Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis

Abstract

Background: To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.

Results: Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05).

Conclusion: Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

Figure 1. Correlation between IC₅₀ fold increase and IC₅₀ values of control cell lines.

Linear regression models determined that higher values of IC₅₀ were associated with lower values of fold change (logarithm scale slope of: -0.11 (standard error: 0.02), P < 0.0001, R²= 0.58). Each IC₅₀ value is the mean of experiments performed in triplicate.

Figure 2. Average doubling time of parental (control) and BLM-resistant sub-clones.

Mean doubling time ± standard error of the mean (SEM, n=3) was reported. The mean doubling time (measured in hours) of the parental lines was shorter than that of BLM-resistant sub-clones in all seven cell lines. * P<0.05 compared to parental.

Figure 3. Effects of 3-week discontinuation of maintenance BLM treatment on IC₅₀ (µg/ml).

Experiments were performed in triplicate. Log IC₅₀ comparisons were performed. Three (HOP_0.05, NT2_0.1, and NCCIT_1.5) of the seven cell lines had significant reductions in IC₅₀ values following three weeks of BLM-free maintenance. * P<0.05 for comparisons between BLM resistant sub-clones and their corresponding counterparts with three weeks of treatment break.

Figure 4. Effects of 3-week discontinuation of maintenance BLM treatment on doubling time.

Experiment was performed in triplicate. Three (HOP_0.05, NT2_0.1, NCCIT_1.5) of the seven cell BLM-resistant lines exhibited significant decrease in doubling time following three weeks of BLM-free treatment. * P<0.05 compared to after removal BLM for 3 weeks cell lines.

Figure 5. DNA damage in Olive Tail Moment (OTM) pre- and post- high dose BLM treatment assessed by comet assay.

Experiments were run in triplicates. Cells were subject to high dose BLM exposure (corresponding to ten times their respective maintenance concentrations) for 24 hours. OTM was used for DNA fragmentation (damage) quantification, and was calculated as: OTM = (Tail.mean - Head.mean) × (Tail %DNA)/100. Comet assay revealed greater increase in DNA fragmentation (expressed in OTM levels) after BLM treatment in all parental lines.* P<0.05 for comparison between cell lines prior and after high dose BLM treatment. All parental lines exhibited significant increase in DNA damage. # P<0.05 for comparison between parental and resistant cell lines at baseline (pre-treatment). All BLM-resistant lines except for HOP_0.05 exhibited increased DNA damage at baseline compared to their parental lines. & P<0.05 for comparison between resistant cell lines and parental cell line post BLM treatment. Less DNA damage (compared to their parental lines) post- BLM treatment was found in five of seven BLM-resistant cell lines (SF_0.4, HOP_0.1, NT2_0.1, NCCIT_1.5, and H322M_2.5).

Figure 6. γ-H2AX formation pre- and post- high dose BLM treatment assessed by flow cytometry.

Experiments were run in triplicate. Cells were subject to high dose BLM exposure (corresponding to ten times their respective maintenance concentrations) for 24 hours. Flow cytometric detection of BLM-induced γ-H2AX foci formation were then obtained in a subset of four cell lines (ACHN, HOP, NCCIT and H322M). * P<0.05 for comparison between cell lines prior and after high dose BLM treatment. All parental lines exhibited significant increase in formation of γ-H2AX. # P<0.05 for comparison between parental and resistant cell lines at baseline (pre-treatment). One of four BLM-resistant cell lines (NCCIT_1.5) had greater γ-H2AX formation than its parental counterpart at baseline. & P<0.05 for comparison between resistant and parental cell lines following BLM treatment. Two of four BLM-resistant cell lines (HOP_0.1 and NCCIT1._5.) revealed significantly less γ-H2AX formation than their parental counterparts post BLM treatment, with a third line (H322M_2.5) being borderline significant (p=0.054).

Figure 7. Summary of % G2/M phase distribution pre- and post- high dose BLM treatment in all 7 parental/resistant pairs.

High dose BLM treatment corresponds to ten times the corresponding maintenance concentration for each cell line. Mean ± SEM (n=3) values are reported. * P<0.05 for the comparison between cell lines prior to and after high dose BLM treatment. All parental lines exhibited significant increases in G2/M cell cycle distribution. # P<0.05 for comparison between parental and resistant cell lines at baseline (pre-treatment). Three of seven BLM-resistant cell lines (HOP_0.05, NCCIT_1.5, and H322M_2.5) exhibited increased % G2/M distribution at baseline compared to their parental cell lines. & P<0.05 for comparison of % G2/M distribution between parental and resistant cell lines after BLM treatment. Less % G2/M distribution than parental lines was found in five out of seven BLM-resistant cell lines (SF_0.4, NT2_0.1, NCCIT_1.5, H322M_2.5, and MB231_3.0) after BLM treatment.

Figure 8. Time course measurement of % G2/M distribution in four parental/resistant cell line pairs at 0 (baseline) 4, 8, 12, 20, and 24 hours following high dose BLM treatment.

Experiments were run in triplicate. G2/M distribution was found to be higher in parental lines (compared to resistant sub-clones) 8 hours after BLM treatment.

Figure 9. Percent cell apoptosis pre- and post- high dose BLM exposure in four parental/resistant cell line pairs.

* P<0.05 for comparison between cell lines prior to and after high dose BLM treatment. All parental lines but no resistant lines exhibited significant increases in apoptosis post- BLM treatment. & P<0.05 for comparison between resistant and parental cell line following BLM treatment. Less cell apoptosis was found in three (HOP_0.05, NCCIT_1.5, and H322M_2.5) of four BLM-resistant lines, when compared to their parental lines.