title: Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level.
abstract: Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells.
authors.affiliation: Institute of Cell Biology (Cancer Research), West German Cancer Center Essen, University of Essen Medical School, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Thomale
authors.forename: F
authors.initials: F
authors.lastname: Seiler
authors.forename: M R
authors.initials: MR
authors.lastname: Müller
authors.forename: S
authors.initials: S
authors.lastname: Seeber
authors.forename: M F
authors.initials: MF
authors.lastname: Rajewsky
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D001752:Blast Crisis / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004273:DNA, Neoplasm / Q000378:metabolism; D004351:Drug Resistance / Q000502:physiology; D006150:Guanine Nucleotides / Q000378:metabolism*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009607:Nitrosourea Compounds / Q000633:toxicity; D019853:O(6)-Methylguanine-DNA Methyltransferase; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism
chemical_list: D000477:Alkylating Agents; D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D006150:Guanine Nucleotides; D009607:Nitrosourea Compounds; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase