THE ROLE OF NAD(P)H-QUINONE OXIDOREDUCTASE IN QUINONE-MEDIATED P21 INDUCTION IN HUMAN COLON-CARCINOMA CELLS

This study examines the role of NAD(P)H:quinone acceptor oxidoreductas e (NQOR) (EC 1.6.99.2) in the metabolism of aziridinylbenzoquinones an d the ensuing formation of reactive oxygen species in the induction of the cell cycle inhibitor p21 (WAF1, Cip1, or sdi1) in human colon car cinoma cells. The aziridinylibenzoquinones used were 2,5-diaziridinyl- 1,4-benzoquinone (DZQ) and arboethoxyamino)-3,6-diaziridinyl-1,4-benzo quinone (AZQ). The cell lines used in this study, BE and HT29 human co lon carcinoma cell lines, are devoid of and overexpress NQOR activity, respectively. The rate of reduction of the above quinones in BE cells proceeded at similar rates (similar to 170 nmol/min/mg protein) and, expectedly, it was not affected by the NQOR inhibitor, dicumarol. The metabolism of DZQ in HT29 cells was largely accomplished by NQOR (simi lar to 94%), whereas that of AZQ was accomplished by dicumarol-insensi tive reductases. The metabolism of DZQ, in HT29 cells was accompanied by H2O2 formation, which was similar to 10-fold higher than that ensui ng from the activation of AZQ. In agreement with these data, the produ ction of H2O2 during the activation of DZQ by purified NQOR was simila r to 10-fold higher than that of AZQ. The formation of H2O2 during the metabolism of aziridinylbenzoquinones in BE cells was 24- to 57-fold lower than that in HT29 cells. At variance with HT29 cells, H2O2 forma tion by BE cells was insensitive to the catalase inhibitor sodium azid e. The bioactivation of AZQ and DZQ in BE cells yielded O-2(.-) and HO . as detected by spin trapping/EPR, the intensity of the former adduct being similar to 2-fold higher than that of the latter. These signals were insensitive to dicumarol. The metabolism of DZQ in HT29 cells yi elded mainly HO. and a modest contribution of O-2(.-)(ratio HO./O-2(.- ) similar to 10), whereas that of AZQ yielded a HO<bullet>/O-2(.-) sim ilar to 2. The effect of dicumarol on the free radical pattern obtaine d during DZQ metabolism resulted in a strong inhibition (80%) of HO. p roduction and a substantial increase of O-2(.)-generation. The metabol ism of DZQ and AZQ in BE cells was associated with a significant incre ase of pal mRNA levels; the former quinone was similar to 2-fold more efficient than the latter. DZQ metabolism in HT29 cells led to an incr ease of p21 mRNA levels 15-fold higher than that observed with AZQ act ivation Dicumarol did not inhibit p21 induction associated with the me tabolism of DZQ in the NQOR-deficient BE cells, whereas the inhibitor decreased p21 induction in HT29 cells by similar to 30%. This modest i nhibition is likely due to the low concentration of dicumarol used, wh ich did not affect p21 constitutive levels in control experiments carr ied out in the absence of the quinone. p21 induction in HT29 cells was also inhibited by DTPA, a metal chelator, and N-acetylcysteine, a pot ent cellular anti-oxidant, suggesting that HO. may serve as an ultimat e mediator for the induction. It may be surmised that the higher effic iency of DZQ in p21 induction may be related to its efficient metaboli sm by NQOR in HT29 cells and the associated high level of reactive oxy gen species. The role of reactive oxygen species in p21 induction was further assessed upon supplementation of cells with H2O2: p21 inductio n in BE cells was 4-fold higher than that in HT29 cells. These finding s suggest that assessment of the role of NQOR and reactive oxygen spec ies in p21 induction requires careful consideration of the cell genoty pe. (C) 1997 Academic Press.