Skip to main content
NCBI home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1989 Sep;60(3):332–338. doi: 10.1038/bjc.1989.281

Hydroethidine: a fluorescent redox probe for locating hypoxic cells in spheroids and murine tumours.

P L Olive 1
PMCID: PMC2247196  PMID: 2789941

Abstract

The fluorescent redox probe hydroethidine was accumulated and metabolised about five times faster in aerobic than in hypoxic mammalian cells. Patterns of fluorescence in Chinese hamster V79 spheroids also indicated that internal hypoxic cells were less able to metabolise the drug; toxicity was observed in cells only when cell fluorescence exceeded about 500 times background. In medium equilibrated with air or nitrogen, cell accumulation of the stain was rapid, and began to plateau after 30 min; loss of ethidium was initially rapid, with a slower component after 30 min, and transfer of the metabolite ethidium between stained and unstained cells was observed after 2 h co-incubation. Sorting cells from irradiated spheroids on the basis of ethidium fluorescence provided good separation of aerobic radiosensitive and hypoxic radioresistant cells, although separation using the perfusion probe, Hoechst 33342, was superior. Similar experiments with the murine SCCVII squamous cell carcinoma suggested that hydroethidine might be a useful indirect stain for locating hypoxic cells in experimental tumours when used in combination with a perfusion probe such as Hoechst 33342.

Full text

PDF
332

Images in this article

334Figure 5
on p.334

Selected References

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

  1. ALPER T., HOWARD-FLANDERS P. Role of oxygen in modifying the radiosensitivity of E. coli B. Nature. 1956 Nov 3;178(4540):978–979. doi: 10.1038/178978a0. [DOI] [PubMed] [Google Scholar]
  2. Begg A. C., Hodgkiss R. J., McNally N. J., Middleton R. W., Stratford M. R., Terry N. H. Fluorescent markers of hypoxic cells: a comparison of two compounds on three cell lines. Br J Radiol. 1985 Jul;58(691):645–654. doi: 10.1259/0007-1285-58-691-645. [DOI] [PubMed] [Google Scholar]
  3. Bucana C., Saiki I., Nayar R. Uptake and accumulation of the vital dye hydroethidine in neoplastic cells. J Histochem Cytochem. 1986 Sep;34(9):1109–1115. doi: 10.1177/34.9.2426339. [DOI] [PubMed] [Google Scholar]
  4. Chaplin D. J., Durand R. E., Olive P. L. Acute hypoxia in tumors: implications for modifiers of radiation effects. Int J Radiat Oncol Biol Phys. 1986 Aug;12(8):1279–1282. doi: 10.1016/0360-3016(86)90153-7. [DOI] [PubMed] [Google Scholar]
  5. Chaplin D. J., Durand R. E., Olive P. L. Cell selection from a murine tumour using the fluorescent probe Hoechst 33342. Br J Cancer. 1985 Apr;51(4):569–572. doi: 10.1038/bjc.1985.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chapman J. D., Franko A. J., Sharplin J. A marker for hypoxic cells in tumours with potential clinical applicability. Br J Cancer. 1981 Apr;43(4):546–550. doi: 10.1038/bjc.1981.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Durand R. E. Calibration of flow cytometry detector systems. Cytometry. 1981 Nov;2(3):192–193. doi: 10.1002/cyto.990020312. [DOI] [PubMed] [Google Scholar]
  8. Durand R. E., Olive P. L. Enhancement of toxicity from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea in V79 spheroids by a nitrofuran. Cancer Res. 1987 Oct 15;47(20):5303–5309. [PubMed] [Google Scholar]
  9. Durand R. E. Use of Hoechst 33342 for cell selection from multicell systems. J Histochem Cytochem. 1982 Feb;30(2):117–122. doi: 10.1177/30.2.6174559. [DOI] [PubMed] [Google Scholar]
  10. Durand R. E. Use of a cell sorter for assays of cell clonogenicity. Cancer Res. 1986 Jun;46(6):2775–2778. [PubMed] [Google Scholar]
  11. Franko A. J. Misonidazole and other hypoxia markers: metabolism and applications. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1195–1202. doi: 10.1016/0360-3016(86)90257-9. [DOI] [PubMed] [Google Scholar]
  12. Garrecht B. M., Chapman J. D. The labelling of EMT-6 tumours in BALB/C mice with 14C-misonidazole. Br J Radiol. 1983 Oct;56(670):745–753. doi: 10.1259/0007-1285-56-670-745. [DOI] [PubMed] [Google Scholar]
  13. Gosalvez M., Thurman R. G., Chance B., Reinhold H. Regional variation in the oxygenation of mouse mammary tumours in vivo demonstrated by fluorescence of pyridine nucleotide. Br J Radiol. 1972 Jul;45(535):510–514. doi: 10.1259/0007-1285-45-535-510. [DOI] [PubMed] [Google Scholar]
  14. Olive P. L., Chaplin D. J., Durand R. E. Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours. Br J Cancer. 1985 Nov;52(5):739–746. doi: 10.1038/bjc.1985.252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Olive P. L., Chaplin D. J. Oxygen and nitroreductase-dependent binding of AF-2 in spheroids and murine tumors. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1247–1250. doi: 10.1016/0360-3016(86)90269-5. [DOI] [PubMed] [Google Scholar]
  16. Olive P. L., Durand R. E. Characterization of a carbocyanine derivative as a fluorescent penetration probe. Cytometry. 1987 Nov;8(6):571–575. doi: 10.1002/cyto.990080607. [DOI] [PubMed] [Google Scholar]
  17. Olive P. L., Durand R. E. Fluorescent nitroheterocycles for identifying hypoxic cells. Cancer Res. 1983 Jul;43(7):3276–3280. [PubMed] [Google Scholar]
  18. Olive P. L. Evidence suggesting that the mechanism for aerobic and hypoxic cytotoxicity of nitroheterocycles is the same. Int J Radiat Oncol Biol Phys. 1982 Mar-Apr;8(3-4):687–691. doi: 10.1016/0360-3016(82)90713-1. [DOI] [PubMed] [Google Scholar]
  19. Olive P. L., Rasey J. S., Durand R. E. Comparison between the binding of [3H]misonidazole and AF-2 in Chinese hamster V79 spheroids. Radiat Res. 1986 Jan;105(1):105–114. [PubMed] [Google Scholar]
  20. Raleigh J. A., Miller G. G., Franko A. J., Koch C. J., Fuciarelli A. F., Kelly D. A. Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours. Br J Cancer. 1987 Oct;56(4):395–400. doi: 10.1038/bjc.1987.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Saiki I., Bucana C. D., Tsao J. Y., Fidler I. J. Quantitative fluorescent microassay for identification of antiproliferative compounds. J Natl Cancer Inst. 1986 Dec;77(6):1235–1240. [PubMed] [Google Scholar]
  22. Siemann D. W., Keng P. C. Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (II). Cell sorting. Br J Cancer. 1988 Sep;58(3):296–300. doi: 10.1038/bjc.1988.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Taylor Y. C., Rauth A. M. Sulphydryls, ascorbate and oxygen as modifiers of the toxicity and metabolism of misonidazole in vitro. Br J Cancer. 1980 Jun;41(6):892–900. doi: 10.1038/bjc.1980.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Trotter M. J., Chaplin D. J., Durand R. E., Olive P. L. The use of fluorescent probes to identify regions of transient perfusion in murine tumors. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):931–934. doi: 10.1016/0360-3016(89)90889-4. [DOI] [PubMed] [Google Scholar]
  25. Trotter M. J., Chaplin D. J., Olive P. L. Use of a carbocyanine dye as a marker of functional vasculature in murine tumours. Br J Cancer. 1989 May;59(5):706–709. doi: 10.1038/bjc.1989.148. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES