Selective enhancement of ifosfamide-induced toxicity in Chinese hamster ovary cells.
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.
Smith
Sonali M
SM
Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
Ludeman
Susan M
SM
Wilson
Lynette R
LR
Springer
James B
JB
Gandhi
Mihir C
MC
Dolan
M Eileen
ME
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
D017304:Annexin A5; D018906:Antineoplastic Agents, Alkylating; D004273:DNA, Neoplasm; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007069:Ifosfamide