Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Feb 1;100(2):508–513. doi: 10.1083/jcb.100.2.508

Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate

PMCID: PMC2113457  PMID: 2578470

Abstract

Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3 microM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain- and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 mo. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on approximately 12-14 h and/or CE 5 to greater than 50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to greater than 50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.

Full Text

The Full Text of this article is available as a PDF (504.6 KB).

Selected References

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

  1. Albino A. P., Le Strange R., Oliff A. I., Furth M. E., Old L. J. Transforming ras genes from human melanoma: a manifestation of tumour heterogeneity? Nature. 1984 Mar 1;308(5954):69–72. doi: 10.1038/308069a0. [DOI] [PubMed] [Google Scholar]
  2. Barranco S. C., Haenelt B. R., Gee E. L. Differential sensitivities of five rat hepatoma cell lines to anticancer drugs. Cancer Res. 1978 Mar;38(3):656–660. [PubMed] [Google Scholar]
  3. Boon T., Kellermann O. Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line. Proc Natl Acad Sci U S A. 1977 Jan;74(1):272–275. doi: 10.1073/pnas.74.1.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Breznik T., Cohen J. C. Altered methylation of endogenous viral promoter sequences during mammary carcinogenesis. Nature. 1982 Jan 21;295(5846):255–257. doi: 10.1038/295255a0. [DOI] [PubMed] [Google Scholar]
  5. Christy B., Scangos G. Expression of transferred thymidine kinase genes is controlled by methylation. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6299–6303. doi: 10.1073/pnas.79.20.6299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dackowski W., Morrison S. L. Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7091–7095. doi: 10.1073/pnas.78.11.7091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Doerfler W. DNA methylation and gene activity. Annu Rev Biochem. 1983;52:93–124. doi: 10.1146/annurev.bi.52.070183.000521. [DOI] [PubMed] [Google Scholar]
  8. Ehrlich M., Wang R. Y. 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350–1357. doi: 10.1126/science.6262918. [DOI] [PubMed] [Google Scholar]
  9. Fidler I. J., Hart I. R. Biological diversity in metastatic neoplasms: origins and implications. Science. 1982 Sep 10;217(4564):998–1003. doi: 10.1126/science.7112116. [DOI] [PubMed] [Google Scholar]
  10. Frost P., Kerbel R. S. On a possible epigenetic mechanism(s) of tumor cell heterogeneity. The role of DNA methylation. Cancer Metastasis Rev. 1983;2(4):375–378. doi: 10.1007/BF00048568. [DOI] [PubMed] [Google Scholar]
  11. Gunthert U., Schweiger M., Stupp M., Doerfler W. DNA methylation in adenovirus, adenovirus-transformed cells, and host cells. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3923–3927. doi: 10.1073/pnas.73.11.3923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harris M. Induction of thymidine kinase in enzyme-deficient Chinese hamster cells. Cell. 1982 Jun;29(2):483–492. doi: 10.1016/0092-8674(82)90165-9. [DOI] [PubMed] [Google Scholar]
  13. Hoffmann J. W., Steffen D., Gusella J., Tabin C., Bird S., Cowing D., Weinberg R. A. DNA methylation affecting the expression of murine leukemia proviruses. J Virol. 1982 Oct;44(1):144–157. doi: 10.1128/jvi.44.1.144-157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Holliday R. A new theory of carcinogenesis. Br J Cancer. 1979 Oct;40(4):513–522. doi: 10.1038/bjc.1979.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Holliday R., Pugh J. E. DNA modification mechanisms and gene activity during development. Science. 1975 Jan 24;187(4173):226–232. [PubMed] [Google Scholar]
  16. Landolph J. R., Jones P. A. Mutagenicity of 5-azacytidine and related nucleosides in C3H/10T 1/2 clone 8 and V79 cells. Cancer Res. 1982 Mar;42(3):817–823. [PubMed] [Google Scholar]
  17. Nowell P. C. The clonal evolution of tumor cell populations. Science. 1976 Oct 1;194(4260):23–28. doi: 10.1126/science.959840. [DOI] [PubMed] [Google Scholar]
  18. Olsson L., Forchhammer J. Induction of the metastatic phenotype in a mouse tumor model by 5-azacytidine, and characterization of an antigen associated with metastatic activity. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3389–3393. doi: 10.1073/pnas.81.11.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Olsson L., Kaplan H. S. Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5429–5431. doi: 10.1073/pnas.77.9.5429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Olsson L., Kronstrøm H., Cambon-De Mouzon A., Honsik C., Brodin T., Jakobsen B. Antibody producing human-human hybridomas. I. Technical aspects. J Immunol Methods. 1983 Jun 24;61(1):17–32. doi: 10.1016/0022-1759(83)90004-2. [DOI] [PubMed] [Google Scholar]
  21. Olsson L. Phenotypic diversity in leukemia cell populations. Cancer Metastasis Rev. 1983;2(2):153–163. doi: 10.1007/BF00048967. [DOI] [PubMed] [Google Scholar]
  22. Olsson L., Sorensen H. R., Behnke O. Intratumoral phenotypic diversity of cloned human lung tumor cell lines and consequences for analyses with monoclonal antibodies. Cancer. 1984 Nov 1;54(9):1757–1765. doi: 10.1002/1097-0142(19841101)54:9<1757::aid-cncr2820540902>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  23. Ott M. O., Sperling L., Cassio D., Levilliers J., Sala-Trepat J., Weiss M. C. Undermethylation at the 5' end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture. Cell. 1982 Oct;30(3):825–833. doi: 10.1016/0092-8674(82)90287-2. [DOI] [PubMed] [Google Scholar]
  24. Poste G., Doll J., Fidler I. J. Interactions among clonal subpopulations affect stability of the metastatic phenotype in polyclonal populations of B16 melanoma cells. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6226–6230. doi: 10.1073/pnas.78.10.6226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Poste G., Fidler I. J. The pathogenesis of cancer metastasis. Nature. 1980 Jan 10;283(5743):139–146. doi: 10.1038/283139a0. [DOI] [PubMed] [Google Scholar]
  26. Razin A., Riggs A. D. DNA methylation and gene function. Science. 1980 Nov 7;210(4470):604–610. doi: 10.1126/science.6254144. [DOI] [PubMed] [Google Scholar]
  27. Riggs A. D., Jones P. A. 5-methylcytosine, gene regulation, and cancer. Adv Cancer Res. 1983;40:1–30. doi: 10.1016/s0065-230x(08)60678-8. [DOI] [PubMed] [Google Scholar]
  28. Riggs A. D. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14(1):9–25. doi: 10.1159/000130315. [DOI] [PubMed] [Google Scholar]
  29. Shen C. K., Maniatis T. Tissue-specific DNA methylation in a cluster of rabbit beta-like globin genes. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6634–6638. doi: 10.1073/pnas.77.11.6634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stewart C. L., Stuhlmann H., Jähner D., Jaenisch R. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc Natl Acad Sci U S A. 1982 Jul;79(13):4098–4102. doi: 10.1073/pnas.79.13.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weintraub H., Larsen A., Groudine M. Alpha-Globin-gene switching during the development of chicken embryos: expression and chromosome structure. Cell. 1981 May;24(2):333–344. doi: 10.1016/0092-8674(81)90323-8. [DOI] [PubMed] [Google Scholar]
  32. Wigler M., Levy D., Perucho M. The somatic replication of DNA methylation. Cell. 1981 Apr;24(1):33–40. doi: 10.1016/0092-8674(81)90498-0. [DOI] [PubMed] [Google Scholar]
  33. Youssoufian H., Hammer S. M., Hirsch M. S., Mulder C. Methylation of the viral genome in an in vitro model of herpes simplex virus latency. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2207–2210. doi: 10.1073/pnas.79.7.2207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES