ABSTRACT
The aim of the present study was to investigate the effect of transforming growth factor-beta1 (TGF-beta1) on telomerase activity in a panel of human anaplastic thyroid carcinoma (ATC) cell lines. Addition of TGF-beta1 decreased the telomerase activity in HTh 74 and KTC-1 cells, while in C 643 and HTh 7 an increased activity was observed. The decreased telomerase activity appeared to be due to transcriptional repression of the hTERT promoter. Addition of a PI-3 kinase inhibitor (LY294002) abrogated the stimulatory effect of TGF-beta1 on the telomerase activity, indicating the possible involvement of hTERT activation via phosphorylation. Furthermore, the MEK-inhibitor U0126 had similar effects suggesting dual regulatory mechanisms. Interestingly, the cell lines differed genetically in that ATC cell lines responding with increased telomerase activity harbored a p53 mutation. In conclusion, TGF-beta1 exerts opposing effects on telomerase activity in ATC cell lines, possibly reflecting deregulation of TGF-beta1 signaling in a more malignant genotype.
Subject(s)
Telomerase/metabolism , Thyroid Neoplasms/enzymology , Transforming Growth Factor beta/pharmacology , Cell Line, Tumor , Down-Regulation/drug effects , Genotype , Humans , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Mutation/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Promoter Regions, Genetic/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Telomerase/genetics , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Transforming Growth Factor beta1 , Tumor Suppressor Protein p53/geneticsABSTRACT
Enhancement of tumor cell growth and invasiveness by transforming growth factor-beta (TGF-beta) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-beta1. The epithelial tightness was maintained after single stimulation with EGF or TGF-beta1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [(3)H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single-stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast-like morphology and formed multilayers. TGF-beta1 only partially inhibited EGF-induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-beta1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-beta1 to convert normal thyroid epithelial cells to a mesenchymal phenotype.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Epidermal Growth Factor/metabolism , Epithelial Cells/enzymology , Mesoderm/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Neoplasm Invasiveness/genetics , Protein Serine-Threonine Kinases/metabolism , Transforming Growth Factor beta/metabolism , Adherens Junctions/drug effects , Adherens Junctions/metabolism , Adherens Junctions/ultrastructure , Animals , Cadherins/drug effects , Cadherins/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Size/drug effects , Cell Size/physiology , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Claudin-1 , Down-Regulation/drug effects , Down-Regulation/physiology , Drug Interactions/physiology , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/ultrastructure , MAP Kinase Kinase 1 , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Mesoderm/drug effects , Mesoderm/ultrastructure , Microscopy, Electron , Mitogen-Activated Protein Kinase Kinases/drug effects , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Neoplasm Invasiveness/physiopathology , Occludin , Protein Serine-Threonine Kinases/drug effects , Protein Serine-Threonine Kinases/genetics , Sus scrofa , Thyroid Gland/cytology , Thyroid Gland/drug effects , Thyroid Gland/enzymology , Tight Junctions/drug effects , Tight Junctions/metabolism , Tight Junctions/ultrastructure , Transforming Growth Factor beta/pharmacologyABSTRACT
Fibrosis in solid malignancies plays a significant role in tumor pathophysiology. Potential mechanisms for collagen type I deposition in anaplastic thyroid carcinoma (ATC) were investigated using 6 characterized ATC cell lines. Three of these cell lines, which produced collagen type I, had, as a group, a poor tumorigenicity when inoculated in athymic mice. This group of cells generated tumors in 4 of 24 injected animals (17%). Pro-alpha 1(I) collagen mRNA-expressing carcinoma and stromal cells were interdispersed in the tumors generated by these ATC cells. By contrast, the 3 noncollagen-producing ATC cell lines were all tumorigenic with a tumor take of 60% in the whole group. In the latter tumors, pro-alpha 1(I) collagen mRNA-expressing cells were confined to the stromal compartment, well delineated from carcinoma cell islets. To study the influence of ATC cells on collagen type I synthesis by fibroblasts, we used AG 1518 diploid human fibroblasts cultured on poly-(2-hydroxyethyl methacrylate) (poly[HEMA])-coated plates. This culture condition allows the study of the effect of collagen mRNA translation in the regulation of collagen type I synthesis. Conditioned media from the 6 ATC cell lines did not influence collagen synthesis. The ATC cell line KAT-4 stimulated fibroblast synthesis of collagen type I when the two cell types were cocultured on poly[HEMA]-coated substrates. Specific inhibitors of PDGF and TGF-beta reduced the KAT 4 carcinoma cell-induced stimulation of collagen type I synthesis. Our data suggest that collagen type I production by carcinoma cells correlates negatively with tumorigenicity and that the formation of a well-defined stroma is of importance for tumor growth. Furthermore, our data suggest that tumor cells are able to stimulate collagen mRNA translation in stromal fibroblasts in direct cell-cell contact by, at least in part, transferring PDGF or TGF-beta.
