INDUCTION OF P21 MEDIATED BY REACTIVE OXYGEN SPECIES FORMED DURING THE METABOLISM OF AZIRIDINYLBENZOQUINONES BY HCT116 CELLS

Aziridinylbenzoquinones are a group of antitumor agents that elicit cy totoxicity by generating either alkylating intermediates or reactive o xygen species, The mechanism of toxicity may not always, however, invo lve profound damage of cellular constituents, but may involve a cytost atic effect through interference with the cell cycle, In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-b enzoquinone (DZQ) and its C-2,C-5-substituted derivatives: 2,5-bis-(ca rboethoxyamino) (AZQ) and 2,5-bis-2(-hydroxyethylamino) (BZQ). Both DZ Q and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sen sitive activity, whereas that of the latter appeared to be accomplishe d primarily by one-electron transfer reductases, BZQ was not a substra te for the dicoumarol-sensitive enzyme in HCT116 cells, Cellular activ ation of the first two quinones was associated with formation of oxyge n-centered radicals as detected by EPR in conjunction with the spin tr ap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ inv olved hydroxyl radical formation and were strongly inhibited by catala se, whereas those of AZQ showed a strong superoxide anion component se nsitive to superoxide dismutase, These signals were suppressed by N-ac etylcysteine with concomitant production of a thiyl radical adduct, Th is suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ i nduced significantly the expression of p21 in HCT116 cells, but a 10-f old higher concentration of AZQ was required to achieve the level of i nduction elicited by DZQ, BZQ had little effect on p21 expression, p21 induction at both mRNA and protein levels correlated with the inhibit ion of either cyclin-dependent kinase activity or cell proliferation, p21 induction elicited by the above quinones was inhibited by N-acetyl cysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect, Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extr acellular sources of oxygen radicals generated by plasma membrane redu ctases have no influence in the expression of this gene, Hydrogen pero xide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cell s, The latter lacks p53, one of the activators of p21 transcription, t hus suggesting that p21 expression can be accomplished in a p53-indepe ndent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction b y DZQ may be related to its efficient metabolism by NAD(P)H:quinone ox idoreductase activity in HCT116 cells.