Characterization of the Ah receptor and aryl hydrocarbon hydroxylase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benz(a)anthracene in the human A431 squamous cell carcinoma line

Certain human cell lines previously have been shown to exhibit substantial induction of aryl hydrocarbon hydroxylase (AHH, cytochrome P450IA1) when treated in culture with aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz(a)anthracene. Yet the Ah receptor, which is known to mediate the AHH induction process in rodent cells and tissues, has not previously appeared to be present at a significant level in any human cell line. In the human A431 squamous cell carcinoma line we found that cytosolic Ah receptor was present in high concentration (approximately 200 fmol/mg cytosol protein at maximal saturation); this corresponds to approximately 10,000 Ah receptor sites per cell in the human A431 line compared with about 35,000 receptor sites per cell in the mouse Hepa-1 hepatoma cell line in which Ah receptor previously has been extensively characterized. Detection of Ah receptor in A431 cytosol required modification of assay techniques, especially reduction in the amount of charcoal used to adsorb nonspecifically bound radioligand. The specific binding peak from A431 cytosol sedimented approximately 9S on sucrose gradients, the same as the cytosolic receptor from the well-characterized mouse Hepa-1 hepatoma cell line. In addition to [3H]TCDD, specific binding to Ah receptor in A431 cytosol also was detected with [3H]3-methylcholanthrene and with [3H]benzo(a)pyrene as radioligands. A specific [3H]TCDD-Ah receptor complex was extracted from nuclei of A431 cells incubated in culture at 37 degrees C with [3H]TCDD. The nuclear form of Ah receptor sedimented approximately 5S, the same as the nuclear receptor from mouse Hepa-1 cells. AHH activity was induced in A431 cells treated in culture with TCDD or benz(a)anthracene. The maximum level of induced AHH activity that could be achieved in A431 cells was about 20% of the maximally induced level in the mouse Hepa-1 cell line. However, the dose-response curves for AHH induction by TCDD or benz(alpha)anthracene in A431 cells were shifted about one log unit to the right of the curves for Hepa-1 cells. The lower sensitivity of A431 cells to AHH inducers was in proportion to the lower affinity with which cytosolic Ah receptor in A431 cells bound [3H]TCDD. The saturation curve for binding of [3H]TCDD to cytosolic Ah receptor in A431 cells also was shifted about one log unit to the right of the curve for saturation of the cytosolic receptor from mouse Hepa-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of growth by 2,3,7,8-tetrachlorodibenzo-p-dioxin in 5L rat hepatoma cells is associated with the presence of Ah receptor

The role of the Ah receptor in mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in 5L rat hepatoma cells containing TCDD-inducible cytochrome P450IA1 activity and in variants lacking cytochrome P450IA1 and Ah receptor. TCDD inhibited growth of the wild-type 5L cells, but not of the Ah receptor deficient variants. The two strong Ah receptor ligands 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and benz[a]anthracene (BA) exerted toxic effects in 5L cells that resembled those of TCDD. The poor Ah receptor ligand 2,2',4,4'-tetrachlorobiphenyl was not toxic in 5L cells. The concentrations of TCDD, 3,3',4,4'-TCB or BA required for the toxic response were similar to those that elicited P450IA1 induction. The present results suggest strongly that interaction with the Ah receptor is a necessary link in the chain of events leading to toxic effects in 5L cells upon exposure to TCDD.     

Ah receptor mediating induction of aryl hydrocarbon hydroxylase: detection in human lung by binding of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

In laboratory animals and in mouse hepatoma cells in culture the Ah receptor previously has been shown to mediate induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) by 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We examined human lung cytosols to determine whether the Ah receptor was present in human tissues. Cytosol was prepared from grossly normal lung tissue obtained at pulmonary lobectomy for presumed lung cancer from 53 consecutive adult patients including 32 males (42-77 years old) and 21 females (18-81 years old). Ah receptor in the cytosols was identified and quantitated by specific binding of [3H]TCDD after separation by ultracentrifugation on sucrose gradients. Specific binding of [3H]TCDD to a component which met the criteria for Ah receptor was detected in 10 of the 53 specimens. As previously established in tissues from laboratory animals, the specific [3H]TCDD-binding component sedimented approximately 9S. Binding of [3H]TCDD to the 9S component was competitively inhibited by incubation in the presence of 2,3,7,8-tetrachlorodibenzofuran, dibenz(a,h)anthracene, and nonradioactive TCDD, all known to be potent agonists for Ah-receptor-mediated induction of aryl hydrocarbon hydroxylase. Specific Ah receptor also was detected in some specimens by direct binding of [3H]-3-methylcholanthrene. The human population studied exhibited striking heterogeneity in Ah receptor concentrations. Only 10 of the 53 individuals studied had detectable Ah receptor. In specimens with detectable specific binding, the mean concentration of binding sites was 6.9 +/- 1.2 (SE) fmol/mg cytosolic protein. These concentrations are approximately 10-30% of the concentrations of Ah receptor found in lung cytosols from laboratory animals. Our experiments indicate that the Ah receptor can be detected in lung cytosol from some humans and suggest that the regulatory mechanism mediating human cytochrome P1-450 induction may be similar to that in the murine model. Aryl hydrocarbon hydroxylase, the major enzyme induced under control of the Ah receptor, plays an important role in the metabolism of several carcinogens including polycyclic aromatic hydrocarbons such as benzo(a)pyrene. It is possible that differences in the Ah receptor content within the human population may be genetically based and that variation at the Ah receptor level may be an important determinant of individual susceptibility to certain chemically induced cancers.     

Ontogeny of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and its endocrine indepence.

The ontogeny of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor was studied in Sprague-Dawley rats by quantitation of the receptor in liver cytosol using isoelectric focusing in polyacrylamide gel. No differences by sex in the receptor concentration were seen at any of the ages studied. Newborn, 21-day-old, and 42-day-old rats contained significantly more receptor in the liver cytosol than did 56-day-old rats. There was no significant difference in the receptor concentration in liver cytosol from 7-day-old rats compared to that from 56-day-old rats. The maximum receptor concentration was found in cytosol from 21-day-old rats [36.1 +/- 24.0 (S.D.) fmol/mg protein]. Adult rats (56 days old) contained the lowest concentration of receptor (13.3 +/- 6.3 fmol/mg protein). The level of TCDD receptor in liver cytosol from adult rats was not significantly changed by orchiectomy, ovariectomy, adrenalectomy, or hypophysectomy. The maximum for TCDD receptor concentration at puberty corresponds to the reported maximum for the induction of aryl hydrocarbon hydroxylase activity. However, no further conclusion can as yet be drawn concerning the regulation of the TCDD receptor.     

Green tea polyphenols reverse cooperation between c-Rel and CK2 that induces the aryl hydrocarbon receptor, slug, and an invasive phenotype

Exposure to and bioaccumulation of lipophilic environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs), has been implicated in breast cancer. Treatment of female rats with the prototypic xenobiotic PAH 7,12-dimethylbenz(a)anthracene (DMBA) induces mammary tumors with an invasive phenotype. Here, we show that green tea prevents or reverses loss of the epithelial marker E-cadherin on the surface of DMBA-induced in situ cancers. To investigate the mechanism(s) leading to a less invasive phenotype, the effects of the green tea polyphenol epigallocatechin-3 gallate (EGCG) on mammary tumor cells were assessed. EGCG reversed epithelial to mesenchymal transition (EMT) in DMBA-treated NF-kappaB c-Rel-driven mammary tumor cells and reduced levels of c-Rel and the protein kinase CK2. Ectopic coexpression of c-Rel and CK2alpha in untransformed mammary epithelial cells was sufficient to induce a mesenchymal gene profile. Mammary tumors and cell lines derived from MMTV-c-Rel x CK2alpha bitransgenic mice displayed a highly invasive phenotype. Coexpression of c-Rel and CK2, or DMBA exposure induced the aryl hydrocarbon receptor (AhR) and putative target gene product Slug, an EMT master regulator, which could be reversed by EGCG treatment. Thus, activation of c-Rel and CK2 and downstream targets AhR and Slug by DMBA induces EMT; EGCG can inhibit this signaling.     

Suppression of cell cycle progression by flavonoids: dependence on the aryl hydrocarbon receptor.

Some flavonoids are ligands of the aryl hydrocarbon receptor (AHR) and cause cell cycle arrest. The dependency of the cytostatic effects of five flavonoids (flavone, alpha-naphthoflavone, apigenin, 3'-methoxy-4'-nitroflavone and 2'-amino-3'-methoxyflavone) on a functional AHR was examined in AHR-containing rat hepatoma 5L cells and an AHR-deficient cell line (BP8) derived from the 5L line. The potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was cytostatic to the 5L line due to the induction of a G(1) arrest and dramatically elevated steady-state levels of CYP1A1 mRNA. TCDD affected neither the proliferation nor CYP1A1 mRNA contents of BP8 cells. With the exception of apigenin, the flavonoids under study induced G(1) arrest in both 5L and BP8 cells when used at concentrations at which they functioned as AHR agonists, but not antagonists. Apigenin-treated 5L and BP8 cultures primarily arrested in G(2)/M. The AHR-containing murine hepatoma cell line 1c1c7 arrested following exposure to AHR agonist concentrations of flavone and alpha-naphthoflavone, but not TCDD. Unlike the G(1) arrest observed in 5L cultures, the latter two flavonoids caused principally G(2)/M arrest in 1c1c7 cells. These studies demonstrate that the cytostatic activities of flavonoids do not require the AHR and the site of checkpoint arrest with a specific flavonoid can vary with cell type.     

