Sensitivity of Neutral Filter Elution but Not PFGE Can Be Modified by Non-dsb Chromatin Damage

Abstract

Hamster V79 fibroblast cells and human squamous carcinoma cells (Caski) were exposed to ⁶⁰Co radiation and DNA double-strand break (dsb) induction was analysed by DNA elution at neutral pH from polycarbonate filter or out of an agarose matrix in pulsed-field electrophoresis (PFGE). While dsb yields were equal for the two cell lines (using 125-iodine calibration) a reduced responsiveness of filter elution was found for V79 versus Caski cells. This difference could be abolished when additional single-strand breaks (ssb) were introduced by an incubation at 10⁻⁴m H₂O₂ for up to 40 min that itself did not give a response in neutral elution. No such lack of specificity for the detection of dsb was seen in electrophoretic elution where also the influence of peroxide incubation was absent. The presumed potential of ssb to modify dsb detection was paralleled by the kinetics of dsb rejoining: a pronounced transient increase of DNA elution from filters was observed for V79 cells (less prominent with Caski cells) at 15–40 min which is thought to reflect the occurence of secondary ssb from incisions during base damage repair. Rejoining measured by PFGE did not show this behaviour. The results suggest that ssb may aid decondensation of the chromatin during lysis of cells required for an efficient release of dsb fragments when supported on filters, but which depends on cell type and is less critical in electrophoretic elution out of an agarose matrix. This involvement of ssb in the neutral filter elution assay appears to be contrary to published data obtained with different experimental systems. The finding of an increase of DNA elution from filters due to hyperthermia at 45°C is also taken to indicate an involvement of non-dsb chromatin damage in the response of filter elution at neutral pH with V79 but not with Caski cells.