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 cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic 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-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, 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 extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent 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 by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.
Qiu
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Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Forman
H J
HJ
Schönthal
A H
AH
Cadenas
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D000111:Acetylcysteine / Q000494:pharmacology; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D001388:Aziridines / Q000737:chemistry* / Q000378:metabolism; D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D001728:Dicumarol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D001388:Aziridines; D016227:Benzoquinones; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D005609:Free Radicals; C010530:N-acetylalanine; D009249:NADP; D001728:Dicumarol; D006861:Hydrogen Peroxide; D000409:Alanine; D010100:Oxygen; D000111:Acetylcysteine