TGF-beta1 acts as a tumor suppressor of human malignant keratinocytes independently of Smad 4 expression and ligand-induced G(1) arrest.
This study examined the role of TGF-beta1 in human keratinocyte malignancy. Two carcinoma-derived human oral keratinocyte cell lines, BICR 31 and H314, were selected on the basis of their known resistance to TGF-beta1-induced G(1) arrest, the presence of wild type TGF-beta cell surface receptors and normal Ras. Smad 4 protein was undetectable in both cell lines, but Smad 2 and Smad 3 were expressed at levels comparable with a fully TGF-beta responsive cell line, and treatment of the cells with TGF-beta1 resulted in the phosphorylation of Smad 2. Treatment with exogenous TGF-beta1 resulted in a failure to induce transcription from an artificial Smad-dependent promoter and a failure to down-regulate c-myc, but resulted in an up-regulation of AP-1 associated genes (Fra-1, JunB and fibronectin). Transient transfection of Smad 4 into BICR 31 restored TGF-beta1-induced growth inhibition and Smad-dependent transcriptional activation. Protracted treatment of cells with exogenous TGF-beta1 resulted in the attenuation of cell growth in vitro. To over-express TGF-beta1, both cell lines were transfected with latent TGF-beta1 cDNA; neutralization studies of conditioned media demonstrated that whilst the majority of the peptide was in the latent form, a small proportion was present as the active peptide. Cells that over-expressed endogenous TGF-beta1 grew more slowly in vitro compared to both the vector-only controls and cells that did not over-express the peptide. Orthotopic transplantation of cells that over-expressed endogenous TGF-beta1 to the floor of the mouth in athymic mice resulted in marked inhibition of primary tumor formation compared to controls. Expression of a dominant-negative TGF-beta type II receptor in cells that over-expressed endogenous TGF-beta1 resulted in enhanced cell growth in vitro and diminished the tumor suppressor effect of the ligand in vivo, indicating that the endogenous TGF-beta1 was acting in an autocrine capacity. The results demonstrate that over-expression of endogenous TGF-beta1 in human malignant oral keratinocytes leads to growth inhibition in vivo and tumor suppression in vitro by mechanisms that are independent of Smad 4 expression and TGF-beta1-induced G(1) arrest.
Paterson
Ian C
IC
Department of Oral and Dental Science, University of Bristol, Bristol, BS1 2LY, UK. ian.paterson@bristol.ac.uk
Davies
Maria
M
Stone
Andrea
A
Huntley
Suzy
S
Smith
Emily
E
Pring
Miranda
M
Eveson
John W
JW
Robinson
C Max
CM
Parkinson
E Kenneth
EK
Prime
Stephen S
SS
D000818:Animals; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016193:G1 Phase; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008819:Mice, Nude; D009062:Mouth Neoplasms / Q000378:metabolism / Q000473:pathology; D009368:Neoplasm Transplantation; D012334:RNA, Neoplasm / Q000096:biosynthesis; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
D004268:DNA-Binding Proteins; D008024:Ligands; D012334:RNA, Neoplasm; C497796:SMAD4 protein, human; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D025521:Tumor Suppressor Proteins