Determination of spatial patterns of DNA damage and repair in intestinal crypts by multi-cell gel electrophoresis

Abstract

We have developed a method to quantitate DNA strand breaks as a measure of DNA damage and repair in intact, isolated intestinal crypts. The assay is a modified form of the single-cell gel electrophoresis or ‘comet’ assay. By maintaining the spatial relationship between the cells we were able to characterise the repair response and the susceptibility to DNA damage of cells as a function of their position in the crypt. All cells were equally repair competent over the first 30 minutes of the repair of UV-C and gamma-radiation induced lesions. DNA damage was equally distributed following gamma-radiation but following incubation with the topoisomerase II inhibitor etoposide, damage was greater in the lower crypt with an unusual component to the comet tall which was tapered, implying an incremental change in susceptibility by cell position. This tapered component of the comet tail resolved rapidly after removal of etoposide. The pattern of damage produced by hydrogen peroxide was dose dependent with lower doses producing more strand breaks in the base of the crypt-an effect lost at higher doses. The assay has the ability to detect differences between cells in their susceptibility to DNA damage and their subsequent repair response which may vary with their proliferative or differentiative status.