Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1990 Jul 1;111(1):95–102. doi: 10.1083/jcb.111.1.95

Comparative two-dimensional gel analysis and microsequencing identifies gelsolin as one of the most prominent downregulated markers of transformed human fibroblast and epithelial cells

PMCID: PMC2116170  PMID: 2164032

Abstract

A systematic comparison of the protein synthesis patterns of cultured normal and transformed human fibroblasts and epithelial cells, using two-dimensional gel protein analysis combined with computerized imaging and data acquisition, identified a 90-kD protein (SSP 5714) as one of the most striking downregulated markers typical of the transformed state. Using the information stored in the comprehensive human cellular protein database, we found this protein strongly expressed in several fetal tissues and one of them, epidermis, served as a source for preparative two-dimensional gel electrophoresis. Partial amino acid sequences were generated from peptides obtained by in situ digestion of the electroblotted protein. These sequences identified the marker protein as gelsolin, a finding that was confirmed by two-dimensional immunoblotting of human MRC-5 fibroblast proteins using specific antibodies and by coelectrophoresis with purified human gelsolin. These results suggest that an important regulatory protein of the microfilament system may play a role in defining the phenotype of transformed human fibroblast and epithelial cells in culture.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauw G., De Loose M., Inzé D., Van Montagu M., Vandekerckhove J. Alterations in the phenotype of plant cells studied by NH(2)-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4806–4810. doi: 10.1073/pnas.84.14.4806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauw G., Van Damme J., Puype M., Vandekerckhove J., Gesser B., Ratz G. P., Lauridsen J. B., Celis J. E. Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7701–7705. doi: 10.1073/pnas.86.20.7701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boschek C. B., Jockusch B. M., Friis R. R., Back R., Grundmann E., Bauer H. Early changes in the distribution and organization of microfilament proteins during cell transformation. Cell. 1981 Apr;24(1):175–184. doi: 10.1016/0092-8674(81)90513-4. [DOI] [PubMed] [Google Scholar]
  5. Bravo R., Celis J. E. Human proteins sensitive to neoplastic transformation in cultured epithelial and fibroblast cells. Clin Chem. 1982 Apr;28(4 Pt 2):949–954. [PubMed] [Google Scholar]
  6. Bravo R., Small J. V., Fey S. J., Larsen P. M., Celis J. E. Architecture and polypeptide composition of HeLa cytoskeletons. Modification of cytoarchitectural polypeptides during mitosis. J Mol Biol. 1982 Jan 5;154(1):121–143. doi: 10.1016/0022-2836(82)90421-1. [DOI] [PubMed] [Google Scholar]
  7. Bryan J. Gelsolin has three actin-binding sites. J Cell Biol. 1988 May;106(5):1553–1562. doi: 10.1083/jcb.106.5.1553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Celis J. E., Ratz G. P., Madsen P., Gesser B., Lauridsen J. B., Hansen K. P., Kwee S., Rasmussen H. H., Nielsen H. V., Crüger D. Computerized, comprehensive databases of cellular and secreted proteins from normal human embryonic lung MRC-5 fibroblasts: identification of transformation and/or proliferation sensitive proteins. Electrophoresis. 1989 Feb;10(2):76–115. doi: 10.1002/elps.1150100204. [DOI] [PubMed] [Google Scholar]
  9. Chaponnier C., Gabbiani G. Gelsolin modulation in epithelial and stromal cells of mammary carcinoma. Am J Pathol. 1989 Mar;134(3):597–603. [PMC free article] [PubMed] [Google Scholar]
  10. Chaponnier C., Patebex P., Gabbiani G. Human plasma actin-depolymerizing factor. Purification, biological activity and localization in leukocytes and platelets. Eur J Biochem. 1985 Jan 15;146(2):267–276. doi: 10.1111/j.1432-1033.1985.tb08649.x. [DOI] [PubMed] [Google Scholar]
  11. Chaponnier C., Yin H. L., Stossel T. P. Reversibility of gelsolin/actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways. J Exp Med. 1987 Jan 1;165(1):97–106. doi: 10.1084/jem.165.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dieffenbach C. W., SenGupta D. N., Krause D., Sawzak D., Silverman R. H. Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells. J Biol Chem. 1989 Aug 5;264(22):13281–13288. [PubMed] [Google Scholar]
  13. Fenech M., Morley A. A. Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat Res. 1986 Jul;161(2):193–198. doi: 10.1016/0027-5107(86)90010-2. [DOI] [PubMed] [Google Scholar]
  14. Flanagan M. D., Lin S. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem. 1980 Feb 10;255(3):835–838. [PubMed] [Google Scholar]
  15. Garrels J. I., Franza B. R., Jr Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. A two-dimensional gel analysis of SV40-, adenovirus-, and Kirsten murine sarcoma virus-transformed rat cells using the REF52 protein database. J Biol Chem. 1989 Mar 25;264(9):5299–5312. [PubMed] [Google Scholar]
  16. Garrels J. I. The QUEST system for quantitative analysis of two-dimensional gels. J Biol Chem. 1989 Mar 25;264(9):5269–5282. [PubMed] [Google Scholar]
  17. Garrels J. I. Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines. J Biol Chem. 1979 Aug 25;254(16):7961–7977. [PubMed] [Google Scholar]
  18. Godman G. C., Miranda A. F. Cellular contractility and the visible effects of cytochalasin. Front Biol. 1978;46:277–429. [PubMed] [Google Scholar]
  19. Hartwig J. H., Yin H. L. The organization and regulation of the macrophage actin skeleton. Cell Motil Cytoskeleton. 1988;10(1-2):117–125. doi: 10.1002/cm.970100116. [DOI] [PubMed] [Google Scholar]
  20. Hendricks M., Weintraub H. Tropomyosin is decreased in transformed cells. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5633–5637. doi: 10.1073/pnas.78.9.5633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ishikawa R., Yamashiro S., Matsumura F. Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon. J Biol Chem. 1989 May 5;264(13):7490–7497. [PubMed] [Google Scholar]
  22. Kwiatkowski D. J., Mehl R., Yin H. L. Genomic organization and biosynthesis of secreted and cytoplasmic forms of gelsolin. J Cell Biol. 1988 Feb;106(2):375–384. doi: 10.1083/jcb.106.2.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kwiatkowski D. J. Predominant induction of gelsolin and actin-binding protein during myeloid differentiation. J Biol Chem. 1988 Sep 25;263(27):13857–13862. [PubMed] [Google Scholar]
  24. Kwiatkowski D. J., Stossel T. P., Orkin S. H., Mole J. E., Colten H. R., Yin H. L. Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 1986 Oct 2;323(6087):455–458. doi: 10.1038/323455a0. [DOI] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Leavitt J., Gunning P., Kedes L., Jariwalla R. Smooth muscle alpha-action is a transformation-sensitive marker for mouse NIH 3T3 and Rat-2 cells. 1985 Aug 29-Sep 4Nature. 316(6031):840–842. doi: 10.1038/316840a0. [DOI] [PubMed] [Google Scholar]
  27. Leavitt J., Latter G., Lutomski L., Goldstein D., Burbeck S. Tropomyosin isoform switching in tumorigenic human fibroblasts. Mol Cell Biol. 1986 Jul;6(7):2721–2726. doi: 10.1128/mcb.6.7.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matsumura F., Lin J. J., Yamashiro-Matsumura S., Thomas G. P., Topp W. C. Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells. J Biol Chem. 1983 Nov 25;258(22):13954–13964. [PubMed] [Google Scholar]
  29. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  30. Owada M. K., Hakura A., Iida K., Yahara I., Sobue K., Kakiuchi S. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells. Proc Natl Acad Sci U S A. 1984 May;81(10):3133–3137. doi: 10.1073/pnas.81.10.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pollack R., Osborn M., Weber K. Patterns of organization of actin and myosin in normal and transformed cultured cells. Proc Natl Acad Sci U S A. 1975 Mar;72(3):994–998. doi: 10.1073/pnas.72.3.994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shapland C., Lowings P., Lawson D. Identification of new actin-associated polypeptides that are modified by viral transformation and changes in cell shape. J Cell Biol. 1988 Jul;107(1):153–161. doi: 10.1083/jcb.107.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Verderame M., Alcorta D., Egnor M., Smith K., Pollack R. Cytoskeletal F-actin patterns quantitated with fluorescein isothiocyanate-phalloidin in normal and transformed cells. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6624–6628. doi: 10.1073/pnas.77.11.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yin H. L. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays. 1987 Oct;7(4):176–179. doi: 10.1002/bies.950070409. [DOI] [PubMed] [Google Scholar]
  35. Yin H. L., Kwiatkowski D. J., Mole J. E., Cole F. S. Structure and biosynthesis of cytoplasmic and secreted variants of gelsolin. J Biol Chem. 1984 Apr 25;259(8):5271–5276. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES