Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Selective capture of acentric fragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA

Nature Genetics volume 12, pages 65–71 (1996)Cite this article

Abstract

The amplification and overexpression of a number of oncogenes is strongly associated with the progression of a variety of different cancers. We now present a strategy to purify amplified DNA on double minute chromosomes (DMs) to enable analysis of their prevalence and contribution to tumourigenesis. Using cell lines derived from four different tumour types, we have developed a general and rapid method to purify micronuclei that are known to entrap extrachromosomal elements. The isolated DNA is highly enriched in DM sequences and can be used to prepare probes to localize the progenitor single copy chromosomal regions. The capture of DMs by micronuclei appears to be dependent on their lack of a centromere rather than their small size.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Yin, Y., Tainsky, M.A., Bischoff, F.Z., Strong, L.C. & Wahl, G.M. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70, 937–948 (1992).

    Article  CAS  PubMed  Google Scholar 

  2. Livingstone, L.R., White, A., Sprouse, J., Livanos, E., Jacks, T. & Tlsty, T.D. Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53. Cell 70, 923–935 (1992).

    Article  CAS  PubMed  Google Scholar 

  3. Benner, S.E., Wahl, G.M. & Von Hoff, D.D. Double minute chromosomes and homogeneously staining regions in tumours taken directly from patients versus in human tumour cell lines. Anti-Cancer Drugs 2, 11–25 (1991).

    Article  CAS  PubMed  Google Scholar 

  4. Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumours. Science 258, 818–821 (1992).

    Article  CAS  PubMed  Google Scholar 

  5. Kallioniemi, A. et al. Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc. Natl. Acad. Sci. USA 91, 2156–2160 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cowell, J.K. Double minutes and homogeneously staining regions: gene amplification in mammalian cells. Annu. Rev. Genet. 16, 21–69 (1982).

    Article  CAS  PubMed  Google Scholar 

  7. Windle, B., Draper, B.W., Yin, Y., O'Gorman, S. & Wahl, G.M. A central role for chromosome breakage in gene amplification, deletion formation, and amplicon integration. Genes Dev. 5, 160–174 (1991).

    Article  CAS  PubMed  Google Scholar 

  8. Ma, C., Martin, S., Trask, B. & Hamlin, J.L. Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells. Genes Dev. 7, 605–620 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Smith, K.A., Stark, M.B., Gorman, P.A. & Stark, G.R. Fusions near telomeres occur very early in the amplification of CAD genes in Syrian hamster cells. Proc. Natl. Acad. Sci. USA 89, 5427–5431 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Stark, G.R. & Wahl, G.M. Gene amplification. Annu. Rev. Biochem. 53, 447–491 (1984).

    Article  CAS  PubMed  Google Scholar 

  11. Biedler, J.L. & Spengler, B.A. A novel chromosome abnormality in human neuroblastoma and antifolate-resistant Chinese hamster cell lines in culture. J. natn. Cancer Inst. 57, 683–695 (1976).

    Article  CAS  Google Scholar 

  12. Brison, O. Gene amplification and tumour progression. Biochim. biophys. Acta 1155, 25–41 (1993).

    CAS  PubMed  Google Scholar 

  13. Von Hoff, D.D. et al. Elimination of extrachromosomally amplified MYC genes from human tumour cells reduces their tumourigenicity. Proc. Natl. Acad. Sci. USA 89, 8165–8169 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shimizu, N. et al. Loss of amplified c-myc genes in spontaneously differentiated HL-60 cells. Cancer Res. 54, 3561–3567 (1994).

    CAS  PubMed  Google Scholar 

  15. Eckhardt, S.G. et al. Induction of differentiation in HL-60 cells by the reduction of extrachromosomally amplified c-myc . Proc. Natl. Acad. Sci. USA 91, 6674–6678 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Snapka, R.M. & Varshavsky, A. Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea. Proc. Natl. Acad. Sci. USA 80, 7533–7537 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Von Hoff, D.D., Waddelow, T., Forseth, B., Davidson, K., Scott, J. & Wahl, G.M. Hydroxyurea accelerates loss of extrachromosomally amplified genes from tumour cells. Cancer Res. 51, 6273–6279 (1991).

    CAS  PubMed  Google Scholar 

  18. Holt, J.T., Redner, R.L. & Nienhuis, A.W. An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Molec. Cell. Biol. 8, 963–973 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wright, J.A. . et al. DNA amplification is rare in normal human cells. Proc. Natl. Acad. Sci. USA 87, 1791–1795 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tlsty, T.D. Normal diploid human and rodent cells lack a detectable frequency of gene amplification. Proc. Natl. Acad. Sci. USA. 87, 3132–3136 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pathak, S. Cytogenetic analysis in human breast tumours. Cancer Genet. Cytogenet. 8, 125–138 (1986).

    Google Scholar 

  22. Nielsen, J.L. et al. Evidence of gene amplification in the form of double minute chromosomes is frequently observed in lung cancer. Cancer Genet. Cytogenet. 65, 120–124 (1993).

    Article  CAS  PubMed  Google Scholar 

  23. McGill, J.R. et al. Double Minutes are frequently found in ovarian carcinomas. Cancer Genet. Cytogenet. 71, 125–131 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. McGill, J.R. et al. Mapping and characterization of a microdissected colon cancer double minute chromosome. Proc. Am. Ass. Cancer Res. 36, 540 (1995).

    Google Scholar 

  25. Pinkel, D. Visualizing tumour amplification. Nature Genet. 8, 107–108 (1994).

    Article  CAS  PubMed  Google Scholar 

  26. Meltzer, P.S., Guan, X.-Y., Burgess, A. & Trent, J.M. Rapid generation of region specific probes by chromosome microdissection and their application. Nature Genet. 1, 24–28 (1992).

    Article  CAS  PubMed  Google Scholar 

  27. Guan, X.-Y., Meltzer, P.S., Dalton, W.S. & Trent, J.M. Identification of cryptic sites of DNA sequence amplification in human breast cancer by chromosome microdissection. Nature Genet. 8, 155–161 (1994).

    Article  CAS  PubMed  Google Scholar 

  28. Alitalo, K., Schwab, M., Lin, C.C., Varmus, H.E. & Bishop, J.M. Homogeneously staining chromosomal regions contain amplified copies of an abundantly expressed cellular oncogene (c-myc) in malignant neuroendocrine cells from a human colon carcinoma. Proc. Natl. Acad. Sci. USA 80, 1707–1711 (1983).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Quinn, L.A., Moore, G.E., Morgan, R.T. & Woods, L.K. Cell lines from human colon carcinoma with unusual cell products, double minutes, and homogeneously staining regions. Cancer Res. 39, 4914–4924 (1979).

    CAS  PubMed  Google Scholar 

  30. Dhar, V., Searle, B.M., & Athwal, R.S., Transfer of Chinese hamster chromosome 1 to mouse cells and regional assignment of 7 genes: a combination of gene transfer and microcell fusion. Somat. Cell Mol. Genet. 10, 547–559 (1984).

    Article  CAS  PubMed  Google Scholar 

  31. Nusse, M. & Kramer, J. Flow cytometric analysis of micronuclei found in cells after irradiation. Cytometry. 5, 20–25 (1984).

    Article  CAS  PubMed  Google Scholar 

  32. Hartzer, M.K., Pang, Y.-Y.S. & Robson, R.M. Assembly of vimentin in w'fro and its implications concerning the structure of intermediate filaments. J. Mol. Biol. 183, 365–375 (1985).

    Article  PubMed  Google Scholar 

  33. Wahl, G.M. The importance of circular DNA in mammalian gene amplification. Cancer Res. 49, 1333–1340 (1989).

    CAS  PubMed  Google Scholar 

  34. Siebert, P.D. & Larrick, W. PCR MIMICS: Competitive DNA fragments for use as internal standards in quantitative PCR. BioTechniques 14, 244–249 (1993).

    CAS  PubMed  Google Scholar 

  35. Forster, E. Rapid generation of internal standards for competitive PCR by low-stringency primer annealing. BioTechniques 16, 1006–1008 (1994).

    CAS  PubMed  Google Scholar 

  36. Ahmed Rasheed, B.K. et al. Alterations of TP53 gene in human gliomas. Cancer Res. 54, 1324–1330 (1994).

    Google Scholar 

  37. Bigner, S.H., Friedman, H.S., Vogelstein, B., Oakes, W.J. & Bigner, D.D. Amplification of the c-myc gene in human medulloblastoma cell lines and xenografts. Cancer Res. 50, 2347–2350 (1990).

    CAS  PubMed  Google Scholar 

  38. Telenius, H. et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Cnrom. Cancer. 4, 257–263 (1992).

    Article  CAS  Google Scholar 

  39. Taub, R. et al. Translocation of the c-myc gene into the immunoglobulin heavy chain locus in human Burkitt lymphoma and murine plasmacytoma cells. Proc. Natl. Acad. Sci. USA 79, 7837–7841 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Neel, B.G., Jhanwar, S.C., Chaganti, R.S.K. & Hayward, W.S. Two human c-onc genes are located on the long arm of chromosome 8. Proc. Natl. Acad. Sci. USA 79, 7842–7846 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Carine, K., Solu, J., Waltzer, E., Manch-Citron, J., Hamkalo, B.A. & Scheffler, I.E. Chinese hamster cells with a minichnomosome containing the centromere region of human chromosome 1. Somat. Cell Mol. Genet. 12, 479–491 (1986).

    Article  CAS  PubMed  Google Scholar 

  42. Carine, K., Jacquemin-Sablon, A., Waltzer, E., Mascarello, J. & Scheffler, I.E. Molecular characterization of human minichromosomes with centromere from chromosome 1 in human-hamster hybrid cells. Somat. Cell. Mol. Genet. 15, 445–460 (1989).

    Article  CAS  PubMed  Google Scholar 

  43. Fenech, M. & Morley, A.A. Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36 (1985).

    Article  CAS  PubMed  Google Scholar 

  44. Ford, J.H., Schultz, C.J. & Cornell, A.T. Chromosome elimination in micronuclei: a common cause of hypoploidy. Am. J. Hum. Genet. 43, 733–740 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Heddle, J.A. & Carrano, A.V. The DNA content of micronuclei induced in mouse bone marrow by γ-irradiation: evidence that micronuclei arise from acentric chromosomal fragments. Mutat. Res. 44, 63–69 (1977).

    Article  CAS  PubMed  Google Scholar 

  46. Heddle, J.A. et al. The induction of micronuclei as a measure of genotoxicity. Mutat. Res. 123, 61–118 (1983).

    Article  CAS  PubMed  Google Scholar 

  47. Becker, R., Scherthan, H. & Zankl, H. Use of a centromere-specific DNA probe (p82H) in nonisotopic in situ hybridization for classification of micronuclei. Genes Cnrom. Cancer. 2, 59–62 (1990).

    Article  CAS  Google Scholar 

  48. Li, J.C. & Kaminskas, E. Accumulation of DNA strand breaks and methotrexate cytotoxicity. Proc. Natl. Acad. Sci. USA 81, 5694–5698 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Eki, T., Enomoto, T., Murakami, Y., Hanaoka, F. & Yamada, M. Characterization of chromosome aberrations induced by induction at a restrictive temperature in the mouse temperature-sensitive mutant tsFT20 strain containing heat-labile DNA polymerase α. Cancer Res. 47, 5162–5170 (1987).

    CAS  PubMed  Google Scholar 

  50. Collins, S.J. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood 70, 1233–1244 (1987).

    CAS  PubMed  Google Scholar 

  51. Pinkel, D., Straume, T. & Gray, J.W. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc. Natl. Acad. Sci. USA 83, 2934–2938 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Gene Expression Laboratory, The Salk Institute for Biological Studies, 10010N. Torrey Pines Road, La Jolla, California, 92037, USA

    Noriaki Shimizu, Teru Kanda & Geoffrey M. Wahl

  2. Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 724, Japan

    Noriaki Shimizu

Authors
  1. Noriaki Shimizu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teru Kanda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Geoffrey M. Wahl
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shimizu, N., Kanda, T. & Wahl, G. Selective capture of acentric fragments by micronuclei provides a rapid method for purifying extrachromosomally amplified DNA. Nat Genet 12, 65–71 (1996). https://doi.org/10.1038/ng0196-65

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng0196-65

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing