title: Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts.
abstract: This study examined the autocrine production of TGF-beta 1, -beta 2 and -beta 3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-beta 1, 78.0 pg ml-1 for TGF-beta 2 and 1.9 ng ml-1 for TGF-beta 3. Tumour-derived oral keratinocytes predominantly produced less TGF-beta 1 than normal oral epithelial cells; the expression of endogenous TGF-beta 2 was variable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 production was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-beta peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-beta 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory to both TGF-beta 1 and TGF-beta 2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-beta 3, TGF-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-beta isoforms suggests that dysregulation of TGF-beta autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.
authors.affiliation: Department of Oral and Dental Science, University of Bristol, UK.
authors.forename: M S
authors.initials: MS
authors.lastname: Fahey
authors.forename: I C
authors.initials: IC
authors.lastname: Paterson
authors.forename: A
authors.initials: A
authors.lastname: Stone
authors.forename: A J
authors.initials: AJ
authors.lastname: Collier
authors.forename: Y L
authors.initials: YL
authors.lastname: Heung
authors.forename: M
authors.initials: M
authors.lastname: Davies
authors.forename: V
authors.initials: V
authors.lastname: Patel
authors.forename: E K
authors.initials: EK
authors.lastname: Parkinson
authors.forename: S S
authors.initials: SS
authors.lastname: Prime
mesh_terms: D002460:Cell Line; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D016158:Genes, p53 / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000494:pharmacology; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000494:pharmacology
chemical_list: D009363:Neoplasm Proteins; D016212:Transforming Growth Factor beta