Barbara C. Spink

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.

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.

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.

Long-term estrogen exposure promotes carcinogen bioactivation, induces persistent changes in gene expression, and enhances the tumorigenicity of MCF-7 human breast cancer cells

The cumulative exposure to estrogens is an important determinant in the risk of breast cancer, yet the full range of mechanisms involving estrogens in the genesis and progression of breast cancer remains a subject of debate. Interactions of estrogens and environmental toxicants have received attention as putative factors contributing to carcinogenesis. Mechanistic studies have demonstrated interactions between estrogen receptor alpha (ERalpha) and the aryl hydrocarbon receptor (AhR), with consequences on the genes that they regulate. Many studies of ERalpha and AhR-mediated effects and crosstalk between them have focused on the initial molecular events. In this study, we investigated ERalpha- and AhR-mediated effects in long-term estrogen exposed (LTEE) MCF-7 human breast cancer cells, which were obtained by continuous culturing for at least 12 weeks in medium supplemented with 1 nM of 17beta-estradiol (E(2)). With these LTEE cells and with parallel control cells cultured without E(2) supplementation, we performed an extensive study of cytochrome P450 (CYP) induction, carcinogen bioactivation, global gene expression, and tumorigenicity in immunocompromised mice. We found that LTEE cells, in comparison with control cells, had higher levels of AhR mRNA and protein, greater responsiveness for AhR-regulated CYP1A1 and CYP1B1 induction, a 6-fold higher initial level of benzo(a)pyrene-DNA adducts as determined by liquid chromatography tandem mass spectrometry, marked differences in the expression of numerous genes, and a higher rate of E(2)-dependent tumor growth as xenografts. These studies indicate that LTEE causes adaptive responses in MCF-7 cells, which may reflect processes that contribute to the overall carcinogenic effect of E(2).

Expression of the aryl hydrocarbon receptor is not required for the proliferation, migration, invasion, or estrogen-dependent tumorigenesis of MCF-7 breast cancer cells.

The AhR was initially identified as a ligand‐activated transcription factor mediating effects of chlorinated dioxins and polycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvement of the AhR in cell‐cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis of MCF‐7 human breast cancer cells. In these studies, the properties of MCF‐7 cells were compared with those of two MCF‐7‐derived sublines: AHR100, which express minimal AhR, and AhRexp, which overexpress AhR. Quantitative PCR, Western immunoblots, 17β‐estradiol (E2) metabolism assays, and ethoxyresorufin O‐deethylase assays showed the lack of AhR expression and AhR‐regulated CYP1 expression in AHR100 cells, and enhanced AhR and CYP1 expression in AhRexp cells. In the presence of 1 nM E2, rates of cell proliferation of the three cell lines showed an inverse correlation with the levels of AhR mRNA. In comparison with MCF‐7 and AhRexp cells, AHR100 cells produced more colonies in soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despite the lack of significant AhR expression, AHR100 cells retained the ability to form tumors in severe combined immunodeficient mice when supplemented with E2, producing mean tumor volumes comparable to those observed with MCF‐7 cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression, the proliferation, invasion, migration, anchorage‐independent growth, and estrogen‐stimulated tumor formation of MCF‐7 cells do not require the AhR.

Persistent and non-persistent changes in gene expression result from long-term estrogen exposure of MCF-7 breast cancer cells

Life-long estrogen exposure is recognized as a major risk factor for the development of breast cancer. While the initial events in the regulation of gene expression by estrogen have been described in detail, far less is known of the role of estrogen in the long-term regulation of gene expression. In this study, we investigated the effects of long-term exposure of MCF-7 breast cancer cells to 1nM 17β-estradiol on gene expression with the goal of distinguishing between gene expression that is continually reliant on estrogen receptor (ER) function as opposed to secondary and persistent effects that are downstream of ER. To assess the direct involvement of ER in the differential gene expression of long-term estrogen exposed (LTEE) cells in comparison with that of control cells, we exposed cultures to the selective estrogen receptor modulator raloxifene (RAL). cDNA microarray analysis showed that exposure to RAL inhibited expression of numerous characterized estrogen-regulated genes, including PGR, GREB1, and PDZK1. Genes that were increased in expression in LTEE cells yet were unaffected by RAL exposure included the aryl hydrocarbon receptor (AHR) and numerous other genes that were not previously reported to be regulated by estrogen. Epigenetic regulation was evident for the AHR gene; AhR transcript levels remained elevated for several cell passages after the removal of estrogen. Signal transducer and activator of transcription 1 (STAT1); STAT1-regulated genes including ISG15, IFI27, and IFIT1; and MHC class I genes were also up-regulated in LTEE cells and were unaffected by RAL exposure. STAT1 is commonly overexpressed in breast and other cancers, and is associated with increased resistance to radiation and chemotherapy. This is the first study to relate estrogen exposure to increased STAT1 expression in breast cancer cells, an effect that may represent an additional role of estrogen in the pathogenesis of breast cancer.

Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC)n, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC)2 alleles were observed; however, in western gorilla, (GGGGC)n alleles with n=2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC)n was n=4>5≫2, 6. When frequencies of the (GGGGC)n alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC)2 was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC)n short tandem repeats are inherited, and that the (GGGGC)2 allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility.