DNA Excision Repair Profiles of Normal and Leukemic Human Lymphocytes: Functional Analysis at the Single-Cell Level

Abstract

Recent evidence has linked cellular DNA repair capacity to the chemosensitivity of cancer cells to alkylating agents. Using single-cell gel electrophoresis (“comet assay”), we have analyzed the induction and differential processing of DNA damage in human lymphocytes derived from healthy donors and from patients with chronic lymphatic leukemia (CLL) after exposure to N-ethyl-N-nitrosourea in vitro.

The extent of comet formation in lymphocytes after N-ethyl-N-nitrosourea exposure appears to depend predominantly on the processing of DNA repair intermediates, because strand breaks in plasmid DNA were not induced by ethylation before the addition of nuclear proteins. Although the initial level of a specific alkylation product (O⁶-ethylguanine) in nuclear DNA was uniform, different dose-response curves were obtained for the comet size in individual cell samples immediately after exposure, with small intercellular variation. The individual kinetics of DNA repair varied significantly between specimens derived from both healthy individuals and CLL patients; for the DNA repair half-time (t_1/2), large difference was found.

Pretreatment of cells with methoxyamine as a DNA repair modifier blocking the base excision repair pathway revealed a quite similar extent of base excision repair-independent DNA incision in almost all normal lymphocyte samples. In contrast, this portion varied relatively and absolutely to a great extent among individual samples of CLL lymphocytes, suggesting a loss of stringent control of DNA repair processes in these cells. The comet assay can thus be used to gain information about inter-individual variation in the efficiency of different DNA repair processes in small samples of normal cells and their malignant counterparts.