Homologous Recombination Repair Protein Rad51 Research Articles

Heat Shock Protein Family A Member 1 Promotes Intracellular Amplification of Hepatitis B Virus Covalently Closed Circular DNA.

Hepatitis B virus (HBV) contains a partially double-stranded relaxed circular DNA (rcDNA) genome that is converted into a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocyte by cellular DNA repair machinery. cccDNA associates with nucleosomes to form a minichromosome that transcribes RNA to support the expression of viral proteins and reverse transcriptional replication of viral DNA. In addition to the de novo synthesis from incoming virion rcDNA, cccDNA can also be synthesized from rcDNA in the progeny nucleocapsids within the cytoplasm of infected hepatocytes via the intracellular amplification pathway. In our efforts to identify cellular DNA repair proteins required for cccDNA synthesis using a chemogenetic screen, we found that B02, a small-molecule inhibitor of DNA homologous recombination repair protein RAD51, significantly enhanced the synthesis of cccDNA via the intracellular amplification pathway in human hepatoma cells. Ironically, neither small interfering RNA (siRNA) knockdown of RAD51 expression nor treatment with another structurally distinct RAD51 inhibitor or activator altered cccDNA amplification. Instead, it was found that B02 treatment significantly elevated the levels of multiple heat shock protein mRNA, and siRNA knockdown of HSPA1 expression or treatment with HSPA1 inhibitors significantly attenuated B02 enhancement of cccDNA amplification. Moreover, B02-enhanced cccDNA amplification was efficiently inhibited by compounds that selectively inhibit DNA polymerase α or topoisomerase II, the enzymes required for cccDNA intracellular amplification. Our results thus indicate that B02 treatment induces a heat shock protein-mediated cellular response that positively regulates the conversion of rcDNA into cccDNA via the authentic intracellular amplification pathway. IMPORTANCE Elimination or functional inactivation of cccDNA minichromosomes in HBV-infected hepatocytes is essential for the cure of chronic hepatitis B virus (HBV) infection. However, lack of knowledge of the molecular mechanisms of cccDNA metabolism and regulation hampers the development of antiviral drugs to achieve this therapeutic goal. Our findings reported here imply that enhanced cccDNA amplification may occur under selected pathobiological conditions, such as cellular stress, to subvert the dilution or elimination of cccDNA and maintain the persistence of HBV infection. Therapeutic inhibition of HSPA1-enhanced cccDNA amplification under these pathobiological conditions should facilitate the elimination of cccDNA and cure of chronic hepatitis B.

Downregulation of CDC20 Increases Radiosensitivity through Mcl-1/p-Chk1-Mediated DNA Damage and Apoptosis in Tumor Cells.

Radiotherapy is an important modality for the local control of human cancers, but the radioresistance induced by aberrant apoptotic signaling is a hallmark of cancers. Restoring the aberrant apoptotic pathways is an emerging strategy for cancer radiotherapy. In this study, we determined that targeting cell division cycle 20 (CDC20) radiosensitized colorectal cancer (CRC) cells through mitochondrial-dependent apoptotic signaling. CDC20 was overexpressed in CRC cells and upregulated after radiation. Inhibiting CDC20 activities genetically or pharmacologically suppressed the proliferation and increased radiation-induced DNA damage and intrinsic apoptosis in CRC cells. Mechanistically, knockdown of CDC20 suppressed the expression of antiapoptotic protein Mcl-1 but not other Bcl-2 family proteins. The expressions of CDC20 and Mcl-1 respond to radiation simultaneously through direct interaction, as evidenced by immunoprecipitation and glutathione S-transferase (GST) pull-down assays. Subsequently, decreased Mcl-1 expression inhibited the expression level of phosphorylated checkpoint kinase 1 (p-Chk1), thereby resulting in impaired DNA damage repair through downregulating the homologous recombination repair protein Rad51 and finally causing apoptotic signaling. In addition, both CDC20 and Chk1 inhibitors together, through in vivo studies, confirmed the radiosensitizing effect of CDC20 via inhibiting Mcl-1 and p-Chk1 expression. In summary, our results indicate that targeting CDC20 is a promising strategy to improve cancer radiotherapy.

Targeting PARP and autophagy evoked synergistic lethality in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), one of the most lethal malignancies worldwide, has limited efficient therapeutic options. Here, we first demonstrated that simultaneously targeting poly (ADP-ribose) polymerase (PARP) and autophagy could evoke striking synergistic lethality in HCC cells. Specifically, we found that the PARP inhibitor Niraparib induced cytotoxicity accompanied by significant autophagy formation and autophagic flux in HCC cells. Further experiments showed that Niraparib induced suppression of the Akt/mTOR pathway and activation of the Erk1/2 cascade, two typical signaling pathways related to autophagy. In addition, the accumulation of reactive oxygen species was triggered, which was involved in Niraparib-induced autophagy. Blocking autophagy by chloroquine (CQ) in combination with Niraparib further enhanced cytotoxicity, induced apoptosis and inhibited colony formation in HCC cells. Synergistic inhibition was also observed in Huh7 xenografts in vivo. Mechanistically, we showed that autophagy inhibition abrogated Niraparib-induced cell-cycle arrest and checkpoint activation. Cotreatment with CQ and Niraparib promoted the formation of γ-H2AX foci while inhibiting the recruitment of the homologous recombination repair protein RAD51 to double-strand break sites. Thus, the present study developed a novel promising strategy for the management of HCC in the clinic and highlighted a potential approach to expand the application of PARP inhibitors.

Linifanib (ABT-869), enhances cytotoxicity with poly (ADP-ribose) polymerase inhibitor, veliparib (ABT-888), in head and neck carcinoma cells

PARP inhibitors (PARPi) may provide an opportunity to gain selective killing of tumor cells which have deficiencies in cellular DNA repair systems. We previously demonstrated linifanib (ABT-869), a multi-receptor tyrosine kinase inhibitor of VEGF and PDGF receptor families, radiosensitized Head and Neck Squamous Cell Carcinoma cells (HNSCC) via inhibiting STAT3 activation. Given that STAT3 can modulate DNA damage response (DDR) pathway, in this study, we evaluate the effects of linifanib to enhance cytotoxicity with the PARPi, veliparib (ABT-888), in HNSCC. UMSCC-22A and UMSCC-22B cells were treated with linifanib (ABT-869) and veliparib (ABT-888). Cell viability, cytotoxicity, apoptosis induction, DNA single strand break (SSB) and double strand break (DSB) damages were examined by MTT assay, colony formation assay, flow cytometry and comet assay. In addition, the expression of DNA homologous recombination repair protein Rad51, γH2AX, a double strand break marker and cleaved PARP, an apoptotic cell death marker, were assessed using western immunoblotting. Combination treatment resulted in more cell growth inhibition, induction of apoptosis, DNA damages and double strand breaks, lower expression of Rad51, increase γH2AX expression and PARP cleavage. These data suggest the possibility of combining targeted therapeutic such as linifanib with veliparib to augment the inhibition of cell growth and apoptosis via synthetic lethality in HNSCC cells. Thus, it may provide a novel therapeutic strategy and improve efficacy and outcome in HNSCC.

Genistein Enhances the Radiosensitivity of Breast Cancer Cells via G2/M Cell Cycle Arrest and Apoptosis

The aim of the present study was to investigate the radiosensitizing effect of genistein, and the corresponding mechanisms of action on breast cancer cells with different estrogen receptor (ER) status. Human breast cancer cell lines such as MCF-7 (ER-positive, harboring wild-type p53) and MDA-MB-231 (ER-negative, harboring mutant p53) were irradiated with X-rays in the presence or absence of genistein. Cell survival, DNA damage and repair, cell cycle distribution, cell apoptosis, expression of proteins related to G2/M cell cycle checkpoint and apoptosis were measured with colony formation assays, immunohistochemistry, flow cytometry and western blot analysis, respectively. Genistein showed relatively weak toxicity to both cell lines at concentrations in the range of 5–20 μM. Using the dosage of 10 μM genistein, the sensitizer enhancement ratios after exposure to X-rays at a 10% cell survival (IC10) were 1.43 for MCF-7 and 1.36 for MDA-MB-231 cells, respectively. Significantly increased DNA damages, arrested cells at G2/M phase, decreased homologous recombination repair protein Rad51 foci formation and enhanced apoptotic rates were observed in both cell lines treated by genistein combined with X-rays compared with the irradiation alone. The combined treatment obviously up-regulated the phosphorylation of ATM, Chk2, Cdc25c and Cdc2, leading to permanent G2/M phase arrest, and up-regulated Bax and p73, down-regulated Bcl-2, finally induced mitochondria-mediated apoptosis in both cell lines. These results suggest that genistein induces G2/M arrest by the activation of the ATM/Chk2/Cdc25C/Cdc2 checkpoint pathway and ultimately enhances the radiosensitivity of both ER+ and ER- breast cancer cells through a mitochondria-mediated apoptosis pathway.

Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F+RT). This study was conducted to assess the effects of fulvestrant alone vs. F+RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F+RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6Gy ± fulvestrant. The effects of F+RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F+RT was 0.885±0.013 vs. 0.622±0.029 @2Gy, 0.599±0.045 vs. 0.475±0.054 @4Gy, and 0.472±0.021 vs. 0.380±0.018 @6Gy RT (p=0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F+RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p<0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F+RT compared with irradiation alone. F+RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F+RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.

Functional Connection between Rad51 and PML in Homology-Directed Repair

The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology-directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following γ-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon γ-irradiation. Consistent with the lack of RPA and BrdU foci, γ-irradiation fails to induce Chk1 activation, when PML is depleted. Taken together, we have discovered a novel functional connection between PML and the homologous recombination-mediated repair machinery, which might contribute to PML tumor suppressor activity.

Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51

BackgroundEsophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells.Principal FindingsBerberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance.Conclusions/SignificanceBerberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored.

Multiple DNA Damage Signaling and Repair Pathways Deregulated by Simian Virus 40 Large T Antigen

We demonstrated previously that expression of simian virus 40 (SV40) large T antigen (LT), without a viral origin, is sufficient to induce the hallmarks of a cellular DNA damage response (DDR), such as focal accumulation of gamma-H2AX and 53BP1, via Bub1 binding. Here we expand our characterization of LT effects on the DDR. Using comet assays, we demonstrate that LT induces overt DNA damage. The Fanconi anemia pathway, associated with replication stress, becomes activated, since FancD2 accumulates in foci, and monoubiquitinated FancD2 is detected on chromatin. LT also induces a distinct set of foci of the homologous recombination repair protein Rad51 that are colocalized with Nbs1 and PML. The FancD2 and Rad51 foci require neither Bub1 nor retinoblastoma protein binding. Strikingly, wild-type LT is localized on chromatin at, or near, the Rad51/PML foci, but the LT mutant in Bub1 binding is not localized there. SV40 infection was previously shown to trigger ATM activation, which facilitates viral replication. We demonstrate that productive infection also triggers ATR-dependent Chk1 activation and that Rad51 and FancD2 colocalize with LT in viral replication centers. Using small interfering RNA (siRNA)-mediated knockdown, we demonstrate that Rad51 and, to a lesser extent, FancD2 are required for efficient viral replication in vivo, suggesting that homologous recombination is important for high-level extrachromosomal replication. Taken together, the interplay of LT with the DDR is more complex than anticipated, with individual domains of LT being connected to different subcomponents of the DDR and repair machinery.

566 Phase 1 study of CT-2103/carboplatin in patients with solid tumors

The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.

565 Phase I combination study of oral vinorelbine (VRL) and oral cyclophosphamide (CTX) in patients with metastatic breast cancer (MBC)

The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT increases breast cancer cell radiosensitivity compared with radiation alone. These findings have salient implications for designing clinical trials using fulvestrant and radiation therapy.