DYSREGULATION OF AUTOCRINE TGF-BETA ISOFORM PRODUCTION AND LIGAND RESPONSES IN HUMAN TUMOR-DERIVED AND HA-RAS-TRANSFECTED KERATINOCYTES ANDFIBROBLASTS

This study examined the autocrine production of TGF-beta 1, -beta 2 an d -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) h uman 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 va riable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 producti on was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was d etected by reverse transcription-polymerase chain reaction (RT-PCR) bu t the protein was not detected in conditioned media, most probably bec ause of the low detection limits of the ELISA for this isoform. Neutra lisation experiments indicated that the latent TGF-beta peptide was se creted in keratinocyte conditioned medium. Seven tumour-derived kerati nocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined fo r their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-bet a 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory t o 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 TCF-beta 3. TG F-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associ ated fibroblasts, but the tumour-associated fibroblasts showed less re sponse to the ligands than their normal counterparts following prolong ed treatment with each isoform. The results demonstrate variable autoc rine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenot ype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable respons e of the tumour-derived keratinocytes and contiguous fibroblasts to th e TGF-beta isoforms suggests that dysregulation of TGF-beta autocrine and paracrine networks are common characteristics of squamous epitheli al malignancy.