RESUMO
Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues.
Assuntos
Asparagina/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Polimorfismo Genético/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/genética , Serina/genética , Alelos , Substituição de Aminoácidos/genética , Animais , Hidrocarboneto de Aril Hidroxilases , Células COS , Chlorocebus aethiops , Citocromo P-450 CYP1B1 , Sistema Enzimático do Citocromo P-450/biossíntese , Estabilidade Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
Tobacco-associated carcinogens are catalyzed by microsomal epoxide hydrolase (mEH). Combinations of the Y113H and H139R polymorphic EPHX1 variants have been assumed to alter the enzyme activity and thus the risk of squamous cell head and neck cancer (SCCHN). Based on in vitro data, a putative low, medium and high mEH activity has been associated with combinations of these genotypes, and the respective activity categories have been frequently used in the estimation of risks for smoking-related cancers. We investigated the SCCHN risk for EPHX1 genotypes among 280 cases and 289 controls. We could not detect main effects of the EPHX1 genotypes, but a smaller risk of the 139HR genotype in smokers (odds ratio, OR, 0.57; 95% confidence interval, CI, 0.34-0.95). We could not confirm an increase of the SCCHN risk for genotype combinations according to a putative medium and high enzyme activity (OR 1.28, 95% CI 0.84-1.96; OR 0.98, 95% CI 0.58-1.64, respectively), but a significant heterogeneity of the estimated risks for the singular genotypes within these categories among smokers ( P=0.02). Further, p53 mutations among smoking cases were less frequent in the group with a putative high enzyme activity, although insignificant due to small numbers (OR 0.54, 95% CI 0.13-2.17). This supports uncertainties in categorizing genotypes with respect to limited enzyme activity data, especially when taken from in vitro experiments.