Expression of CYP1A1 and CYP1B1 depends on cell-specific factors in human breast cancer cell lines: role of estrogen receptor status

William G.R. Angus1, Michele C. Larsen2 and Colin R. Jefcoate1,2,3

1 Department of Pharmacology and
2 Environmental Toxicology Center, University of Wisconsin, 3770 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The impact of estrogen receptor (ER) was examined for expression and activity of cytochrome P4501B1 (CYP1B1) and cytochrome P4501A1 (CYP1A1) in two pairs of ER+/ER human breast epithelial cell lines derived from single lineages, and representing earlier (T47D) or later (MDA-MB-231) stages of tumorigenesis. Acute loss of ER was evaluated using the anti-estrogen ICI 182,780 (ICI). In all lines, CYP1B1 was expressed constitutively and was induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), whereas CYP1A1 was expressed only following induction. Expression of each CYP (with or without TCDD) was greater in T47D cells than MDA cells. The ER impacted expression of these genes in opposite directions. The ER phenotype was associated with less TCDD-induced CYP1A1 expression, but greater basal and induced CYP1B1 expression. A 48 h treatment of ER+ cells with ICI did not revert the P450 expression pattern to that of ER cells. Based on activities of recombinant enzyme and expression levels, differences in 7,2-dimethylbenz [a]anthracene (DMBA) metabolism between the cell lines were consistent with differences in CYP1A1 and CYP1B1 expression. In T47D lines, basal microsomal DMBA metabolism was primarily due to CYP1B1, based on regioselective metabolite distribution and inhibition by anti-CYP1B1 antibodies (>80%). Metabolism in TCDD-induced microsomes was mostly due to CYP1A1 and was inhibited by anti-CYP1A1 antibody (>50%). TCDD-induced MDA+ cells demonstrated CYP1A1 activity, whereas TCDD-induced MDA cells displayed CYP1B1 activity. Aryl hydrocarbon receptor (AhR) levels, but not AhR nuclear translocator protein (ARNT) levels were highly dependent on cell type; AhR was high and ER-independent in MDA, and low and ER-linked in T47D. AhR levels were insensitive to ICI. ER does not directly modulate the expression of CYP1A1, CYP1B1 or AhR. Indeed, factors that have replaced ER in growth regulation during clonal selection predominate in this regulation. Characteristics unique to each cell line, including ER status, determine CYP1A1 and CYP1B1 expression.

Abbreviations: AHH, aryl hydrocarbon hydroxylase; AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator protein; CYP1A1, cytochrome P4501A1; CYP1B1, cytochrome P4501B1; DMBA, 7,12-dimethylbenz[a]anthracene; ECL, enhanced chemiluminescence; ER, estrogen receptor; FBS, fetal bovine serum; HMEC, human mammary epithelial cells; ICI, ICI 182,780; MDA+, S30 cells; MDA, MDA-MB-231 cells; PAH, polycyclic aromatic hydrocarbon; T47D+, T47D:A18 cells; T47D, T47D:C4:2W cells; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cytochrome P4501B1 (CYP1B1) is found in steroid-responsive mesodermal tissues, such as the breast, uterus and prostate (1,2). Like cytochrome P4501A1 (CYP1A1), CYP1B1 can be induced by polycyclic aromatic hydrocarbons (PAHs) through a mechanism involving the aryl hydrocarbon receptor (AhR) (3,4). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the archetypal agonist for AhR-mediated gene induction, induces the expression of CYP1B1 and CYP1A1 in human breast epithelial MCF7 cells, although CYP1B1 alone is expressed constitutively (3). CYP1B1 is also present basally in normal human breast epithelial cells, while CYP1A1 is below the lower limit of detectability or present at very low levels (<0.03 to 0.16 pmol/mg microsomal protein; 5).

The functional involvement of CYP1B1 in PAH metabolism has been demonstrated in MCF7 cells (3). Metabolism of the potent rodent mammary carcinogen 7,12-dimethylbenz[a] anthracene (DMBA; 6–9) by microsomes from uninduced MCF7 cells resulted in a different metabolite distribution from that observed for CYP1A1, which is further inhibited by anti-CYP1B1 antibodies, but not anti-CYP1A1 antibodies (3). By contrast, microsomes from TCDD-induced MCF7 cells display a CYP1A1 pattern of DMBA metabolites, which is completely inhibited by anti-CYP1A1 antibodies. DMBA, like other PAHs, requires metabolic activation through P450 cytochromes to a dihydrodiolepoxide to initiate carcinogenesis (1012). Hence, activation of PAHs by CYP1A1 and CYP1B1 could be an important pathway for breast cancer initiation.

