title: Expression of Smad4 in the FaDu cell line partially restores TGF-beta growth inhibition but is not sufficient to regulate fibronectin expression or suppress tumorigenicity.
abstract: Mutations of the Smad4 gene, a member of a group of TGF-beta signal transduction components, occur in several types of cancer suggesting that its inactivation significantly affects TGF-beta responsiveness in these tumors. To further investigate the role of Smad4 with respect to TGF-beta signaling and carcinogenesis, we re-expressed the Smad4 gene in the Smad4-deficient cancer cell line FaDu by microcell-mediated chromosome transfer (MMCT) and retroviral infection to closely approximate physiological protein levels. The Smad4-expressing FaDu clones were then evaluated for TGF-beta responsiveness to assess the role of Smad4 in TGF-beta-induced growth inhibition and target gene regulation. We found that the re-expression of the Smad4 gene by either method partially restored TGF-beta responsiveness in FaDu cells with respect to both growth inhibition and expression of p21WAF1/CIP1 and p15INK4B. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. In contrast, the re-expression of Smad4 by either method failed to suppress tumorigenicity. These results suggest that in addition to a homozygous deletion of Smad4, FaDu cells contain additional defects within the TGF-beta signaling pathway, thereby limiting the extent of TGF-beta responsiveness upon Smad4 re-expression and perhaps accounting for the inability to induce p15INK4B to a high level. They also demonstrate the advantages of providing a physiological extracellular environment, when assessing TGFbeta responsiveness.
authors.affiliation: Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA. bhummer@som.umaryland.edu
authors.forename: B Timothy
authors.initials: BT
authors.lastname: Hummer
authors.forename: Christopher
authors.initials: C
authors.lastname: Bartlett
authors.forename: Erin
authors.initials: E
authors.lastname: Henry
authors.forename: Bernard E
authors.initials: BE
authors.lastname: Weissman
mesh_terms: D015197:Carcinogenicity Tests; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D015179:Colorectal Neoplasms; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D006822:Hybrid Cells; D009924:Organ Culture Techniques; D010766:Phosphorylation; D012190:Retroviridae / Q000235:genetics; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
chemical_list: C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D005353:Fibronectins; D006131:Growth Inhibitors; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins