Advertisement

Radiation and Environmental Biophysics

, Volume 33, Issue 1, pp 35–44 | Cite as

Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. II. Repair

  • M. Kapiszewska
  • I. Szumiel
  • C. S. Lange
Article

Abstract

In the preceding paper we described the properties of nucleoids analyzed with the fluorescent halo assay at pH 6.9 and 9, as well as in the presence of reducing and chelating agents and after X-irradiation. We found analogies between the properties of type I and II nucleoids, as examined by Lebkowski and Laemmli (1982), and nucleoids analyzed with the fluorescent halo assay. We concluded that radiation-inflicted damage at two levels of DNA folding is measured at pH 6.9 and 9. In this paper we examined repair of damage to the nucleoid structure as assayed by the fluorescent halo method in X-irradiated L5178Y (LY) sublines; R (radiation resistant,D0=1.4 Gy) and S (radiation sensitive,D0=0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI; 0.5 to 50 µg/ml) at pH 6.9 and 9. The ability of DNA to be rewound at 10–50 µg/ml of PI was impaired by X-irradiation and partly restored during 90-min post-irradiation incubation, indicating damage to the superhelical structure and its partial restoration. The exponential time constants for repair were 10.1 min (LY-S, 6 Gy), 11.2 min (LY-R, 12 Gy), and 20.3 min (LY-s, 12 Gy) when measured at pH 9. In X-irradiated (12 Gy) LY-S cells, slower restoration of DNA supercoiling was observed at pH 9 than at pH 6.9. The presence of labile lesions at pH 9 did not prevent restoration of the higher-order DNA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.

Keywords

Iodide Propidium Iodide Environmental Physic Radiation Resistant Exponential Time 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alexander P, Mikulski ZB (1961) Mouse lymphoma cells with different radiosensitivities. Nature 195:572–573Google Scholar
  2. Beer JZ, Budzicka E, Niepokojczycka E, Rosiek O, Szumiel I, Walicka M (1983) Loss of tumorigenicity with simultaneous changes in radio- and photosensitivity during in vitro growth of L5178Y murine lymphoma cells. Cancer Res 43:4736–4742Google Scholar
  3. Beer JZ, Mencl J, Horng M-F, Gregg EC, Evans HH (1985) Effects of low dose rate (0.003–0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells. Int J Radiat Biol 48:609–619Google Scholar
  4. Budzicka E, Niepokojczycka E, Szumiel I, Zernik-Kobak M (1985) Radiosensitivity and sensitivity to drugs of colchicine-resistant variants of L5178Y lymphoma cells. Nukleonika 30:1–16Google Scholar
  5. Cress AE, Kurath KM, Hendrix MJC, Bowden GT (1989) Nuclear protein organization and the repair of radiation damage. Carcinogenesis 10:939–943Google Scholar
  6. Evans HH, Ricanati M, Horng M-F (1987) Deficiency in DNA repair in mouse lymphoma strain L5178Y-S. Proc Natl Acad Sci USA 84:7562–7566Google Scholar
  7. Flick MB, Warters RL, Yasui LS, Krisch RE (1989) Measurement of radiation-induced DNA damage using gel electrophoresis or neutral filter elution shows an increased frequency of DNA strand breaks after exposure to pH 9.6. Radiat Res 119:452–456Google Scholar
  8. Frankenberg-Schwager ML (1990) Induction, repair and biological relevance of radiation induced DNA lesions in eukaryotic cells. Radiat Environ Biophys 29:273–292Google Scholar
  9. George AM, Sabovljev SA, Hart LE, Cramp WA, Harros G, Hornsey S (1987) DNA quaternary structure in the radiation sensitivity of human lymphocytes - a proposed role of copper. Br J Cancer 55 (suppl 8):141–144Google Scholar
  10. Herrlich P, Angle P, Rahmsdorf HJ, Mallick U, Poting A, Hieber L, Lucke-Huhle C, Schorpp M (1986) The mammalian genetic stress response. Adv Enzyme Regul 25:485–504Google Scholar
  11. Hittelman WN, Pandita T, Jayanth RV, Wlodek D (1991) The potential role of chromatin organization in the induction and repair of chromosome aberrations (abstract). In: Chapman JD, Dewey WC, Whitmore GF (eds) Radiation research, twentieth-century perspective. (Congress Abstracts, vol 1) Academic Press, San Diego, p 55Google Scholar
  12. Iliakis G (1991) The role of DNA double strand breaks in ionizing radiation-induced killing of eukaryotic cells. BioEssays 13:641–648Google Scholar
  13. Iliakis G, Mehta R, Jackson M (1992) Level of DNA double strand break rejoining in Chinese hamster xrs5 cells is dose dependent: implications for the mechanism of radio sensitivity. Int J Radiat Biol 61:315–321Google Scholar
  14. Johanson K-J, Wlodek D, Szumiel I (1982) DNA repair and replication in radiation-sensitive and resistant mouse lymphoma cellsγ-irradiated under aerobic and hypoxic conditions. Int J Radiat Biol 41:261–270Google Scholar
  15. Kampinga HH, Wright WD, Konings AWT, Roti Roti JL (1988) The interaction of heat and radiation affecting the ability of nuclear DNA to undergo supercoiling changes. Radiat Res 116:114–123Google Scholar
  16. Kapiszewska M, Lange CS (1988) The effect of reduced temperature and/or starvation conditions on the radiosensitivity and repair of potentially lethal damage and sublethal damage in L5178Y-R and L5178Y-S cells. Radiat Res 113:458–472Google Scholar
  17. Kapiszewska M, Wright WD, Lange CS, Roti Roti JL (1989) DNA supercoiling changes in nucleoids from irradiated L5178Y-S and -R cells. Radiat Res 119:569–575Google Scholar
  18. Kapiszewska M, Szumiel I, Lange CS (1992) Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. I. Initial lesions. Radiat Environ Biophys 31:311–322Google Scholar
  19. Kaufmann W (1989) Pathways of human cell post-replication repair. Carcinogenesis 10:1–11Google Scholar
  20. Korner I, Walicka M, Malz M, Beer JZ (1977) DNA repair in two L5178Y cell lines with different X-ray sensitivities. Studia Biophys 61:141–149Google Scholar
  21. Krisch RE, Flick MB, Trumbore CN (1991) Radiation chemical mechanisms of single- and double-strand break formation in irradiated SV40 DNA. Radiat Res 126:251–259Google Scholar
  22. Lebkowski JS, Laemmli UK (1982) Evidence for two levels of DNA folding in histone-depleted HeLa interphase nuclei. J Mol Biol 156:309–324Google Scholar
  23. Ostashevsky JY (1989) A model relating cell survival to DNA fragment loss and unrepaired double-strand breaks. Radiat Res 118:437–466Google Scholar
  24. Ostashevsky JY (1990) Prediction of cell survival curves from DNA double-strand break repair data for low- and high-LET radiation. Int J Radiat Biol 57:523–536Google Scholar
  25. Schwartz JL, Vaughan ATM (1989) Association among DNA/chromosome break rejoining rates, chormatin structure alterations, and radiation sensitivity in human tumor cell lines. Cancer Res 49:5045–5057Google Scholar
  26. Siddiqi MA, Bothe E (1987) Single- and double-strand break formation in DNA irradiated in aqueous solution: dependence on dose and scavenger concentration. Radiat Res 112:449–463Google Scholar
  27. Szumiel I, Wlodek D, Johanson KJ, Sundell-Bergman S (1984) ADP-ribosylation and postirradiation cellular recovery in two strains of L5178Y cells. Br J Cancer 49:33–38Google Scholar
  28. Vaughan ATM, Loviscek KL, Gordon DJ (1991) Cell killing by H2O2: DNA damage and chromatin relaxation (abstract). In: Chapman JD, Dewey WC, Whitmore GF (eds) Radiation research, a twentieth-century perspective. Congress Abstracts, vol 1. Academic Press, San Diego, p 124Google Scholar
  29. Wheeler KT, Wierowski JV (1983a) DNA repair kinetics in irradiated undifferentiated and terminally differentiated cells. Radiat Environ Biophys 22:3–19Google Scholar
  30. Wheeler KT, Wierowski JV (1983b) DNA accessibility: a determinant of mammalian cell differentiation? Radiat Res 93:312–318Google Scholar
  31. Wierowski JV, Thomas RR, Wheeler KT (1984) DNA repair kinetics in mammalian cells following split-dose irradiation. Radial Res 98:242–253Google Scholar
  32. Wlodek D, Hittelman WN (1987)The repair of double-strand DNA breaks correlates with the radiosensitivity of L5178Y-S and L5178Y-R cells. Radiat Res 112:146–155Google Scholar
  33. Wlodek D, Hittelman WN (1988a) The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. I. Initial damage. Radiat Res 115:550–565Google Scholar
  34. Wlodek D, Hittelman WN (1988b) The relationship of DNA and chromosome damage to survival of synchronized X-irradiated L5178Y cells. II. Repair. Radiat Res 115:566–575Google Scholar
  35. Wlodek D, Olive P (1990) Physical basis for DNA double strand break detection using neutral filter elution. Radiat Res 124:326–333Google Scholar
  36. Wlodek D, Olive PL (1992) Neutral filter elution detects differences in chromatin organization which can influence cellular radiosensitivity. Radiat Res 132:242–247Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • M. Kapiszewska
    • 1
  • I. Szumiel
    • 2
  • C. S. Lange
    • 3
  1. 1.Institute of Molecular BiologyJagiellonian UniversityKrakówPoland
  2. 2.Institute of Nuclear Chemistry and TechnologyWarszawaPoland
  3. 3.Department of Radiation OncologySUNY-Health Science Center at BrooklynBrooklynUSA

Personalised recommendations