* Laboratory of Cytokinetics, Institute of Biophysics, Královopolská 135, 612 65 Brno, Czech Republic; and
Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic
Received May 31, 2002; accepted August 12, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: PAHs; MAPkinases; estrogenicity; cell cycle; ER.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PAHs have been reported to possess both estrogenic and antiestrogenic properties in various experimental settings. Today, PAHs are regarded mostly as antiestrogens, principally due to their ability to activate the Ah receptor, which leads to suppression of estrogen response element (ERE)-controlled gene expression via AhR-estrogen receptor (ER) cross-talk, increase in estradiol metabolism, and/or simultaneous downregulation of ER levels (reviewed in Safe, 2001
). However, recently, several PAHs, including benzo[a]pyrene (BaP) and benz[a]anthracene (BaA), or their oxygenated derivatives, have been shown to act as estrogenic compounds in ER-regulated reporter gene assays (Charles et al., 2000
; Fertuck et al., 2001b
; Machala et al., 2001a
). The in vivo estrogenicity of PAHs remains controversial; while some early studies suggested weak estrogenicity of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in a rat uterotrophic assay (Morreal et al., 1979
), others have found no evidence of in vivo estrogenicity of BaP and its metabolites (Fertuck et al., 2001b
).
It has been suggested that estrogenicity of PAHs is potentially the nongenotoxic component of their carcinogenic effects via increased organ-specific cell proliferation (Santodonato, 1997). The stimulation of cell cycle progression and cellular proliferation is a process that is likely to be associated with nongenotoxic, chemical-induced carcinogenesis (Foster, 1997
). A number of xenoestrogenic compounds have been shown to act as effective mitogens in ER
-expressing cells such as MCF-7 cells (Soto et al., 1995
). Although it has been generally accepted that estrogen-mediated gene regulation is responsible for hormone-dependent cell proliferation, the exact mechanisms of cell cycle control by estrogens are still not fully understood (reviewed in Foster et al., 2001
). Both estrogens and peptide growth factors modulate levels, cellular redistribution, and activities of key cell-cycle regulators (reviewed in Pestell et al., 1999
). Moreover, several rapid, so-called "nongenomic effects" of estrogens have been reported, including activation of two mitogen-activated protein kinases, ERK1/2, which participate in cell signaling associated with the induction of proliferation. However, while some authors have found a rapid and transient ERK1/2 activation within several minutes following estradiol addition (Improta-Brears et al., 1999
; Migliaccio et al., 1996
), others have not observed such response in breast cancer cells (Caristi et al., 2001
; Joel et al., 1998
; Lobenhofer and Marks, 2000
). The cross-talk between peptide growth factor receptors and ER
can be even more complex, since ER
may be activated in a ligand-independent manner by a number of mechanisms, among which regulation of ER
phosphorylation by growth factors plays a significant role (reviewed in Cenni and Picard, 1999
; Kato et al., 2000
). An example thereof is the phosphorylation of ER
at serine 118 by activated ERK1/2. The phosphorylation of this serine residue has been shown to induce estrogen-like responses, such as proliferation of mammary and uterine epithelial cells and other estrogen-dependent cells and tissues (Arnold et al., 1995
; Kato et al., 1995
).
Taken together, the potential role of PAHs in regulation of ER activity, as well as its impact on cell proliferation, still remain unclear. Both estrogens and growth factors employ some similar pathways regulating cellular proliferation. ERK1/2 rank with the major components of pathways controlling cell proliferation, differentiation, and apoptosis, and their activation was suggested as playing a role in estrogen-induced mitogenesis. The present study investigated the effects of PAHs on ER-dependent reporter construct activation and ERK1/2 activation in human breast carcinoma MCF-7 cells with respect to modulation of cell cycle progression. The aim was to define a mode of action that could potentially contribute to carcinogenic effects of PAHs in estrogen-dependent tissues.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reporter gene assay.
MVLN cells stably transfected with the p-Vit-tk-Luc plasmid (Pons et al., 1990) were used for the detection of ER-mediated activity. The cell line was chosen due both to its consistence of response to 17ß-estradiol and its sensitivity to various types of phytoestrogens and xenoestrogens, being comparable to other established estrogenicity bioassays such as E-SCREEN (Guttendorf and Westendorf, 2001
). The assays were run in 96-well cell culture plates. Briefly, 24 h after seeding, the medium was replaced with a medium supplemented with dextran/charcoal-treated serum for another 24 h. The cells were then dosed either with the tested compounds or with 17ß-estradiol calibration standards, prepared in dimethylsulfoxide (DMSO), and diluted with a serum-free medium, and were then incubated for a given time period (6 or 24 h). Maximum concentrations of DMSO did not exceed 0.1% (v/v). Cytotoxicity of PAHs was not observed at concentrations up to 20 µM (higher concentrations were not used because of a limited solubility of some compounds) as verified by MTT assay. In experiments where various types of inhibitors were used, these were applied to cells 90 min before the addition of the studied compounds. The concentrations of both effectors and inhibitors were based on available data.
After exposure, the medium was removed, cells were washed and lysed with a low salt lysis buffer (10 mM Tris, 2 mM DTT, 2 mM 1,2-diamin cyclic hexane-N,N,N,N-tetraacetic acid, pH 7.8), and the plates were frozen at 80°C. Luciferase activity was then measured in the microplate luminometer Luminoscan (Labsystems, Turku, Finland) using the Luciferase Monitoring Kit (Labsystems).
Western blot analyses.
MCF-7 cells were serum-starved for 24 h and then treated with selected compounds for the time indicated. After the incubation, the cells were washed with PBS and lysed with the sodium dodecyl sulfate (SDS) lysis buffer containing protease and phosphatase inhibitors (10% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 nM aprotinin, 1 µM leupeptin, 1 µM antipain, 100 µM Na3VO4, and 5 mM NaF). Thirty µg of total protein per sample was separated on 12% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. Activated ERK1/2 was detected with a phospho-specific polyclonal antibody and compared with total ERK1/2 expression, detected with a polyclonal antibody directed against a different epitope of ERK1/2. After incubation with primary and secondary antibodies, detection was performed with the ECLPlus Western blotting detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).
ER phosphorylation was detected using the method described by Joel et al. (1998
). Briefly, thirty micrograms of total protein per sample was separated on a 7% polyacrylamide gel until 35 kDa standard run off the gel, and transferred onto a polyvinylidene difluoride membrane, probed with antihuman ER
murine monoclonal antibody, and incubated with a secondary antibody conjugated with horseradish peroxidase. The acrylamide/bis-acrylamide ratio was changed to 99:1 in order to improve the separation of phosphorylated and unphosphorylated forms of ER
. The secondary antibody was detected using the ECLPlus. A Model GS-670 Imaging Densitometer with Molecular Analyst Software (Bio-Rad Laboratories, Hercules, CA) was used to determine the relative amounts of total ER
.
Cell cycle analysis.
MCF-7 cells were seeded in 12-well plates at an initial concentration of 24,000 cells per well. Cells were allowed to attach for 24 h, synchronized by serum withdrawal for 72 h, and treated with estradiol, BaA, or combinations thereof with antiestrogen ICI 182,780. After a 24-h incubation, cells were harvested, washed once with PBS, and then fixed in 5 ml of 70% ethanol at 4°C. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1M Tris-HCl, pH 8.0; 0.1% Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 µg/ml; RNase A 50 Kunitz units/ml) and incubated at 37°C for 30 min (Vindelov, 1977). Cells were then analyzed on FACSCalibur, using a 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and CELLQuestTM software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME).
Statistics.
Data were expressed as means ± SD for at least three independent repeats and analyzed by the nonparametric Mann-Whitney U test or Kruskal-Wallis analysis of variance (ANOVA). A p value of less than 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to the above reports, it has been shown that PAHs and/or their hydroxy- and oxy-derivatives can weakly activate ER-dependent reporter constructs in vitro (Charles et al., 2000; Fertuck et al., 2001b
; Machala et al., 2001a
). In the present study, we found that BaA and BaP can act as estrogenic compounds in the MCF-7 cell line stably transfected with the luciferase reporter gene, although at significantly higher concentrations than does estradiol. Metabolites of both BaP and BaA have been shown to act as estrogens (Charles et al., 2000
; Fertuck et al., 2001b
; Schneider et al., 1976
). Moreover, a number of other PAHs, namely fluorene, fluoranthene, pyrene, chrysene, phenanthrene, and anthracene, acted as very weak inducers of ER-mediated activity in this study. The effects of PAHs were affected by their metabolism; while luciferase activity continued to rise following estradiol treatment of cells, the activity 24 h after addition of PAHs decreased or altogether disappeared when compared with ER-inducing potencies after a 6-h treatment (Fig. 1
). This effect is apparently similar to AhR-mediated response, where metabolic clearance of PAHs, in contrast to highly persistent polyhalogenated aromatics, plays an important role in their time-dependent effects on this transcription factor (Jones et al., 2000
; Machala et al., 2001b
). Previously, no ER-mediated activity has been reported for the compounds found to be weakly active in MVLN cells (Fertuck et al., 2001a
). However, this study used a chimeric Gal4 receptor-reporter system, which lacks ER activation function-1 (AF-1), while we employed a stably transfected reporter gene system under the control of endogenous ER
. Another factor that might potentially contribute to the observed differences could have been the incubation period, as in the case of AhR-mediated activity of PAHs (Jones et al., 2000
; Machala et al., 2001b
). Nevertheless, our results do not exclude the possibility that the full-length ER expression is necessary for induction of these weak estrogenic effects in MVLN cells. The concentrations of PAHs required to observe a significant ER activation are relatively high, seemingly above their environmental levels. However, it has been shown recently that weak environmental estrogens can, at least in vitro, produce significant mixture effects when combined at concentrations below their NOECs (Silva et al., 2002
). Therefore, as exposure to PAHs is always an example of a complex mixture exposure, mixture effects should be taken into consideration in their risk assessment.
These facts have led us to explore in more detail the effects of PAHs on ER-dependent reporter construct and ER, which is the predominant form of ER in MCF-7 cells (Burow et al., 2000
). ICI 182,780 belongs among "type II" antiestrogens that act as competitive inhibitors of ER and block the transport of newly synthesized ER into the nucleus, leading thus to its subsequent proteosomal degradation (MacGregor and Jordan, 1998
). The current hypotheses of its action also suggest that although ICI 182,780-bound ER dimers still bind to EREs, ICI 182,780 effectively blocks the association with cofactors necessary for the formation of a transcriptionally active protein complex (MacGregor and Jordan, 1998
). While ICI 182,780 fully inhibited estradiol-induced, ER-mediated transcriptional activity, we found a significantly higher luciferase activity in samples treated with a combination of PAHs and ICI 182,780 than in those treated with the antiestrogen alone. These results indicated that PAHs could perhaps still initiate the ER-mediated transcription by affecting the association of the ICI-ER
complex with cofactors. Moreover, estrogenic PAHs significantly enhanced the effects of estradiol, the cause of which could be related to different effects of these compounds on ER
(Fig. 6
). ER
can be activated in a ligand-independent manner by a number of mechanisms, among which the regulation of ER
phosphorylation by growth factors plays a significant role (Cenni and Picard, 1999
; Kato et al., 2000
). The activation functions in ER
, AF-1 and AF-2, can be activated by phosphorylation of the receptor at several, mostly serine, residues mediated by various protein kinases (reviewed in Cenni and Picard, 1999
). However, from results gained after pretreatment of MVLN cells with specific inhibitors of ERK1/2 activation, it appears that PI3K, PKC, PKA, or Src-family kinases are not involved in effects of PAHs. Moreover, specific inducers of activity of the kinases mentioned above, such as EGF, TPA, forskolin, or Br-cAMP, neither elicited significant luciferase activity nor enhanced the effects of estradiol in MVLN cells model. Hence, ER
phosphorylation by these kinases is probably not involved in the observed effects. This finding does not exclude the possibility that PAHs could affect the interaction between ER
and one or more of its cofactors, or other signaling pathways leading to modulation of estrogenic response. Both cell cycle regulators such as cyclin D1 (Cenni and Picard, 1999
) and altered interaction with coactivators may transactivate ER
(Kato et al., 2000
).
