The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide)
DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2) isolated from Walker 256 rat carcinoma cells can convert CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) to a cytotoxic DNA interstrand cross-linking agent. This is achieved by reduction of the 4-nitro group of CB 1954 to produce the hydroxylamino species, a bioactivation which accounts for the much greater sensitivity of Walker cells to CB 1954 when compared with other cells which are unable to carry out this reduction (Knox et al., Biochem Pharmacol 37: 4661-4669 and 4671-4677, 1988). As predicted from their measured DT diaphorase activities a number of rat hepatoma and hepatocyte cell lines were also shown to be sensitive to CB 1954. However, no CB 1954-sensitive cell lines of human origin were found, although levels of DT diaphorase similar to those in the sensitive rat cells were present in these cells. The human cells were as sensitive as rat cells to the active form of CB 1954 (5-(aziridin-1-yl)-4-hydroxyla mino-2-nitrobenzamide). DT diaphorase, purified to homogeneity from human Hep G2 cells, did metabolize CB 1954 to this 4-hydroxylamino product, but the rate of CB 1954 reduction and thus production of the cytotoxic product, was much lower than that of purified Walker enzyme (ratio of Kcat = 6.4). In addition, CB 1954 could be considered an inhibitor of, rather than a substrate for, the human form of DT diaphorase. The purified rat and human DT diaphorases possessed otherwise similar biochemical and molecular properties. These findings explain the decreased sensitivity towards CB 1954 of human cell lines when compared to rat cell lines.
Boland
M P
MP
Molecular Pharmacology Unit, Institute of Cancer Research, Sutton, Surrey, U.K.
Knox
R J
RJ
Roberts
J J
JJ
D000818:Animals; D001388:Aziridines / Q000378:metabolism / Q000494:pharmacology*; D002279:Carcinoma 256, Walker / Q000201:enzymology; D002460:Cell Line / Q000187:drug effects / Q000201:enzymology; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D016660:NAD(P)H Dehydrogenase (Quinone); D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D001388:Aziridines; C100099:tretazicar; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases