Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements - PubMed (original) (raw)

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

R A El-Awady et al. Nucleic Acids Res. 2001.

Abstract

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G(0) fibroblasts were either preheated (45 degrees C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 x 10(-3) dsb/Gy/MB: Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12-16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.

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Figures

Figure 1

Cell survival. Cells were either preheated at 45°C for 20 min (open circles) or mock treated (closed circles). Cells were then irradiated on ice and immediately plated for colony formation. Survival of irradiated cells was normalized to survival of their non-irradiated counterparts.

Figure 2

Induction of dsb by X-rays. Cells were embedded in agarose, irradiated on ice and lysed. DNA was then digested with _Not_I and resolved in a pulsed field gel. (A) The gel stained with ethidium bromide (top) was then blotted and hybridized against the DXS53 probe (bottom). Signals were detected using a phosphorimager. (B) Intensity profiles for the entire lanes 1 and 5.

Figure 3

From relative band intensities dsb induction rate was calculated for non-heated and preheated cells (circles and squares) and fitted by linear regression. Alternatively, intensity profiles of lanes of the Southern blot (as shown in Fig. 2B) were used to calculate dsb rate (see text), giving 2-fold higher rates (straight lines without symbols).

Figure 4

Repair of dsb. Fibroblasts were irradiated, incubated for repair and prepared for two assays. (A) Restitution of _Not_I fragments as a measure of correct rejoining. Lane 1, 0 Gy, restriction buffer; lane 2, 0 Gy, _Not_I; lanes 3–9, 50 Gy, repair as indicated, _Not_I; lanes 10 and 11, S.pombe and S.cerevisiae chromosomes as size markers. (B) Band destruction and restitution were quantified using a phosphorimager and expressed as remaining damage (squares). The overall rejoining was measured using CFGE (circles). Both data sets, as well as their differences (dashed line, see text), were fitted by non-linear regression.

Figure 5

Repair of dsb after heat treatment (45°C, 20 min). Experiments, analysis and blots are as described in the legend to Figure 4. (A) Lane 1, 0 Gy, _Not_I; lanes 2–9, 50 Gy, repair, _Not_I; lane 10, marker.

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Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements - PubMed (original) (raw)