A natural substrate for CYP1B1 could mediate important physiological functions, since (i) the expression of CYP1B1 is under hormonal control in many steroidogenic tissues, (ii) CYP1B1 displays selective expression in the mouse embryo cells (13), and (iii) there is a reported linkage between CYP1B1 mutations and congenital glaucoma in humans (14). Importantly, human CYP1B1 has been linked to the conversion of 17ß-estradiol to 4-hydroxyestradiol (15,16), in addition to the bioactivation of many PAHs. Estradiol 4-hydroxylation activity is elevated in breast and uterine tumors, relative to surrounding tissues. This suggests that there may be more functional CYP1B1 in these tumors and that there may be a role for CYP1B1 in tumorigenesis, in addition to mutagen activation (17).

Some commonly used human mammary cell lines, including MCF7 and T47D cells express the estrogen receptor ER+ (estrogen receptor {alpha}; 18,19), while others, such as MDA-MB-231 (MDA), lack the receptor completely (ER) (20). The ER has been implicated in the induced expression and activity of CYP1A1 through a deficiency in aryl hydrocarbon hydroxylase (AHH) activity (a measure of CYP1A1 activity) in ER-negative MDA cells, which was restored by transient transfection of ER into these cells (21). Another report has demonstrated a lack of TCDD-inducible AHH activity in several additional ER breast tumor cell lines, regardless of their capacity to bind TCDD (22). It was concluded that ER status plays an important role in the TCDD-induction of CYP1A1 in human breast epithelial cells. In contrast to these findings, however, we have recently observed TCDD-induction of CYP1A1 and CYP1B1 mRNA in primary human breast epithelial cells, which are ER (5), suggesting that the ER may not be necessary for TCDD-induction of CYPs in human breast cells. We therefore addressed whether ER status differentially influences CYP1B1 expression relative to CYP1A1 expression in human breast cancer cell lines.

Cell lines lacking ER differ from ER+ cells in more respects than merely the lack of this receptor. In an effort to minimize differences in cell lineage with respect to ER status, we have paired cell lines which derive from the same background, but differ in ER status. An ER clone T47D:C4:2W (T47D), has previously been derived from normal ER+ T47D cells by long-term culture in estrogen-free medium (23). MDA cells, which normally lack ER, have been paired with the S30 variant (MDA+), which resulted from stable transfection of the ER into these cells (24). T47D cell lines depict a classic epithelial cell morphology, while the MDA cells are morphologically fibroblast-like and representative of a more advanced malignant and metastatic nature. These cell lineages, therefore, introduce differences in many additional regulatory processes, such as growth factor and plasma membrane receptor expression. For example, recent studies suggest that unusually low levels of Ah receptor nuclear translocator protein (ARNT) in some cell lines may also contribute to low CYP1A1 induction (25). Hence, we have examined whether AhR and ARNT levels differ in these cell lines, and whether ER status affects their expression.

In an additional test of the direct role of the ER in CYP1B1 and CYP1A1 expression, the effect of a rapid loss of ER activity was examined through treatment of the ER+ lines with the `pure' anti-estrogen ICI 182,780 (ICI). ICI suppresses ER activity by inhibiting ER dimer formation and prevents shuttling of the ER into the nucleus. These actions, respectively, eliminate interaction with the DNA and decrease levels of the protein (26,27). This rapid change in ER status differs fundamentally from the generation of separate clones, which have adapted slowly to growth in altered estrogen signaling environments. These combinations of cells and treatments were used to evaluate whether ER status or other aspects of the cell phenotype are more influential in determining CYP expression.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MDA-MB-231 cells (20) were purchased from ATCC (Rockville, MD). The cell lines T47D:A18, T47D:C4:2W and S30 were generous gifts from Drs V.Craig Jordan and John Pink (Northwestern University, University of Wisconsin Comprehensive Cancer Center, Madison, WI; 23,24). ICI was obtained from Dr Alan Wakling (Zeneca Pharmaceuticals, Manchester, UK). The human CYP1B1 antibody used in these studies was a generous gift from Dr Craig Marcus (University of New Mexico) and Dr William Greenlee (University of Massachusetts); the Ah receptor monoclonal antibody was obtained from Dr Gary Perdew (Pennsylvania State University, Hershey, PA). Polyclonal antibody to ARNT was a gift from Dr Rick Pollenz. Antibody to ER was purchased from Santa Cruz Biologicals (HC-20, Santa Cruz, CA). The antibodies for CYP1A1, and inhibitory antibodies for CYP1A1 and CYP1B1 were generated in this laboratory (3). TCDD was purchased from Accustandard (New Haven, CT). [{alpha}-32P]dCTP was purchased from NEN DuPont (Boston, MA). Reagents purchased from GIBCO (Grand Island, NY) included Dulbecco's Modified Eagle Medium/Ham's F12 (DMEM/F12), RPMI 1640 and agarose. Cell culture was carried out using Falcon flasks and Corning plates. Fetal bovine serum (FBS) was obtained from Gemini (Calabasas, CA) or HyClone (Logan, UT), and dextran charcoal stripped FBS was purchased from HyClone (Logan, UT). Reagents purchased from the Sigma Chemical Co. (St Louis, MO) included glucose-6-phosphate, DMBA, nicotinamide dinucleotide phosphate (NADP), HEPES, insulin, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (TEMED), dimethylsulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, soybean trypsin inhibitor, aurin tricarboxylic acid (ATA), salmon sperm DNA, diethyl pyrocarbonate (DEPC), penicillin/streptomycin solution and phenol red-free DMEM/F12. Sodium dodecyl sulfate (SDS) was purchased from BioRad, Inc. (Hercules, CA). Proteinase K was obtained from Fisher Chemical (LaJolla, CA). Formamide was purchased from Ambion (Austin, TX). Oligo dT was obtained from Calbiochem (LaJolla, CA). The PRIME IT II kit was purchased from Stratagene (LaJolla, CA). Nylon (Hybond N+) and nitrocellulose membranes (Hybond ECL), Hyperfilm ECL film and enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Arlington Heights, IL). BCA protein determination kit was purchased from Pierce (Rockford, IL). Recombinant human CYP1A1 and CYP1B1 protein were obtained from Gentest (Worburg, MA). Other reagents used for these studies were of the highest grade possible.

Cell culture
All cells were maintained in monolayer culture in 75 or 175 cm2 flasks in a humidified 5% CO2 atmosphere. MDA cells were grown in DMEM/F12 with 10 mM HEPES, 5% FBS and penicillin/streptomycin. The MDA+ cells, which are growth inhibited by estrogens (24), were grown in phenol red-free DMEM/F12 containing 15 mM HEPES and 5% dextran charcoal stripped FBS. T47D+ and T47D cells were cultured in phenol red-free RPMI 1640 with 10% FBS, 6 ng/ml insulin and penicillin/streptomycin. All cells were passaged using 0.5% trypsin at 80–90% confluence.

Hybridization analysis of mRNA
Cells cultured in 150 mm plates were treated or kept as controls for 24 h with 10–7 M ICI, then for an additional 24 h with or without 10–8 M TCDD in the continued presence or absence of ICI. Controls were treated for 24 h with DMSO (0.1%). Cells were harvested and poly A+ RNA isolated according to the technique of Badley et al. (28). Briefly, cells were washed with PBS (0.01 M phosphate buffer in 2.7 mM KCl and 137 mM NaCl, pH 7.4) containing 20 µM ATA and lysed in a buffer consisting of 0.2 M NaCl, 0.2 M Tris-HCl, pH 7.5, 0.15 mM MgCl2 2% SDS, 200 µg/ml proteinase K and 20 µM ATA. Lysates were sheared with a 23-gauge needle and incubated for 2 h at 45°C, then agitated with oligo dT, eluted in DEPC-treated water and precipitated with ethanol. The RNA was resuspended in sterile DEPC-treated H2O and quantitated by reading absorbance at 260 nm.

Poly A+ RNA was electrophoresed through a formaldehyde-containing 1% agarose gel. Capillary action was used to transfer the RNA to nylon membrane, and RNA was cross-linked to the membrane by UV. Membranes were prehybridized in a buffer of 6x SSC, 5x Denhardt's reagent, 0.1% SDS, 10 µg/ml salmon sperm DNA and 50% formamide at 42°C for at least 2 h. Membranes were then incubated with probes for ß-actin, human CYP1A1 and human CYP1B1 as described below. Probes were randomly labeled using [{alpha}-32P]dCTP (50 µCi) following the manufacturer's instructions for the Prime-It II kit. Specific activity was >=106 c.p.m./ml of probe. Non-specific hybridization was removed by sequential washing with 2x SSPE + 0.5% SDS, 1x SSPE + 0.5% SDS, 0.5x SSPE + 0.5% SDS and 0.25x SSPE + 0.25% SDS at hybridization temperature for 15 min. Signals were visualized by autoradiography and quantitated by densitometry on a laser scanner.

A 1.4 kb cDNA probe from the 3' end of the human CYP1A1 was obtained from Dr Lynne Allen-Hoffman (University of Wisconsin, Madison, WI). The CYP1B1 cDNA probe was from a 3.1 kb fragment encoding the open reading frame of human CYP1B1 and was obtained from Dr William Greenlee (University of Massachusetts).

Microsomal metabolism of DMBA
Microsomes were generated by differential centrifugation as previously described (29) from T47D+, T47D, MDA and MDA+ cells treated with or without 10–7 M ICI for 24 h, then with or without TCDD for an additional 24 h in the presence/absence of ICI. Microsomal metabolism was performed using 0.2 or 1 mg of TCDD-induced and basal microsomal protein, respectively, as described by Christou et al. (3). Samples were incubated for 15 min at 37°C and 1.5 µM DMBA was used in a 1 ml reaction volume. Inhibition studies were carried out using chicken anti-CYP1B1 (IgY) at 5 mg/mg microsomal protein or rabbit anti-CYP1A1 (IgG) antibodies (3) at 10 mg/mg microsomal protein. The reaction mixtures containing the antibodies and pre-immune serum were incubated at room temperature for 40 min prior to the 15 min incubations with DMBA at 37°C.

Cell lysates and western immunoblots
Confluent cells in 60 mm plates, treated as previously described for ICI and TCDD, were harvested by scraping in PBS and pelleted. A lysis buffer consisting of 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM sodium orthovanadate, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml soy bean trypsin inhibitor and 1 mM PMSF was added to each pellet. Following 10 min on ice, samples were sheared using a pipette tip and centrifuged at 10 000 g for 30 min at 4°C. Supernatants were transferred to new tubes, an aliquot removed for protein determination and frozen at –80°C.

Cellular proteins were separated on SDS-PAGE containing 7.5% acrylamide as per Laemmli (30) and transferred to nitrocellulose membrane. Membranes were probed for CYP1A1, CYP1B1, AhR, ER and ARNT. Reactive proteins were visualized using the ECL procedure according to manufacturer's instructions.

Protein levels in cellular extracts and microsomal preparations were determined using the BCA method as per manufacturer's instructions using bovine serum albumin as a standard.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
These studies were undertaken to examine the direct involvement of the ER in the expression and activity of CYP1A1 and CYP1B1 in human mammary epithelial cells. The expression of CYP1A1 and CYP1B1 mRNA and protein, and the associated metabolism of DMBA has been examined in two pairs of human mammary cell lines, each pair providing a comparison between ER+ and ER status (T47D+, T47D, MDA+, MDA). Additionally, the anti-estrogen ICI was used to determine if CYP expression patterns in ER+ cells could be shifted to those observed in ER cells. Cells were examined under basal conditions and following 24 h treatment with 10–8 M TCDD.

We have measured expression in confluent cells with and without a 24 h exposure to TCDD (10–8 M). Quantitation was made from three separate culture experiments for each line. The levels of CYP mRNA and protein were measured by, respectively, northern hybridization (Figure 1Go) and western immunoblots (Figure 2Go), each with fully selective probes. We found that the changes in ER status in both types of breast cell resulted in systematic effects on CYP1B1 and CYP1A1 expression, but in opposite directions. Similar results were obtained for both mRNA and protein. The basal expression of CYP1B1 mRNA, which was very apparent in each line, was four or five times lower in the ER+ line than the ER counterpart (T47D ER+/ER = 0.28 ± 0.07; MDA ER+/ER = 0.2 ± 0.1). Similar differences were seen in basal CYP1B1 protein expression, although the levels in MDA cells were much lower and difficult to quantitate (Figure 2Go). Following a 24 h TCDD treatment, CYP1B1 expression increased substantially in each cell line, but by a larger factor in ER+ cells. In T47D cells, the larger stimulus in ER+ cells as measured by either protein or mRNA (mRNA ER+21 ± 5 versus ER 4 ± 1.5; protein ER+ 23 ± 15 versus ER 3 ± 0.2) completely removed the sensitivity to ER status for both mRNA and protein. In MDA cells, the deviation in the induction factors in ER+ cells was less marked (mRNA 10 ± 4 versus 6 ± 2) leaving levels of CYP1B1 mRNA and protein about 2-fold higher in ER cells. CYP1A1 was only observable after induction by TCDD, but here the opposite effect of ER status was seen. TCDD-induced levels of both CYP1A1 mRNA and protein were each two- to three-fold greater in ER+ cells. It can also be seen (Figures 1 and 2GoGo) that by either measurement expression of both of these CYPs is much greater for the equivalent ER status in T47D than in MDA cells (three-fold for mRNA, four-fold for protein). This indicates a strong influence of lineage-dependent, ER-independent factors that act similarly on transcription of either basal or induced CYP1B1, and induced CYP1A1. The close parallels between mRNA and protein indicate that each of these regulatory differences largely arises at the level of transcription.




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Fig. 1. Expression of CYP1A1 and CYP1B1 mRNA in T47D and MDA cells treated with or without TCDD. (A) Five micrograms mRNA from T47D or MDA cells treated with or without 10–8 M TCDD for 24 h was northern blotted, and probed for CYP1A1 or CYP1B1 as described in Materials and methods. Image is representative of three separate measurements. (B) Bar graphs represent relative levels of CYP1A1 or CYP1B1 message found in 5 µg mRNA as determined by densitometry (mean ± SEM). In the top graph, bars represent only TCDD-induced CYP1A1 mRNA levels; in the bottom graph, solid bars represent basal CYP1B1 mRNA and hatched bars depict TCDD-induced CYP1B1 mRNA levels. Levels of mRNA were normalized to TCDD-induced T47D+ cells for CYP1A1 and to basal T47D+ cells for CYP1B1, which were each assigned a value of 1. Inset: basal levels of CYP1B1 mRNA shown in enlarged scale. Ordinate measured in arbitrary units. Actin standardization was consistent (<10% variation) within a cell type, however, MDA cells contained only 70% of T47D actin levels (data not shown).

 



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Fig. 2. Relative CYP1A1 and CYP1B1 protein levels in T47D and MDA cells treated with or without TCDD. (A) Protein (30 µg) from solubilized T47D or MDA cells treated with or without 10–8 M TCDD for 24 h was immunoblotted for CYP1A1 or CYP1B1 as described in Materials and methods. Image is representative of at least two separate experiments. (B) Bar graphs represent relative levels of CYP1A1 or CYP1B1 protein found in 30 µg total cellular protein as determined by densitometry (mean ± SEM). In the top graph, bars represent only TCDD-induced CYP1A1 protein levels; in the bottom graph, solid bars represent basal CYP1B1 protein and hatched bars depict TCDD-induced CYP1B1 protein levels. Protein levels were normalized to TCDD-induced T47D+ cell levels (CYP1A1) or basal T47D+ cell levels (CYP1B1), each assigned a value of 1. Inset: basal levels of CYP1B1 protein shown in enlarged scale. Ordinate measured in arbitrary units.

 
Functional activity
The functional activity of CYP1A1 and CYP1B1 from cellular microsomal protein has been calculated relative to recombinant CYP1A1 and CYP1B1 protein standards (M.L.Larsen, in preparation) as shown in Table IGo. Dihydrodiol ratios (5,6/8,9 or 10,11/8,9) was used as reference parameters to determine if the metabolism pattern was CYP1A1-like or CYP1B1-like. Based on metabolism by the recombinant human cytochromes, a 5,6/8,9 ratio of 0.6 – 0.7 indicated a pure CYP1B1 activity, while a ratio of 0.1–0.2 was typical for CYP1A1. A higher 10,11/8,9 ratio also typified CYP1B1, rather than CYP1A1 activity (1.0 versus 0.1). The DMBA metabolizing activity of CYP1A1 was demonstrated to be about six times that for an equivalent amount of CYP1B1 protein (5).


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Table I. Generation of DMBA metabolites from microsomal proteins
 
Consistent with the measurement of cytochrome P450 protein expression, the T47D lines had a much greater DMBA-metabolizing activity than the equivalent MDA cells (6–13-fold). Basal T47D cells, irrespective of ER status and TCDD-induced MDA cells provided indistinguishable ratios typical of CYP1B1 metabolism (5,6/8,9 ratio = 0.5; 10,11/8,9 ratio = 0.2). Basal MDA+ products were essentially background, being similar in pattern of distribution and picomolar amount per milligram of protein to what is observed in cell-free blanks (M.L.Larsen, unpublished results). Meanwhile, the elevated activity observed in MDA cells was consistent with CYP1B1 participation. Under basal conditions, the T47D cells had 50% greater activity than T47D+ cells or TCDD-induced MDA cells, which, in turn, were four- to eight-fold more active than basal MDA cells. These findings were consistent with levels of protein expression.

TCDD treatment shifted the metabolite pattern in T47D cells and MDA+ cells, in contrast to MDA cells, to a CYP1A1 distribution (5,6/8,9 = 0.25; 10,11/8,9 = 0.1). The TCDD-induced metabolic activity of the T47D+ cells was equivalent to that of T47D cells, while TCDD-induced MDA+ cells exhibited 10 times less activity. The differences in TCDD-induced activity between T47D cells and MDA cells parallels the relative expression of the much more active CYP1A1.

Inhibition studies
Inhibitory antibodies were used to determine the contribution of CYP1A1 and CYP1B1 to the DMBA-metabolism profile in T47D+ cells (Table IIGo). Under basal conditions, the anti-CYP1B1 antibody inhibited overall metabolism by 84%, while the anti-CYP1A1 antibody had no effect. In contrast, the anti-CYP1A1 antibody inhibited greater than 50% of the TCDD-induced DMBA metabolism (Table IIGo). The distribution of metabolites removed from the reaction by the anti-CYP1B1 treatment was evaluated by subtraction of anti-CYP1B1 metabolite values from pre-immune metabolite values. This indicated that ratios of dihydrodiols were very similar to those found for recombinant CYP1B1 (5,6/8,9 = 0.9; 10,11/8,9 = 0.4). A similar subtraction was used to demonstrate the activity of CYP1A1 (5,6/8,9 = 0.15; 10,11/8,9 = 0.07). The residual activity from these TCDD-induced microsomes after removal of the CYP1A1 contribution with this antibody treatment was consistent with induced CYP1B1 activity (5,6/8,9 = 0.4; 10,11/8,9 = 0.14). The remaining metabolites that are unaffected by either antibody indicate a residual basal metabolic activity that may be due to other P450s present in breast epithelial cells (31) or to a peroxidative mechanism. This activity seems to contribute to the very low basal activity in MDA+ cells.


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Table II. Antibody inhibition of DMBA metabolism
 
Specific enzyme content and specific activity
To be able to compare the relative rates of DMBA-metabolizing activity in cell lines with primary cells (5), a turnover value (P450/h) was calculated for CYP1A1 and CYP1B1 in each line, based on the specific enzyme content, the total DMBA metabolizing activity, and the fractions of total activity inhibited by specific antibodies under both basal and induced conditions (Table IIIGo). The products of distribution of residual activity after 50% inhibition of TCDD-induced microsomes with anti-CYP1A1 antibodies indicates approximately equal contributions of residual CYP1A1 and CYP1B1 activity. This indicates a CYP1A1 turnover of 24 pmol/mg/h (363 pmol/mg/h; 15 pmol CYP1A1) and a CYP1B1 activity of 3 pmol/mg/h (121 pmol/mg/h; 43 pmol CYP1B1). For uninduced microsomes, the CYP1B1 turnover was higher at about 18 pmol/mg/h (35 pmol/mg/h; 2 pmol CYP1B1). CYP1B1 activities for uninduced microsomes for other cells were somewhat lower (4–6 pmol/mg/h). Taken together, these CYP1B1 activities are close to the basal activity reported for primary breast epithelial cells (6.0 pmol/mg/h) (5). TCDD-induced T47D contained about as much CYP1B1 as CYP1A1. Thus, the product distribution was indistinguishable from equivalent T47D+ microsomes. Based on the P450 content, this same product distribution is obtained with specific activities of about 40 pmol/mg/h for CYP1A1 and 4 pmol/mg/h for CYP1B1. Taken together, this indicates turnover number for CYP1A1 that are 8–10 times larger than for CYP1B1, but reasonably consistent with those in primary human breast epithelial cells. Specific activities following TCDD-induction in MDA+ and MDA cells were 2–4 times lower than in T47D cells.


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Table III. Specific content and turnover of CYP1A1 and CYP1B1 in T47D and MDA cells
 
Effects of anti-estrogen and expression of ER
Levels of ER protein in the four lines were verified by immunoblotting (Figure 3Go). As expected, MDA and T47D cells expressed no ER protein. Treatment with ICI for 48 h decreased the amount of ER protein in both the T47D+ and MDA+ cells by 70%. This treatment of the ER+ cell lines with 10–7 M ICI had little effect on CYP1A1 message or protein. Treatment with ICI also had minimal effects on CYP1B1 mRNA and protein. Thus, this 48 h treatment with ICI failed to reproduce the differences in CYP expression observed between ER+ and ER lines (data not shown).



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Fig. 3. Estrogen receptor content in T47D and MDA cell clones. ER+ cells were treated with or without 10–7 M ICI for 24 h , followed by treatment with or without 10–8 M TCDD for an additional 24 h in the continued presence/absence of ICI. ER cells were treated for TCDD only. A 30 µg protein sample from whole-cell solubilizations was immunoblotted for ER as described in Materials and methods.

 
AhR and ARNT expression
AhR, which mediates the induction of CYP1A1 and CYP1B1 by TCDD, was present in all four lines (Figure 4Go). The AhR levels in the MDA lines was about seven-fold greater than in the T47D lines. The ER+ clones of both lineages demonstrated approximately 3-fold greater levels of AhR than the ER clones. The 24 h TCDD treatment decreased the amount of AhR in each cell line, which was consistent with previous observations in mouse hepatoma cell lines that TCDD induces a translocation of the receptor to the nucleus followed by down-regulation (32).



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Fig. 4. Ah receptor and ARNT content in T47D and MDA cell clones. Cells were treated with or without 10–8 M TCDD for 24 h and 30 µg protein from whole-cell solubilizations was immunoblotted for AhR or ARNT as described in Materials and methods.

 
Levels of ARNT protein, which forms a heterodimer with AhR, and is necessary for binding to xenobiotic response elements, were greater in T47D than MDA cell lines. No apparent effect of the ER on ARNT protein expression was observed. Furthermore, TCDD treatment had no effect on levels of ARNT protein in any of these cell lines (Figure 4Go).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of CYP1A1 and CYP1B1 has been characterized in two pairs of ER+ and ER cell lines which were derived from T47D and MDA-MB-231, and which represent earlier and later stage tumor cells, respectively. As in other cell lines (3), we show that CYP1B1 is expressed in the absence of an external stimulus, whereas CYP1A1 is only expressed after treatment with an AhR agonist, TCDD. CYP1B1 is also stimulated by TCDD, but by a smaller factor than CYP1A1. CYP1A1 expression (TCDD-induced) was greater, and CYP1B1 expression (basal) was lower, in the ER+ clones of both cell types compared with the ER clones. TCDD induction of CYP1B1 largely removed the ER-linked constraints on CYP1B1 expression.

The contributions of CYP1A1 and CYP1B1 to the metabolism of DMBA parallel the levels of protein expression. This has been resolved by determining differences in regioselectivity of DMBA metabolites and by selective antibody inhibition. CYP1B1-like metabolic activity predominated under basal conditions, as previously reported for other human mammary epithelial cells (3,5). Most of the TCDD-induced activity was due to CYP1A1, even though CYP1B1 protein levels were comparable, due to the much greater metabolic activity of CYP1A1 for DMBA (5). By contrast, the strong suppression of CYP1A1 expression in MDA cells resulted in TCDD-induced activity which was almost entirely attributable to CYP1B1. These data are consistent with the dominant contributions of, respectively, TCDD-induced CYP1A1 and CYP1B1 to estradiol metabolism in T47D and MDA cells (33).

The rapid removal of ER activity by anti-estrogen ICI has been used to distinguish between direct effects of ER and indirect consequences of changes in ER status. We have found that loss of the ER from ER+ clones of either lineage following 48 h of treatment with 10–7 M ICI (26,27) had no effect on the distinctive expression patterns of CYP1A1 and CYP1B1. Thus, while the chronic presence or absence of the ER may influence the expression of CYP1A1 and CYP1B1, the regulatory pattern is not a direct result of transcriptional regulation of these genes by ER. This insensitivity to ICI establishes that these differences are attributable to processes that, at most, respond very slowly to changes in ER signaling. These slow changes are consistent with extensive reprogramming of cell signaling and of the organization of genes in chromatin structure, such as is likely in the growth selection of T47D and MDA+ clones.

Two separate sets of changes in cell regulation likely account for differences between the two lineages and the accompanying alteration in ER status. First, changes in growth regulation occur in vivo during the tumorigenic processes culminating in the initial genesis of the T47D and MDA cell lines. Secondly, subsequent changes occur during the selection, in culture, of the variants with altered ER status (i.e. T47D and MDA+). For example, the T47D and MDA lineages differ substantially with respect to the expression of the CYPs, AhR and ARNT, with only minor differences in CYP expression attributable to ER status. The levels of AhR are far higher in MDA lines than T47D lines, but relatively little difference exists between ER+ and ER clones. Levels of ARNT are lower in MDA lines than T47D lines, regardless of ER status. Furthermore, while both lineages show modest and opposite effects of ER status on expression of CYP1A1 and CYP1B1, the basal and TCDD-induced levels of both enzymes are set much higher in the T47D lines. These differences are apparently linked to facets of the signaling phenotype that are not sensitive to ER status. The shift in AhR, ARNT and CYP expression which we describe here seems to be general to multiple lines within the early and late categories (WGRA, unpublished observations).

Interline differences in the expression of AhR did not relate in any way to the ability of TCDD to induce CYP1A1 (34) and CYP1B1 (3,4). Thus, the very low AhR protein levels in T47D cells, as compared with MDA cells, were not linked to lower AhR activity, as evidenced by induction of both CYP1A1 and CYP1B1 by TCDD. Interestingly, the approximately seven-fold differences in AhR protein expression between T47D lines and MDA lines were inversely related to mRNA and protein expression of CYP1A1 or CYP1B1. In addition, the much higher AhR expression in the MDA lines may diminish the availability of ARNT for heterodimerization with other PAS domain containing proteins (35). The reverse of this would be true in T47D cells.

The growth of T47D+ cells is highly stimulated by estrogens and, therefore, much of the growth-related signaling is directly dependent on ER. MDA cells, like other ER breast cell lines, replaces this growth activation with ER-independent processes, frequently involving autocrine growth factors. Several of the growth factor signaling differences between T47D+ and MDA cells are associated with both ER status and lineage, and therefore also differ in the paired line with opposite ER status. In order to understand the changes in CYP regulation, we need to know which of these signaling changes are directly dependent on ER, and therefore rapidly reversed by ICI, and which signaling changes are more imprinted, and therefore are slowly responsive or resistant to ICI. Currently, the mechanisms associated with the generation of these irreversible differences are poorly understood.

MDA and T47D+ cells are thus regulated through different growth factor signaling cascades. For example, ErbB2/neu, an estrogen-repressed ErbB kinase family member, which is expressed at higher levels in these MDA cells than the T47D+ cells (WGRA, unpublished). The expression of ErbB2/neu is elevated within 48 h in ER+ cells by treatment with ICI or estrogen-stripped media, thus suggesting that this change in ErbB kinase expression is either directly controlled by ER or involves an ER-sensitive regulatory factor (36). Based on this ICI sensitivity, the difference in ErbB2 expression between the ER+ and ER lines is insufficient to account for differences in CYP expression observed in these studies. EGF receptor (EGFr, ErbB1) is also much more highly expressed in MDA cells than T47D+, and this is typical of ER tumors and cell lines (3739). This difference in EGFr expression may also be subject to an ER-linked repressive mechanism (40). However, the response of these receptors to EGF in MDA cells is low in part due to expression of a less active form of the receptor (e.g. through extensive PKC-mediated phosphorylation; 37,39,41), and in part due to the involvement of the ER in the EGFr signaling cascade (4244). MDA cells also produce much greater amounts of the estrogen-suppressed growth inhibitory protein TGFß, and the corresponding receptors, than T47D+ cells (4549). On the other hand, T47D+ cells produce large amounts of IGF-I and IGF-II, which are poorly expressed in MDA cells (50). High expression of IGFs is reversed by ICI, through reversal of an ER-dependent stimulation mechanism. MDA cells appear to be regulated by a balance of stimuli through the ErbB pathway and inhibition though the TGFß pathway. T47D+ cells demonstrate stimulation through low levels of EGFr in conjunction with the ER and by IGFs. Growth stimulatory signaling through these pathways in ER+ cells are often rapidly blocked by ICI (and possibly TCDD/AhR acting as an anti-estrogen), indicating that they are not contributors to the linkage between ER status and CYP expression. There must be further differences in signaling that allow ER-transfected MDA+ cells to survive. These cells must exhibit very different cell cycle controls from other ER+ cells, since they are growth inhibited by estrogens. This phenomenon has also been observed in other ER lines stably transfected with the ER (5153).

Previous work has shown that transient transfection of ER into MDA cells enhances CYP1A1 promoter activity in an estrogen-dependent manner (21). This implies a more direct cross-talk between ER and AhR-enhanced transcription than we are reporting here. However, it is now recognized that transiently transfected receptors can produce effects that are not realized by receptors when they are more completely integrated into chromatin structure or do not interact correctly with co-activators (5255). The transiently expressed receptors are not encumbered in the same way by these complex nuclear interactions that are typically generated slowly. Thus, the ER when fully integrated into the MDA+ cells, does not directly stimulate the natural CYP1A1 promoter in these cells, even though this promoter in MDA cells is accessible to the transfected ER. Similar differences in promoter site accessibility has been demonstrated with respect to ErbB2/neu. Estrogen repression of ErbB2/neu expression occurs in an ER-dependent manner at a DNase I hypersensitive site that is accessible in ER+ ZR75.1 cells, but not in MDA cells transfected with the ER (36). Hence, this work recommends caution in extrapolating such transient transfection results to natural gene regulation.

Current work is being directed toward these signaling differences between ER+ and ER cells from the same lineages. One goal is to identify other genes of this slow response type and to determine whether longer treatments of ER+ cells with ICI will eventually cause a reversion of an ER+ expression pattern to that of the ER cells. The mechanism of such changes would depend on the time course for reprogramming slower responding steps (kinases, nuclear regulatory factors, chromatin, etc.) in this shift from ER+ to ER signaling.

These studies establish that AhR regulation is partially suppressed in ER cells, and may become elevated in more metastatic cells. This examination of two AhR-responsive genes suggests that factors linked to ER-status and to cell lineage exert distinct and near additive effects on CYP1A1 and CYP1B1 expression. Induction of CYP1A1 is, indeed, lower in ER cells relative to the ER+ counterpart of the same lineage. This change affects CYP1B1 in the opposite direction, but primarily through elevated basal mRNA in ER cells. TCDD activation of AhR seems to elevate CYP1B1 expression up to a maximal level, which is less sensitive to ER-linked factors. Our CYP expression patterns suggest that there are a set of lineage-dependent factors, perhaps impacted by EGF receptor status or response to insulin-like growth factors or transforming growth factors, that are shared by these two T47D lines that allow high expression of both of these AhR-sensitive genes, and a different set of lineage-dependent factors that is shared by the two MDA lines, and that provide low expression of these genes. This is not unreasonable since many of the phenotypic characteristics are shared by the respective clones from each line, but the phenotypes of T47D versus MDA lines differ dramatically. A summary of the impact on CYP, AhR, and ARNT expression of lineage versus ER status is depicted in Figure 5Go.



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Fig. 5. Summary of effects of lineage versus ER status. For genes described here, effects of lineage are comparable with or are greater than the effect of ER status. Indirect effects of ER, which are not reversed by ICI are modest. Strong sensitivity would be predicted to parallel direct effect of ER that is reversible by ICI.

 
More recent work in this laboratory has explored the concept of altered CYP and AhR expression for a wide range of breast cell lines. Early progression cell lines, such as ZR75.1 and MCF7, exhibit similar patterns of AhR expression and regulation of CYP1A1 as observed in T47D+. Late progression lines show generally suppressed expression of both CYP genes and increased expression of AhR. In this context, the changes seen in T47D and MDA cells with change of ER status are small. We are now examining whether this change in AhR activity between early and late progression lines is seen for other AhR-responsive processes.


    Acknowledgments
 
We thank Drs V.Craig Jordan and John Pink for kindly providing the two T47D cell lines and the S30 cell line used in these studies. We also thank Paul Hanlon, for assistance in the isolation of mRNA, and Leonardo Ganem, for assistance in preparation of this manuscript. This work was presented at the Society of Toxicology Annual Meeting, Cincinnati, OH, 1997, and was supported by NIEHS grant 144EN46, DOD Breast Cancer Research grant DAMD17-94-J-4054 (CRJ) and NRSA 1-F32-ES05733-01(WA).


    Notes
 
3 To whom correspondence should be addressed Email: jefcoate{at}facstaff.wisc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 31, 1998; revised January 26, 1999; accepted February 5, 1999.