Expression of CYP1B1 but not CYP1A1 by primary cultured human mammary stromal fibroblasts constitutively and in response to dioxin exposure: role of the Ah receptor

The expression of CYP1B1 in human mammary fibroblasts (HMFs) was characterized as a potential modulator of their individual function as well as effects on adjacent mammary epithelia. We have used these characteristics to explore the diversity of fibroblast cells isolated from reduction mammoplasty patients and from different breast locations in breast cancer patients (tumors, peripheral to tumor and skin). These parameters have also been used to examine differences between two donors. The results have shown that while none of these HMFs expressed a detectable CYP1A1 protein basally or in response to TCDD, they all expressed CYP1B1 constitutively at similar levels (0.5-0.9 pmol/mg microsomal proteins) and they were induced by TCDD (up to 5-fold) consistent with mediation by the Ah receptor (AhR). DMBA metabolism by HMFs exhibited high proportions of 5,6-, 10,11- and 3,4-dihydrodiols, a profile that is typical of human CYP1B1 regioselectivity. RT-PCR followed by Southern blot analyses demonstrated that CYP1B1 mRNA expression in HMFs parallels levels of respective microsomal proteins. The AhR is expressed in these HMFs as two cytosolic forms (approximately 106 and 104 kDa) and a substantial proportion of the 104 kDa form was localized to the nucleus even prior to TCDD treatment. In all HMFs isolated directly from collagenase digested breast tissues the AhR is expressed at levels 10-fold lower than in breast epithelial cells. However, HMFs that were isolated after serial passaging of mammary epithelial cultures had shown much higher levels of the AhR expression and more dramatic TCDD-induced down-regulation (>80% in 24 h) associated with more efficient nuclear translocation. These differences suggested the presence of two functionally distinct subtypes of HMFs: interstitial stromal fibroblasts that are readily released by collagenase digestion of breast tissues, and lobular stromal fibroblasts which are more tightly associated with the breast epithelia.      

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4- hydroxylation were highly elevated following exposure to TCDD. In MDA- MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2- hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR- mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.      

Effect of transient expression of the oestrogen receptor on constitutive and inducible CYP1A1 in Hs578T human breast cancer cells.

Hs578T human breast cancer cells are an oestrogen receptor (ER)-negative cell line. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in formation of a 6.9 S nuclear aryl hydrocarbon (Ah) receptor complex, which bound to a [32P]dioxin-responsive element in a gel electrophoretic mobility shift assay. However, TCDD does not induce CYP1A1 gene expression or chloramphenicol acetyl transferase (CAT) activity in cells transiently transfected with pRNH11c or pMCAT5.12, which are Ah-responsive plasmids derived from the 5''-flanking region of the human and murine CYP1A1 genes respectively. Restoration of Ah responsiveness was investigated by co-transfecting Hs578T cells with pRNH11c or pMCAT5.12 and plasmids that express the ER (hER), Ah receptor (AhR) and AhR nuclear translocator (Arnt) proteins. ER expression resulted in significantly increased basal CAT activity; however, TCDD did not induce CAT activity in the transiently transfected cells. Expression of the AhR or Arnt proteins did not alter basal or inducible CAT activity. Expression of N- or C-terminal truncated ER in Hs578T resulted in differential regulation of Ah responsiveness. In Hs578T cells transiently expressing the ER, which contains C-terminal deletions (amino acids 282-595), basal CAT activity was also increased; however, Ah responsiveness was not restored. In contrast, transient expression of N-terminal-deleted (amino acids 1-178) ER resulted in a marked decrease in basal CAT activity but a restoration of Ah responsiveness. These results suggest that basal and inducible CAT activity in Hs578T cells transiently transfected with pRNH11c is modulated differentially by ER domains that are present in the N- and C-terminal regions of the ER.     

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

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.     

Expression of cytochrome P-450 1A1 in human fetal adrenal cells

It is well known that cytochrome P-450 (CYP) 1A was thought to be responsible for activation of the majority of precarcinogens and premutagens in human liver. The level of CYP1A may serve as a potential indicator of carcinogenesis.[1] Therefore, study of CYP1A expression in human fetal liver and extrahepatic tissue still seems to be important with respect to the possible toxicological significance. Our previous work demonstrated the greater activities of drug-metabolizing enzymes in human…     

Aryl hydrocarbon hydroxylase represents CYP1B1, and not CYP1A1, in human freshly isolated white cells: trimodal distribution of Japanese population according to induction of CYP1B1 mRNA by environmental dioxins.

The expression level of mRNAs for cytochrome P450 (CYP) 1A1 and 1B1 in freshly prepared white cells from 72 subjects exposed to dioxins at waste incinerators was investigated. The amounts of CYP1B1 mRNA ranged from 0.16 to 671 molecules/10(7) molecules of 18S rRNA, whereas the amounts of CYP1A1 mRNA were <6 molecules/10 ng total RNA, indicating that CYP1A1 was not induced to a detectable level by environmentally exposed dioxins. The inducibility of CYP1B1 mRNA in leukocytes, defined as the ratio of CYP1B1 mRNA to the plasma concentration of dioxins, varied among the subjects. It was found that the subjects showed trimodal distribution according to inducibility: 39 (54.2%), 25 (34.7%), and 8 (11.1%) of 72 subjects were judged as poor, intermediate, and high responders to environmental dioxins, respectively. The amounts of CYP1B1 mRNA in leukocytes of the intermediate and high responders were highly correlated with the plasma concentrations of dioxins (P < 0.05 and <0.01). These results suggest that CYP1B1 with polymorphic inducibility by dioxins is involved in aromatic hydrocarbon hydroxylase activities in human lymphocytes.     

Differential induction of CYP1A1 and CYP1B1 by benzo[a]pyrene in oral squamous cell carcinoma cell lines and by tobacco smoking in oral mucosa

Polyaromatic hydrocarbons, including benzo[a]pyrene (BP), are major tobacco carcinogens. Their carcinogenic effects require metabolic activation by cytochrome p450 (CYP) enzymes. Relative CYP isoform expression is related to tissue-specific tobacco-related squamous cell carcinoma (SCC) susceptibility. There have been conflicting reports regarding relative CYP1A1 and CYP1B1 oral expression, and information regarding CYP1B1 expression in oral tissues is limited. To quantify BP- and tobacco-induced CYP1A1 and CYP1B1 expression in oral SCC cells and oral mucosa. Study Design: Real-time qPCR was performed to measure (1) BP-induced CYP1A1 and CYP1B1 mRNA expression in seven oral/other head and neck SCC cell lines (2) CYP1A1 and CYP1B1 mRNA expression in gingiva from 22 smokers and 24 nonsmokers. SCC lines exhibited either similar induction of both isoforms or preferential CYP1A1 induction (CYP1A1-to-CYP1B1 ratios 0.8–4.3). In contrast, gingival tissues from smokers exhibited preferential CYP1B1 induction. Marked interindividual variation in CYP1A1 and CYP1B1 expression was observed among smokers. In vitro conditions may not account for factors that modulate expression in vivo. Interindividual variation in inducible CYP1A1 and CYP1B1 expression may account in part for variation in tobacco-related oral SCC risk.     

Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.     

乳腺癌CYP1B1的表达对紫杉醇耐药性的影响

  目的  探讨乳腺癌细胞CYP1B1表达与紫杉醇耐药产生的相关性。   方法  以乳腺癌细胞系MCF-7为研究对象, 经TCDD诱导MCF-7表达CYP1B1, 观察CYP1B1表达后对紫杉醇药物的细胞毒效应的影响。将紫杉醇联合CYP1B1的拮抗剂ANF使用, 观察ANF是否可逆转CYP1B1对紫杉醇的耐药作用。采用RT-PCR测定诱导后CYP1B1 mRNA水平, 采用细胞免疫化学染色和蛋白印迹技术测定诱导后CYP1B1蛋白水平, MTS比色法测定紫杉醇对细胞增殖的抑制强度。   结果  TCDD可诱导MCF-7细胞表达CYP1B1, 其mRNA和蛋白表达水平与TCDD浓度具有明显的剂量依赖性(P < 0.05);而单独紫杉醇不能诱导MCF-7细胞表达CYP1B1。当MCF-7细胞经TCDD诱导表达CYP1B1后, 紫杉醇浓度在0.01~0.1μg/mL时, 紫杉醇对MCF-7细胞的抑制率与对照组相比明显下降(P <0.05)。使用CYP1B1的特异性拮抗剂ANF后, MCF-7细胞对紫杉醇的敏感性升高, 细胞抑制率与对照组比较无显著性差异(P>0.05)。   结论   CYP1B1表达后可导致细胞产生对紫杉醇耐药。      

Estrogen regulates Ah responsiveness in MCF-7 breast cancer cells

Cytochrome P450 (CYP)1A1 and CYP1B1, which are under the regulatory control of the aryl hydrocarbon (Ah) receptor (AhR), catalyze the metabolic activation of numerous procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. There is evidence of cross-talk between estrogen receptor alpha (ERalpha)- and AhR-mediated signaling in breast and endometrial cells. To further examine these interactions, we investigated the short- and long-term effects of E2 exposure on Ah responsiveness in MCF-7 human breast cancer cells. Short-term exposure to 1 nM E2 elevated the ratio of the 4- to 2-hydroxylation pathways of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced E2 metabolism and the ratio of the induced CYP1B1 to CYP1A1 mRNA levels, as determined by real-time PCR. Cells maintained long-term (9-12 months) in low-E2 medium progressively lost Ah responsiveness, as indicated by diminished rates of TCDD-induced E2 metabolism and ethoxyresorufin O-deethylase activity, and the reduced expression of the CYP1A1 and CYP1B1 mRNAs and proteins levels. These E2-deprived cells showed elevated levels of ERalpha mRNA, depressed levels of AhR mRNA, and unchanged levels of the AhR nuclear translocator mRNA. Transient transfection studies using a CYP1B1-promoter-luciferase reporter construct showed that reduced CYP1B1 promoter activity in E2-deprived cells could be restored by co-transfection with an AhR expression construct, indicating that AhR expression was limiting in these cells. The reduced Ah responsiveness of E2-deprived cells was reversed by culture for four passages in medium supplemented with 1 nM E2; ERalpha and AhR mRNAs returned to near-normal levels and the inducibility of the CYP1A1 and CYP1B1 mRNAs, proteins, and E2 metabolic activities by TCDD was restored. These studies indicate that the continued presence of estrogen is required to maintain high levels of AhR expression and inducibility of the procarcinogen-bioactivating enzymes, CYP1A1 and CYP1B1, in MCF-7 cells.     

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1- 12-3 recognized only one protein of identical mobility on the two- dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.      

Aberrant CYP1A1 Induction: Discrepancy of CYP1A1 mRNA and Aryl Hydrocarbon Hydroxylase Activity in Mutant Cells of Mouse Hepatoma Line, Hepa-1

We have isolated new benzo[α]pyrene-resistant clones, cl-21 and cl-32, of the mouse hepatoma line, Hepa-1. CYP1A1-dependent aryl hydrocarbon hydroxylase activity is not inducible by 2,3,7,8-tetrachlorodibenzo- p -dioxin or 3-methylcholanthrene in these two cell lines. However, mRNA of CYP1A1 is inducible in cl-21 and cl-32 cells, as in the wild-type cells, in spite of an undetectable level of cytosolic Ah receptor. The cl-21 cDNA of Cypla-1 was found to have a single mutation leading to an amino acid substitution from Leu (118) to Arg (118). However, the CYP1A1 protein band was not detected on Western immunoblots. The cDNA of cl-32 was found to have a single mutation leading to an amino acid change from Arg (359) to Trp (359). The presence of the mature protein in cl-32 was confirmed by Western blot analysis. Somatic cell hybridization experiments demonstrated that the phenotype of cl-21 and cl-32 is recessive and that these clones belong to the same complementation group. These data suggest that there may be a non-Ah receptor-mediated mechanism of CYP1A1 induction.