Co-expression of human CYP1A1 and a human analog of cytochrome P450-EF in response to 2,3,7,8-tetrachloro-dibenzo-p-dioxin in the human mammary carcinoma-derived MCF-7 cells.
Cultured human mammary carcinoma (MCF-7) cells exhibited constitutive cytochrome P450-dependent metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) (45-75 pmol/mg microsomal protein). Exposure of the cells to 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), which is known to induce CYP1A1, not only resulted in a 30-fold increase in the total microsomal metabolism of DMBA but produced substantial differences in the distribution of DMBA metabolites formed. This suggested that different cytochrome P450 (P450) forms predominated in untreated and induced cells. Comparative studies with TCDD-induced human hepatoblastoma (HepG2) and skin cell carcinoma (SCC-13) cells and also recombinantly expressed human CYP1A1, confirmed that the DMBA-metabolite profile in TCDD-induced MCF-7 cells was that of human CYP1A1. This distribution, however, differed substantially from the regioselectivity of rat CYP1A1 and mouse Cyp1a-1. Rabbit antibodies to rat CYP1A1 completely inhibited the DMBA-metabolizing activity of TCDD-induced MCF-7 cells but had no inhibitory effect on constitutive DMBA metabolism which was, however, completely inhibited by chicken antibodies to the novel P450 in mouse embryo fibroblasts (P450-EF). Anti-P450-EF inhibited only 10% of the DMBA-metabolizing activity in the TCDD-induced MCF-7 cell microsomes. Microsomes from untreated MCF-7 cells expressed a 52 kDa protein that was immunodetectable by rabbit anti-P450-EF and failed to express immunodetectable levels of human CYP1A1. DMBA metabolism, therefore, switches from P450-EF in uninduced microsomes to CYP1A1 in TCDD-induced microsomes. The mobility of the P450-EF-like protein in MCF-7 cells was higher than that of P450-EF from C3H/10T1/2CL8 (10T1/2) cells (55 kDa). The 52 kDa protein from MCF-7 cells was induced approximately 8-fold by TCDD while CYP1A1 immunodetectable protein was increased to much higher levels. The SCC-13 cell line exhibited a similar pattern of expression of a 52 kDa P450-EF-like protein and CYP1A1. HepG2 cells expressed the highest levels of CYP1A1 in response to TCDD without expression of the 52 kDa protein.
Christou
M
M
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Savas
U
U
Spink
D C
DC
Gierthy
J F
JF
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D001943:Breast Neoplasms / Q000201:enzymology*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D012867:Skin / Q000201:enzymology; D014407:Tumor Cells, Cultured; D010130:p-Aminohippuric Acid / Q000378:metabolism
D007527:Isoenzymes; D000072317:Polychlorinated Dibenzodioxins; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; D010130:p-Aminohippuric Acid