Normal and Malignant Prostate Epithelial Cells Differ in Their Response to Hepatocyte Growth Factor/Scatter Factor
2001; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)61729-4
ISSN1525-2191
AutoresGlenn A. Gmyrek, Marc Walburg, Craig P. Webb, Hsiao‐Man Ivy Yu, Xueke You, E. Darracott Vaughan, George F. Vande Woude, Beatrice S. Knudsen,
Tópico(s)Fibroblast Growth Factor Research
ResumoHepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer. Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer. Hepatocyte growth factor/scatter factor (HGF/SF) 1Stoker M Perryman M An epithelial scatter factor released by embryo fibroblasts.J Cell Sci. 1985; 77: 209-223Crossref PubMed Google Scholar, 2Nakamura T Nishizawa T Hagiya M Seki T Shimonishi M Sugimura A Tashiro K Shimizu S Molecular cloning and expression of human hepatocyte growth factor.Nature. 1989; 342: 440-443Crossref PubMed Scopus (2087) Google Scholar has been implicated in the communication between stromal and epithelial cells of many different organs. 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Despite an understanding of the Met expression pattern in normal and malignant PrECs, the precise function of HGF/SF-Met in the normal prostate and in prostate cancer is not known. To determine whether HGF/SF responsiveness is altered as a result of oncogenic transformation of prostate epithelial cells, we compared the HGF/SF-induced proliferation and migration of normal PrEC and prostate cancer cells. Whereas HGF/SF stimulation of normal primary PrECs resulted in growth inhibition and differentiation, transformed prostate cancer cells proliferated on HGF/SF stimulation. This is the first evidence that a single stromal factor can induce differentiation of primary cultured PrECs and that oncogenic transformation can potentially alter the responsiveness of epithelial cells to stromal-derived growth factors. The anti-BrdU monoclonal antibody was purchased from DAKO (Carpinteria, CA), the anti-human HGF/SF monoclonal from Sigma (St. Louis, MO) and the 4G10 anti-phosphotyrosine from Upstate Biotechnology (Lake Placid, NY), hMet (C-28) polyclonal antibodies were obtained from Santa Cruz (Santa Cruz, CA), MAPK and phospho-MAPK antibodies were purchased from New England Biolabs (Beverly, MA). The anti-HGF serum was raised in rabbits immunized with rHGF/SF.34Webb CP Hose CD Koochekpour S Jeffers M Oskarsson M Sausville E Monks A Vande Woude GF The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network.Cancer Res. 2000; 60: 342-349PubMed Google Scholar Growth factor reduced (GFR)-MatriGel was purchased from Becton Dickinson, (Bedford, MA) and polylysine from Sigma and diluted 1:100 in distilled water. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin were obtained from Sigma. Recombinant HGF/SF (rHGF/SF) was purified as described.28Rong S Oskarsson M Faletto D Tsarfaty I Resau JH Nakamura T Rosen E Hopkins RF Vande Woude GF Tumorigenesis induced by coexpression of human hepatocyte growth factor and the human met protooncogene leads to high levels of expression of the ligand and receptor.Cell Growth Differ. 1993; 4: 563-569PubMed Google Scholar DU145 cells were obtained from the American Type Culture Collection and cultured in RPMI medium with 10% fetal calf serum (FCS) (Gemini, Calabasas, CA). Madin Darbey canine kidney (MDCK) cells were grown in Dulbecco's modified Eagle's medium, 25 mmol/L HEPES pH 7.5, 10% FCS and used in the scatter assay as previously described.34Webb CP Hose CD Koochekpour S Jeffers M Oskarsson M Sausville E Monks A Vande Woude GF The geldanamycins are potent inhibitors of the hepatocyte growth factor/scatter factor-met-urokinase plasminogen activator-plasmin proteolytic network.Cancer Res. 2000; 60: 342-349PubMed Google Scholar Prostate tissue was obtained from surgical specimens with Institutional Review Board approval. Briefly, a 0.5 × 0.5 × 0.3 cm piece was collected in transfer medium (50% HAM, 50% F12 supplemented with penicillin and streptomycin) from the posterior prostate, from the side with a negative biopsy result. One- to 8-mm3 pieces were incubated with collagenase (250 U/ml), hyaluronidase (325 U/ml) and 5% FCS for 18 hours, washed on a 100-μm filter (Falcon cell striever, Becton Dickinson, Franklin Lakes, NJ, catalog no. 2360) and plated in 10-cm dishes in 5 ml of culture medium. Epithelial cells grew in PrEGM (Clonetics, Baltimore, MD), supplied as a base medium without growth factors (referred to as base medium PrEGM) to be reconstituted with growth factors, or in keratinocyte-SF medium (GIBCO BRL, Rockville, MD). Outgrowth from the tissue pieces was observed after 5 to 6 days. Cells were first passaged after 12 to 14 days by consecutive incubations with phosphate-buffered saline containing/1 mmol/L ethylene diaminetetraacetic acid (EDTA), cell dissociation buffer (GIBCO BRL) and 0.12% trypsin/EDTA. For subsequent passages, the trypsin/EDTA was reduced to 0.06%. Trypsinized cells were plated at 1 × 104 cells per cm2 and grown to 80% confluence. Cells were propagated for four passages. Cells were characterized after outgrowth from tissue pieces that were placed on glass coverslips or after the first passage. Table 1 summarizes the immunohistochemical and reverse transcription-polymerase chain reaction profile of the cultured cells. Most epithelial cells stained positive with the K903 mAb which binds high molecular weight cytokeratins (CK5 and CK14) as well as with Cam 5.2 mAb which binds low molecular weight CK (CK8 and 18) and focally positive for vimentin. The specificity of antibodies for individual cytokeratins was ascertained by negative staining of stromal cells as well as by different reactivities of antibodies of the same isotype. The K903, CK18, androgen receptor and desmin antibodies of the IgG1 isotype and the Cam 5.2, vimentin and SMA antibodies of the IgG2a isotype showed specific staining patterns, consistent with specific binding of individual antibodies to the respective antigenic determinants. Expression of the androgen receptor or Bcl-2 was not detectable by immunohistochemistry. The vimentin positive cells had epithelial and not stromal cell morphology and expressed cytokeratins. Such cells were also observed in vivo, at the tips of hyperplastic papillae54Knudsen BS Harpel PC Nachman RL Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells.J Clin Invest. 1987; 80: 1082-1089Crossref PubMed Scopus (136) Google Scholar and therefore represents an in vivo existing cell population and not a culture artifact.Table 1Immunohistochemical and Reverse Transcription-Polymerase Chain Reaction Expression Profile of Cultured Primary Epithelial and Stromal Cells from the ProstateAntibody/PCR primersCompanyDilutionCultured PrECCultured PrSCProstate frozen section control*Cells that stained positive in the frozen sections from the prostate: PrBEC, prostate basal epithelial cells, PrSEC, prostate secretory epithelial cells which comprise most epithelial cells.K903 (CK5+ CK14)†Cytokeratin.DAKO1:200+n.d.PrBECCK5 (RT-PCR)‡PCR primers for CK5: forward primer: GTC ACC AAC TTG CTG CCA AG; reverse primer: GTT CCT GGT GGA GCA AGA GAA C.−/+−/++Cam 5.2 (CK8+ CK18)Becton Dickinson1:1+n.d.PrSECCK18Santa Cruz1:100+n.d.PrSECAndrogen receptorBioGenex1:1−−PrSEC, PrSCVimentinDAKO1:200+/−+PrSEC, PrSCDesminDAKO1:100n.d.−PrSCSMADAKO1:100n.d.+PrSCMinute pieces of prostate tissue were placed on coverslips for explant cultures of stromal and epithelial cells. Colonies that formed after 3 weeks were fixed and stained with the antibodies specified. Bound antibodies were detected colorimetrically.* Cells that stained positive in the frozen sections from the prostate: PrBEC, prostate basal epithelial cells, PrSEC, prostate secretory epithelial cells which comprise most epithelial cells.† Cytokeratin.‡ PCR primers for CK5: forward primer: GTC ACC AAC TTG CTG CCA AG; reverse primer: GTT CCT GGT GGA GCA AGA GAA C. Open table in a new tab Minute pieces of prostate tissue were placed on coverslips for explant cultures of stromal and epithelial cells. Colonies that formed after 3 weeks were fixed and stained with the antibodies specified
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