Estradiol induces ER phosphorylation, which modulates estradiol-induced transcriptional activation (Joel et al., 1998
; Kato et al., 1995
). The results of this study indicate that BaA could induce ER
phosphorylation in a similar manner as estradiol, albeit to a lower extent (Fig. 5
). In accordance with earlier studies showing that phosphorylation of Ser118 is involved in the regulation of transcriptional activity of AF-1 in response to EGF (Joel et al., 1998
; Kato et al., 1995
), EGF also induced transient ER
phosphorylation. The time profile of ERK1/2 phoshorylation after EGF treatment correlated with the time profile and the rate of ER
phosphorylation (Fig. 6
). Several authors have suggested that estradiol, among other "nongenomic" effectors, can also induce a rapid ERK1/2 activation in MCF-7 cells (Improta-Brears et al., 1999
; Migliaccio et al., 1996
). On the other hand, results of other studies have not found a significant activation of ERK1/2 following estradiol treatment in this cell line (Caristi et al., 2001
; Joel et al., 1998
; Lobenhofer and Marks, 2000
). It has been shown that ERK1/2 are not responsible for the ER
phosphorylation on Ser118 in response to estradiol, and that another kinase or kinases must be involved in this process (Joel et al., 1998
). In the present study, neither estradiol nor BaA induced ERK1/2 phosphorylation significantly (Fig. 6
). Similar results were obtained also for BaP and fluoranthene (data not shown). The weak nonspecific effect observed may have been caused by cell manipulation (Caristi et al., 2001
). Thus, together with the results reported in the previous paragraph, ERK1/2 do not seem to be involved in the effects of BaA.
Estrogens are known to induce G0/G1-S transition in breast cancer cells and a number of cell cycle regulatory proteins have been shown to be affected by estradiol treatment during the early cell cycle events (reviewed in Foster et al., 2001). As it has been reported that PAHs may exert antiestrogenic effects by blocking estradiol-mediated postconfluent cell growth (Arcaro et al., 1999
), the effect of BaA on cell cycle progression was investigated. Both estradiol and BaA were found to induce G0/G1 S-transition in a similar manner, although the combination of both compounds did not increase percentage of S-phase cells (Table 3
). Their action was inhibited by synthetic antiestrogen ICI 182,780, which also blocked cell cycle progression more efficiently than serum starvation. These results seem to contradict published data on antiestrogenicity of AhR ligands (Arcaro et al., 1999
; Wang et al., 1998
). However, unlike the weakly estrogenic PAHs, persistent planar AhR inducers are not metabolized to compounds that would stimulate ER (Charles et al., 2000
; Fertuck et al., 2001b
) and may block the cell cycle progression through AhR-mediated mechanisms (Wang et al., 1998
). Also, the effects of PAHs on subconfluent serum-deprived MCF-7 cell model used in the present study could have differed from the model using postconfluent, fourteen-day foci formation as an endpoint (Arcaro et al., 1999
).
Taken together, a number of PAHs may act as weak estrogens in hormone-responsive human breast cancer cells. Importantly, the same compounds belong mostly among moderate and weak inducers (BaA, BaP, pyrene, phenanthrene, fluoranthene) or noninducers (fluorene, anthracene) of AhR-mediated activity (Machala et al., 2001b). Therefore, their respective antiestrogenic potency, mediated through the activation of AhR, could be counterbalanced by their direct ER-mediated activity. However, only BaA and BaP seem to have sufficient potency to act as weakly estrogenic compounds. Importantly, the ER-inducing PAHs, such as BaA, BaP, and fluoranthene, significantly potentiated the ER-mediated response to natural estrogen treatment. The most effective compound, BaA, as judged from the results of the assay of ER-dependent reporter construct activation, induced ER
phosphorylation in a similar manner to estradiol, although to a lesser extent. It was found that, like estradiol, BaA does not directly induce significant ERK1/2 phosphorylation, which, consequently, does not seem to play the key role either in phosphorylation of ER
or in a stimulation of cell-cycle progression observed after BaA treatment. Further studies are necessary to elucidate the exact mode(s) of action of PAHs, or to establish whether this phenomenon is of in vivo relevance and could play a promoting role in their tissue-specific carcinogenicity.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arnold, S. F., Obourn, J. D., Jaffe, H., and Notides, A. C. (1995). Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: Consequence on DNA binding. J. Steroid Biochem. Mol. Biol. 55, 163172.[ISI][Medline]
Bláha, L., Kapplová, P., Vondráek, J., Upham, B., and Machala, M. (2002). Inhibition of gap-junctional intercellular communication by environmentally occurring polycyclic aromatic hydrocarbons. Toxicol. Sci. 65, 4351.
Burow, M. E., Weldon, C. B., Chiang, T. C., Tang, Y., Collins-Burow, B. M., Rolfe, K., Li, S., McLachlan, J. A., and Beckman, B. S. (2000). Differences in protein kinase C and estrogen receptor , ß expression, and signaling correlate with apoptotic sensitivity of MCF-7 breast cancer cell variants. Int. J. Oncol. 16, 11791187.[ISI][Medline]
Callahan, M. A., Slimak, M. W., Gabel, N. W., May, I. P., Flower, C. F., Freed, J. R., Jennings, P., DuPree, R. I., Whitmore, F. C., Maestri, B., Mabey, W. R., Holt, B. R., and Gould, C. (1979) Water-related environmental fate of 129 priority pollutants. In EPA-440/479029a and b, Vols. I and II. U.S. EPA, Springfield, VA.
Caristi, S., Galera, J. L., Matarese, F., Imai, M., Caporali, S., Cancemi, M., Altucci, L., Cicatiello, L., Teti, D., Bresciani, F., and Weisz, A. (2001). Estrogens do not modify MAP kinase-dependent nuclear signaling during stimulation of early G(1) progression in human breast cancer cells. Cancer Res. 61, 63606366.
Cenni, B., and Picard, D. (1999). Ligand-independent activation of steroid receptors: New roles for old players. Trends Endocrinol. Metab. 10, 4146.[ISI][Medline]
Chaloupka, K., Krishnan, V., and Safe, S. (1992). Polynuclear aromatic hydrocarbon carcinogens as antiestrogens in MCF-7 human breast cancer cells: Role of the Ah receptor. Carcinogenesis 13, 22332239.[Abstract]
Charles, G. D., Bartels, M. J., Zacharewski, T. R., Gollapudi, B. B., Freshour, N. L, and Carney, E. W. (2000). Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor- reporter gene assay. Toxicol. Sci. 55, 320326.
Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990). Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265, 52675272.
Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998). Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. 273, 1862318632.
Fertuck, K. C., Kumar, S., Sikka, H. C., Matthews, J. B., and Zacharewski, T. R. (2001a). Interaction of PAH-related compounds with the and ß isoforms of the estrogen receptor. Toxicol. Lett. 121, 167177.[ISI][Medline]
Fertuck, K. C., Matthews, J. B., and Zacharewski, T. R. (2001b). Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. Toxicol. Sci. 59, 231240.
Foster, J. R. (1997). The role of cell proliferation in chemically induced carcinogenesis. J. Comp. Pathol. 116, 113144.[ISI][Medline]
Foster, J. S., Henley, D. C., Ahamed, S., and Wimalasena, J. (2001). Estrogens and cell-cycle regulation in breast cancer. Trends Endocrinol. Metab. 12, 320327.[ISI][Medline]
Guttendorf, B., and Westendorf, J. (2001). Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens, and xenoestrogens. Toxicology 166, 7989.[ISI][Medline]
Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette, W. H., Weringer, E. J., Pollok, B. A., and Connelly, P. A. (1996). Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T-cell activation. J. Biol. Chem. 271, 695701.
Improta-Brears, T., Whorton, A. R., Codazzi, F., York, J. D., Meyer, T., and McDonnell, D. P. (1999). Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc. Natl. Acad. Sci. U.S.A. 96, 46864691.
Joel, P. B., Traish, A. M., and Lannigan, D. A. (1998). Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J. Biol. Chem. 273, 1331713323.
Jones, J. M., Anderson, J. W., and Tukey, R. H. (2000). Using the metabolism of PAHs in a human cell line to characterize environmental samples. Environ. Toxicol. Pharmacol. 8, 119126.[ISI][Medline]
Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., et al. (1995). Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270, 14911494.[Abstract]
Kato, S., Masuhiro, Y., Watanabe, M., Kobayashi, Y., Takeyama, K. I., Endoh, H., and Yanagisawa, J. (2000). Molecular mechanism of a cross-talk between oestrogen and growth factor signalling pathways. Genes Cells 5, 593601.
Lobenhofer, E. K., and Marks, J. R. (2000). Estrogen-induced mitogenesis of MCF-7 cells does not require the induction of mitogen-activated protein kinase activity. J. Steroid Biochem. Mol. Biol. 75, 1120.[ISI][Medline]
MacGregor, J. I., and Jordan, V. C. (1998). Basic guide to the mechanisms of antiestrogen action. Pharmacol. Rev. 50, 151196.
Machala, M., Ciganek, M., Bláha, L., Minksová, K., and Vondráek, J. (2001a). Aryl hydrocarbon receptor-mediated and estrogenic activities of oxygenated polycyclic aromatic hydrocarbons and azaarenes originally identified in extracts of river sediments. Environ. Toxicol. Chem. 20, 27362743.[ISI][Medline]
Machala, M., Vondráek, J., Bláha, L., Ciganek, M., and Ne
a, J. V. (2001b). Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay. Mutat. Res. 497, 4962.[ISI][Medline]
Migliaccio, A., Di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E., and Auricchio, F. (1996). Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J. 15, 12921300.[Abstract]
Morreal, C. E., Schneider, S. L., Sinha, D. K., and Bronstein, R. E. (1979). Estrogenic properties of 3,9-dihydroxy-7,12-dimethylbenz[a]anthracene in rats. J. Natl. Cancer Inst. 62, 15851588.[ISI][Medline]
Pestell, R. G., Albanese, C., Reutens, A. T., Segall, J. E., Lee, R. J., and Arnold, A. (1999). The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation. Endocr. Rev. 20, 501534.
Piskorska-Pliszczynska, J., Keys, B., Safe, S., and Newman, M. S. (1986). The cytosolic receptor-binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons. Toxicol. Lett. 34, 6774.[ISI][Medline]
Pons, M., Gagne, D., Nicolas, J. C., and Mehtali, M. (1990). A new cellular model of response to estrogens: A bioluminescent test to characterize (anti)-estrogen molecules. Biotechniques 9, 450459.[ISI][Medline]
Rummel, A. M., Trosko, J. E., Wilson, M. R., and Upham, B. L. (1999). Polycyclic aromatic hydrocarbons with bay-like regions inhibited gap junctional intercellular communication and stimulated MAPK activity. Toxicol. Sci. 49, 232240.[Abstract]
Safe, S. (2001). Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 120, 17.[ISI][Medline]
Santodonato, J. (1997). Review of the estrogenic and antiestrogenic activity of polycyclic aromatic hydrocarbons: Relationship to carcinogenicity. Chemosphere 34, 835848.[ISI][Medline]
Schneider, S. L., Alks, V., Morreal, C. E., Sinha, D. K., and Dao, T. L. (1976). Estrogenic properties of 3,9-dihydroxybenz[a]anthracene, a potential metabolite of benz[a]anthracene. J. Natl. Cancer Inst. 57, 13511354.[ISI][Medline]
Silva, E., Rajapakse, N., and Kortenkamp, A. (2002). Something from "nothing"eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. 36, 17511756.[ISI][Medline]
Soto, A. M., Sonnenschein, C., Chung, K. L., Fernandez, M. F., Olea, N., and Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. Environ. Health Perspect. 103(Suppl. 7), 113122.[ISI][Medline]
Tannheimer, S. L., Ethier, S. P., Caldwell, K., and Burchiel, S. W. (1998). Benzo[a]pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis 19, 12911297.[Abstract]
Tannheimer, S. L., Lauer, F. T., Lane, J., and Burchiel, S. W. (1999). Factors influencing elevation of intracellular Ca2+ in the MCF-10A human mammary epithelial cell line by carcinogenic polycyclic aromatic hydrocarbons. Mol. Carcinog. 25, 4854.[ISI][Medline]
Toullec, D., Pianetti, P., Coste, H., Bellevergue, P., Grand-Perret, T., Ajakane, M., Baudet V., Boissin, P., Boursier, E., Loriolle, F., et al. (1991). The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 266, 1577115781.
Vindelov, L. L. (1977). Flow microfluorimetric analysis of nuclear DNA in cells from solid tumors and cell suspensions. A new method for rapid isolation and staining of nuclei. Virchows Arch. B Cell Pathol. 24, 227242.[ISI]
Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 52415248.
Wakeling, A. E. (1995). Use of pure antioestrogens to elucidate the mode of action of oestrogens. Biochem. Pharmacol. 49, 15451549.[ISI][Medline]
Wang, W., Smith, R., III, and Safe, S. (1998). Aryl hydrocarbon receptor-mediated antiestrogenicity in MCF-7 cells: Modulation of hormone-induced cell cycle enzymes. Arch. Biochem. Biophys. 356, 239248.[ISI][Medline]
WHO (1998). Selected Non-Heterocyclic Polycyclic Aromatic Hydrocarbons, Vol. 202. World Health Organization, Geneva.