Highly specific cytochrome P450-like enzymes for all-trans-retinoic acid in T47D human breast cancer cells.
When human breast cancer T47D cells were treated with all-trans-retinoic acid (RA), the RA 4- and 18-hydroxylase activities were induced in microsomes in a time-dependent manner, indicating that these cells readily metabolized RA into more polar compounds, such as all-trans-4-hydroxy-RA and all-trans-18-hydroxy-RA. In contrast, T47D cells treated for 12 h with xenobiotics, such as phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, and dimethylsulfoxide, showed lower levels of catalytic activities for 4- and 18-hydroxylases. The induction of 4- and 18-hydroxylase activities appears to be regulated at the level of transcriptional control (basal level). Competitive assays demonstrated that inhibitors and substrates for 1A, 2A, 3A, 2B, and 2C cytochrome P450 (P450 subfamilies), all-trans-retinol, and all-trans-retinal showed no inhibition of RA metabolism, but other retinoic acid derivatives competed highly with RA. The RA-inducible 4- and 18-hydroxylases showed high specificity for RA and high levels of catalytic activities, with Km and maximum velocity values for 4-hydroxylase equal to 99 nmol/L and 0.26 pmol/min.mg protein, respectively, and those for 18-hydroxylase equal to 65 nmol/L and 0.18 pmol/min.mg protein. Cell-free metabolism of RA required microsomes from RA-treated cells and NADPH, and was inhibited by liarozole, an inhibitor of P450. These data suggest that RA-inducible 4- and 18-hydroxylases may be novel P450 isozymes.
Han
I S
IS
College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Choi
J H
JH
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D012176:Retinoids / Q000378:metabolism; D013379:Substrate Specificity; D014212:Tretinoin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D012176:Retinoids; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases