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. 1984 Mar;3(3):659–663. doi: 10.1002/j.1460-2075.1984.tb01863.x

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

R Schäfer, S Griegel, M A Dubbert, K Willecke
PMCID: PMC557403  PMID: 6714229

Abstract

DNA fragments (0.5-4.5 kb) of normal human lymphocytes induced pre-neoplastic mouse NIH/3T3 cells after transfection to grow in soft agar medium at low efficiency (0.0007 colonies/micrograms DNA/10(6) cells). In secondary transfections high mol. wt. DNA (greater than 20 kb) of cells transformed by DNA fragments induced neoplastic transformation with high efficiency (0.16-1.1 soft agar colonies/micrograms DNA/10(6) cells). These results confirm previous data obtained by others with chicken and mouse donor DNA. We describe here that independent secondary transformants harbored human Alu repetitive DNA sequences on similar restriction fragments and formed progressively growing tumors in BALB/c mice or nude mice. The corresponding primary transformants were not tumorigenic, however, and the ability to proliferate in semi-solid agar medium was gradually lost when the cells were grown as non-confluent monolayers. Furthermore, in contrast to secondary transformants, DNA from primary transformants showed only relatively weak hybridization to a human Alu repetitive DNA probe. We conclude that in primary transformants the transformed phenotype is expressed in an unstable fashion whereas secondary transformants appear to be stably transformed.

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Selected References

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

  1. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  2. Blobel G., Potter V. R. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154(3757):1662–1665. doi: 10.1126/science.154.3757.1662. [DOI] [PubMed] [Google Scholar]
  3. Cooper G. M. Cellular transforming genes. Science. 1982 Aug 27;217(4562):801–806. doi: 10.1126/science.6285471. [DOI] [PubMed] [Google Scholar]
  4. Cooper G. M., Okenquist S., Silverman L. Transforming activity of DNA of chemically transformed and normal cells. Nature. 1980 Apr 3;284(5755):418–421. doi: 10.1038/284418a0. [DOI] [PubMed] [Google Scholar]
  5. Degnen G. E., Miller I. L., Eisenstadt J. M., Adelberg E. A. Chromosome-mediated gene transfer between closely realted strains of cultured mouse cells. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2838–2842. doi: 10.1073/pnas.73.8.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  7. Duesberg P. H. Retroviral transforming genes in normal cells? Nature. 1983 Jul 21;304(5923):219–226. doi: 10.1038/304219a0. [DOI] [PubMed] [Google Scholar]
  8. Houck C. M., Rinehart F. P., Schmid C. W. A ubiquitous family of repeated DNA sequences in the human genome. J Mol Biol. 1979 Aug 15;132(3):289–306. doi: 10.1016/0022-2836(79)90261-4. [DOI] [PubMed] [Google Scholar]
  9. Huttner K. M., Barbosa J. A., Scangos G. A., Pratcheva D. D., Ruddle F. H. DNA-mediated gene transfer without carrier DNA. J Cell Biol. 1981 Oct;91(1):153–156. doi: 10.1083/jcb.91.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klobutcher L. A., Miller C. L., Ruddle F. H. Chromosome-mediated gene transfer results in two classes of unstable transformants. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3610–3614. doi: 10.1073/pnas.77.6.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Murray M. J., Cunningham J. M., Parada L. F., Dautry F., Lebowitz P., Weinberg R. A. The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors. Cell. 1983 Jul;33(3):749–757. doi: 10.1016/0092-8674(83)90017-x. [DOI] [PubMed] [Google Scholar]
  12. Perucho M., Goldfarb M., Shimizu K., Lama C., Fogh J., Wigler M. Human-tumor-derived cell lines contain common and different transforming genes. Cell. 1981 Dec;27(3 Pt 2):467–476. doi: 10.1016/0092-8674(81)90388-3. [DOI] [PubMed] [Google Scholar]
  13. Perucho M., Hanahan D., Wigler M. Genetic and physical linkage of exogenous sequences in transformed cells. Cell. 1980 Nov;22(1 Pt 1):309–317. doi: 10.1016/0092-8674(80)90178-6. [DOI] [PubMed] [Google Scholar]
  14. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  15. Roberts J. M., Axel R. Gene amplification and gene correction in somatic cells. Cell. 1982 May;29(1):109–119. doi: 10.1016/0092-8674(82)90095-2. [DOI] [PubMed] [Google Scholar]
  16. Robins D. M., Ripley S., Henderson A. S., Axel R. Transforming DNA integrates into the host chromosome. Cell. 1981 Jan;23(1):29–39. doi: 10.1016/0092-8674(81)90267-1. [DOI] [PubMed] [Google Scholar]
  17. Rowe W. P., Pugh W. E., Hartley J. W. Plaque assay techniques for murine leukemia viruses. Virology. 1970 Dec;42(4):1136–1139. doi: 10.1016/0042-6822(70)90362-4. [DOI] [PubMed] [Google Scholar]
  18. Rubin C. M., Houck C. M., Deininger P. L., Friedmann T., Schmid C. W. Partial nucleotide sequence of the 300-nucleotide interspersed repeated human DNA sequences. Nature. 1980 Mar 27;284(5754):372–374. doi: 10.1038/284372a0. [DOI] [PubMed] [Google Scholar]
  19. Russell W. C., Newman C., Williamson D. H. A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature. 1975 Feb 6;253(5491):461–462. doi: 10.1038/253461a0. [DOI] [PubMed] [Google Scholar]
  20. Scangos G. A., Huttner K. M., Juricek D. K., Ruddle F. H. Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability. Mol Cell Biol. 1981 Feb;1(2):111–120. doi: 10.1128/mcb.1.2.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schäfer R., Hoffmann H., Willecke K. Suppression of tumorigenicity in hybrids of tumorigenic Chinese hamster cells and diploid mouse fibroblasts: dependence on the presence of at least three different mouse chromosomes and independence of hamster genome dosage. Cancer Res. 1983 May;43(5):2240–2246. [PubMed] [Google Scholar]
  22. Shih C., Padhy L. C., Murray M., Weinberg R. A. Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature. 1981 Mar 19;290(5803):261–264. doi: 10.1038/290261a0. [DOI] [PubMed] [Google Scholar]
  23. Shih C., Shilo B. Z., Goldfarb M. P., Dannenberg A., Weinberg R. A. Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5714–5718. doi: 10.1073/pnas.76.11.5714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shih C., Weinberg R. A. Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 1982 May;29(1):161–169. doi: 10.1016/0092-8674(82)90100-3. [DOI] [PubMed] [Google Scholar]
  25. Teng C. S., Teng C. T., Allfrey V. G. Studies of nuclear acidic proteins. Evidence for their phosphorylation, tissue specificity, selective binding to deoxyribonucleic acid, and stimulation effects on transcription. J Biol Chem. 1971 Jun 10;246(11):3597–3609. [PubMed] [Google Scholar]
  26. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weinberg R. A. Oncogenes of spontaneous and chemically induced tumors. Adv Cancer Res. 1982;36:149–163. doi: 10.1016/s0065-230x(08)60424-8. [DOI] [PubMed] [Google Scholar]
  28. Wigler M., Pellicer A., Silverstein S., Axel R. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell. 1978 Jul;14(3):725–731. doi: 10.1016/0092-8674(78)90254-4. [DOI] [PubMed] [Google Scholar]
  29. Willecke K., Klomfass M., Mierau R., Döhmer J. Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells. Mol Gen Genet. 1979 Feb 26;170(2):179–185. doi: 10.1007/BF00337794. [DOI] [PubMed] [Google Scholar]
  30. Willecke K., Lange R., Krüger A., Reber T. Cotransfer of two linked human genes into cultured mouse cells. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1274–1278. doi: 10.1073/pnas.73.4.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]

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