Metabolism of the skin tumor promoter butylated hydroxy-toluene hydroperoxide (2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone; BHTOOH) to reactive intermediates is required for tumor promotion by this compound. In particular, an electrophilic quinone methide is known to mediate both in vivo tumor promotion as well as in vitro cytotoxicity by BHTOOH. In the present study, the role of this reactive intermediate in the induction of ornithine de-carboxylase (ODC), a gene strongly associated with tumor promotion, was investigated in cultured keratinocytes. BHTOOH stimulates a time-dependent increase in ODC enzyme activity, paralleled by ODC mRNA induction, suggesting transcriptional regulation of ODC by BHTOOH. Depletion of intracellular glutathione caused a 5-fold potentiation of keratinocyte sensitivity to BHTOOH. Concordantly, ODC induction by BHTOOH could be completely inhibited by soluble thiol compounds. These results suggest that ODC induction is mediated by a thiol-reactive metabolite of BHTOOH. The iron-specific chelator desferal blocked ODC induction by BHTOOH, indicating that formation of this intermediate is iron-dependent. Substitution of the 4-methyl group of BHTOOH with alkyl groups of incrementally larger size is known to reduce accordingly quinone methide production; comparative study of these BHTOOH analogs demonstrated a corresponding loss of potency for ODC induction, indicating that BHT-quinone methide mediates the in vitro induction of ODC by BHTOOH. Finally, kinase inhibitor studies suggested a role for protein kinase C in the induction of ODC by BHTOOH. Taken together, these results provide insight into the cellular mechanisms through which the reactive electrophile BHT-quinone methide can mediate alterations in gene expression, such as occur in tumor promotion in vivo.
ASJC Scopus subject areas
- Cancer Research