Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Sep;78(9):5593–5597. doi: 10.1073/pnas.78.9.5593

Sequence of events in the transformation process in cells infected with a temperature-sensitive transformation mutant of Moloney murine sarcoma virus.

R L Brown, J P Horn, L Wible, R B Arlinghaus, B R Brinkley
PMCID: PMC348797  PMID: 6946495

Abstract

Normal rat kidney cells infected with the temperature-sensitive transformation mutant of Moloney murine sarcoma virus were used to study the biochemical and morphological changes that occur during transformation. The infected cells exhibited a normal morphology at the nonpermissive temperature (39 degrees C) and a transformed morphology at the permissive temperature (33 degrees C). A new viral protein was detected 2 hr after shift to the permissive temperature as a polyprotein with an estimated Mr of 85,000 (p85). Scanning electron microscopy of the cells within 5 hr after shifting them to the permissive temperature showed that they became smaller and rounded with numerous elongated microvilli. In an earlier study, changes in hexose uptake were found to occur 8-12 hr after the shift [Horn, J. P., Wood, T. G., Blair, D. G. & Arlinghaus, R. B. (1980) Virology 105, 516-525]. By 48 hr, the cells had the morphology of a fully transformed cell. Concomitant with the changes in the morphology were alterations in the cytoplasmic microtubule complex. At the nonpermissive temperature, the complex consisted of a lacy network of microtubules. Within 5 hr at the permissive temperature, the lacy network was still present but the microtubules were more diffusely stained and less discernible. By 48 hr, the microtubules were so diffuse that the lacy network could not be recognized. Alterations in the F-actin cables did not occur until 24 hr after shifting the cells to the permissive temperature. Enucleation of the cells at the nonpermissive temperature and shifting the cytoplasts to the permissive temperature did not result in the synthesis of detectable p85 or in any alteration of the cytoplast morphology or microtubule complex, suggesting that the temperature-sensitive lesion affects some event occurring in the nucleus.

Full text

PDF
5595

Images in this article

5594Image
on p.5594
5594Image
on p.5594
5595Image
on p.5595
5595Image
on p.5595
5595Image
on p.5595
5596Image
on p.5596
5596Image
on p.5596
5597Image
on p.5597

Selected References

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

  1. Beug H., Claviez M., Jockusch B. M., Graf T. Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells. Cell. 1978 Aug;14(4):843–856. doi: 10.1016/0092-8674(78)90340-9. [DOI] [PubMed] [Google Scholar]
  2. Bishop J. M. Retroviruses. Annu Rev Biochem. 1978;47:35–88. doi: 10.1146/annurev.bi.47.070178.000343. [DOI] [PubMed] [Google Scholar]
  3. Blair D. G., Hull M. A., Finch E. A. The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus. Virology. 1979 Jun;95(2):303–316. doi: 10.1016/0042-6822(79)90486-0. [DOI] [PubMed] [Google Scholar]
  4. Brinkley B. R., Fuller E. M., Highfield D. P. Cytoplasmic microtubules in normal and transformed cells in culture: analysis by tubulin antibody immunofluorescence. Proc Natl Acad Sci U S A. 1975 Dec;72(12):4981–4985. doi: 10.1073/pnas.72.12.4981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burger M. M. Surface changes in transformed cells detected by lectins. Fed Proc. 1973 Jan;32(1):91–101. [PubMed] [Google Scholar]
  6. Burr J. G., Dreyfuss G., Penman S., Buchanan J. M. Association of the src gene product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3484–3488. doi: 10.1073/pnas.77.6.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Edelman G. M., Yahara I. Temperature-sensitive changes in surface modulating assemblies of fibroblasts transformed by mutants of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2047–2051. doi: 10.1073/pnas.73.6.2047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fuller G. M., Brinkley B. R. Structure and control of assembly of cytoplasmic microtubules in normal and transformed cells. J Supramol Struct. 1976;5(4):497(349)–514(366). doi: 10.1002/jss.400050407. [DOI] [PubMed] [Google Scholar]
  9. Gail M. H., Boone C. W. Cytochalasin effects on BALB-3T3 fibroblasts: dose-dependent, reversible alteration of motility and cytoplasmic cleavage. Exp Cell Res. 1971 Sep;68(1):226–228. doi: 10.1016/0014-4827(71)90610-0. [DOI] [PubMed] [Google Scholar]
  10. Horn J. P., Wood T. G., Blair D. G., Arlinghaus R. B. Partial characterization of a moloney murine sarcoma virus 85,000-dalton polypeptide whose expression correlates with the transformed phenotype in cells infected with a temperature-sensitive mutant virus. Virology. 1980 Sep;105(2):516–525. doi: 10.1016/0042-6822(80)90052-5. [DOI] [PubMed] [Google Scholar]
  11. Horn J. P., Wood T. G., Murphy E. C., Jr, Blair D. G., Arlinghaus R. B. A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus. Cell. 1981 Jul;25(1):37–46. doi: 10.1016/0092-8674(81)90229-4. [DOI] [PubMed] [Google Scholar]
  12. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  13. Lucas J. J., Szekely E., Kates J. R. The regeneration and division of mouse L-cell karyoplasts. Cell. 1976 Jan;7(1):115–122. doi: 10.1016/0092-8674(76)90261-0. [DOI] [PubMed] [Google Scholar]
  14. MACPHERSON I., MONTAGNIER L. AGAR SUSPENSION CULTURE FOR THE SELECTIVE ASSAY OF CELLS TRANSFORMED BY POLYOMA VIRUS. Virology. 1964 Jun;23:291–294. doi: 10.1016/0042-6822(64)90301-0. [DOI] [PubMed] [Google Scholar]
  15. Naso R. B., Arcement L. J., Arlinghaus R. B. Biosynthesis of Rauscher leukemia viral proteins. Cell. 1975 Jan;4(1):31–36. doi: 10.1016/0092-8674(75)90130-0. [DOI] [PubMed] [Google Scholar]
  16. Pollack R. E., Burger M. M. Surface-specific characteristics of a contact-inhibited cell line containing the SV40 viral genome. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1074–1076. doi: 10.1073/pnas.62.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Puck T. T. Cyclic AMP, the microtubule-microfilament system, and cancer. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4491–4495. doi: 10.1073/pnas.74.10.4491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Puck T. T., Waldren C. A., Hsie A. W. Membrane dynamics in the action of dibutyryl adenosine 3':5'-cyclic monophosphate and testosterone on mammalian cells. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1943–1947. doi: 10.1073/pnas.69.7.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Risser R., Pollack R. A nonselective analysis of SV40 transformation of mouse 3T3 cells. Virology. 1974 Jun;59(2):477–489. doi: 10.1016/0042-6822(74)90457-7. [DOI] [PubMed] [Google Scholar]
  21. Sen A., Todaro G. J. A murine sarcoma virus-associated protein kinase: interaction with actin and microtubular protein. Cell. 1979 Jun;17(2):347–356. doi: 10.1016/0092-8674(79)90161-2. [DOI] [PubMed] [Google Scholar]
  22. Sen A., Todaro G. J., Blair D. G., Robey W. G. Thermolabile protein kinase molecules in a temperature-sensitive murine sarcoma virus pseudotype. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3617–3621. doi: 10.1073/pnas.76.8.3617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. TEMIN H. M., RUBIN H. Characteristics of an assay for Rous sarcoma virus and Rous sarcoma cells in tissue culture. Virology. 1958 Dec;6(3):669–688. doi: 10.1016/0042-6822(58)90114-4. [DOI] [PubMed] [Google Scholar]
  24. TODARO G. J., GREEN H., GOLDBERG B. D. TRANSFORMATION OF PROPERTIES OF AN ESTABLISHED CELL LINE BY SV40 AND POLYOMA VIRUS. Proc Natl Acad Sci U S A. 1964 Jan;51:66–73. doi: 10.1073/pnas.51.1.66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Willingham M. C., Pastan I., Shih T. Y., Scolnick E. M. Localization of the src gene product of the Harvey strain of MSV to plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1980 Apr;19(4):1005–1014. doi: 10.1016/0092-8674(80)90091-4. [DOI] [PubMed] [Google Scholar]
  27. Wood T. G., Peltier-Horn J., Robey W. G., Blair D. G., Arlinghaus R. B. Characterization of virus-specified proteins present in NRK cells infected with a temperature-sensitive transformation mutant of Moloney murine sarcoma virus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):747–754. doi: 10.1101/sqb.1980.044.01.080. [DOI] [PubMed] [Google Scholar]
  28. Wulf E., Deboben A., Bautz F. A., Faulstich H., Wieland T. Fluorescent phallotoxin, a tool for the visualization of cellular actin. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4498–4502. doi: 10.1073/pnas.76.9.4498. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES