TIMP-2, a growth-stimulatory protein from SV40-transformed human fibroblasts.
The mechanisms by which the early genes of simian virus 40 (SV40) transform human cells are unclear; however, this is clearly a multistep process involving a number of cellular and genetic changes. An early change following expression of the SV40 genes is growth under reduced serum conditions, which could be consistent with the production of autocrine/paracrine growth factors. HSF4-T12 is a human fibroblast cell line produced by transfection of primary cells with the genes for large T and small t antigens. A progressive stepwise transformation was observed with in vitro culture, eventually resulting in a tumorigenic cell line. Serum-free defined medium conditioned by HSF4-T12 was able to stimulate growth of normal human fibroblasts as determined by growth curve and [3H]-thymidine incorporation assays. Purification of this activity by heparin affinity chromatography and nondenaturing polyacrylamide gel electrophoresis resulted in a single band of approximately 21 kDa on a nonreducing, denaturing gel. A partial 14-amino acid sequence was found to share 100% homology with a region of tissue inhibitor of metalloproteinases-2 (TIMP-2). Western blot analysis with anti-TIMP-2 antiserum confirmed this identification, and addition of this same antiserum to HSF4-T12-conditioned medium resulted in inhibition of stimulatory activity.
Department of Pathology, Northwestern University Medical School/VA Lakeside Medical Center, Chicago, Illinois 60611.
J A
JA
Nemeth
C L
CL
Goolsby
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis*; D017386:Sequence Homology, Amino Acid; D013539:Simian virus 40 / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2; D014316:Tritium
D009363:Neoplasm Proteins; D014316:Tritium; D019716:Tissue Inhibitor of Metalloproteinase-2; D004247:DNA; D008666:Metalloendopeptidases; D013936:Thymidine