Isolation and characterization of mutant cell lines defective in transforming growth factor beta signaling.
To isolate and characterize effector molecules of the transforming growth factor beta (TGFbeta) signaling pathway we have used a genetic approach involving the generation of stable recessive mutants, defective in their TGFbeta signaling, which can subsequently be functionally complemented to clone the affected genes. We have generated a cell line derived from a hypoxanthine-guanine phosphoribosyltransferase negative (HPRT-) HT1080 clone that contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to a TGFbeta-responsive promoter. This cell line proliferates or dies in the appropriate selection medium in response to TGFbeta. We have isolated three distinct TGFbeta-unresponsive mutants following chemical mutagenesis. Somatic cell hybrids between pairs of individual TGFbeta-unresponsive clones reveal that each is in a distinct complementation group. Each mutant clone retains all three TGFbeta receptors yet fails to induce a TGFbeta-inducible luciferase reporter construct or TGFbeta-mediated plasminogen activator inhibitor-1 (PAI-1) expression. Two of the three have an attenuated TGFbeta-induced fibronectin response, whereas in the other mutant the fibronectin response is intact. These TGFbeta-unresponsive cells should allow selection and identification of signaling molecules through functional complementation.
Hocevar
B A
BA
Department of Cell Biology, Cleveland Clinic Research Institute, OH 44195, USA.
Howe
P H
PH
D002460:Cell Line; D002999:Clone Cells; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005353:Fibronectins / Q000494:pharmacology; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis; D016296:Mutagenesis; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis
D005353:Fibronectins; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D008156:Luciferases; D007041:Hypoxanthine Phosphoribosyltransferase; D001616:beta-Galactosidase