficacy. We thus undertook a preliminary investigation of BG's effects on ifosfamide and ifosfamide derivatives in vitro.
  
  
  METHODS
  BG's effect on ifosfamide toxicity was studied in CHO cells transfected with O6-alkylguanine-DNA alkyltransferase (AGT) (CHOwtAGT) or control plasmid pcDNA3 (CHOpcDNA) using five ifosfamide derivatives and two control compounds: 4-hydroperoxyifosfamide (4HI), isophosphoramide mustard (IPM), phenylketoifosfamide (PKIF), 4-hydroperoxydidechloroifosfamide (4HDI), chloroacetaldehyde (CAA), didechloroisophosphoramide mustard (d-IPM), didechlorophenylketoifosfamide (d-PKIF). To further explore the mechanism of interaction, BG's effect on apoptosis (annexin V-FITC) and cell cycle distribution in cells exposed to ifosfamide was also analyzed.
  
  
  RESULTS
  BG substantially enhanced cytotoxicity induced only by agents that produce IPM (4HI, IPM, PKIF) in both CHOwtAGT and CHOpcDNA cell lines. BG did not modulate 4HDI or CAA cytotoxicity. The addition of BG to IPM in CHO cells increased the percentage of apoptotic cells from 5.5% to 28.9% at 72 h after treatment. Cell cycle analysis showed that BG exposure was associated with G1 arrest. At 16 h following treatment with IPM, PKIF, or phosphoramide mustard (PM), BG increased the percentage of cells in G1 from 16-20% to 29-64%.
  
  
  CONCLUSIONS
  BG's ability to increase 4HI-, IPM-, and PKIF-mediated cytotoxicity in cells devoid of AGT activity suggests a novel AGT-independent mode of action that is associated with increased apoptosis and may involve G1 arrest. BG selectively enhanced IPM toxicity without enhancement of acrolein and CAA toxicity. The data strongly support further investigation into combinations of BG and nitrogen mustards.
authors.affiliation: Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
authors.forename: Sonali M
authors.initials: SM
authors.lastname: Smith
authors.forename: Susan M
authors.initials: SM
authors.lastname: Ludeman
authors.forename: Lynette R
authors.initials: LR
authors.lastname: Wilson
authors.forename: James B
authors.initials: JB
authors.lastname: Springer
authors.forename: Mihir C
authors.initials: MC
authors.lastname: Gandhi
authors.forename: M Eileen
authors.initials: ME
authors.lastname: Dolan
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D016466:CHO Cells; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004273:DNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity*; D006207:Half-Life; D007069:Ifosfamide / Q000031:analogs & derivatives / Q000737:chemistry / Q000633:toxicity*; D007700:Kinetics; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014162:Transfection
chemical_list: D017304:Annexin A5; D018906:Antineoplastic Agents, Alkylating; D004273:DNA, Neoplasm; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007069:Ifosfamide