TY - JOUR
T1 - Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A
AU - Gu, Yongchuan
AU - Patterson, Adam V.
AU - Atwell, Graham J.
AU - Chernikova, Sophia B.
AU - Brown, J. Martin
AU - Thompson, Larry H.
AU - Wilson, William R.
PY - 2009/6
Y1 - 2009/6
N2 - PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparingsensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D -/- cells but not of XPD-/- or DNA-PKCS -/- cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restartingcollapsed forks. However, even under anoxia, the hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D-/- cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D-/- cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linkingby its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity.
AB - PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparingsensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D -/- cells but not of XPD-/- or DNA-PKCS -/- cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restartingcollapsed forks. However, even under anoxia, the hypersensitivity of XPF-/-, ERCC1-/-, and Rad51D-/- cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D-/- cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linkingby its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity.
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U2 - 10.1158/1535-7163.MCT-08-1209
DO - 10.1158/1535-7163.MCT-08-1209
M3 - Article
C2 - 19509245
AN - SCOPUS:67649354921
VL - 8
SP - 1714
EP - 1723
JO - Molecular Cancer Therapeutics
JF - Molecular Cancer Therapeutics
SN - 1535-7163
IS - 6
ER -