Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems

Abstract

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

Keywords: BER, base excision repair; CLB, chlorambucil; Chlorambucil; DNA repair kinetics; ICL, interstrand crosslink; Interstrand crosslink; MS, mass spectrometry; Mass spectrometry; Monoalkylated DNA adducts; NER, nucleotide excision repair; NM, Nitrogen mustard; Nitrogen mustard; PBMCs, peripheral blood mononuclear cells; PI, propidium iodide; RPE-1, human retinal pigment epithelial; SD, standard deviation; VH10, human foreskin fibroblasts; dG, 2'-deoxyguanosine; hTERT, human telomerase reverse transcriptase; rFADU, reverse fluorometric analysis of alkaline DNA unwinding.

Fig. 1

General reaction scheme of a NM with guanine and the major DNA lesions induced. From with some modifications.

Fig. 2

LC-MS chromatograms of the main DNA adducts formed after treatment with CLB. A. Calf thymus DNA was treated with 500 µM CLB for 4 h at room temperature and the DNA monoadducts formed (N3-CLB-Ade and N7-CLB-Gua) as well as their non-reacted counterparts (dA and dG) were detected using UPLC-MS/MS. The adducts were separated on a BEH C18 column using a water-acetonitrile gradient. Shown is a representative chromatogram. B. Structural formulae of the two monoadducts N3-CLB-Ade (m/z 385.19) and N7-CLB-Gua (m/z 411.20). The arrow indicates the fragmentation point after CID in the MS of the two adducts and the mass-to-charge ratio of the detected CLB-derived fragment (m/z 250.14).

Fig. 3

Method development for the detection of ICLs in adherent cells using the rFADU assay. HaCaT cells were treated for 1 h at 37 °C with HN2 solutions (A) or CLB solutions (B) at the concentrations indicated. After treatment, the cells were X-irradiated on ice with 25 Gy. The amount of ICLs was determined via the rFADU assay. Each column represents the mean of 3 independent experiments ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA test with the Dunnett’s post-test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001. (C) Collagen-coating test experiment with RPE-1 cells treated with CLB. RPE-1 cells were treated with the doses of CLB indicated above, and the induction of the ICLs was measured via the rFADU assay. The treatment was performed with cells growing on culture dishes, which were coated with collagen or not coated. Three independent experiments were performed and the mean ± the SD is shown. Statistical analysis was performed using the one-way ANOVA test with the Dunnett’s post-test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 4

Cellular effects of CLB treatment in the human ovarian carcinoma cell line A2780. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. A2780 cells were treated with CLB for 1 h at 37 °C and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 4 independent experiments, except 500 µM CLB (n = 3). Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C, and ICLs were monitored using the rFADU assay. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Time-course analysis of CLB-induced DNA ICLs. Cells were treated with 50 µM CLB for 1 h at 37 °C, and ICLs were monitored using the reverse automated FADU assay. Treated cells were allowed to recover for the time points indicated, up to 51 h. Data represents the mean ± SD from 4 independent experiments, except 28 h and 32.5 h (n = 3). Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (E) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 100 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 30 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments, except 3 h and 21.5 h (n = 3). Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation in C. and E. (F) Cytotoxicity as well as proliferation status of cells after the treatment with the indicated treatment concentrations of CLB were monitored as a function of time. A2780 cells were stained with 10 µM CFSE and after 24 h, the cells were treated for 1 h with 50 µM (△) or 100 µM of CLB (▲). Treated cells were allowed to recover for the time points indicated, and the samples were additionally stained with PI before flow cytometric analysis. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 5

Cellular effects of CLB treatment in the human osteosarcoma U2OS cell line. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. U2OS cells were treated with CLB for 1 h at 37 °C and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C and ICLs were monitored using the reverse automated FADU assay. Data represents the mean ± SD from 4 independent experiments, except for 2.5 µM and 5 µM (n = 3). Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Time-course analysis of CLB-induced DNA ICLs. Cells were treated with 100 µM CLB for 1 h at 37 °C and ICLs were monitored using the rFADU assay. Treated cells were allowed to recover for the time points indicated, up to 51 h. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (E) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 200 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 30 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation in C. and E. (F) Cytotoxicity as well as proliferation status of cells after the treatment with the indicated treatment concentrations of CLB were monitored as a function of time. U2OS cells were stained with 10 µM CFSE and after 24 h, the cells were treated for 1 h with 100 µM (△) or 200 µM of CLB (▲). Treated cells were allowed to recover for the time points indicated, and the samples were additionally stained with PI before the flow cytometric analysis. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 6

Cellular effects of CLB treatment in the hTERT immortalized RPE-1 cell line. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. RPE-1 cells were treated with CLB for 1 h at 37 °C and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 4 independent experiments, except for 200 µM H₂O₂ (n = 3). Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C and ICLs were monitored using the reverse automated FADU assay. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Time-course analysis of CLB-induced DNA ICLs. Cells were treated with 200 µM CLB for 1 h at 37 °C and ICLs were monitored using the rFADU assay. Treated cells were allowed to recover for the time points indicated, up to 51 h. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 5 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (E) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 300 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 30 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation in C. and E. (F) Cytotoxicity as well as proliferation status of cells after the treatment with the indicated treatment concentrations of CLB were monitored as a function of time. RPE-1 cells were stained with 10 µM CFSE and after 24 h, the cells were treated for 1 h with 200 µM (△) or 300 µM of CLB (▲). Treated cells were allowed to recover for the time points indicated and the samples were additionally stained with PI before the flow cytometric analysis. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 7

Cellular effects of CLB treatment in the hTERT immortalized VH10 cell line. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. VH10 cells were treated with CLB for 1 h at 37 °C, and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C and ICLs were monitored using the rFADU assay. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Time-course analysis of CLB-induced DNA ICLs. Cells were treated with 100 µM CLB for 1 h at 37 °C and ICLs were monitored using the rFADU assay. Treated cells were allowed to recover for the time points indicated, up to 51 h. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (E) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 400 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 30 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation in C. and E. (F) Cytotoxicity as well as proliferation status of cells after the treatment with the indicated treatment concentrations of CLB were monitored as a function of time. VH10 cells were stained with 10 µM CFSE and after 24 h, the cells were treated for 1 h with 100 µM (△) or 400 µM of CLB (▲). Treated cells were allowed to recover for the time points indicated, and the samples were additionally stained with PI before the flow cytometric analysis. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 8

Cellular effects of CLB treatment in the hTERT immortalized podocytes. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. Podocytes were treated with CLB for 1 h at 37 °C, and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C and ICLs were monitored using the reverse automated FADU assay. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Time-course analysis of CLB-induced DNA ICLs. Cells were treated with 50 µM CLB for 1 h at 37 °C, and ICLs were monitored using the rFADU assay. Treated cells were allowed to recover for the time points indicated, up to 51 h. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 5 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (E) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 200 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 30 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation in C. and E. (F) Cytotoxicity as well as proliferation status of cells after the treatment with the indicated treatment concentrations of CLB were monitored as a function of time. Podocytes were stained with 10 µM CFSE and after 24 h, the cells were treated for 1 h with 50 µM (△) or 200 µM of CLB (▲). Treated cells were allowed to recover for the time points indicated and the samples were additionally stained with PI before the flow cytometric analysis. Data represents the mean ± SD from 3 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.

Fig. 9

Cellular effects of CLB treatment in human PBMCs. (A) Cytotoxicity of the indicated doses of CLB was detected using the annexin V/PI staining and flow cytometry. PBMCs were treated with CLB for 1 h at 37 °C and the cell death status was analyzed 24 h later. ‘Dead’ refers to annexin V/PI positive cells, ‘apoptotic’ to annexin V positive/PI negative cells and ‘viable’ to annexin V/PI negative cells. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using two-way-ANOVA followed by a post hoc Dunnett‘s test. (B) Dose-dependent induction of ICLs after the treatment with CLB. Cells were treated with the indicated doses of CLB for 1 h at 37 °C and ICLs were monitored using the rFADU assay. Data represents the mean ± SD from 5 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (C) Dose-response relationship of the treatment with CLB and the amount of monoalkylated DNA adducts. Cells were treated with the indicated doses of CLB for 1 h at 37 °C. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 6 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. (D) Time-course analysis of CLB-induced monoalkylated adducts in DNA (N7-CLB-Gua and N3-CLB-Ade). Cells were treated with 10 µM CLB for 1 h at 37 °C and were allowed to recover for the time points indicated, up to 50 h. The amount of N7-CLB-Gua and N3-CLB-Ade was monitored using MS. Data represents the mean ± SD from 4 independent experiments. Statistical analysis was performed using one-way-ANOVA followed by a post hoc Dunnett‘s test. For clarity, the display of the statistically significant differences of data points was omitted in the phase of adduct formation. *p˂0.05, * *p˂ 0.01, * **p˂ 0.001, * ** *p˂ 0.0001.