外周血中芳香烃受体及芳香烃代谢酶活性与乳腺癌关系的研究

背景与目的：大量研究表明,活化的芳香烃受体(aryl hydrocarbon receptor,AhR)可能参与了由多环芳烃诱导的乳腺癌的发生,AhR作为公认的配体激活的转录因子,可诱导多种酶表达。本研究旨在探讨芳香烃受体和芳香烃代谢酶在人体内蛋白水平与乳腺癌及其病理指标间的关系。方法：以1∶1配对进行病例对照研究,共收集65对血样,采用酶联免疫吸附法检测血液中芳香烃受体及芳香烃代谢酶蛋白水平,同时收集病理资料。计量资料使用秩和检验进行统计分析,相关性分析使用Spearman检验。结果：秩和检验发现对照组与病例组间醛脱氢酶(aldehyde dehydrogenase,ALDH)、细胞色素(cytochrome P450,CYP450)差异无统计学意义(Z=0.196,P=0.845;Z=0.269,P=0.788);AhR、醌氧化还原酶(quinone oxido-ereductase 1,NQO1)、谷胱甘肽-S-转移酶(glutatnione-S-transferases,GST)对照组平均浓度低于病例组,差异有统计学意义(Z=1.956,P=0.041;Z=2.627,P=0.009;Z=3.272,P=0.001)。相关性分析结果显示,病例组中AhR与GST和NQO1相关性较高(r=0.665,P<0.01;r=0.704,P<0.01)。以病理指标(ER、PR、HER-2)-、+、++、+++分组,发现以雌激素受体ER分组的AhR、GST蛋白表达水平差异有统计学意义(P<0.05)。结论：实验结果显示芳香烃受体及芳香烃代谢酶蛋白水平对乳腺癌发生可能有重要影响,检测酶蛋白水平可能对乳腺癌的早期发现有帮助,对治疗方案的选择及预后有一定意义。     

Interaction of the aryl hydrocarbon receptor ligand 6-methyl-1,3,8-trichlorodibenzofuran with estrogen receptor alpha

The polycyclic aromatic hydrocarbon 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) is related to the industrial byproduct dioxin and is a weak agonist and partial antagonist at the aryl hydrocarbon receptor (AhR). Tamoxifen is used for the treatment and prevention of breast cancer and interferes with the interaction of estrogen with estrogen receptor alpha (ER). The combination of MCDF and tamoxifen lowered the effective dose of both drugs required to inhibit 7,12-dimethylbenz(a)anthracene-induced mammary tumor growth in rats and protected against the estrogenic effects of tamoxifen on the uterus in rats (A. McDougal et al., Cancer Res 2001;61:3902-7), pointing to the potential use of MCDF in breast cancer treatment. Potential AhR-ER cross-talk is evidenced by the antiestrogenic activity of MCDF and the degradative effect of MCDF on ER protein levels. Our studies confirmed that MCDF degraded the ER. MCDF displayed antiestrogenic activity at higher concentrations in MCF-7 human breast cancer cells, but MCDF alone (10(-6) M) stimulated the growth of MCF-7 cells. MCDF also activated an estrogen response element (ERE)-luciferase reporter and increased mRNA levels of the estrogen-responsive gene transforming growth factor (TGF)-alpha. The estrogenic effects of MCDF are ER dependent because they were blocked by the pure antiestrogen ICI 182,780. MCDF induced ER-coactivator interaction in glutathione S-transferase pull-down assays and the formation of an ER.ERE complex in gel mobility shift assays, further indicating that the estrogenic actions of MCDF are mediated by the ER. In addition, knockdown of the AhR with small interfering RNA did not affect MCDF-induced ERE-luciferase activity. Overall, these data support the conclusion that MCDF is a partial agonist at the ER. This study provides the first evidence for the direct interaction of the ER with MCDF and challenges the view that MCDF is simply an AhR-specific ligand.     

The role of signal transduction in cancer treatment and drug resistance.

Drug resistance in the treatment of cancer still remains a major clinical challenge, in part due to an insufficient understanding of the pathways by which these drugs interact with the mechanisms underlying cellular behaviour and cancer pathogenesis. Signal transduction involves cell differentiation, proliferation and cell death with alterations in these mechanisms being involved in the pathogenesis of cancer. It has been postulated that such pathways could be linked to anti-cancer drug resistance. Recently, novel approaches to overcome anti-cancer drug resistance through manipulation of signal transduction pathways, have been introduced in clinical trials. In this article we present a review of the current understanding in the field of signal transduction and the existing evidence for its role in drug resistance. We also discuss its clinical relevance with regard to overcoming drug resistance.     

Identification of kaempferol as an inhibitor of cigarette smoke-induced activation of the aryl hydrocarbon receptor and cell transformation

The aryl hydrocarbon receptor (AHR) is a cytosolic receptor which upon activation by its agonists, translocates into the nucleus and forms a dimer with ARNT (aryl hydrocarbon nuclear translocator). The AHR/ARNT dimer regulates the expression of its target genes by binding to DNA recognition elements termed dioxin responsive elements (DREs). Many AHR agonists, like the polyaromatic hydrocarbons and polyhalogenated hydrocarbons are known human carcinogens. Human exposure to these compounds is common due to their presence in air pollution and cigarette smoke. Interestingly, many dietary constituents that have chemo preventative properties have been found to also act as antagonists of the AHR pathway. Thus, a chemopreventive approach that may be effective in decreasing the incidences of many human cancers may involve a dietary regimen that includes a number of these naturally occurring AHR antagonists. With this idea in mind, we have assayed the ability of 15 flavonoids to inhibit AHR activated reporter activity and selected kaempferol for further analysis. Kaempferol proved to be capable of inhibiting binding of agonist and agonist-induced formation of the AHR/ARNT DNA-binding complex and upregulation of the AHR target gene, CYP1A1. Using an in vitro paradigm of events that are thought to occur during cigarette-smoke-induced lung cancer, we found that kaempferol also inhibited the ability of cigarette smoke condensate to induce growth of immortalized lung epithelial (BEAS-2B) cells in soft agar. Taken together, these results illustrate the promise associated with the use of flavonoids, that inhibit both AHR signaling and the carcinogenic actions of AHR agonists, for chemopreventive purposes.     

Affinities for the aryl hydrocarbon receptor, potencies as aryl hydrocarbon hydroxylase inducers and relative toxicities of polychlorinated biphenyls. A congener specific approach

Polychlorinated biphenyls (PCBs) are nonplanar aromatic xenobioticsthat are not structurally related to polychlorinated dibenzo-p-dioxins(PCDDs) and dibenzofurans (PCDFs), yet, some PCBs are potentligands for the aryl hydrocarbon receptor (AhR), active inducersof aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufinO-deethylase (EROD), and elicit toxicological responses in animalssimilar to PCDDs and PCDFs. We report new methodologies forquantifying the affinities of PCBs for AhR and correspondingpotencies as AHH and EROD inducers. The models show that lipophilicities,electon affinities, entropies and electronic energy gaps ofPCBs are key physicochemical properties controlling their AhR,AHH and EROD activities. Using 3,3',4,4'-tetrachlorobiphenyl(TCB) as the reference compound, it is shown that PCBs havinghigher electron affinities, lower lipophilicities and entropiesthan TCB are potent ligands for rat hepatic AhR. In addition,the congeners having higher binding affinities to AhR and smallerenergy gaps than TCB are potent AHH and EROD inducers in rathepatoma cells in culture. The reported models qualitativelyexplain and quantify AhR, AHH and EROD activities of all 209-PCBsand related xenobiotics, e.g. PCDDs and PCDFs. Furthermore,we demonstrated that AhR and AHH activities of PCBs relativeto 2,3,7,8-tetrachlorodibenzo-p-dioxin correlate with correspondingin vivo relative toxicities in animals as well as assigned toxicequivalency factors. The reported methodologies are likely tobe useful for identifying potentially toxic aromatic xenobioticsin mammals, and minimizing the need for animal testing.     

Evidence that Argos is an antagonistic ligand of the EGF receptor

Argos, the inhibitor of the Drosophila epidermal growth factor (EGF) receptor, remains the only known extracellular inhibitor of this family of receptors in any organism. The functional domain of Argos includes an atypical EGF domain and it is not clear whether it binds to the EGF receptor or if it acts via a distinct receptor to reduce Egfr activity indirectly. Here we present two lines of evidence that strongly suggest that Argos directly interacts with the EGF receptor. First, Argos is unable to inhibit a chimeric receptor that contains an extracellular domain from an unrelated RTK, indicating the need for the EGF receptor extracellular domain. Second, Argos can inhibit the Drosophila EGF receptor even when expressed in human cells, implying that no other Drosophila protein is necessary for inhibition. We also report that Argos and the Drosophila activating ligand, Spitz, can influence mammalian RTK activation, albeit in a cell-type specific manner. This includes the first evidence that Argos can inhibit signalling in mammalian cells, raising the possibility of engineering an effective human EGF receptor/ErbB antagonist. Oncogene (2000) 19, 3560 - 3562