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.