Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1984 Jan;49(1):162–170. doi: 10.1128/jvi.49.1.162-170.1984

Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

D T Rowe, P E Branton, S P Yee, S Bacchetti, F L Graham
PMCID: PMC255437  PMID: 6690708

Abstract

We have established a library of hamster cells transformed by adenovirus 5 DNA fragments comprising all (XhoI-C, 0 to 16 map units) or only a part (HindIII-G, 0 to 7.8 map units) of early region 1 (E1: 0 to 11.2 map units). These lines have been analyzed in terms of content of viral DNA, expression of E1 antigens, and capacity to induce tumors in hamsters. All cells tested were found to express up to eight proteins encoded within E1A (0 to 4.5 map units) with apparent molecular weights between 52,000 (52K) and 25K. Both G and C fragment-transformed lines expressed a 19K antigen encoded within E1B (4.5 to 11.2 map units), whereas an E1B 58K protein was detected in C fragment-transformed, but not G-fragment-transformed, lines. No clear distinction could be drawn between cells transformed by HindIII-G and by XhoI-C in terms of morphology or tumorigenicity, suggesting that the E1B 58K antigen plays no major role in the maintenance of oncogenic transformation, although possible involvement of truncated forms of 58K cannot be ruled out. Sera were collected from tumor-bearing animals and examined for ability to immunoprecipitate proteins from infected cells. The relative avidity of sera for different proteins was characteristic of the cell line used for tumor induction, and the specificity generally reflected the array of viral proteins expressed by the corresponding transformed cells. However, one notable observation was that even though all transformed lines examined expressed antigens encoded by both the 1.1- and 0.9-kilobase mRNAs transcribed from E1A, tumor sera made against these lines only precipitated products of the 1.1-kilobase message. Thus, two families of E1A proteins, highly related in terms of primary amino acid sequence, appear to be immunologically quite distinct.

Full text

PDF
165

Images in this article

165Image
on p.165
166Image
on p.166
167Image
on p.167
167Image
on p.167
168Image
on p.168

Selected References

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

  1. Aleström P., Akusjärvi G., Perricaudet M., Mathews M. B., Klessig D. F., Pettersson U. The gene for polypeptide IX of adenovirus type 2 and its unspliced messenger RNA. Cell. 1980 Mar;19(3):671–681. doi: 10.1016/s0092-8674(80)80044-4. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Structure of the adenovirus 2 early mRNAs. Cell. 1978 Jul;14(3):695–711. doi: 10.1016/0092-8674(78)90252-0. [DOI] [PubMed] [Google Scholar]
  3. Bernards R., Houweling A., Schrier P. I., Bos J. L., Van der Eb A. J. Characterization of cells transformed by Ad5/Ad12 hybrid early region I plasmids. Virology. 1982 Jul 30;120(2):422–432. doi: 10.1016/0042-6822(82)90042-3. [DOI] [PubMed] [Google Scholar]
  4. Bos J. L., Polder L. J., Bernards R., Schrier P. I., van den Elsen P. J., van der Eb A. J., van Ormondt H. The 2.2 kb E1b mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets. Cell. 1981 Nov;27(1 Pt 2):121–131. doi: 10.1016/0092-8674(81)90366-4. [DOI] [PubMed] [Google Scholar]
  5. Chow L. T., Broker T. R., Lewis J. B. Complex splicing patterns of RNAs from the early regions of adenovirus-2. J Mol Biol. 1979 Oct 25;134(2):265–303. doi: 10.1016/0022-2836(79)90036-6. [DOI] [PubMed] [Google Scholar]
  6. Downey J. F., Rowe D. T., Bacchetti S., Graham F. L., Bayley S. T. Mapping of a 14,000-dalton antigen to early region 4 of the human adenovirus 5 genome. J Virol. 1983 Feb;45(2):514–523. doi: 10.1128/jvi.45.2.514-523.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Esche H., Mathews M. B., Lewis J. B. Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses. J Mol Biol. 1980 Sep 25;142(3):399–417. doi: 10.1016/0022-2836(80)90279-x. [DOI] [PubMed] [Google Scholar]
  8. Esche H., Siegmann B. Expression of early viral gene products in adenovirus type 12-infected and -transformed cells. J Gen Virol. 1982 May;60(Pt 1):99–113. doi: 10.1099/0022-1317-60-1-99. [DOI] [PubMed] [Google Scholar]
  9. Flint S. J., Sambrook J., Williams J. F., Sharp P. A. Viral nucleic acid sequences in transformed cells. IV. A study of the sequences of adenovirus 5 DNA and RNA in four lines of adenovirus 5-transformed rodent cells using specific fragments of the viral genome. Virology. 1976 Jul 15;72(2):456–470. doi: 10.1016/0042-6822(76)90174-4. [DOI] [PubMed] [Google Scholar]
  10. Freeman A. E., Black P. H., Vanderpool E. A., Henry P. H., Austin J. B., Huebner R. J. Transformation of primary rat embryo cells by adenovirus type 2. Proc Natl Acad Sci U S A. 1967 Sep;58(3):1205–1212. doi: 10.1073/pnas.58.3.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freeman A. E., Black P. H., Wolford R., Huebner R. J. Adenovirus type 12-rat embryo transformation system. J Virol. 1967 Apr;1(2):362–367. doi: 10.1128/jvi.1.2.362-367.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallimore P. H. Interactions of adenovirus type 2 with rat embryo cells. Permissiveness, transformation and in vitro characteristics of adenovirus transformed rat embryo cells. J Gen Virol. 1974 Nov;25(2):263–273. doi: 10.1099/0022-1317-25-2-263. [DOI] [PubMed] [Google Scholar]
  13. Gallimore P. H., McDougall J. K., Chen L. B. In vitro traits of adenovirus-transformed cell lines and their relevance to tumorigenicity in nude mice. Cell. 1977 Apr;10(4):669–678. doi: 10.1016/0092-8674(77)90100-3. [DOI] [PubMed] [Google Scholar]
  14. Gallimore P. H., Paraskeva C. A study to determine the reasons for differences in the tumorigenicity of rat cell lines transformed by adenovirus 2 and adenovirus 12. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):703–713. doi: 10.1101/sqb.1980.044.01.075. [DOI] [PubMed] [Google Scholar]
  15. Gallimore P. H. Tumour production in immunosuppressed rats with cells transformed in vitro by adenovirus type 2. J Gen Virol. 1972 Jul;16(1):99–102. doi: 10.1099/0022-1317-16-1-99. [DOI] [PubMed] [Google Scholar]
  16. Gallimore P. H. Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA IN NINE LINES OF TRANSFORMED RAT CELLS USING SPECIFIC FRAGMENTS OF THE VIRAL GENOME;. J Mol Biol. 1974 Oct 15;89(1):49–72. doi: 10.1016/0022-2836(74)90162-4. [DOI] [PubMed] [Google Scholar]
  17. Gingeras T. R., Sciaky D., Gelinas R. E., Bing-Dong J., Yen C. E., Kelly M. M., Bullock P. A., Parsons B. L., O'Neill K. E., Roberts R. J. Nucleotide sequences from the adenovirus-2 genome. J Biol Chem. 1982 Nov 25;257(22):13475–13491. [PubMed] [Google Scholar]
  18. Girvitz S. C., Bacchetti S., Rainbow A. J., Graham F. L. A rapid and efficient procedure for the purification of DNA from agarose gels. Anal Biochem. 1980 Aug;106(2):492–496. doi: 10.1016/0003-2697(80)90553-9. [DOI] [PubMed] [Google Scholar]
  19. Graham F. L., Abrahams P. J., Mulder C., Heijneker H. L., Warnaar S. O., De Vries F. A., Fiers W., Van Der Eb A. J. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and simian virus 40. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):637–650. doi: 10.1101/sqb.1974.039.01.077. [DOI] [PubMed] [Google Scholar]
  20. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Graham F. L., van der Eb A. J. Transformation of rat cells by DNA of human adenovirus 5. Virology. 1973 Aug;54(2):536–539. doi: 10.1016/0042-6822(73)90163-3. [DOI] [PubMed] [Google Scholar]
  23. Green M., Brackmann K. H., Cartas M. A., Matsuo T. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region. J Virol. 1982 Apr;42(1):30–41. doi: 10.1128/jvi.42.1.30-41.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Green M., Wold W. S., Büttner W. Integration and transcription of group C human adenovirus sequences in the DNA of five lines of transformed rat cells. J Mol Biol. 1981 Sep 25;151(3):337–366. doi: 10.1016/0022-2836(81)90001-2. [DOI] [PubMed] [Google Scholar]
  25. Halbert D. N., Spector D. J., Raskas H. J. In vitro translation products specified by the transforming region of adenovirus type 2. J Virol. 1979 Sep;31(3):621–629. doi: 10.1128/jvi.31.3.621-629.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Houweling A., van den Elsen P. J., van der Eb A. J. Partial transformation of primary rat cells by the leftmost 4.5% fragment of adenovirus 5 DNA. Virology. 1980 Sep;105(2):537–550. doi: 10.1016/0042-6822(80)90054-9. [DOI] [PubMed] [Google Scholar]
  27. Jochemsen H., Daniëls G. S., Hertoghs J. J., Schrier P. I., van den Elsen P. J., van der EB A. J. Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis. Virology. 1982 Oct 15;122(1):15–28. doi: 10.1016/0042-6822(82)90373-7. [DOI] [PubMed] [Google Scholar]
  28. Johansson K., Persson H., Lewis A. M., Pettersson U., Tibbetts C., Philipson L. Viral DNA sequences and gene products in hamster cells transformed by adenovirus type 2. J Virol. 1978 Sep;27(3):628–639. doi: 10.1128/jvi.27.3.628-639.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Lassam N. J., Bayley S. T., Graham F. L. Tumor antigens of human Ad5 in transformed cells and in cells infected with transformation-defective host-range mutants. Cell. 1979 Nov;18(3):781–791. doi: 10.1016/0092-8674(79)90131-4. [DOI] [PubMed] [Google Scholar]
  31. Levinson A. D., Levine A. J. The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis. Cell. 1977 Aug;11(4):871–879. doi: 10.1016/0092-8674(77)90298-7. [DOI] [PubMed] [Google Scholar]
  32. Lewis A. M., Jr, Breeden J. H., Wewerka Y. L., Schnipper L. E., Levine A. S. Studies of hamster cells transformed by adenovirus 2 and the nondefective Ad2-SV40 hybrids. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):651–656. doi: 10.1101/sqb.1974.039.01.078. [DOI] [PubMed] [Google Scholar]
  33. Lewis A. M., Jr, Cook J. L. Spectrum of tumorigenic phenotypes among adenovirus 2-, adenovirus 12-, and simian virus 40-transformed Syrian hamster cells defined by host cellular immune-tumor cell interactions. Cancer Res. 1982 Mar;42(3):939–944. [PubMed] [Google Scholar]
  34. Lewis J. B., Mathews M. B. Viral messenger RNAs in six lines of adenovirus-transformed cells. Virology. 1981 Dec;115(2):345–360. doi: 10.1016/0042-6822(81)90116-1. [DOI] [PubMed] [Google Scholar]
  35. MCBRIDE W. D., WIENER A. IN VITRO TRANSFORMATION OF HAMSTER KIDNEY CELLS BY HUMAN ADENOVIRUS TYPE 12. Proc Soc Exp Biol Med. 1964 Apr;115:870–874. doi: 10.3181/00379727-115-29060. [DOI] [PubMed] [Google Scholar]
  36. Mak S., Mak I., Smiley J. R., Graham F. L. Tumorigenicity and viral gene expression in rat cells transformed by Ad 12 virions or by the EcoRI c fragment of Ad 12 DNA. Virology. 1979 Oct 30;98(2):456–460. doi: 10.1016/0042-6822(79)90568-3. [DOI] [PubMed] [Google Scholar]
  37. Matsuo T., Wold W. S., Hashimoto S., Rankin A., Symington J., Green M. Polypeptides encoded by transforming region E 1b of human adenovirus 2: immunoprecipitation from transformed and infected cells and cell-free translation of E 1b-specific mRNA. Virology. 1982 Apr 30;118(2):456–465. doi: 10.1016/0042-6822(82)90366-x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. POPE J. H., ROWE W. P. IMMUNOFLUORESCENT STUDIES OF ADENOVIRUS 12 TUMORS AND OF CELLS TRANSFORMED OR INFECTED BY ADENOVIRUSES. J Exp Med. 1964 Oct 1;120:577–588. doi: 10.1084/jem.120.4.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Perricaudet M., Akusjärvi G., Virtanen A., Pettersson U. Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses. Nature. 1979 Oct 25;281(5733):694–696. doi: 10.1038/281694a0. [DOI] [PubMed] [Google Scholar]
  41. Persson H., Katze M. G., Philipson L. Control of adenovirus early gene expression: accumulation of viral mRNA after infection of transformed cells. J Virol. 1981 Nov;40(2):358–366. doi: 10.1128/jvi.40.2.358-366.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Raska K., Jr, Gallimore P. H. An inverse relation of the oncogenic potential of adenovirus-transformed cells and their sensitivity to killing by syngeneic natural killer cells. Virology. 1982 Nov;123(1):8–18. doi: 10.1016/0042-6822(82)90290-2. [DOI] [PubMed] [Google Scholar]
  43. Rowe D. T., Graham F. L., Branton P. E. Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells. Virology. 1983 Sep;129(2):456–468. doi: 10.1016/0042-6822(83)90183-6. [DOI] [PubMed] [Google Scholar]
  44. Rowe D. T., Yee S. P., Otis J., Graham F. L., Branton P. E. Characterization of human adenovirus type 5 early region 1A polypeptides using antitumor sera and an antiserum specific for the carboxy terminus. Virology. 1983 Jun;127(2):253–271. doi: 10.1016/0042-6822(83)90142-3. [DOI] [PubMed] [Google Scholar]
  45. Ruben M., Bacchetti S., Graham F. L. Integration and expression of viral DNA in cells transformed by host range mutants of adenovirus type 5. J Virol. 1982 Feb;41(2):674–685. doi: 10.1128/jvi.41.2.674-685.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schaffhausen B. S., Silver J. E., Benjamin T. L. Tumor antigen(s) in cell productively infected by wild-type polyoma virus and mutant NG-18. Proc Natl Acad Sci U S A. 1978 Jan;75(1):79–83. doi: 10.1073/pnas.75.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schrier P. I., Van Den Elsen P. J., Hertoghs J. J., Van Der Eb A. J. Characterization of tumor antigens in cells transformed by fragments of adenovirus type 5 DNA. Virology. 1979 Dec;99(2):372–385. doi: 10.1016/0042-6822(79)90016-3. [DOI] [PubMed] [Google Scholar]
  48. Shiroki K., Handa H., Shimojo H., Yano S., Ojima S., Fujinaga K. Establishment and characterization of rat cell lines transformed by restriction endonuclease fragments of adenovirus 12 DNA. Virology. 1977 Oct 15;82(2):462–471. doi: 10.1016/0042-6822(77)90019-8. [DOI] [PubMed] [Google Scholar]
  49. Smart J. E., Lewis J. B., Mathews M. B., Harter M. L., Anderson C. W. Adenovirus type 2 early proteins: assignment of the early region 1A proteins synthesized in vivo and in vitro to specific mRNAs. Virology. 1981 Jul 30;112(2):703–713. doi: 10.1016/0042-6822(81)90315-9. [DOI] [PubMed] [Google Scholar]
  50. Van der Eb A. J., Mulder C., Graham F. L., Houweling A. Transformation with specific fragments of adenovirus DNAs. I. Isolation of specific fragments with transforming activity of adenovirus 2 and 5 DNA. Gene. 1977;2(3-4):115–132. doi: 10.1016/0378-1119(77)90012-9. [DOI] [PubMed] [Google Scholar]
  51. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Williams J. F. Oncogenic transformation of hamster embryo cells in vitro by adenovirus type 5. Nature. 1973 May 18;243(5403):162–163. doi: 10.1038/243162a0. [DOI] [PubMed] [Google Scholar]
  53. Wilson M. C., Sawicki S. G., Salditt-Georgieff M., Darnell J. E. Adenovirus type 2 mRNA in transformed cells: map positions and difference in transport time. J Virol. 1978 Jan;25(1):97–103. doi: 10.1128/jvi.25.1.97-103.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yee S. P., Rowe D. T., Tremblay M. L., McDermott M., Branton P. E. Identification of human adenovirus early region 1 products by using antisera against synthetic peptides corresponding to the predicted carboxy termini. J Virol. 1983 Jun;46(3):1003–1013. doi: 10.1128/jvi.46.3.1003-1013.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. van der Eb A. J., van Ormondt H., Schrier P. I., Lupker J. H., Jochemsen H., van den Elsen P. J., DeLeys R. J., Maat J., van Beveren C. P., Dijkema R. Structure and function of the transforming genes of human adenoviruses and SV40. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 1):383–399. doi: 10.1101/sqb.1980.044.01.043. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES