Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1997;76(2):180–188. doi: 10.1038/bjc.1997.360

Apoptosis and 1-methyl-2-nitroimidazole toxicity in CHO cells.

C B Brezden 1, R A McClelland 1, A M Rauth 1
PMCID: PMC2223929  PMID: 9231917

Abstract

The time course and characteristics of the selective hypoxic cytotoxicity of the 2-nitroimidazole model compound 1-methyl-2-nitroimidazole (INO2) were analysed during prolonged time periods (up to 5 days post treatment). When control populations were seeded at the same cell density as drug-treated cells, they entered confluency at day 3 and underwent apoptosis at day 5, which appeared to be mediated by an autocrine mechanism. In subsequent studies of drug-treated cells, the seeding density of treated cells was adjusted to avoid this cell confluency effect. Treatment with a low INO2 concentration (2.5 mM) resulted in apoptotic DNA fragmentation (ladders), which was observed 4-5 days after an acute 6-h hypoxic drug exposure. In contrast, at a high INO2 concentration (40 mM) for 2 h, which was equitoxic to the low concentration, no characteristic DNA ladders were observed. Fluorescence microscopy revealed apoptotic bodies and pyknotic nuclei 5 days following hypoxic 2.5 mM INO2 exposure, whereas 40 mM INO2 hypoxic treatment produced cellular ghosts devoid of DNA 5 days after exposure, consistent with the DNA ladder results. However, characteristic apoptotic morphology was previously observed immediately after the acute hypoxic exposure of 40 mM INO2. Cell cycle analysis and DNA fragmentation as measured by the TdT assay suggested that dose-dependent differences in the apoptotic response occur post exposure after an equitoxic acute hypoxic exposure to either the low or the high INO2 concentration. This dose-dependent differential in response may be attributed to the degree of initial DNA damage as measured by the comet assay.

Full text

PDF
180

Images in this article

181Figure 1
on p.181
182Figure 2
on p.182
183Figure 3
on p.183
183Figure 4
on p.183

Selected References

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

  1. Bleehen N. M., Maughan T. S., Workman P., Newman H. F., Stenning S., Ward R. The combination of multiple doses of etanidazole and pimonidazole in 48 patients: a toxicity and pharmacokinetic study. Radiother Oncol. 1991;20 (Suppl 1):137–142. doi: 10.1016/0167-8140(91)90201-q. [DOI] [PubMed] [Google Scholar]
  2. Brezden C. B., Rauth A. M. Differential cell death in immortalized and non-immortalized cells at confluency. Oncogene. 1996 Jan 4;12(1):201–206. [PubMed] [Google Scholar]
  3. Bérubé L. R., Farah S., McClelland R. A., Rauth A. M. Effect of 1-methyl-2-nitrosoimidazole on intracellular thiols and calcium levels in Chinese hamster ovary cells. Biochem Pharmacol. 1991 Nov 6;42(11):2153–2161. doi: 10.1016/0006-2952(91)90351-5. [DOI] [PubMed] [Google Scholar]
  4. Colombel M., Olsson C. A., Ng P. Y., Buttyan R. Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle. Cancer Res. 1992 Aug 15;52(16):4313–4319. [PubMed] [Google Scholar]
  5. Cumano A., Paige C. J., Iscove N. N., Brady G. Bipotential precursors of B cells and macrophages in murine fetal liver. Nature. 1992 Apr 16;356(6370):612–615. doi: 10.1038/356612a0. [DOI] [PubMed] [Google Scholar]
  6. Demarcq C., Bunch R. T., Creswell D., Eastman A. The role of cell cycle progression in cisplatin-induced apoptosis in Chinese hamster ovary cells. Cell Growth Differ. 1994 Sep;5(9):983–993. [PubMed] [Google Scholar]
  7. Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994 Feb;124(4):619–626. doi: 10.1083/jcb.124.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hu Q., Kavanagh M. C., Newcombe D., Hill R. P. Detection of hypoxic fractions in murine tumors by comet assay: comparison with other techniques. Radiat Res. 1995 Dec;144(3):266–275. [PubMed] [Google Scholar]
  9. Höckel M, Schlenger K, Mitze M, Schäffer U, Vaupel P. Hypoxia and Radiation Response in Human Tumors. Semin Radiat Oncol. 1996 Jan;6(1):3–9. doi: 10.1053/SRAO0060003. [DOI] [PubMed] [Google Scholar]
  10. KARNOVSKY M. J. THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY. J Cell Biol. 1964 Nov;23:217–232. doi: 10.1083/jcb.23.2.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  12. Koch C. J., Kruuv J. The effect of extreme hypoxia on recovery after radiation by synchronized mammalian cells. Radiat Res. 1971 Oct;48(1):74–85. [PubMed] [Google Scholar]
  13. McGahon A. J., Martin S. J., Bissonnette R. P., Mahboubi A., Shi Y., Mogil R. J., Nishioka W. K., Green D. R. The end of the (cell) line: methods for the study of apoptosis in vitro. Methods Cell Biol. 1995;46:153–185. doi: 10.1016/s0091-679x(08)61929-9. [DOI] [PubMed] [Google Scholar]
  14. Meredith J. E., Jr, Fazeli B., Schwartz M. A. The extracellular matrix as a cell survival factor. Mol Biol Cell. 1993 Sep;4(9):953–961. doi: 10.1091/mbc.4.9.953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moulder J. E., Rockwell S. Hypoxic fractions of solid tumors: experimental techniques, methods of analysis, and a survey of existing data. Int J Radiat Oncol Biol Phys. 1984 May;10(5):695–712. doi: 10.1016/0360-3016(84)90301-8. [DOI] [PubMed] [Google Scholar]
  16. Noss M. B., Panicucci R., McClelland R. A., Rauth A. M. Preparation, toxicity and mutagenicity of 1-methyl-2-nitrosoimidazole. A toxic 2-nitroimidazole reduction product. Biochem Pharmacol. 1988 Jul 1;37(13):2585–2593. doi: 10.1016/0006-2952(88)90250-x. [DOI] [PubMed] [Google Scholar]
  17. Overgaard J. Clinical evaluation of nitroimidazoles as modifiers of hypoxia in solid tumors. Oncol Res. 1994;6(10-11):509–518. [PubMed] [Google Scholar]
  18. Overgaard J, Horsman MR. Modification of Hypoxia-Induced Radioresistance in Tumors by the Use of Oxygen and Sensitizers. Semin Radiat Oncol. 1996 Jan;6(1):10–21. doi: 10.1053/SRAO0060010. [DOI] [PubMed] [Google Scholar]
  19. Pollard J. W., Pacey J., Cheng S. V., Jordan E. G. Estrogens and cell death in murine uterine luminal epithelium. Cell Tissue Res. 1987 Sep;249(3):533–540. doi: 10.1007/BF00217324. [DOI] [PubMed] [Google Scholar]
  20. Sutherland R. M. Cell and environment interactions in tumor microregions: the multicell spheroid model. Science. 1988 Apr 8;240(4849):177–184. doi: 10.1126/science.2451290. [DOI] [PubMed] [Google Scholar]
  21. Sutherland R. M., Keng P., Conroy P. J., McDermott D., Bareham B. J., Passalacqua W. In vitro hypoxic cytotoxicity of nitroimidazoles: uptake and cell cycle phase specificity. Int J Radiat Oncol Biol Phys. 1982 Mar-Apr;8(3-4):745–748. doi: 10.1016/0360-3016(82)90726-x. [DOI] [PubMed] [Google Scholar]
  22. Wasserman T. H., Phillips T. L., Johnson R. J., Gomer C. J., Lawrence G. A., Sadee W., Marques R. A., Levin V. A., VanRaalte G. Initial United States clinical and pharmacologic evaluation of misonidazole (Ro-07-0582), an hypoxic cell radiosensitizer. Int J Radiat Oncol Biol Phys. 1979 Jun;5(6):775–786. doi: 10.1016/0360-3016(79)90061-0. [DOI] [PubMed] [Google Scholar]
  23. Wilson R. E., Keng P. C., Sutherland R. M. Drug resistance in Chinese hamster ovary cells during recovery from severe hypoxia. J Natl Cancer Inst. 1989 Aug 16;81(16):1235–1240. doi: 10.1093/jnci/81.16.1235. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES