title: Pulmonary fibroblasts stimulate the proliferation of cell lines from human lung adenocarcinomas.
abstract: Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.
authors.affiliation: Experimental Oncology Laboratory, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Cekanova
authors.forename: Thomas
authors.initials: T
authors.lastname: Masi
authors.forename: Howard K
authors.initials: HK
authors.lastname: Plummer
authors.forename: Mourad
authors.initials: M
authors.lastname: Majidi
authors.forename: Peter
authors.initials: P
authors.lastname: Fedorocko
authors.forename: Hildegard M
authors.initials: HM
authors.lastname: Schuller
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology*; D066258:Amphiregulin; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D066255:EGF Family of Proteins; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology*; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000276:immunology; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology; D008168:Lung / Q000166:cytology*; D008175:Lung Neoplasms / Q000473:pathology*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D016211:Transforming Growth Factor alpha / Q000037:antagonists & inhibitors / Q000276:immunology
chemical_list: C587848:AREG protein, human; D066258:Amphiregulin; D019138:Antibodies, Blocking; C587849:Areg protein, mouse; D017077:Culture Media, Conditioned; D019927:Cyclin E; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D013778:Tetrazolium Salts; D013844:Thiazoles; D016211:Transforming Growth Factor alpha; D019938:Cyclin D1; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; C022616:thiazolyl blue