Reproductive Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received August 17, 2001; accepted November 19, 2001
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ABSTRACT |
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Key Words: adenovirus; transduction; screening; assay; androgen receptor; human; MDA, CV-1.
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INTRODUCTION |
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In this endeavor we have developed several in vitro approaches to screen for AR activity using gene expression assays. Historically, this type of in vitro assay involves the transient transfection of a tester cell line with a plasmid base receptor and the reporter, followed by chemical exposure and measurement of the modulation of gene expression. These types of assays require relatively large numbers of cells and costly transfection material, with each transfection event introducing yet another source of experimental variation. The KB2 assay, a significant improvement on this type of assay system, was presented recently (Wilson et al., 2002). In the aforementioned assay, we describe the effects of androgens and antiandrogens in a stably transformed cell line that we developed (MDA-KB2), which expresses the human AR (hAR) and an AR-responsive promoter linked to a luciferase reporter gene (MMTV-luc). The main advantage of the KB2 assay is that it employs a genetically modified cell line, which eliminates the effort and inherent variability associated with repeated transient transfections. The approach taken in this study was to attempt to deliver the genes via replication-defective adenovirus (transduction). Viral transduction is a precise and reproducible way of delivering genes in a cost-effective manner that should increase the robustness of the response to androgens and antiandrogens as compared to the transformed MDA-KB2 cell line and should reduce the variability as compared to transient transfected AR assays.
Herein, we describe the responses of several androgens and antiandrogens in two AR-responsive in vitro assays. These assays used transduced MDA-453 and CV-1 cells, cell lines employed extensively in our laboratory. The chemicals and concentrations used in the current study are some that we have extensive experience with and that were utilized in the phenotypically transformed, AR-responsive KB2 cells (Wilson, et al., 2002). In this study we have assessed the ability of an adenovirus to transduce the human breast cancer line MDA-MB-453 (AR+, GR+, PR-, ERa-, weak ERb+) with a luciferase gene regulated by the androgen- and glucocorticoid-inducible hormone response element found in the mouse mammary tumor virus (MMTV) LTR. Utilization of MDA cells, which already contained an endogenous AR, simplified the development of this assay by reducing the transduction requirements to a single reporter gene. In addition, we also assessed the ability of an adenovirus to transduce the CV-1 (African green monkey kidney cells: AR-, GR-, ER-, PR-) (Coutte et al., 1994
; Fenton et al., 1997
; Vamvakopoulous et al., 1993) with both the MMTV-luc reporter and the human androgen receptor (hAR) genes. The responses of each assay to AR agonists, AR antagonists, and GR agonists are described (Table 1
).
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MATERIALS AND METHODS |
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Ad5hAR was generated by subcloning the hAR expression cassette of pCMVhAR into pE1sp1A (see Fig. 1). pCMVhAR was digested with BrsB1 and Fsp1. Digestion liberated a blunt-ended, 5124-bp fragment (bp 22 to 5145) containing the hAR cDNA flanked by promoter and polyadenylation signals. The fragment was blunt-end cloned into the Eco RV site of pE1sp1A in an E1-antiparallel orientation yielding plasmid p
E1sp1AhAR. pBHG 11(7 µg) and p
E1sp1AhAR (9 µg) were mixed and applied to a 35-mm dish containing a monolayer of 293 using the reduced-CO2 transfection method (Sambrook, et al., 1989
). After overnight transfection, the cells were washed twice in medium, then incubated in 5% CO2 in medium containing reduced (5%) serum, and fed when medium became acidic. After 6 weeks, some cultures presented with cytopathic effects consistent with adenovirus infection. Fluids from infected cultures were saved and subjected to plaque assays for purification. Isolated clones were collected, replaqued twice, and the viral DNA was isolated, and the insert identity and orientation confirmed by restriction analysis (Sambrook et al., 1989
).
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Initial viral stocks contained about 1 x 109 plaque-forming units (pfu)/ml. Virus was propagated by inoculating 150-mm dishes containing 293 cells which were 80% confluent. Medium was removed, inocula instilled, and dishes rocked every 15 min for 1 h. Then 20 ml of fresh media was added. The cells were incubated 23 days until 95% of the cells exhibited cytopathic effect. Progeny virus remains associated with cell debris. Cells were scraped off the dish and the cells and media centrifuged at 700 x g for 5 min. The cell pellet was suspended in 1/20th of its original volume in spent media containing 10% glycerol, suspended, and subjected to 3 cycles of freeze-thawing to release the virions from the cell debris. Virus was dispensed in 50-µl aliquots and frozen. Virus was assayed on 293 cells by standard plaque assay (Graham and Prevec, 1991). Briefly, 35-mm cluster dishes were seeded with 293 cells at least 24 h prior to the assay. When monolayers were 80% confluent, medium was removed and 200 µl of serial 10-fold dilutions of the virus added. Dishes were rocked every 15 min for 1 h. Inoculum was removed and agarose overlay added. Dishes were placed at 4°C for 10 min., then incubated for 6 days at 37°C at 100% humidity in 5% CO2. Overlay was prepared by making 2x growth medium, warming it to 37°C, and mixing it with 37°C, 2% aqueous SeaPlaque low gelling temp agarose (FMC Corp., Rockland, ME), mixing, incubating at 37°C for 10 min, and adding 3 ml overlay/well. After incubating for 6 days, 2 ml additional overlay containing 50 µg/ml neutral red was added and dishes incubated overnight. Plaques appeared as light areas on a red background. Values were reported as plaque-forming units (pfu)/ml. Each value was the mean of at least 2 dishes.
Transduction assay.
Transduction assays were performed in 96-well plates. Twenty-four h prior to transduction, 5 x 104 cells were plated per well. Medium was removed and replaced with 20 µl of control medium or medium with diluted virus. MDA cells (which contain endogenous AR) were transduced with Ad/mLuc7 reporter virus at a multiplicity of infection (MOI) of 50 (i.e., 50 virions per cell). CV-1 cells (which lack native AR) were transduced with Ad/mLuc7 at a MOI of 50 and Ad5hAR at a MOI of one. Dishes were rocked every 15 min for 1 h, incubated 3 additional h, then 200 µl of medium or medium and test chemicals were added to each well, followed by a 48-h incubation. Plates were washed 2x with PBS, pH 7.4, decanted, and 25 µl cell culture lysis reagent (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N`,N` tetra-acetic acid, 10% glycerol, 1% Triton X-100 (Promega Corp., Madison WI)] added, and incubated 30 min., or until cells lysed. Plates were either frozen at 80°C, or assayed immediately for luciferase activity. Each well received 25 µl reaction buffer (25 mM glycylglycine, 15 mM MgCl2, 5 mM ATP, 0.1 mg/ml BSA, pH 7.8), followed by 25 µl 1-mM D-luciferin 5 s later. Luciferase activity was quantitated in an MLX microtiter plate luminometer (Dynex Tech, Chantilly, VA) and data expressed in relative light units (RLU).
Chemicals.
All chemicals were purchased from Sigma (purity >99%; St. Louis, MO) unless stated otherwise. The antiestrogen, ICI 182780 (ICI) was supplied by ICI Pharmaceuticals (Macclesfield, England). Hydroxyflutamide (OHF) was provided by R.O. Neri at Schering Corp. (Bloomfield, NJ). (4-[2,2-Dichloro-1-(4-hydroxyphenyl)vinyl]phenol), (OH-DDE: lot c1f03065, purity = 100%) was purchased from SPECS and BioSPECS B.V. (Rijswijk, Netherlands). Vinclozolin metabolite, M2, was obtained from BASF Ag and metabolite, M1, was synthesized from vinclozolin and purified as previously described (Kelce et al., 1994). Synthesis of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE), was previously described (Waller et al., 1996
). The chemicals and dosage levels selected for these 2 assays were chosen because they produce agonist and/or antagonist responses in the MDA-KB2 and CV-1 cell lines in our laboratory (Wilson et al., 2002
).
Data collection and analysis.
The data were collected from several independent experiments, with 34 replicates/plates per experiment. For each cell line, the individual experiments were (1) 5 -dihydrotestosterone (DHT) dose response; (2) medroxyprogesterone acetate (MPA) dose response; (3) 17-ß-estradiol (E2) dose response with and without the antiandrogen hydroxyflutamide (OHF) or the antiestrogen ICI; (4) dose response with dexamethasone, a synthetic corticosteroid (DEX); (5) different doses of the vinclozolin metabolites M1 and M2, with and without 0.1 nM DHT; (6) dose response with OH-DDE with and without 0.1 nM DHT; and (7) dose-response effects of HPTE, the estrogenic and antiandrogenic metabolite of the insecticide methoxychlor (Gaido et al., 2000
) with and without 0.1 nM DHT. A replicate was a 96-well plate, which included 48 independent observations of the media control (plus Et-OH, the dosing solution) and all other treatment groups. Hence, the design is a randomized, complete block design (the term block being equivalent to a plate, referred to herein as a replicate).
Data were analyzed by two-way ANOVA using PROC GLM available with SAS version 6.08 on the U.S. EPA`s IBM mainframe. Relative light units (RLU), fold, and log10 fold data were analyzed in a GLM model, which included the concentrations and replicates (most chemicals being run in 3 replicate assays). Using "replicates" as a blocking factor in the analysis has the effect of "normalizng" the data for overall differences from plate to plate, on average.
Luciferase response.
Statistically significant effects (p < 0.01, F statistic) were examined using the LSMEANS procedure (t-test). Means and standard errors (SE) were calculated using PROC means. In this regard, the SE in the tables are not corrected for replicate variation. For androgen agonists, which stimulate luciferase expression, treatments were compared to the media/ethanol control group, while androgen antagonists, which block DHT-induced luciferase expression, were compared to the 0.1 nM DHT group. Relative light units were converted to fold induction above the media value for each replicate, which in turn were log10 transformed (to correct for heterogeneity of variance, the SD being proportional to the means) for statistical analysis.
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RESULTS |
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MPA and DEX were less effective in the transduced CV-1 cells than in the MDA-453 cells. MPA induced luc expression by about 10-fold at 0.010.05 µM, which was the maximum induction attained (Fig. 4, Table 2
). Dexamethasone induced luc by 320-fold, a level that was only about 5% of the value seen in the MDA cells at 100 nM (Table 2
).
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DISCUSSION |
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In the CV-1 cells (over all replicates), 0.1 nM DHT induced luciferase activity 45-fold (log10 fold = 1.55 vs. 0 for media control) and displayed an intra-assay CV of 20% for fold induction and a CV of 5% for log10 fold data. When 0.1 nM DHT was coadministered with 1 µM hydroxyflutamide in these replicates, luciferase activity was reduced by about 80% to 8.2-fold (log10 = 0.84), which is still significantly above the media control value. Maximal luc induction of about 63.8-fold was attained at 1 nM DHT.
Across replicates using the MDA 453 transduced cells, 0.1 nM DHT induced luc by 23.7-fold (intra-assay CV of 34%) while log10 fold induction was 1.198 (intra-assay CV of 10%). Ten to 100 nM DHT induced maximal luc expression of 100- to 116-fold, respectively.
Consistent with published studies (Table 1), several chemicals displayed mixed activity: they exhibited behavior of AR antagonists at lower concentrations and agonists at higher concentrations (Table 3
). M2, M1 (reported by Wong et al., 1995
), and OH-DDE displayed weak agonist activity at higher concentrations, effects that typically were more apparent in the CV-1 cell line. In this cell line, the relative potency of these chemicals as agonists was M2 (17-fold) > M1 (9.1-fold) = OH-DDE (8.4-fold) > HPTE (which displayed statistically significant but negligible agonist activity of 2.5-fold). In the MDA cells, M2 induced luc by almost 5-fold.
There did not appear to be a major advantage of one cell line over the other. E2 induced luciferase in transduced CV-1 cells to a greater degree at lower concentrations than it did in MDA cells. Furthermore, the effectiveness of HPTE as an AR antagonist was more apparent in the CV cells than in the MDA cells. In addition, the mixed agonist/antagonist activity of the compounds was more evident in the CV-1 cell assay than in the MDA 453 cell line. On the other hand, MPA, which is both an AR and GR agonist, was more potent in inducing luciferase in the MDA 453 cells, which have GR as well as AR activity, than in the CV-1 cell line, which has little or no functional levels of GR. Similarly, dexamethasone, a potent GR agonist, induced luc by 248-fold in the MDA cells but only by 9.3-fold in the CV-1 cell (Table 2). These results suggest that MPA and DEX are acting, at least in part, via GR in the MDA cells. If the objective were to screen for both AR and GR activities, then the MDA cell assay would be more useful, while the transduced CV-1 cells provide greater specificity in displaying AR-mediated responses.
Transduction, the delivery of genes to cells and tissues by replication-deficient viruses, has been studied extensively (Graham, 2000; Haddada et al., 1995
; Hitt et al., 1997
; Hartig and Hunter, 1998a
; Hunter and Hartig, 2000
). While the technology has only recently been applied to toxicology studies (Hartig and Hunter, 1998b
), it is a basic and well understood technique of molecular biology. Like transfection, it allows for the delivery of genetic material to the target cells. Unlike transfection, in which the DNA is delivered in a dynamic chemical solution or suspension, in transduction the DNA is packaged within the protein coat of a virus particle that has lost the capacity to replicate. Owing to the virus`s stable particulate nature, the dose of a gene (i.e., virus) to a cell can be easily quantified and duplicated with precision. Because the virus is replication-defective, it presents no hazard of infection. In fact during this study, the only exposure risk that required special management was to the chemicals and pesticides being tested. Transduction and transfection techniques require similar facilities and both can be accomplished fairly simply, requiring only basic laboratory equipment (e.g., a tissue culture hood and a CO2 incubator).
Cell lines stably transformed with steroid hormone receptor and/or reporters have recently become useful for evaluating xenobiotic perturbations of transcription activation (Terouanne et al., 2000; Wilson et al., 2002). These cell lines were derived via transfection, followed by antibiotic selection and clonal expansion. Ideally, this methodology produces a stable cell population that will respond uniformly to exogenous stimuli. However, a considerable investment of resources is required to produce each cell line, and if it becomes desirable to change the parental cell line, then another round of transfection and antibiotic selection is necessary. In contrast, any cell permissive to transduction can be substituted for any other cell, modifying the assay only minimally as required for the optimum culture condition of the new cell line (i.e., medium formulation, etc.).
Conclusions.
Adenovirus transduction provides a valuable method for delivering exogenous genes. The behavior of the transduced genes can be utilized to assess endocrine-disrupting chemicals. The androgen- and glucocorticoid-regulated luciferase gene (MMTV promoter) responds similarly to chemical stimulus whether it is delivered by transfection or transduction, or is stably integrated into the cellular genome. Transduction utilizes the innate cellular entry mechanisms of the parental virus. Because adenovirus can enter a wide variety of cells, this method should allow the efficient and cost-effective delivery of genes to various cell lines with different compliments of endogenous receptors and co factors. The ability to easily transduce numerous cell lines should facilitate the studies of chemical/receptor interaction.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at MD 67,ORD, NHEERL, RTP, NC 27711. Fax: (919) 541-4017. E-mail: hartig.phillip{at}epa.gov.
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REFERENCES |
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Coutte, B., Le Ricousse, C., Fortin, D., Rafestin-Oblin, M. E., and Richard Foy, H. (1994). The establishment of the long terminal repeat of the mouse mammary tumor virus into CV-1 cells allows a functional analysis of steroid receptors. Biochem. Biophys. Acta 1219, 607612.[ISI][Medline]
Fenton, M. A., Shuster, T. D., Fertig, A. M., Taplin, M. E., Kolvenbag, G., Bubley, G. J., and Balk, S. P. (1997). Functional characterization of mutant androgen receptors from androgen-independent prostate cancer. Clin. Cancer Res 3, 13831388.[Abstract]
Gaido, K. W., Maness, S. C., McDonnell, D. P., Dehal, S. S., Kupfer, D., and Safe, S. (2000). Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: Structure-activity studies. Mol. Pharmacol. 58, 852858.
Graham, F. L. 2000. Adenovirus vectors for high-efficiency gene transfer into mammalian cells. Immunol. Today 21, 426428.[ISI][Medline]
Graham, F. L., and Prevec, L. (1991). Manipulation of adenovirus vectors. Methods Mol. Biol. 7, 109128.
Haddada, H., Cordier, L., and Perricaudet, M. (1995). Gene therapy using adenovirus vectors. Curr. Top. Microbiol. Immunol. 199, 297306.[ISI][Medline]
Hartig, P. C., and Hunter, E. S., III (1998a). Gene delivery to the neurulating embryo during culture. Teratology 58, 103112.[ISI][Medline]
Hartig, P. C., and Hunter, E. S., III (1998b). Effects of over-expression of human p53 on arsenite-induced dysmorphology on mouse embryos in culture. Teratology 75, 215.
Hitt, M. M., Addison, C. L., and Graham, F. L. (1997). Human adenovirus vectors for gene transfer into mammalian cells. Gene Ther. 40, 137206.
Hunter, E. S., III, and Hartig, P. (2000). Transient modulation of gene expression in the neurulation-staged mouse embryo. Ann. N.Y. Acad. Sci. 919, 278283.
Kelce, W. R., Monosson, E., Gamcsik, M. P., Laws, S. C., and Gray, L. E., Jr. (1994). Environmental hormone disruptors: Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol. Appl. Pharmacol. 126, 276285.[ISI][Medline]
Kemppainen, J. A., Langley, E., Wong, C. I., Bobseine, K., Kelce, W. R., and Wilson, E. M. (1999). Distinguishing androgen receptor agonists and antagonists: Distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. Mol. Endocrinol. 13, 440454.
Kontula, K., Paavonen, T., Luukkainen, T., and Andersson, L. C. (1983). Binding of progestins to the glucocorticoid receptor. Correlation to their glucocorticoid-like effects on in vitro functions of human mononuclear leukocytes. Biochem. Pharmacol. 32, 15111518.[ISI][Medline]
Lubahn, D. B., Joseph, D. R., Sar, M., Tan, J., Higgs, H. N., Larson, R. E., French, F. S., and Wilson, E. M. (1988). The human androgen receptor: Complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol. Endocrinol. 2, 12651275.[Abstract]
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Shih, W., Mears, T., Bradley, D. J., Parandoosh, Z., and Weinberger, C. (1991). An adenoviral vector system for functional identification of nuclear receptor ligands. Mol. Endocrinol. 5, 300309.[Abstract]
Vamvakopoulous, N. C., and Chrousos, G. P. (1993). Evidence of direct estrogenic regulation of human corticotropin-releasing hormone gene expression. Potential implications for the sexual dimophism of the stress response and immune/inflammatory reaction. J. Clin. Invest. 92, 18961902.[ISI][Medline]
Waller, C. L., Juma, B. W., Gray, L.E., Jr., and Kelce, W. R., (1996). Three-dimensional quantitative structure-activity relationships for androgen receptor ligands. Toxicol. Appl. Pharmacol. 37, 219227.
Wilson, V. S., Bobseine, K. L., Lambright C. R., and Gray, L. E., Jr. (2002). A novel cell line, MDA-kb2, that stably expresses an androgen and glucocorticoid responsive reporter for the detection of hormone receptor agonists and antagonists. Toxicol. Sci. 66, 6981.
Wong, C., Kelce, W. R., Sar, M., and Wilson, E. M. (1995). Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 1999820003.
Zhang, Z., Lundeen, S. G., Zhu, Y., Carver, J. M., and Winneker R. C. (2000). In vitro characterization of trimegestone: A new potent and selective progestin. Steroids 65, 637643.[ISI][Medline]