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

Alagpulinsa DA, Ayyadevara S, Shmookler Reis RJ - Front Oncol (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: McClellan Veterans Medical Center, Central Arkansas Veterans Healthcare System , Little Rock, AR , USA ; Department of Geriatrics, University of Arkansas for Medical Science , Little Rock, AR , USA.

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.

No MeSH data available.


Related in: MedlinePlus

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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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.

Mentions: Soon after a DSB is formed, histone H2AX (a variant of H2A) in the region of the break becomes phosphorylated on serine 139. The resulting “γH2AX” sites facilitate recruitment of repair components and chromatin-modulating factors to the DSB vicinity, and consequently nuclear foci that bind antibody to γH2AX are widely used as DSB markers (43). RAD51 foci, in contrast, mark sites where thousands of RAD51 monomers, detectable by immunostaining, have bound single-stranded DNA overhangs at DSBs (32); they thus indicate sites of HR repair for DSBs. The two signals largely colocalize in untreated MM.1S cells with moderate levels of DNA damage (DMSO images, Figure 4A).


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

Alagpulinsa DA, Ayyadevara S, Shmookler Reis RJ - Front Oncol (2014)

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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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.
Mentions: Soon after a DSB is formed, histone H2AX (a variant of H2A) in the region of the break becomes phosphorylated on serine 139. The resulting “γH2AX” sites facilitate recruitment of repair components and chromatin-modulating factors to the DSB vicinity, and consequently nuclear foci that bind antibody to γH2AX are widely used as DSB markers (43). RAD51 foci, in contrast, mark sites where thousands of RAD51 monomers, detectable by immunostaining, have bound single-stranded DNA overhangs at DSBs (32); they thus indicate sites of HR repair for DSBs. The two signals largely colocalize in untreated MM.1S cells with moderate levels of DNA damage (DMSO images, Figure 4A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: McClellan Veterans Medical Center, Central Arkansas Veterans Healthcare System , Little Rock, AR , USA ; Department of Geriatrics, University of Arkansas for Medical Science , Little Rock, AR , USA.

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.

No MeSH data available.


Related in: MedlinePlus