DNA excision repair profiles of normal and leukemic human lymphocytes: functional analysis at the single-cell level.

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 (t1/2), 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 interindividual variation in the efficiency of different DNA repair processes in small samples of normal cells and their malignant counterparts.

DNA repair and cellular resistance to alkylating agents in chronic lymphocytic leukemia.

The time course of the formation and persistence of repair-induced DNA lesions such as single-strand breaks (SSBs) were determined in isolated lymphocytes derived from 32 patients with chronic lymphocytic leukemia (CLL) using the single-cell gel electrophoresis (SCGE, "comet") assay. After pulse-exposure to N-ethyl-N-nitrosourea (EtNU), the initial amount of SSBs (t0 SCGE values) and the time periods required to reduce DNA damage by 50% (t50% SCGE values) were determined in nuclear DNA of individual cells. The t0 SCGE and t50% SCGE values varied interindividually between CLL specimens by factors of 16.6 and 8.2, respectively. Regarding cell-to-cell variation, no major subpopulations with significantly different DNA repair capacities were observed in cell specimens from a given patient. In addition, a monoclonal antibody-based immunocytological assay was used to determine the elimination kinetics for the cytotoxic alkylation product O6-ethylguanine from nuclear DNA. A strong correlation was observed between the relative times for SSB repair and the elimination of O6-ethylguanine from nuclear DNA. Because SCGE and immunocytological assay measure different steps of DNA repair, this observation suggests coordinated regulation of the respective repair pathways. With regard to chemosensitivity profiles, a "fast" repair phenotype corresponded to enhanced in vitro resistance to EtNU, 1,3-bis(2-chloroethyl)-1-nitrosourea, or chlorambucil. Accelerated SSB repair and pronounced in vitro resistance to chlorambucil, 1,3-bis(2-chloroethyl)-1-nitrosourea, and EtNU were found in lymphocytes from CLL patients nonresponsive to chemotherapy with alkylating agents. Distinct DNA repair processes thus mediate resistance to alkylating agents in CLL lymphocytes.

Induction and mechanism of DNA single- and double-strand breaks by tetracycline/Cu(II) in the absence of light.

In the absence of light, tetracycline (TC) induced single- and double-strand breaks in PM2 DNA at micromolar concentrations in combination with CuCl2, whereas TC or CuCl2 alone had no effect. Strand break formation was completely suppressed by catalase and the specific Cu(I) scavenger neocuproine. The extent of strand break formation depended on the ratio of Cu(II):TC. At a ratio of > or = 2 most DNA damage was observed. The influence of the kind of Cu(II)/TC complexation on DNA strand break formation is discussed. The DNA damage in PM2 DNA provoked by TC/CuCl2 was indirectly detected also in human fibroblasts by the induction of DNA repair. The results are discussed with regard to human risk from TC/Cu(II).

Capacity of individual chronic lymphatic leukemia lymphocytes and leukemic blast cells for repair of O6-ethylguanine in DNA: relation to chemosensitivity in vitro and treatment outcome.

The elimination kinetics of the alkylation product O6-ethylguanine (O6eGua) from nuclear DNA were determined in individual lymphocytes or blast cells isolated from 27 patients with chronic lymphatic leukemia (CLL) and 26 patients with de novo acute myeloid leukemia (AML). A monoclonal antibody-based immunocytological assay was used for quantification of O6eGua in DNA of individual cells after pulse exposure of cells to N-ethyl-N-nitrosourea (EtNU). In cell specimens from a given patient, no major subpopulations with significantly different capacities for repair of O6eGua were observed. The time required to remove 50% of induced O6eGua residues varied interindividually between 0.5 and 8.4 h in CLL lymphocytes and between 0.8 and 6.3 h in leukemic blast cells. An inverse relationship was found between the rate of removal of O6eGua from DNA and the chemosensitivity of cells to EtNU, 1,3-bis(2-chloroethyl)-1-nitrosourea or mafosfamide in vitro. High rates of O6eGua repair and pronounced resistance to mafosfamide, 1,3-bis(2-chloroethyl)-1-nitrosourea, and EtNU in vitro were found in samples from 8 CLL patients nonresponsive to chemotherapy with alkylating agents. In AML patients treated with anthracyclines and 1-beta-D-arabinofuranosylcytosine, no relation was found between DNA repair capacity and treatment outcome. However, increased P-glycoprotein expression was observed between specimens derived from AML patients who had failed to reach complete remission (n = 12) after chemotherapy versus responsive patients (n = 14). DNA repair rate was not related to chemosensitivity to Adriamycin and 1-beta-D-arabinofuranosylcytosine in vitro, nor were cellular glutathione content, glutathione S-transferases activity, or P-glycoprotein expression.

A comparison of the DNA-damaging, the cytotoxic and genotoxic properties of tetracycline in human fibroblasts in the presence and absence of light.

The reduction of the colony-forming ability, the induction of DNA strand breaks and DNA repair were determined in human fibroblasts after treatment with tetracycline (TC) in the presence and absence of light. In all experiments human fibroblasts were more sensitive to incubations of TC in the light than in the dark. Induction of DNA single-strand breaks and DNA repair were detected in the cells after a 1-h incubation with TC under light but not in the dark. In contrast to these results, TC induced single-strand breaks in isolated PM2 DNA in the dark, however, to a lower extent than in the presence of light. In both cases strand break formation was totally suppressed by adding catalase. The formation of a TC-derived radical by ESR and a decomposition product by UV-vis spectroscopy was observed in the presence and absence of light; their rate of formation in the dark was much smaller than in the light.