John F. Gierthy

The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17β-estradiol 4-hydroxylase

Rates of microsomal 17 beta-estradiol (E2) hydroxylation at the C-2, -4, -6 alpha, and -15 alpha positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 alpha and -15 alpha positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low Km 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E2 catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low Km hydroxylation of E2 at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE2) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE2 formation predominantly over 4-MeOE2. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E2 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.

Induction of cytochrome P450 1B1 and catechol estrogen metabolism in ACHN human renal adenocarcinoma cells

The catechol estrogen metabolites of 17beta-estradiol (E2), 2-hydroxyestradiol (OHE2) and 4-OHE2, differ in hormonal properties and carcinogenic potential. In Syrian hamster kidney, 4-OHE2 induces clear-cell carcinoma whereas 2-OHE2 does not, and an E2 4-hydroxylase appears to be involved in E2-induced carcinogenesis in these animals. Specific E2 4-hydroxylase activity has been observed in extrahepatic tissues from several species. In humans, cytochrome P450 1B1 (CYP1B1) appears to be an extrahepatic E2 4-hydroxylase under the regulatory control of the aromatic hydrocarbon receptor (AhR). As an initial approach to investigating CYP1B1 expression and E2 4-hydroxylase activity in human kidney, we used the ACHN cell line, derived from a human renal adenocarcinoma. In untreated ACHN cells, a very low level of CYP1B1 mRNA expression was observed and CYP1B1 protein could not be detected; however, in ACHN cells exposed to the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), CYP1B1 mRNA levels were elevated 28-fold, and the CYP1B1 protein was detected by immunoblot analysis. Exposure of ACHN cells to TCDD resulted in minimal induction of the CYP1A1 mRNA, and the CYP1A1 protein was not detectable prior to or after exposure to TCDD. E2 hydroxylase activity could not be detected with microsomes from untreated ACHN cells, although activities at C-4 and, to a lesser extent, at C-2 of E2 were observed with microsomes from TCDD-treated ACHN cells. In experiments with intact ACHN cells, elevated rates of formation of 4-methoxyestradiol (MeOE2) and 2-MeOE2 were observed in response to treatment with TCDD. The EC50 for induction of the CYP1B1 mRNA was 1.5 nM TCDD; EC50s for the stimulation of 2- and 4-MeOE2 formation were 0.68 and 1.1 nM TCDD. These results indicate that the ACHN cell line may be a useful in vitro model system to study the regulation of CYP1B1 expression and the cytotoxic effects associated with E2 4-hydroxylation.

Inhibition of postconfluent focus production in cultures of MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Summary2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent inducer of differentiation and an antiestrogen, is shown to suppressin vitro postconfluent cell accumulation in the estrogen-dependent MCF-7 human breast tumor cell line. This dose-responsive suppression is apparent by 14 days of exposure with an EC50 between 10−10 and 10−11 M TCDD, and is characterized by reduced cell density (approximately 60% of controls after 14 days). This was attributed to a reduced formation in TCDD-treated cultures of multicellular foci which are chracteristic of cancer cell growthin vitro (less than 1/mm2 compared to control levels of 40/mm2). Preconfluent cell growth and viability of MCF-7 cells is not affected by 10−9 M TCDD. These results suggest that the principle of TCDDs activity may be useful in the study and possibly the management of estrogen-dependent breast tumors.

Reversible inhibition of in vitro epithelial cell proliferation by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Subconfluent cultures of a mouse epithelial cell line, which after prolonged subculturing exhibited an elevated saturation density as compared to the original cell line, were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cultures of cells with or without TCDD grew at equal rates until confluency was reached. At confluency, cultures treated with as little as 10(-11) M TCDD showed a decline in cell proliferation relative to controls as demonstrated by cell enumeration and supported by reduced [3H]thymidine incorporation (both by liquid scintillation spectrometry of whole culture and autoradiography of individual cells). After 14 days of exposure, the saturation density of the treated culture was about 50% of the control culture. This TCDD-induced, increased sensitivity to density-dependent inhibition of replication ( DDIR ) was accompanied by a change from a fusiform morphology in the high-saturation-density control cells to a flat cobblestone appearance in the treated low-saturation-density cells. The nondividing cultures treated for 14 days with 10(-11) M TCDD had the same viability as control cultures. Upon trypsin suspension and reseeding , these formerly quiescent cultures were again capable of growing to high cell density and of again showing susceptibility to TCDD-induced changes in cell growth and morphology. Evidence is presented to suggest that this reversible increase in sensitivity to DDIR and the morphological change are not a consequence of cell growth inhibition. This system may provide the basis for an in vitro model to study the effect of TCDD on the control of replication of these cells.

A peptide derived from α-fetoprotein prevents the growth of estrogen-dependent human breast cancers sensitive and resistant to tamoxifen

An 8-mer peptide (EMTOVNOG) derived from α-fetoprotein was compared with tamoxifen for activity against growth of human breast cancer xenografts implanted in immune-deficient mice. Both peptide and tamoxifen prevented growth of estrogen-receptor-positive MCF-7 and T47D human breast cancer xenografts. A subline of MCF-7, made resistant to tamoxifen by a 6-month exposure to this drug in culture, was found to be resistant to tamoxifen in vivo. Peptide completely prevented the xenograft growth of this tamoxifen-resistant subline of MCF-7. Neither peptide nor tamoxifen was effective in slowing the xenograft growth of the estrogen-receptor-negative MDA-MB-231 human breast cancer. A worrisome side effect of tamoxifen is its hypertrophic effect on the uterus. In this study, tamoxifen was shown to stimulate the growth of the immature mouse uterus in vivo, and the peptide significantly inhibited tamoxifens uterotrophic effect. The mechanism of action of peptide is different from that of tamoxifen in that the peptide does not interfere with the binding of [3H]estradiol to the estrogen receptor. In conclusion, α-fetoprotein-derived peptide appears to be a novel agent that interferes with the growth of tamoxifen-sensitive as well as tamoxifen-resistant estrogen-receptor-positive human breast cancers; it inhibits the uterotrophic side effect of tamoxifen and, thus, it may be useful in combination with or in place of tamoxifen for treatment of estrogen-receptor-positive human breast cancers.

Effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, 12‐O‐tetradecanoylphorbol‐13‐acetate and 17β‐estradiol on estrogen receptor regulation in MCF‐7 human breast cancer cells

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF‐7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER‐ICA), and on ER function by competitive binding assays under conditions of saturating 17β‐estradiol (E2). Comparative studies were conducted with E2 and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E2 and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER‐ICA after 72 h exposure to 1 nM E2 and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [3H]E2 under saturating conditions (10 nM E2) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [3H]E2 was depressed by 49% in MCF‐7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1‐h treatment with 5 μM α‐naphthoflavone, which inhibits TCDD‐induced, P450‐mediated, E2 metabolism, and subsequent E2 depletion. In conclusion, while TPA and E2 effectively down‐regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF‐7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF‐7 cells by TCDD.

Stimulation of 17β‐estradiol metabolism in MCF‐7 cells by bromochloro‐ and chloromethyl‐substituted dibenzo‐p‐dioxins and dibenzofurans: Correlations with antiestrogenic activity

Mixed halo- and haloalkyl-substituted dibenzo-p-dioxins (DD) and dibenzofurans (DF) are known environmental contaminants, although there is limited information on the toxic effects of these compounds in human cells. In this study antiestrogenicity, a property of 2,3,7,8-Cl4-DD, was investigated with a series of bromochloro- and chloromethyl-substituted DDs and DFs. The effects of these compounds on the metabolism of 17 beta-estradiol (E2) and on the estrogen-dependent formation of multicellular foci in cultures of MCF-7 human breast cells were examined. Pretreatment of MCF-7 cells with 2,3,7,8-Cl4-DD induced pathways of E2 metabolism involving cytochrome P-450-catalyzed hydroxylation, methylation of the catechol estrogens, and conjugation. Several Br-Cl3-DD and Br2-Cl2-DD congeners with halogen substitution at the 2, 3, 7, and 8 positions also stimulated E2 metabolism with similar potency to that of 2,3,7,8-Cl4-DD; however, compounds with substitution of a methyl group for a halogen at any of these positions did not stimulate the metabolism of E2. For the series of compounds tested in MCF-7 cultures, a close correlation was observed between the antiestrogenicity as measured by the inhibition of estrogen-dependent postconfluent growth that results in focus formation and the efficacy with which the compounds stimulated the metabolism of E2. 2,3,7,8-TetrahaloDDs with one or two bromine atoms at these positions were highly antiestrogenic as determined by their inhibition of estrogen-dependent focus formation, whereas the methyl-substituted polychlorinated DDs and DFs investigated did not inhibit focus formation. These results indicate that the 2,3,7,8-substituted mixed halo-substituted DDs and DFs are of importance when the biologic effects of halogenated DD and DF congeners are considered, and provide additional evidence for the role of increased metabolism of E2 in the antiestrogenic effects of halogenated DDs and DFs.

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells.

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4‐hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β‐estradiol (E2) 2‐ and 4‐hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), which downregulates ERα, and the high‐affinity AhR ligand, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF‐7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF‐7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4‐ to 2‐hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998.

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture

A coculture system was developed to investigate the interactions between MCF‐10A breast epithelial cells and MCF‐7 breast cancer cells stably expressing the green fluorescent protein (MCF‐7‐GFP). Studies with this MCF‐10A/MCF‐7‐GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF‐7‐GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF‐7‐GFP and markedly increased MCF‐10A cell proliferation, greatly inhibited MCF‐7‐GFP cell proliferation in MCF‐10A/MCF‐7‐GFP cocultures. 17β‐Estradiol, which stimulated MCF‐7‐GFP but not MCF‐10A cell proliferation in monoculture, inhibited MCF‐7‐GFP cell proliferation in MCF‐10A/MCF‐7‐GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF‐10A/MCF‐7‐GFP cellular interactions were observed in real time that resulted in the formation of acinus‐like structures. These results indicate a role of normal epithelial cells in inhibiting tumor‐cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

In vitro bioassay for dioxinlike activity based on alterations in epithelial cell proliferation and morphology.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to induce changes in morphology and proliferation characteristics of a nonkeratinizing derivative (XBF) of a keratinizing epithelial cell line (XB), cloned from a mouse teratoma, when cocultured with irradiated feeder cells. Polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs), and pesticides (24 compounds in total) were tested for their ability to induce these effects. The results indicated that, for the representative compounds tested, these changes are relatively specific for-and that the XBF cells are extremely sensitive to--PCDDs and PCDFs. TCDD was the most potent congener tested, capable of inducing the effects at a concentration as low as 10(-11) M. The activities of other tested PCDDs and PCDFs ranged from 10(-1) to 10(-3) of TCDD activity. The PCBs, PAHs, and pesticides had lower activities ranging from 10(-3) to 10(-6) that of TCDD. This assay system using XBF cells cocultured with irradiated 3T3 fibroblast feeder cells was examined as a possible in vitro screening assay for dioxinlike activity by testing benzene extracts of soot from a fire involving a PCB-containing transformer. The results were compared to a high-resolution gas chromatographic/mass spectrometric analysis for total PCDFs in the same samples. This comparison showed a good correlation, suggesting that the XBF-3T3 system has potential for use as a semiquantitative assay for dioxinlike activity.