Document Detail


Demonstration of the activation of prodrug CB 1954 using human DT-diaphorase mutant Q104Y-transfected MDA-MB-231 cells and mouse xenograft model.
MedLine Citation:
PMID:  11361019     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The rat form of DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase; EC 1.6.99.2) is more effective than the human form in activating prodrugs such as CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Our site-directed mutagenesis study has revealed that residue 104 (Tyr in the rat enzyme and Gln in the human enzyme) is an important residue responsible for the catalytic differences between the rat and the human enzymes in the activation of CB 1954 (S. Chen et al., 1997, J. Biol. Chem. 272, 1437-1439). The human mutant Q104Y is capable of reducing CB 1954 at a rate identical to that of the wild-type rat DT-diaphorase. In the present study, we prepared both the wild-type human DT-diaphorase- and the mutant Q104Y-expressing MDA-MB-231 breast cancer cell lines using the cDNA transfection method. The MDA-MB-231 cell line is homozygous for a P187S mutation in the DT-diaphorase gene and has no detectable DT-diaphorase activity. Stable clones for the wild-type transfected cells had the DT-diaphorase activity ranged from 0.1 to 3.8 micromol of DCIP reduced/min/mg of protein and the clones for Q104Y transfected cells had the activity ranged from 0.06 to 1.58 micromol of DCIP reduced/min/mg of protein. Furthermore, in contrast to the cells transfected with only expression vector that were not sensitive to CB 1954 treatment, the wild-type and Q104Y-expressing cells were capable of the reductive activation of CB 1954, resulting in cell eradication. Our data showed that cell killing by CB 1954 followed a dose and incubation-time dependent manner. It was also found that the cell survival upon the treatment of CB 1954 was related to the expressed DT-diaphorase activity in these cells. In the presence of 75 microM CB 1954, a 50% cell killing was achieved in cells containing Q104Y and the wild-type DT-diaphorase with the activity at approximately 0.67 and 3.8 micromol of DCIP reduced/min/mg of protein, respectively. These results agree well with those of the in vitro enzyme assays that show that Q104Y is significantly more active than the wild-type DT-diaphorase in the activation of CB 1954. Finally, the in vivo activation of CB 1954 was demonstrated with a nude mouse model using Q104Y-transfected MDA-MB-231 cells. These studies reveal that DT-diaphorase can activate CB 1954, and human Q104Y mutant enzyme is more active than the wild-type enzyme in the intracellular reductive activation of CB 1954.
Authors:
K Wu; E Eng; R Knox; S Chen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Archives of biochemistry and biophysics     Volume:  385     ISSN:  0003-9861     ISO Abbreviation:  Arch. Biochem. Biophys.     Publication Date:  2001 Jan 
Date Detail:
Created Date:  2001-05-21     Completed Date:  2001-06-21     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0372430     Medline TA:  Arch Biochem Biophys     Country:  United States    
Other Details:
Languages:  eng     Pagination:  203-8     Citation Subset:  IM    
Affiliation:
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Aziridines / pharmacology*
Cell Survival / drug effects
Cloning, Molecular
DNA, Complementary / metabolism
Dihydrolipoamide Dehydrogenase / genetics*
Dose-Response Relationship, Drug
Homozygote
Humans
Mice
Mice, Nude
Mutagenesis
Mutation*
Prodrugs / pharmacology*
Time Factors
Transfection
Tumor Cells, Cultured
Grant Support
ID/Acronym/Agency:
CA 65767/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Aziridines; 0/DNA, Complementary; 0/Prodrugs; 21919-05-1/5-(1-aziridinyl)-2,4-dinitrobenzamide; EC 1.8.1.4/Dihydrolipoamide Dehydrogenase

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