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Immortalized Mouse Mammary Fibroblasts Lacking Dioxin Receptor Have Impaired Tumorigenicity in a Subcutaneous Mouse Xenograft Model

2005; Elsevier BV; Volume: 280; Issue: 31 Linguagem: Inglês

10.1074/jbc.m504538200

ISSN

1083-351X

Autores

Sonia Mulero‐Navarro, Eulalia Pozo‐Guisado, Pedro A. Pérez–Mancera, Alberto Álvarez, Inmaculada Catalina‐Fernández, Emilia Hernández‐Nieto, Javier Sáenz‐Santamaría, Natalia J. Martinez, José M. Rojas, Isidro Sánchez‐García, Pedro M. Fernández‐Salguero,

Tópico(s)

Cancer, Hypoxia, and Metabolism

Resumo

Although the dioxin receptor, the aryl hydrocarbon receptor (AhR), is considered a major regulator of xenobiotic-induced carcinogenesis, its role in tumor formation in the absence of xenobiotics is still largely unknown. Trying to address this question, we have produced immortalized cell lines from wild-type (T-FGM-AhR+/+) and mutant (T-FGM-AhR-/-) mouse mammary fibroblasts by stable co-transfection with the simian virus 40 (SV-40) large T antigen and proto-oncogenic c-H-Ras. Both cell lines had a myofibroblast phenotype and similar proliferation, doubling time, SV-40 and c-H-Ras expression and activity, and cell cycle distribution. AhR+/+ and AhR-/- cells were also equally able to support growth factor- and anchorage-independent proliferation. However, the ability of T-FGM-AhR-/- to induce subcutaneous tumors (leimyosarcomas) in NOD/SCID-immunodeficient mice was close to 4-fold lower than T-FGM-AhR+/+. In culture, T-FGM-AhR-/- had diminished migration in collagen-I and decreased lamellipodia formation. VEGFR-1/Flt-1, a VEGF receptor that regulates cell migration and blood vessel formation, was also down-regulated in AhR-/- cells. Signaling through the ERK-FAK-PKB/AKT-Rac-1 pathway, which contributes to cell motility and invasion, was also significantly inhibited in T-FGM-AhR-/-. Thus, the lower tumorigenic potential of T-FGM-AhR-/- could result from a compromised adaptability of these cells to the in vivo microenvironment, possibly because of an impaired ability to migrate and to respond to angiogenesis. Although the dioxin receptor, the aryl hydrocarbon receptor (AhR), is considered a major regulator of xenobiotic-induced carcinogenesis, its role in tumor formation in the absence of xenobiotics is still largely unknown. Trying to address this question, we have produced immortalized cell lines from wild-type (T-FGM-AhR+/+) and mutant (T-FGM-AhR-/-) mouse mammary fibroblasts by stable co-transfection with the simian virus 40 (SV-40) large T antigen and proto-oncogenic c-H-Ras. Both cell lines had a myofibroblast phenotype and similar proliferation, doubling time, SV-40 and c-H-Ras expression and activity, and cell cycle distribution. AhR+/+ and AhR-/- cells were also equally able to support growth factor- and anchorage-independent proliferation. However, the ability of T-FGM-AhR-/- to induce subcutaneous tumors (leimyosarcomas) in NOD/SCID-immunodeficient mice was close to 4-fold lower than T-FGM-AhR+/+. In culture, T-FGM-AhR-/- had diminished migration in collagen-I and decreased lamellipodia formation. VEGFR-1/Flt-1, a VEGF receptor that regulates cell migration and blood vessel formation, was also down-regulated in AhR-/- cells. Signaling through the ERK-FAK-PKB/AKT-Rac-1 pathway, which contributes to cell motility and invasion, was also significantly inhibited in T-FGM-AhR-/-. Thus, the lower tumorigenic potential of T-FGM-AhR-/- could result from a compromised adaptability of these cells to the in vivo microenvironment, possibly because of an impaired ability to migrate and to respond to angiogenesis. Many studies over the past decade have characterized the aryl hydrocarbon receptor (AhR) 1The abbreviations used are: AhR, aryl hydrocarbon receptor; CRIB, Rac-1 binding domain; DAPI, 4,6-diamino-2-phenylindol; ERK, mitogenic extracellular signal-regulated kinase; MMP, matrix metalloproteinase; PKB/AKT, protein kinase B; RBD, Ras binding domain; SV-40, simian virus 40 large T antigen; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; T-FGM-AhR+/+, transformed mammary gland primary fibroblasts from AhR+/+ mice; T-FGM-AhR-/-, transformed mammary gland primary fibroblasts from AhR-/- mice; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PI3K, phosphatidylinositol 3-kinase; FAK, focal adhesion kinase; ANOVA, analysis of variance; MAPK, mitogen-activated protein kinase; FBS, fetal bovine serum; Hif-1α, hypoxia-inducible factor 1α gene; Ang-1, angio-poietin-1 gene; Epo, erythropoietin gene. 1The abbreviations used are: AhR, aryl hydrocarbon receptor; CRIB, Rac-1 binding domain; DAPI, 4,6-diamino-2-phenylindol; ERK, mitogenic extracellular signal-regulated kinase; MMP, matrix metalloproteinase; PKB/AKT, protein kinase B; RBD, Ras binding domain; SV-40, simian virus 40 large T antigen; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; T-FGM-AhR+/+, transformed mammary gland primary fibroblasts from AhR+/+ mice; T-FGM-AhR-/-, transformed mammary gland primary fibroblasts from AhR-/- mice; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PI3K, phosphatidylinositol 3-kinase; FAK, focal adhesion kinase; ANOVA, analysis of variance; MAPK, mitogen-activated protein kinase; FBS, fetal bovine serum; Hif-1α, hypoxia-inducible factor 1α gene; Ang-1, angio-poietin-1 gene; Epo, erythropoietin gene. as a regulator of the toxic and carcinogenic responses to environmental contaminants such as dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) (1.Fernandez-Salguero P.M. Hilbert D.M. Rudikoff S. Ward J.M. Gonzalez F.J. Toxicol. Appl. Pharmacol. 1996; 140: 173-179Crossref PubMed Scopus (699) Google Scholar, 2.Shimizu Y. Nakatsuru Y. Ichinose M. Takahashi Y. Kume H. Mimura J. Fujii-Kuriyama Y. Ishikawa T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 779-782Crossref PubMed Scopus (533) Google Scholar, 3.Nebert D.W. Dalton T.P. Okey A.B. Gonzalez F.J. J. Biol. Chem. 2004; 279: 23847-23850Abstract Full Text Full Text PDF PubMed Scopus (974) Google Scholar, 4.Safe S. Toxicol. Lett. 2001; 120: 1-7Crossref PubMed Scopus (274) Google Scholar, 5.Abbott B.D. Held G.A. Wood C.R. Buckalew A.R. Brown J.G. Schmid J. Toxicol. Sci. 1999; 47: 62-75Crossref PubMed Scopus (47) Google Scholar). Although very important, this xenobiotic-related activity of the AhR does not appear to be its only function in the organism. The large degree of conservation of this receptor among species (6.Hahn M.E. Chem. Biol. Interact. 2002; 141: 131-160Crossref PubMed Scopus (504) Google Scholar), its constitutive pattern of expression during development and in adult tissues (7.Abbott B.D. Birnbaum L.S. Perdew G.H. Dev. Dyn. 1995; 204: 133-143Crossref PubMed Scopus (164) Google Scholar), and the many phenotypic alterations found in mice lacking AhR expression (1.Fernandez-Salguero P.M. Hilbert D.M. Rudikoff S. Ward J.M. Gonzalez F.J. Toxicol. Appl. Pharmacol. 1996; 140: 173-179Crossref PubMed Scopus (699) Google Scholar, 8.Abbott B.D. Schmid J.E. Pitt J.A. Buckalew A.R. Wood C.R. Held G.A. Diliberto J.J. Toxicol. Appl. Pharmacol. 1999; 155: 62-70Crossref PubMed Scopus (172) Google Scholar, 9.Fernandez-Salguero P. Pineau T. Hilbert D.M. McPhail T. Lee S.S. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Crossref PubMed Scopus (934) Google Scholar, 10.Fernandez-Salguero P.M. Ward J.M. Sundberg J.P. Gonzalez F.J. Vet. Pathol. 1997; 34: 605-614Crossref PubMed Scopus (301) Google Scholar, 11.Lahvis G.P. Lindell S.L. Thomas R.S. McCuskey R.S. Murphy C. Glover E. Bentz M. Southard J. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10442-10447Crossref PubMed Scopus (310) Google Scholar, 12.Mimura J. Yamashita K. Nakamura K. Morita M. Takagi T.N. Nakao K. Ema M. Sogawa K. Yasuda M. Katsuki M. Fujii-Kuriyama Y. Genes Cells. 1997; 2: 645-654Crossref PubMed Scopus (541) Google Scholar, 13.Schmidt J.V. Bradfield C.A. Annu. Rev. Cell Dev. Biol. 1996; 12: 55-89Crossref PubMed Scopus (796) Google Scholar) have provided strong support for the involvement of the AhR in cell physiology independent of xenobiotic metabolism.Novel mechanisms of activation have been described that may associate the AhR to endogenous functions. Thus, this receptor is activated by the natural compounds indirubin and indigo (14.Adachi J. Mori Y. Matsui S. Takigami H. Fujino J. Kitagawa H. Miller 3rd, C.A. Kato T. Saeki K. Matsuda T. J. Biol. Chem. 2001; 276: 31475-31478Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), diosmin and diosmetin (15.Ciolino H.P. Wang T.T. Yeh G.C. Cancer Res. 1998; 58: 2754-2760PubMed Google Scholar), and by metabolites produced by the aspartate aminotransferase (16.Bittinger M.A. Nguyen L.P. Bradfield C.A. Mol. Pharmacol. 2003; 64: 550-556Crossref PubMed Scopus (84) Google Scholar). An AhR repressor was identified that regulates AhR activity by binding and sequestering the aryl hydrocarbon receptor nuclear translocator (ARNT) (17.Mimura J. Ema M. Sogawa K. Fujii-Kuriyama Y. Genes Dev. 1999; 13: 20-25Crossref PubMed Scopus (431) Google Scholar). Additional work has also shown that protein kinase C cooperates with ligand binding for receptor activation (18.Carrier F. Owens R.A. Nebert D.W. Puga A. Mol. Cell. Biol. 1992; 12: 1856-1863Crossref PubMed Scopus (152) Google Scholar, 19.Long W.P. Pray-Grant M. Tsai J.C. Perdew G.H. Mol. Pharmacol. 1998; 53: 691-700Crossref PubMed Scopus (137) Google Scholar) and that proteasome inhibition activates the AhR in mouse embryo primary fibroblasts in the absence of xenobiotics (20.Santiago-Josefat B. Fernandez-Salguero P.M. J. Mol. Biol. 2003; 333: 249-260Crossref PubMed Scopus (23) Google Scholar, 21.Santiago-Josefat B. Pozo-Guisado E. Mulero-Navarro S. Fernandez-Salguero P.M. Mol. Cell. Biol. 2001; 21: 1700-1709Crossref PubMed Scopus (61) Google Scholar).Among the physiological functions that could require AhR activity, the regulation of the cell cycle and the control of cell proliferation are the best analyzed (reviewed in Ref. 22.Puga A. Xia Y. Elferink C. Chem. Biol. Interact. 2002; 141: 117-130Crossref PubMed Scopus (144) Google Scholar); yet, the role of this receptor in cell proliferation is still controversial because it can promote or block cell cycle progression. Several studies support the AhR as an oncoprotein: (i) AhR-defective (AhR-D) mouse hepatoma cells had a prolonged duplication time because of a delayed G1/S transition (23.Ma Q. Whitlock Jr., J.P. Mol. Cell. Biol. 1996; 16: 2144-2150Crossref PubMed Scopus (243) Google Scholar); (ii) mouse embryo fibroblasts (MEF) lacking AhR entered senescence much earlier than wild-type cells (24.Alexander D.L. Ganem L.G. Fernandez-Salguero P. Gonzalez F. Jefcoate C.R. J. Cell Sci. 1998; 111: 3311-3322Crossref PubMed Google Scholar) and had a diminished proliferation rate and increased levels of transforming growth β-1 (TGFβ-1) (25.Elizondo G. Fernandez-Salguero P. Sheikh M.S. Kim G.Y. Fornace A.J. Lee K.S. Gonzalez F.J. Mol. Pharmacol. 2000; 57: 1056-1063PubMed Google Scholar, 26.Santiago-Josefat B. Mulero-Navarro S. Dallas S.L. Fernandez-Salguero P.M. J. Cell Sci. 2004; 117: 849-859Crossref PubMed Scopus (44) Google Scholar); (iii) the AhR was relevant in DNA synthesis mediated by p300 and the adenovirus E1A (27.Tohkin M. Fukuhara M. Elizondo G. Tomita S. Gonzalez F.J. Mol. Pharmacol. 2000; 58: 845-851Crossref PubMed Scopus (38) Google Scholar); and finally, (iv) constitutive activation of this receptor increased hepatocarcinogenesis in transgenic B6C3F1 mice (28.Moennikes O. Loeppen S. Buchmann A. Andersson P. Ittrich C. Poellinger L. Schwarz M. Cancer Res. 2004; 64: 4707-4710Crossref PubMed Scopus (192) Google Scholar). The AhR also has tumor suppressor activity. It arrests cell proliferation at the G1/S transition in 5L rat hepatoma cells through interaction with hypophosphorylated retinoblastoma (pRb) protein (29.Ge N.L. Elferink C.J. J. Biol. Chem. 1998; 273: 22708-22713Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In mouse hepatoma Hepa-1 and in human breast cancer MCF-7 cells, TCDD induced AhR binding and displacement of p300 from E2F-dependent promoters (30.Marlowe J.L. Knudsen E.S. Schwemberger S. Puga A. J. Biol. Chem. 2004; 279: 29013-29022Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). A constitutively active AhR induced apoptosis in Jurkat T cells by arresting cell cycle progression at G1 (31.Ito T. Tsukumo S. Suzuki N. Motohashi H. Yamamoto M. Fujii-Kuriyama Y. Mimura J. Lin T.M. Peterson R.E. Tohyama C. Nohara K. J. Biol. Chem. 2004; 279: 25204-25210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and AhR activation by ligand binding blocked cell cycle by increasing p27Kip1 expression (32.Kolluri S.K. Weiss C. Koff A. Gottlicher M. Genes Dev. 1999; 13: 1742-1753Crossref PubMed Scopus (304) Google Scholar). In this context, a recent report has shown that while the AhR had growth inhibitory activity in epithelial MCF-7 cells, it promoted proliferation in HepG2 hepatoma cells, further suggesting that the contribution of this receptor to proliferation is cell type-dependent (33.Abdelrahim M. Smith 3rd, R. Safe S. Mol. Pharmacol. 2003; 63: 1373-1381Crossref PubMed Scopus (138) Google Scholar). Thus, although the contribution of the AhR to cell proliferation is strongly supported, its role in tumor development in the absence of xenobiotics remains largely unknown.It is increasingly recognized that the stroma plays a major role in determining the rate of tumor growth and invasiveness in vivo (34.Noel A. Foidart J.M. J. Mammary Gland Biol. Neoplasia. 1998; 3: 215-225Crossref PubMed Scopus (83) Google Scholar, 35.Weaver V.M. Fischer A.H. Peterson O.W. Bissell M.J. Biochem. Cell Biol. 1996; 74: 833-851Crossref PubMed Scopus (168) Google Scholar). Fibroblasts, as one of the most abundant cell types in connective tissue, regulate the synthesis, degradation, and remodeling of the extracellular matrix. Fibroblasts are particularly important in diseases such as breast cancer in which their conversion to smooth muscle α-actin-expressing myofibroblasts is considered a stromal reaction to the invading epithelial tumor cells (desmoplastic reaction) (36.Ronnov-Jessen L. Petersen O.W. Bissell M.J. Physiol. Rev. 1996; 76: 69-125Crossref PubMed Scopus (638) Google Scholar, 37.Elenbaas B. Weinberg R.A. Exp. Cell Res. 2001; 264: 169-184Crossref PubMed Scopus (443) Google Scholar). Because secretion of cytokines, proteases, and growth factors by stromal fibroblasts promotes proliferation of tumor cells (38.Frisch S.M. Francis H. J. Cell Biol. 1994; 124: 619-626Crossref PubMed Scopus (2749) Google Scholar), therapeutic targeting of stromal components constitutes a relevant tool to deprive tumor cells from critical factors needed for their growth, proliferation, and migration. In this context, it is interesting to note that the livers of AhR-/- mice had thickening of the stromal tissue surrounding the portal triads with increased staining for the fibroblast markers vimentin and smooth muscle α-actin (39.Corchero J. Martin-Partido G. Dallas S.L. Fernandez-Salguero P.M. Int. J. Exp. Pathol. 2004; 85: 295-302Crossref PubMed Scopus (40) Google Scholar), as well as altered vascular architecture (11.Lahvis G.P. Lindell S.L. Thomas R.S. McCuskey R.S. Murphy C. Glover E. Bentz M. Southard J. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10442-10447Crossref PubMed Scopus (310) Google Scholar).In an attempt to analyze the contribution of the AhR to tumor development in absence of xenobiotics, we have immortalized primary mammary gland fibroblasts from AhR+/+ and AhR-/- mice by stable co-transfection with the SV-40 large T-antigen and proto-oncogenic c-H-Ras. Although both cell lines had similar proliferation rates, cell cycle distribution and a transformed phenotype in vitro, the ability of T-FGM-AhR-/- to induce subcutaneous tumors in immunodeficient mice was markedly reduced. These results suggest that the absence of AhR could compromise the ability of myofibroblasts to adapt to the in vivo microenvironment and identify this receptor as a potential therapeutic target for the treatment of human cancers of fibroblastic origin.EXPERIMENTAL PROCEDURESChemicals and Reagents—TaqDNA polymerase and MMLV reverse transcriptase were from Ecogen and from Ambion, respectively. Mouse β-actin and secondary rabbit-TRITC antibodies, DAPI, enzymes, and hormones were obtained from Sigma-Aldrich. Collagenase, Dulbecco's modified Eagle's medium/F12 (DMEM/F12), OPTI-MEM, Lipofectamine Plus reagent, neomycin sulfate, and trypsin-EDTA were obtained from Invitrogen. Fetal bovine serum (FBS) was from Bio-Whittaker and was heat-inactivated before use. Complete protease inhibitor mixture was purchased from Roche Applied Science. Supersignal chemiluminescence substrate was obtained from Pierce. [methyl-3H]Thymidine was obtained from PerkinElmer Life Sciences. Protein A/G plus agarose, antibodies against p-Tyr, cyclin D1, cyclin E, p21Cip1, p27Kip1, p-PKB/AKT, PKB/AKT, p-ERK, ERK, H-pan-Ras, and secondary anti-mouse-FITC were from Santa Cruz Biotechnology. Anti-Rac-1 and anti-FAK antibodies were obtained from Transduction Laboratories. Antibodies for SV-40 large T antigen, VEGFR-1, and VEGFR-2 were from NeoMarkers. Smooth muscle α-actin 1A4 and desmin antibodies were from Anacrom Diagnósticos. Anti-vimentin was obtained from DAKO and anti-CD49f/α6 integrin from Immunotech.Mice and Primary Culture of Mammary Gland Fibroblasts—AhR-null and wild-type control mice of the same genetic background (C57BL6/N × 129/sV) were produced as previously described (9.Fernandez-Salguero P. Pineau T. Hilbert D.M. McPhail T. Lee S.S. Kimura S. Nebert D.W. Rudikoff S. Ward J.M. Gonzalez F.J. Science. 1995; 268: 722-726Crossref PubMed Scopus (934) Google Scholar). Animals were housed in a germ-free facility in accordance with the Animal Care and Use Guidelines of the University of Extremadura. Mice were genotyped by restriction fragment length polymorphism of tail DNA as described (26.Santiago-Josefat B. Mulero-Navarro S. Dallas S.L. Fernandez-Salguero P.M. J. Cell Sci. 2004; 117: 849-859Crossref PubMed Scopus (44) Google Scholar). Mammary gland stromal fibroblasts were isolated from 8 to12-week old virgin female mice of each genotype. Briefly, mice were killed, and the mammary glands quickly removed, washed twice in Hank's salt solution, and finely minced. Tissues were digested by incubation at 37 °C for 4 h in Hank's containing 10% FBS, 3500 units/ml collagenase, and 2000 units/ml hyaluronidase. The resulting cell suspension was briefly centrifuged, and the pellet resuspended in DMEM/F12 and filtered through a 41-μm nylon mesh. Single fibroblasts passing through the mesh were collected, centrifuged, and resuspended in DMEM/F12 containing 10% FBS, 2 mm l-glutamine, 50 μg/ml gentamycin, 11 mm d-glucose, 5 μg/ml insulin, 125 units prolactin, and 2 μg/ml hydrocortisone. Cells were cultured at 37 °C in a 5% CO2 atmosphere until 80-90% confluence and stored in liquid nitrogen. Aliquots from these primary cultures were propagated for 1-2 passages and used for transfection at 30-40% confluence.Stable Co-transfection with SV-40 Large T Antigen and AU5-c-H-Ras—Stable co-transfection was performed on AhR-null and wild type mammary fibroblasts using the Lipofectamine Plus reagent and 0.5 μg of EcoRI-linearized DNA. Plates were transfected with either pSV3Neo (SV-40), pCEFL-AU5-c-H-Ras (proto-oncogenic H-Ras) or a 1:1 mix of both DNA constructs. Proto-oncogenic (cellular) human H-Ras was used rather than the oncogenic, hyperactivated protein, because previous studies had revealed that while AhR activity remained unaffected by expression of the H-Ras proto-oncogene it was inhibited by the oncogenic protein (40.Reiners Jr., J.J. Jones C.L. Hong N. Clift R.E. Elferink C. Mol. Carcinog. 1997; 19: 91-100Crossref PubMed Scopus (44) Google Scholar). Stable transfectants were obtained after 4-5 weeks of selection in complete medium containing 400 μg/ml neomycin (G418). After isolation and re-plating, clones were considered to be at passage 1.Hematoxylin Staining and Immunocytochemistry—After removing the medium, plates were washed with PBS, fixed in cold methanol for 10 min at -20 °C and air-dried. For hematoxylin staining, cells were hydrated in PBS and incubated for 3 min in 50% Harris's hematoxylin. After an additional wash in PBS, cultures were photographed using a Nikon E600 microscope. For immunocytochemistry, cells were permeabilized with three 5-min washes in PBS containing 0.05% Triton X-100 (PBS-T). Blocking was performed for 30 min at room temperature in PBS containing 2% bovine serum albumin and 10% goat serum. Cells were incubated for 16 h at 4 °C with primary anti-vimentin, anti-smooth muscle α-actin 1A4, anti-CD49f/α6 integrin, anti-SV-40, anti-AU5, or anti-VEGFR-1 antibodies. After washing in TBS-T, anti-mouse-FITC or anti-rabbit-TRITC secondary antibodies were added, and incubation continued at room temperature for 1 h. Plates were finally washed in PBS as above. Nuclei were stained with DAPI for 5 min and cells observed and photographed using a Nikon E600 fluorescence microscope.Cell Proliferation, Cell Number, and Duplication Time—Cell proliferation was determined by measuring the rate of DNA synthesis. Cultures growing in 24-well plates were incubated with 1 μCi of [methyl-3H]thymidine (specific activity, 7 Ci/mmol) for 2 h. Labeling medium was removed and cells fixed for 2 h at room temperature in 1 ml of methanol/acetic acid (1:1). Fixed cells were washed with 80% ethanol and incubated in 0.05% trypsin-EDTA for 30 min at 37 °C. Cells were then lysed for 5 min at room temperature by the addition of 1% (w/v) SDS and incorporated thymidine quantitated in a Beckman LS 3801 liquid scintillation counter. The number of cells attached to the plates was determined in parallel cultures after trypsinization (see above) and cell counting using a hemocytometer. For some experiments, cells were grown in DMEM/F12 medium without FBS. Duplication time was determined from the slope of a semilogarithmic plot representing log of cell number against time.Flow Cytometry Analysis—Cell cycle distribution and ploidy status of the cultures were determined by flow cytometry DNA analysis. Cells were released from the plates by the addition of 0.25% trypsin, washed in PBS, fixed at 4 °C in 70% cold ethanol, and treated with RNase (10 mg/ml) for 30 min at 37 °C. DNA content per cell was determined in a Cyan flow cytometer (DAKO Cytomation) after staining with propidium iodide (50 μg/ml) for 15 min at room temperature in the dark. For cell cycle analysis, only signals from single cells were considered (10,000 cells/sample).Cell Lysates and Immunoblotting—Cells were washed with PBS and scraped from the plates at 4 °C in lysis buffer (50 mm Tris-HCl pH 7.5, 2 mm EGTA, 10 mm β-glycerophosphate, 5 mm sodium pyrophosphate, 50 mm sodium fluoride, 0.1 mm sodium orthovanadate, 1% Triton X-100, and Complete protease inhibitor mixture). For PKB/AKT and ERK proteins, the following lysis buffer was used: 20 mm Tris-HCl, pH 7.5, 1% Triton X-100, 10% glycerol, 137 mm NaCl, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml trypsin inhibitor, 20 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4. Lysed cultures were centrifuged at 15000 × g for 15 min at 4 °C (with prior 10-min shaking at 4 °C for PKB/AKT and ERK). Protein concentration was determined in the supernatants using the Coomassie Plus protein assay reagent (Pierce) and bovine serum albumin as standard. For immunoblotting, 15 μg of protein were denatured, separated on 8% or 12% SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked for 2 h at room temperature in TBS-T (50 mm Tris-HCl, pH 7.5, 10 mm NaCl, 0.5% Tween 20) containing 5% nonfat milk and incubated for another 2 h with the corresponding primary antibodies (H-pan-Ras, AU5, cyclin D, cyclin E, p21, p27, β-actin, PKB/AKT, pAKT, ERK, pERK, Rac-1, VEGFR-1, and VEGFR-2). After washing in TBS-T, blots were incubated with the horseradish peroxidase-coupled secondary antibody for 1 h at room temperature. Following additional washing in TBS-T, the SuperSignal chemiluminescence substrate was added, and the blots were exposed and developed using the Molecular Imager FX System (Bio-Rad).Immunoprecipitation—Cells were lysed on ice for 15 min with IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 10 mm β-glycerophosphate, 1 mm dithiothreitol, and Complete protease inhibitor mixture. Lysates were centrifuged and protein concentration determined in the supernatants as indicated above. An amount of 0.8-1 mg of protein was immunoprecipitated by overnight incubation at 4 °C with 2 μg of anti-SV-40 or anti-FAK antibodies, followed by 2-h incubation at 4 °C with protein A/G plus agarose. Immunoprecipitates were washed four times in IP buffer, denatured, separated on 8% SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked as indicated above and incubated overnight at 4 °C with antibodies against p53 (1:1000), Rb (1:300), or pTyr (1:1000). After washing and incubation with the horseradish peroxidase-coupled secondary antibody, the SuperSignal chemiluminescence substrate was added and membranes exposed and developed using a Molecular Imager FX System.Subcutaneous Mouse Xenograft Model for AhR+/+ and AhR-/- Myofibroblasts—Male or female NOD.CB17 PRKDC/J mice (Charles River Laboratories) at 6 weeks of age were used for this assay. T-FGM-AhR+/+ and T-FGM-AhR-/- myofibroblasts were grown and passed at 50-60% confluence. At the time of injection, cells were washed with PBS, trypsinized, and counted. Aliquots of 250 μl of PBS containing 1.5 × 106 cells were injected subcutaneously per flank in each mouse. Mice were sacrificed at 7-8 weeks after injection and tumors measured and processed for immunohistochemistry as indicated below. Tumor volume was calculated from the equation: tumor volume = x2y/2, where x and y correspond to the width and thickness of the tissue.Immunohistochemistry—Tumors were fixed in 5% buffered formalin, dehydrated in serially diluted ethanol, embedded in paraffin, and 5-μm serial sections obtained and mounted on immunohistochemically pretreated slides. Sections were then dewaxed in xylene and gradually rehydrated to deionized distilled water. Antigen unmasking was performed by incubating the slides at 100 °C for 5 min in 10 mm citrate buffer, pH 6.0. Incubation with antibodies against smooth muscle α-actin 1A4, desmin, or vimentin was carried out for 1 h at room temperature using an automatic immunostainer. Protein-antibody complexes were revealed by avidin-biotin complex (ABC) staining.Soft Agar Assay—Colony formation in soft agar was analyzed in 60-mm cell culture dishes coated with two layers of agarose. A 1:5 dilution of cell culture-grade agarose was made in prewarmed culture medium for a final concentration of 1% agar, and 4 ml of this solution added to each culture plate. Agarose was allowed to solidify at room temperature for 10 min and plates placed in the CO2 incubator for pH equilibration. A 1:10 dilution of agarose was also prepared in prewarmed culture medium for a final concentration of 0.5%. T-FGM-AhR+/+ and T-FGM-AhR-/- cells were trypsinized and serially diluted to 103, 5 × 103, 104, or 105 cells/ml. A 100-μl aliquot of each cell dilution was mixed with 900 μl of 0.5% agarose to give final concentrations of 102, 5 × 102, 103, and 104 cells/ml. These cell dilutions were poured on top of the 1% agarose layer previously solidified in each agar plate. Cells were fed twice per week by the addition of 400 μl of complete culture medium. After 22 days, clones were stained with crystal violet and counted under the microscope.Cell Migration and Wound Closure Assays—Migration assays were performed using Biocoat 24-well collagen I culture inserts and matrigel invasion chambers (BD Biosciences). A total of 7000 cells were seeded into the upper compartment of each well. Complete culture medium was placed in both lower and upper chambers. After 48 h, inserts were removed from the plates, washed in PBS, fixed in 70% ethanol, and incubated for 15 min with 10 ng/ml RNase in PBS. Next, cells were stained with 50 μg/ml propidium iodide at room temperature in the dark and migration-analyzed in a 2100 Radiance confocal microscope (Bio-Rad). For wound closure assays, cells were allowed to reach confluence in serum-containing medium. Wounds were performed in the plates with the aid of a 1-ml pipette tip, and cultures were incubated for 18 h in serum-free medium. Closure was monitored by microscopy after staining the cells with hematoxylin.Matrix Metalloproteinase Activity—MMP activity was determined in conditioned culture medium from T-FGM-AhR+/+ and T-FGM-AhR-/- by gelatin zymography. Cells were grown in OPTI-MEM for 60 h and culture medium recovered and centrifuged at 10,000 × g for 15 min at 4 °C. Cells attached to the plates at the time of the experiment were trypsinized and counted. A volume of medium equivalent to the same number of cells was mixed with non-reducing Laemmli's sample buffer (62.2 mm Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 0.025% bromphenol blue) and applied to 8% SDS-PAGE gels polymerized in the presence of 1% gelatin. After electrophoresis, the gels were washed three times in a solution containing 2.5% Triton X-100 to eliminate the SDS and to allow reconstitution of the proteins. MMP activity was stimulated by incubating the gels at 37 °C for 16 h in reaction buffer (50 mm Tris-HCl, pH 6.8, 150 mm NaCl, 5 mm CaCl2, and 0.05% sodium azide). The position of the MMPs was visualized by staining the gels in Coomassie Brilliant Blue G-250 solution.Rhodamine Phalloidin Staining—Complete medium of cultures at 20-30% confluence was replaced by serum-free DMEM/F12 for 18 h. Cells were then fixed in 4% paraformaldehyde in PBS for 5 min at 4 °C and permeabilized in

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