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. 2014 Oct 30:4:289.
doi: 10.3389/fonc.2014.00289. eCollection 2014.

A Small-Molecule Inhibitor of RAD51 Reduces Homologous Recombination and Sensitizes Multiple Myeloma Cells to Doxorubicin

David A Alagpulinsa  1 , Srinivas Ayyadevara  1 , Robert Joseph Shmookler Reis  1
Affiliations

A Small-Molecule Inhibitor of RAD51 Reduces Homologous Recombination and Sensitizes Multiple Myeloma Cells to Doxorubicin

David A Alagpulinsa et al. Front Oncol. .

Abstract

We previously reported high expression of RAD51 and increased homologous recombination (HR) rates in multiple myeloma (MM) cells, and showed that genomic instability and disease progression are commensurate with HR levels. Moreover, high RAD51 expression in vivo is associated with chemoresistance and poor patient survival. Doxorubicin (DOX) is one of the most widely used drug treatments in MM chemotherapy. DOX is cytotoxic because it induces DNA double-strand breaks, which can be repaired by RAD51-mediated HR; activation of this pathway thus contributes to resistance. To investigate the role of RAD51 in MM drug resistance, we assessed the ability of B02, a small-molecule inhibitor of RAD51, to enhance DOX sensitivity of MM cells. Combining low-toxicity doses of DOX and B02 resulted in significant synthetic lethality, observed as increased apoptosis and reduced viability compared to either agent alone, or to the product of their individual effects. In contrast, the combination did not produce significant synergy against normal human CD19(+) B cells from peripheral blood. DOX induced RAD51 at both mRNA and protein levels, while arresting cells in S and G2. DOX treatment also increased the number of RAD51 foci, a marker of HR repair, so that the fraction of cells with ≥5 foci rose fourfold, whereas γH2AX foci rose far less, implying that most new breaks are repaired. When B02 treatment preceded DOX exposure, the induction of RAD51 foci was severely blunted, whereas, γH2AX foci rose significantly relative to basal levels or either agent alone. In MM cells carrying a chromosomally integrated reporter of HR repair, DOX increased HR events while B02 inhibition of RAD51 blocked the HR response. These studies demonstrate the crucial role of RAD51 in protecting MM cells from genotoxic agents such as DOX, and suggest that specific inhibition of RAD51 may be an effective means to block DNA repair in MM cells and thus to enhance the efficacy of chemotherapy.

Keywords: B02; H2AX; RAD51; chemoresistance; doxorubicin; homologous recombination; multiple myeloma; recombinase.

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Figures

Figure 1
Figure 1
Rad51 inhibitors potentiate doxorubicin (DOX) toxicity in myeloma cells but not in normal B cells. (A,B) Interaction of DOX with anti-Rad51 siRNA for toxicity to myeloma cells in mass culture. Cell lines MM.1S (A) and H929 (B) were treated with DOX at 0–160 nM, 24 h after transfection with plasmid expressing either a Rad51-specific siRNA (4) or a scrambled control siRNA. After a further 72 h, cell survival and proliferation were estimated by the WST-1 viable-cell assay (Clontech). (C,D) Interaction of DOX with B02, a small-molecule inhibitor of RAD51, for toxicity to myeloma cells in mass culture. Cell lines MM.1S (C) and H929 (D) were treated with DOX at 0–160 nM, ±B02 at 10 μM; viable-cell number was assessed by WST-1 assay after 72 h. (E,F) Effect of DOX ± B02 on colony formation at low density. MM cell lines MM.1S (E) and H929 (F) were treated with vehicle, DOX (80 nM), B02 (10 μM), or both. Clonogenic survival was assessed in soft agar as described in “Materials and Methods” section. Mean ± SEM is shown for each treatment group, normalized to untreated cells and combined from three independent experiments. (G) Viability of normal CD19+ B cells from human peripheral blood was assessed by WST-8 assay (Sigma CCK-8). For comparisons of treatment groups connected by brackets, *, **, ***, and **** indicate p < 0.05, 0.01, 0.001, and 0.0001, respectively, by heteroscedastic 2-tailed t-tests. Significance of synthetic lethality was also tested, by comparing the percentage viability for the combination (B02 + DOX), to the percentage viability predicted by multiplying the surviving fractions after each treatment alone. The coefficient of variation (CoV) for each product of two treatment survivals is the geometric mean of the two individual CoVs. The predicted mean and SD were contrasted to the actual combined-treatment values by a one-tailed heteroscedastic t test; +, ++, and +++ indicate synergy p values of <0.05, <0.01, and <0.001, respectively.
Figure 2
Figure 2
The RAD51 inhibitor B02 potentiates doxorubicin-induced apoptosis in MM cells. Myeloma cell lines H929 (A,C) and MM.1S (B,D) were treated 72 h with vehicle (DMSO), B02 (10 μM), DOX (80 nM), or B02 plus DOX. (A,B) The percent of cells undergoing apoptosis was assessed by dual staining with propidium iodide (y axis in each panel) and FITC-tagged antibody to Annexin V (x axis in each panel). Apoptotic cells are defined by Annexin V content only, and thus are quantitated as the sum of the two right quadrants in each FACS panel. Scatter plots are representative of triplicate samples in each of two independent experiments, comprising 10,000 cells per run scored by flow cytometry. All six replicates of each condition produced similar results. (C,D) Combined data are summarized as the mean ± SEM of six data points from two independent experiments (each with n = 3). *,**Pairs of treatment vs. control groups, connected by brackets, differed significantly (*p < 0.05; **p < 0.01). +++, Synthetic lethality was significant (p < 0.0003), based on a one-tailed heteroscedastic t test comparing the surviving (non-apoptotic) fraction for B02 + DOX combined, vs. the product of the individual surviving fractions after exposure to either agent alone (each corrected for the “background” or uninduced level of apoptosis in cells exposed only to DMSO).
Figure 3
Figure 3
Doxorubicin effects on MM.1S cells: induction of Rad51 mRNA and protein, and cell-cycle arrest in S and G2. Incubation of MM.1S cells with 250 or 500 nM DOX for the indicated periods of time leads to (A) increased Rad51 mRNA levels as assayed by qRT-PCR; (B,C) a dose- dependent increase in RAD51 protein level, as shown in western blots; and (D,E) cell-cycle arrest, chiefly in G2. (A) Data from three independent experiments combined, shown as means ± SEM. Significance of differences between either DOX group vs. DMSO controls (black bars, set to a value of 1), by two-tailed t test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (B) A representative western blot probed with primary antibodies to RAD51 and β-actin. (C) Summary of three independent experiments, combined as mean ± SEM (*p < 0.05 or **p < 0.01 relative to vehicle treatment alone). (D) Shift in cell-cycle distribution, indicating arrest in S and G2 phases, determined by FACS analysis of relative DNA content per cell (based on fluorescence of DNA with intercalated propidium iodide, in permeabilized cells). Mean values ± SEM are shown for triplicate treatments per experiment for 2–3 biological replicates (total n = 6 or 9). Unadjusted significance of differences, relative to the same cell-cycle phase of cells exposed only to DMSO: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (E) Cells treated with DOX or vehicle were stained with propidium iodide and Alexa Fluor 488-conjugated antibody to phosphorylated (ser10) histone H3, to determine the fraction of G2/M-arrested cells that are in mitosis (M).
Figure 4
Figure 4
B02 inhibits DOX-induced formation of RAD51 foci, increases persistence of γH2AX foci, and inhibits HR repair of I-SceI-induced chromosomal DSBs in MM cells. (A–C) MM.1S cells, exposed 24 h to DMSO, B02 (20 μM), DOX (160 nM), or B02 + DOX, were examined by immunofluorescence to identify foci, and DAPI staining to define nuclei. (A) Representative images of RAD51 and γH2AX foci in cells exposed to chemicals indicated at left. (B) Mean percent of cells with ≥5 RAD51 or γH2AX foci, ±SEM, after the exposures indicated; data were combined from three experiments. ****p < 0.0001 for the effect of each drug treatment, relative to DMSO (vehicle) alone. (C) Mean fluorescence (integrated pixel intensity per nucleus) ±SEM, of RAD51 and γH2AX foci after the drug exposures indicated. ****p < 0.0001, as in (B). (D–F) B02 inhibits HR repair of I-SceI-induced chromosomal DSBs in MM cells. (D) Scheme of HR at a cleaved I-SceI site within the integrated DR-GPF locus. Chromosomal DSBs are first introduced at the single insertion site of the DR-GFP reporter, via cleavage at a unique I-SceI site by site-specific endonuclease introduced by adenovirus infection. HR repair of these DSBs creates intact GFP genes, detected by flow cytometry. (E) Examples of flow-cytometric analysis of MM.1S-DR-GFP cells, wherein GFP fluorescence (x axis signal) beyond the control boundary (segmented line) indicates HR repair. Background signal (1.8% of cells), defined in cells without I-SceI introduction, rose to ~24% after I-SceI expression. Lower panels show results for I-SceI-exposed cells +B02 (~5% GFP+) or +DOX (~93% GFP+). (F) Summary of combined data from runs such as those illustrated in (E), for cells without I-SceI infection (mock), cells treated with vehicle (DMSO), 20-μM B02, or 160-nM DOX for 24 h after transient infection with I-SceI expression adenovirus (AdNUGS24i). HR data combined from three experiments are presented as means ± SEM. Statistical significance between groups (each n = 3) by two-tailed t-tests: ****p < 0.0001.
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