Feasibility of measuring radiation-induced DNA double strand breaks and their repair by pulsed field gel electrophoresis in freshly isolated cells from the mouse RIF-1 tumor

MAWH vanWaarde, AJ vanAssen, AWT Konings, HH Kampinga

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    Abstract

    Purpose: To examine the technical feasibility of pulsed field gel electrophoresis (PFGE) as a predictive assay for the radioresponsiveness of tumors. Induction and repair of DNA double strand breaks (DSBs) in a freshly prepared cell suspension from a RIF-1 tumor (irradiated ex vivo) was compared with DSB induction and repair in exponentially growing RIF-1 cells in culture (irradiated in vitro).

    Methods and Materials: A murine RIF-1 tumor grown in vivo was digested, and cells were exposed to x-rays (ex vivo) at doses of 1 to 75 Gy. DNA damage was measured using CHEF (clamped homogeneous electric fields) electrophoresis. Repair kinetics were studied at 37 degrees C for 4 h after irradiation, Radiosensitivity was determined by clonogenic assay, and cell cycle distributions by flow cytometry. For comparison, a trypsinized suspension of exponentially growing RIF-1 cells in vitro was run parallel with each ex vivo experiment.

    Results: Induction of DSBs, expressed as % DNA extracted from the plug, was similar in the in vitro and ex vivo irradiated cells, Compared to repair rates in in vitro cultured RIF-1 cells, repair kinetics in a freshly prepared cell suspension from the tumor were decreased, unrelated to differences in radiosensitivity. Differences in repair could not be explained by endogenous DNA degradation, nor by influences of enzymes used for digestion of the tumor. A lower plating efficiency and differences in ploidy (as revealed by how cytometry) were the only reproducible differences between in vivo and in vitro grown cells that may explain the differences in repair kinetics.

    Conclusions: The current results do not support the idea that PFGE is a technique robust enough to be a predictive assay for the radiosensitivity of tumor cells.

    Original languageEnglish
    Pages (from-to)125-134
    Number of pages10
    JournalInternational Journal of Radiation Oncology Biology Physics
    Volume36
    Issue number1
    Publication statusPublished - 1-Aug-1996

    Keywords

    • PFGE
    • ex vivo radiosensitivity
    • DNA double strand breaks
    • DNA repair
    • predictive assay
    • RIF-1 tumor
    • CELLULAR RADIOSENSITIVITY
    • CHEF ELECTROPHORESIS
    • X-RADIATION
    • COMET ASSAY
    • RADIOTHERAPY
    • DAMAGE
    • PREDICTION
    • SURVIVAL
    • INVITRO
    • LINE

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