Advertisement

Detection of c-Sis proto-oncogene transcripts by direct enzyme-labeled cDNA probes and in situ hybridization

  • J. T. McClintock
  • I-J. Chan
  • S. R. Thaker
  • A. Katial
  • F. E. Taub
  • A. E. Aotaki-Keen
  • L. M. Hjelmeland
Regular Papers

Summary

Using in situ hybridization and platelet-derived growth factor (PDGF) cDNA probes labeled with horseradish peroxidase, PDGF-A and -B (c-cis proto-oncogene) mRNA transcripts were identified and localized in proliferating cultures. A human retinal pigment epithelial (RPE) cell line and a glial cell line were treated with either transforming growth factor beta-1 (TGFB1), phorbol-12-myristate-13-acetate (PMA), or thrombin from human plasma and compared for their ability to stimulate the production of PDGF-A and -B. Expression of both PDGF-A and -B transcripts were found to be localized predominantly in the cytoplasm of TGFB1-treated RPE cells, with a portion of these cells displaying a hybridization response in the nuclear region. When compared to PMA- and thrombin-treated cells, TGFB1 stimulated the RPE cell line to yield the greatest amount of detectable PDGF mRNA. In addition, the hybridization response observed in TGFB1-treated cells was shown to be RNA dependent.

Key words

c-sis proto-oncogene mRNA detection horseradish peroxidase-labeled cDNA probes in situ hybridization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aotaki-Keen, A.; Harvey, A. K.; de Juan, E., et al. Primary culture of human retinal glia. Invest. Ophthalmol. Visual Sci. 32:1733–1738; 1991.Google Scholar
  2. Betscholtz, C.; Johnsson, A.; Heldin, C.-H., et al. cDNA sequence and chromosomal localization of human platelet derived growth factor A chain and its expression in tumor cell lines. Nature 320:695–699; 1986.CrossRefGoogle Scholar
  3. Bressler, J. P.; Grotendorst, G. R.; Levitov, C., et al. Chemotaxis of rat brain astrocytes to platelet derived growth factor. Brain Res. 344:249–254; 1985.PubMedCrossRefGoogle Scholar
  4. Collins, T.; Ginsburg, D.; Boss, J., et al. Cultured human endothelial cells express platelet derived growth factor B chain: cDNA cloning and structural analysis. Nature 316:748–750; 1985.PubMedCrossRefGoogle Scholar
  5. Gospodarowicz, D.; Moran, J. S. Growth factors in mammalian cell culture. Annu. Rev. Biochem. 45:531–558; 1976.PubMedCrossRefGoogle Scholar
  6. Grotendorst, G. R.; Chang, T.; Seppa, S. E. J., et al. Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J. Cell. Physiol. 113:261–266; 1982.PubMedCrossRefGoogle Scholar
  7. Heldin, C.-H.; Westermark, B.; Wasteson, A. Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc. Natl. Acad. Sci. USA 78:3664–3668; 1981.PubMedCrossRefGoogle Scholar
  8. Ishigooka, H.; Aotaki-Keen, A.; Hjelmeland, L. M. Subcellular localization of bFGF in human retinal pigment epithelium in vitro. Exp. Eye Res. In press; 1991.Google Scholar
  9. Johnsson, A.; Heldin, C.-H.; Wasteson, A., et al. The c-sis gene encodes a precursor of the B chain of platelet-derived growth factor. EMBO J. 3:921–928; 1984.PubMedGoogle Scholar
  10. Johnsson, A.; Heldin, C.-H.; Westermark, B., et al. Platelet-derived growth factor: identification of constituent polypeptide chains. Biochem. Biophys. Res. Commun. 104:66–74; 1982.PubMedCrossRefGoogle Scholar
  11. Seppa, H.; Grotendorst, G.; Seppa, S., et al. Platelet-derived growth factor is chemotactic for fibroblasts. J. Cell Biol. 92:584–588; 1982.PubMedCrossRefGoogle Scholar
  12. Shipley, G. D.; Tucker, R. F.; Moses, H. L. Typeβ transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval. Proc. Natl. Acad. Sci. USA 82:4147–4151; 1985.PubMedCrossRefGoogle Scholar
  13. Tucker, R. F.; Shipley, G. D.; Moses, H. L. Growth inhibitor from BSC-1 cells closely related to platelet typeβ transforming growth factor. Science 226:705–707; 1984.PubMedCrossRefGoogle Scholar
  14. Waterfield, M. D.; Scrace, G. T.; Whittle, N., et al. Platelet-derived growth factor is structurally related to the putative transforming protein p28-sis of simian sarcoma virus. Nature 304:35–39; 1983.PubMedCrossRefGoogle Scholar

Copyright information

© Tissue Culture Association 1992

Authors and Affiliations

  • J. T. McClintock
    • 1
    • 2
  • I-J. Chan
    • 2
  • S. R. Thaker
    • 2
  • A. Katial
    • 2
    • 3
  • F. E. Taub
    • 2
    • 3
  • A. E. Aotaki-Keen
    • 2
    • 3
  • L. M. Hjelmeland
    • 2
    • 3
  1. 1.U.S. EPA, OPP, HED, SACBWashington, DC
  2. 2.Digene Diagnostics, Inc.Silver Spring
  3. 3.Department of OphthalmologyUniversity of California-Davis, Medical SchoolSacramento

Personalised recommendations