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. 1973 Apr;70(4):1055–1059. doi: 10.1073/pnas.70.4.1055

Transformation of Chick-Embryo Fibroblasts by Wild-Type and Temperature-Sensitive Rous Sarcoma Virus Alters Adenylate Cyclase Activity

Wayne B Anderson 1, George S Johnson 1, Ira Pastan 1
PMCID: PMC433424  PMID: 4352222

Abstract

The activity of the enzyme adenylate cyclase, a component of the plasma membrane, has been determined in chick-embryo fibroblasts and in cells transformed by either Bryan high-titer strain of Rous sarcoma virus (RSV-BH) or a temperature-sensitive mutant of this virus (RSV-BH-Ta). Adenylate cyclase activity is reduced in cells transformed by the wild-type virus and also by the temperature-sensitive mutant when the cells are grown at the permissive temperature (37°). Transformation results in an altered affinity (Km) for the substrate (Mg ATP). The apparent Km ATP is 0.23 mM in normal cells and 1.1 mM in cells transformed with wild-type virus. The Km ATP of the cells infected with RSV-BH-Ta is 0.67-1.0 mM at 37° and 0.28 mM at 42°. The enzyme from normal cells appears to have two binding sites for Mg++, one at the catalytic site and a second at a regulatory site. Transformation by RSV-BH or RSV-BH-Ta (37°) apparently alters this second Mg++ site. A decrease in adenylate cyclase activity occurs within 10 min after cells infected with RSV-BH-Ta are shifted from 42° to 37°; the activity falls to one-half that of normal cells 30 min after the temperature shift. Our observations indicate that a viral function lowers cyclic AMP content by lowering the activity of adenylate cyclase, probably through some modification of the plasma membrane.

Keywords: ATP, Mg++, cyclic AMP

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Selected References

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

  1. Bader J. P., Brown N. R. Induction of mutations in an RNA tumour virus by an analogue of a DNA precursor. Nat New Biol. 1971 Nov 3;234(44):11–12. doi: 10.1038/newbio234011a0. [DOI] [PubMed] [Google Scholar]
  2. Bader J. P. Temperature-dependent transformation of cells infected with a mutant of Bryan Rous sarcoma virus. J Virol. 1972 Aug;10(2):267–276. doi: 10.1128/jvi.10.2.267-276.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birnbaumer L., Pohl S. L., Rodbell M. Adenyl cyclase in fat cells. 1. Properties and the effects of adrenocorticotropin and fluoride. J Biol Chem. 1969 Jul 10;244(13):3468–3476. [PubMed] [Google Scholar]
  4. Brady R. O., Borek C., Bradley R. M. Composition and synthesis of gangliosides in rat hepatocyte and hepatoma cell lines. J Biol Chem. 1969 Dec 10;244(23):6552–6554. [PubMed] [Google Scholar]
  5. Brown H. D., Chattopadhyay S. K., Morris H. P., Pennington S. N. Adenyl cyclase activity in Morris hepatomas 7777, 7794A, and 9618A. Cancer Res. 1970 Jan;30(1):123–126. [PubMed] [Google Scholar]
  6. Buck C. A., Glick M. C., Warren L. A comparative study of glycoproteins from the surface of control and Rous sarcoma virus transformed hamster cells. Biochemistry. 1970 Nov 10;9(23):4567–4576. doi: 10.1021/bi00825a016. [DOI] [PubMed] [Google Scholar]
  7. Conway A., Koshland D. E., Jr Negative cooperativity in enzyme action. The binding of diphosphopyridine nucleotide to glyceraldehyde 3-phosphate dehydrogenase. Biochemistry. 1968 Nov;7(11):4011–4023. doi: 10.1021/bi00851a031. [DOI] [PubMed] [Google Scholar]
  8. Drummond G. I., Severson D. L., Duncan L. Adenyl cyclase. Kinetic properties and nature of fluoride and hormone stimulation. J Biol Chem. 1971 Jul 10;246(13):4166–4173. [PubMed] [Google Scholar]
  9. Goggins J. F., Johnson G. S., Pastan I. The effect of dibutyryl cyclic adenosine monophosphate on synthesis of sulfated acid mucopolysaccharides by transformed fibroblasts. J Biol Chem. 1972 Sep 25;247(18):5759–5764. [PubMed] [Google Scholar]
  10. Granner D., Chase L. R., Aurbach G. D., Tomkins G. M. Tyrosine aminotransferase: enzyme induction independent of adenosine 3', 5'-monophosphate. Science. 1968 Nov 29;162(3857):1018–1020. doi: 10.1126/science.162.3857.1018. [DOI] [PubMed] [Google Scholar]
  11. Hakomori S. Cell density-dependent changes of glycolipid concentrations in fibroblasts, and loss of this response in virus-transformed cells. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1741–1747. doi: 10.1073/pnas.67.4.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Heidrick M. L., Ryan W. L. Adenosine 3',5'-cyclic monophosphate and contact inhibition. Cancer Res. 1971 Sep;31(9):1313–1315. [PubMed] [Google Scholar]
  13. Hsie A. W., Jones C., Puck T. T. Further changes in differentiation state accompanying the conversion of Chinese hamster cells of fibroblastic form by dibutyryl adenosine cyclic 3':5'-monophosphate and hormones. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1648–1652. doi: 10.1073/pnas.68.7.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hsie A. W., Puck T. T. Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3':5'-monophosphate and testosterone. Proc Natl Acad Sci U S A. 1971 Feb;68(2):358–361. doi: 10.1073/pnas.68.2.358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Johnson G. S., Friedman R. M., Pastan I. Restoration of several morphological characteristics of normal fibroblasts in sarcoma cells treated with adenosine-3':5'-cyclic monphosphate and its derivatives. Proc Natl Acad Sci U S A. 1971 Feb;68(2):425–429. doi: 10.1073/pnas.68.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Johnson G. S., Morgan W. D., Pastan I. Regulation of cell motility by cyclic AMP. Nature. 1972 Jan 7;235(5332):54–56. doi: 10.1038/235054a0. [DOI] [PubMed] [Google Scholar]
  17. Johnson G. S., Pastan I. Role of 3',5'-adenosine monophosphate in regulation of morphology and growth of transformed and normal fibroblasts. J Natl Cancer Inst. 1972 May;48(5):1377–1387. [PubMed] [Google Scholar]
  18. Krishna G., Weiss B., Brodie B. B. A simple, sensitive method for the assay of adenyl cyclase. J Pharmacol Exp Ther. 1968 Oct;163(2):379–385. [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Meezan E., Wu H. C., Black P. H., Robbins P. W. Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry. 1969 Jun;8(6):2518–2524. doi: 10.1021/bi00834a039. [DOI] [PubMed] [Google Scholar]
  21. Mora P. T., Brady R. O., Bradley R. M., McFarland V. W. Gangliosides in DNA virus-transformed and spontaneously transformed tumorigenic mouse cell lines. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1290–1296. doi: 10.1073/pnas.63.4.1290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nakata Y., Bader J. P. Studies on the fixation and development of cellular transformation by Rous sarcoma virus. Virology. 1968 Nov;36(3):401–410. doi: 10.1016/0042-6822(68)90165-7. [DOI] [PubMed] [Google Scholar]
  23. Otten J., Bader J., Johnson G. S., Pastan I. A mutation in a rous sarcoma virus gene that controls adenosine 3',5'-monophosphate levels and transformation. J Biol Chem. 1972 Mar 10;247(5):1632–1633. [PubMed] [Google Scholar]
  24. Otten J., Johnson G. S., Pastan I. Cyclic AMP levels in fibroblasts: relationship to growth rate and contact inhibition of growth. Biochem Biophys Res Commun. 1971 Sep;44(5):1192–1198. doi: 10.1016/s0006-291x(71)80212-7. [DOI] [PubMed] [Google Scholar]
  25. Peery C. V., Johnson G. S., Pastan I. Adenyl cyclase in normal and transformed fibroblasts in tissue culture. Activation by prostaglandins. J Biol Chem. 1971 Sep 25;246(18):5785–5790. [PubMed] [Google Scholar]
  26. Perdue J. F., Kletzien R., Miller K. The isolation and characterization of plasma membrane from cultured cells. I. The chemical composition of membrane isolated from uninfected and oncogenic RNA virus-converted chick embryo fibroblasts. Biochim Biophys Acta. 1971 Dec 3;249(2):419–434. doi: 10.1016/0005-2736(71)90120-9. [DOI] [PubMed] [Google Scholar]
  27. Robison G. A., Butcher R. W., Sutherland E. W. Cyclic AMP. Annu Rev Biochem. 1968;37:149–174. doi: 10.1146/annurev.bi.37.070168.001053. [DOI] [PubMed] [Google Scholar]
  28. Ryan W. L., Heidrick M. L. Inhibition of cell growth in vitro by adenosine 3',5'-monophosphate. Science. 1968 Dec 27;162(3861):1484–1485. doi: 10.1126/science.162.3861.1484. [DOI] [PubMed] [Google Scholar]
  29. Severson D. L., Drummond G. I., Sulakhe P. V. Adenylate cyclase in skeletal muscle. Kinetic properties and hormonal stimulation. J Biol Chem. 1972 May 10;247(9):2949–2958. [PubMed] [Google Scholar]
  30. Sheppard J. R. Difference in the cyclic adenosine 3',5'-monophosphate levels in normal and transformed cells. Nat New Biol. 1972 Mar 1;236(61):14–16. doi: 10.1038/newbio236014a0. [DOI] [PubMed] [Google Scholar]
  31. Sheppard J. R. Restoration of contact-inhibited growth to transformed cells by dibutyryl adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1316–1320. doi: 10.1073/pnas.68.6.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Siddiqui B., Hakomori S. Change of glycolipid pattern in Morris hepatomas 5123 and 7800. Cancer Res. 1970 Dec;30(12):2930–2936. [PubMed] [Google Scholar]
  33. Wu H. C., Meezan E., Black P. H., Robbins P. W. Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. I. Glucosamine-labeling patterns in 3T3, spontaneously transformed 3T3, and SV-40-transformed 3T3 cells. Biochemistry. 1969 Jun;8(6):2509–2517. doi: 10.1021/bi00834a038. [DOI] [PubMed] [Google Scholar]

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