Melphalan-resistant cells have a more rapid kinetics of γH2AX foci formation than sensitive cells.

<p>(A) The number of γ-H2AX foci per cell nucleus (N>13) was manually quantified for each time point and the experiment was performed in triplicate. Corresponding standard deviation bars are indicated. P<0.01 at all time points as calculated using 2-sample t-test with equal variance. (B) Representative confocal images of the cells at different time points subsequent to Melphalan treatment. Foci formation was monitored using γH2AX polyclonal antibody (red) and due to the significant fraction of NFkB protein present in the cytoplasm of these cells, NFkB p65 mouse monoclonal antibody was used as control for cytoplasmic staining (green).</p

An Inverse Switch in DNA Base Excision and Strand Break Repair Contributes to Melphalan Resistance in Multiple Myeloma Cells

<div><p>Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.</p> </div

Assessment of alkali-labile sites in Melphalan sensitive and resistant cells.

<p>(A) Comet assays demonstrated that the resistant LR cells contained a significantly lower number of alkali-labile sites than the sensitive cells both at steady-state and subsequent to high-dose melphalan treatment. Per cent tail DNA of the comets is presented in dot plots with average and corresponding SEM. P values were calculated using an unpaired two-tailed t-test. R: resistant, S: sensitive. 100 comets were randomly selected and evaluated from one experiment at each treatment. (B) Western blot analysis showed that PARP-1 is activated via poly-ADP(ribosyl)ation only in the resistant cells suggesting that SSB repair and potentially also DSB synapsis is enhanced in the resistant cells. (C) Activation of PARP1 by PARylation in the resistant LR5 cells was completely abolished subsequent to treatment with the PARP-1 inhibitor 4-AN. (D) Inhibition of PARP-1 by 4-AN or KU58684 obstructs the proliferation of resistant cells while having a minor effect on the sensitive cells.</p

Steady state expression levels of proteins involved in DNA repair and DNA damage signaling response in 8226-LR5 cells relative to parental cells.

<p>Protein levels were assessed by quantitative western blot analysis using specific antibodies against target proteins. Each bar represent the mean expression ratio of the target protein in the LR5 (resistant) cells relative to the reference value of 1 (dotted line) in the sensitive cells subsequent to normalization against either β-actin or tubulin (to avoid overlapping signals). Quantitative analysis enclosed an average of 3 to 5 biological replicates with standard deviations as indicated. The P values were calculated by one sample two tailed t test against a hypothetical expression ratio set to 1 (no change in expression). >90%, >95% and >98% confidence levels indicated by *, **, and ***, respectively. # Only non-ubiquitinylated FANCD2 was detected in the analyses.</p

Effect of PI3-kinase-like kinase (PIKK)- and CHK1/2 inhibitors on the proliferation of 8226-LR5 cells.

<p>The MTT assay was used to monitor the survival after 72h incubation with the various inhibitors ±2.5 Melphalan. CHK1/2 inhibitor AZD7762 (0.1 µM), DNA-PK inhibitor NU7441 (0.2 µM), ATM inhibitor KU55933 (3 µM), ATR inhibitor VE821 (1 µM). Whereas a moderate inhibition on proliferation was observed with either inhibitor alone, strong inhibition was observed in combination with melphalan for the CHK1/2, DNA-PK and ATR inhibitors. Each bar represents the mean of at least 5 independent experiments with standard deviations as indicated. P-values were calculated using 2-sample t-test with equal variance. >95%, >99% and >99.9% confidence levels indicated by *, **, and ***, respectively.</p

Melphalan resistance in RPMI8226 cells is accompanied by increased S-phase progression and reduced G2/M arrest.

<p>Cell growth rate (A) and cell cycle distribution (B) of sensitive (8226) and resistant (LR5) cells exposed or non-exposed to 2.5 µM Melphalan. (A) Exposure to 2.5 µM Melphalan greatly reduced the proliferation of the sensitive cells, while a minor effect upon the proliferation of resistant cells was observed. (B) Flow cytometric analysis of cell cycle phases shows that 2.5 µM Melphalan caused delayed S-phase progression in sensitive cells followed by G2/M arrest. Conversely, progression throughout S-phase was less affected in the resistant cells exposed to Melphalan. Moreover, the resistant cells were able to overcome the G2/M arrest induced by Melphalan and re-enter the cell cycle. Per cent cells in each of the cell cycle phases are indicated above the histograms. The fraction of cells in subG1 is indicated, but not illustrated in the gated histograms.</p

Level of DNA repair proteins (A) and damage signaling kinases (B) involved in DSB repair in Melphalan-sensitive and -resistant cells.

<p>(A) Quantitative western blot analysis revealed that the NHEJ proteins DNA ligase IV, XRCC4 and RPA2 are significantly and constitutively upregulated in Melphalan-resistant cells. In addition, KU80 and phosphorylated Artemis was consistently, although not significantly upregulated. Exposure to high dose Melphalan did not mediate further alteration of NHEJ proteins in the cell lines. (B) The steady-state levels of the kinase ATR and its phosphorylated form are increased in Melphalan resistant cells. Conversely, ATM is constitutively downregulated in the same cells. Rather than activation of CHK1, increased steady-state phosphorylation of pCHK2 was observed in resistant cells. Notably, high dose Melphalan strongly activates CHK2 in both sensitive and resistant cells but was consistently higher in resistant cells. Moreover, BRCA1, which is slightly induced in resistant cells, becomes markedly reduced in both sensitive and resistant cells after high dose Melphalan indicating temporary downregulation of HRR. Also note the the lack of apparent monoubiquitinylation of FANCD2 (B). β-actin was used as reference for quantitative analysis.</p

DNA glycosylases with dual ss/dsDNA substrate specificity are downregulated in Melphalan-resistant cells.

<p>Levels of DNA glycosylases (A), relative UDG activities (B) and genomic 8-oxodG-content in sensitive and resistant cells prior and after exposure to 50 µM Melphalan. Western blot analysis revealed downregulation of UNG2, MPG and NEIL1 (dual ss/dsDNA-specific glycosylases) in the Melphalan-resistant cells compared to sensitive cells while this was not observed for the dsDNA-specific glycosylases TDG, MBD4 and OGG1, the latter barely above the detection level in the cells (A). In agreement with UNG2 protein levels, activity assays showed that uracil excision activity was significantly higher in sensitive cells against [³H]dUMP-containing ss- and dsDNA substrates. Moreover, high dose Melphalan mediated an apparent increase in UDG activity in both cell lines, although this was only significant (P<0.05) for the resistant cells. Addition of the UNG-specific inhibitor Ugi to the reactions demonstrated that the observed reduced uracil-excision activity in the resistant cell line was mainly accounted for by reduction of UNG-activity. Each bar constitutes an average from four independent experiments, each run in triplicate, with standard deviations as demonstrated. P-values were calculated using unpaired Student t-test (B). A twofold increased steady state level of genomic 8-oxodG was found in the resistant compared to the sensitive cells. Each bar constitutes and average from three independent experiments with standard deviations as indicated (C).</p

Melphalan resistance is accompanied by increased resistance to agents inducing non-bulky BER substrates.

<p>Cell were exposed to MMS (A), H₂O₂ (B), low (C) and high (D) doses of 5-FU, MMC (E) and UVB (F) assessed by the MTT assay. Viability curves showed that the Melphalan-resistant cells are cross-resistant to MMS, H₂O₂, low dose 5-FU and MMC. Conversely, high dose of 5-FU affects the proliferation of resistant cells in higher extent compared to sensitive cells. Exposure to UVB similarly affected growth of both cell lines. Increasing absorbance correlates directly with the number of living cells. Standard deviation bars are indicated.</p

SILAC and mRNA quantitative profiling.

<p>(A), histogram of log₂ SILAC ratios. (B), Venn diagram of DEGs identified in SILAC and mRNA analyses (threshold 1.25 fold change). (C), Scatter plot illustrating fold change relationships between DEGs identified in SILAC and mRNA analyses. (D), Most affected canonical pathways as identified by IPA analysis of SILAC data (threshold 1.25x change). The orange line represents the ratio of the number of genes represented within each pathway to the total number of genes in the pathway. (E), Distribution of DEGs in GO: biological process categories.</p