Homologous Recombination-directed DNA Repair Research Articles

In silico cloning, expression and bioinformatics analysis of NtODB from common tobacco

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.

Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli

Diverse stress insults trigger interactions of PML with nucleolus, however, the function of these PML nucleolar associations (PNAs) remains unclear. Here we show that during induction of DNA damage-induced senescence in human non-cancerous cells, PML accumulates at the nucleolar periphery simultaneously with inactivation of RNA polymerase I (RNAP I) and nucleolar segregation. Using time-lapse and high-resolution microscopy, we followed the genesis, structural transitions and destiny of PNAs to show that: 1) the dynamic structural changes of the PML-nucleolar interaction are tightly associated with inactivation and reactivation of RNAP I-mediated transcription, respectively; 2) the PML-nucleolar compartment develops sequentially under stress and, upon stress termination, it culminates in either of two fates: disappearance or persistence; 3) all PNAs stages can associate with DNA damage markers; 4) the persistent, commonly long-lasting PML multi-protein nucleolar structures (PML-NDS) associate with markers of DNA damage, indicating a role of PNAs in persistent DNA damage response characteristic for senescent cells. Given the emerging evidence implicating PML in homologous recombination-directed DNA repair, we propose that PNAs contribute to sequestration and faithful repair of the highly unstable ribosomal DNA repeats, a fundamental process to maintain a precise balance between DNA repair mechanisms, with implications for genomic integrity and aging.

Class I histone deacetylase inhibition is synthetic lethal with BRCA1 deficiency in breast cancer cells

Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene, which is frequently mutated in breast and ovarian cancers. BRCA1 plays a key role in the homologous recombination directed DNA repair, allowing its deficiency to act as a therapeutic target of DNA damaging agents. In this study, we found that inhibition of the class I histone deacetylases (HDAC) exhibited synthetic lethality with BRCA1 deficiency in breast cancer cells. Transcriptome profiling and validation study showed that HDAC inhibition enhanced the expression of thioredoxin interaction protein (TXNIP), causing reactive oxygen species (ROS)-mediated DNA damage. This effect induced preferential apoptosis in BRCA1−/− breast cancer cells where DNA repair system is compromised. Two animal experiments and gene expression-associated patients’ survival analysis further confirmed in vivo synthetic lethality between BRCA1 and HDAC. Finally, the combination of inhibitors of HDAC and bromodomain and extra-terminal motif (BET), another BRCA1 synthetic lethality target that also works through oxidative stress-mediated DNA damage, showed a strong anticancer effect in BRCA1−/− breast cancer cells. Together, this study provides a new therapeutic strategy for BRCA1-deficient breast cancer by targeting two epigenetic machineries, HDAC and BET.

Abstract 736: Combining PD1- and CTLA4-inhibiting antibodies with cisplatin or PARP inhibition in an attempt to eradicate BRCA1-deficient mouse mammary tumors

Abstract Solid tumors are usually not eradicated by conventional chemotherapy resulting in disease relapse and mortality. An intriguing example is BRCA-associated breast cancer. A genetically engineered mouse model for BRCA1-associated breast cancer (K14Cre;Brca1F/F;p53F/F), which highly resembles disease in human patients, can be used to study therapy escape and residual disease in BRCA-deficient tumors. Due to the BRCA inactivation, the tumors that arise in this model lack homologous recombination-directed DNA repair, an Achilles heel that has provided a therapeutic opportunity to eradicate tumors with DNA damage-inducing agents. Despite repeated sensitivity some residual cancer cells escape the deadly effect of anticancer therapy and lead to disease relapse. A special histopathological feature of BRCA1-mutated tumors in both humans and mice is the presence of infiltrating lymphocytes. This may be explained by the high frequency of genomic alterations of BRCA1-mutated tumors resulting in the generation of many neo-antigens. Our hypothesis is that blocking inhibitory T-cell signaling, using antibodies directed against CTLA4 and PD1, may increase the activity of the T-cell compartment towards a diverse pool of antigens, and successfully eliminate residual tumor cells in combination with DNA damage-inducing drugs. In particular, we will present data of combining CTLA4- and PD1- targeting antibodies with PARP inhibition or cisplatin to eliminate residual tumor cells in this mouse model for BRCA1-mutated breast cancer. Citation Format: Sohvi Blatter, Charlotte Guyader, Aslı Küçükosmanoğlu, Stephan Freriks, Karin de Visser, Piet Borst, Sven Rottenberg. Combining PD1- and CTLA4-inhibiting antibodies with cisplatin or PARP inhibition in an attempt to eradicate BRCA1-deficient mouse mammary tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 736. doi:10.1158/1538-7445.AM2015-736

Inhibition of Both EGFR and IGF1R Sensitized Prostate Cancer Cells to Radiation by Synergistic Suppression of DNA Homologous Recombination Repair

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.

MEN1 Is a Melanoma Tumor Suppressor That Preserves Genomic Integrity by Stimulating Transcription of Genes That Promote Homologous Recombination-Directed DNA Repair

Multiple endocrine neoplasia type 1 is a familial cancer syndrome resulting from loss-of-function mutations in the MEN1 gene. We previously identified the tumor suppressor MEN1 as a gene required for oncogene-induced senescence in melanocytes, raising the possibility that MEN1 is a melanoma tumor suppressor. Here we show that MEN1 expression is lost in a high percentage of human melanomas and melanoma cell lines. We find that melanocytes depleted of MEN1 are deficient in homologous recombination (HR)-directed DNA repair, which is accompanied by increased nonhomologous end-joining activity. Following DNA damage, MEN1 levels increase as a result of phosphorylation by the DNA damage kinase ATM/ATR. Most importantly, we show that MEN1 functions by directly stimulating the transcription of several genes, including BRCA1, RAD51, and RAD51AP1, that encode proteins involved in HR. MEN1 and its coactivator, the mixed-lineage leukemia histone methyltransferase, are recruited to the BRCA1, RAD51, and RAD51AP1 promoters by estrogen receptor 1, resulting in increased histone H3-lysine 4 trimethylation and transcription. Collectively, our results indicate that MEN1 is a melanoma tumor suppressor that functions by stimulating the transcription of genes involved in HR-directed DNA repair.

Inhibition of ERβ Induces Resistance to Cisplatin by Enhancing Rad51–Mediated DNA Repair in Human Medulloblastoma Cell Lines

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin–induced cytotoxicity in human medulloblastoma cell lines.

Activation of HIV-1 LTR by Rad51 in microglial cells

Infection with HIV-1 induces a variety of biological alterations to the host that are beneficial to the life cycle of the virus but may have adverse effects on the host cell. Here we demonstrate that expression of Rad51, a major component of the homologous recombination-directed DNA repair (HRR) pathway, is induced upon HIV-1 infection of microglial cells. Activation of Rad51 expression positively impacts on HIV-1 LTR transcription through a region of the viral promoter known for binding the inducible transcription factor NF-κB. Rad51 showed the ability to form a complex with the p65 subunit of NF-κB and regulate the level of p65 interaction with LTR DNA encompassing the κB motif. This study provides evidence for reciprocal interaction of HIV-1 and a host DNA repair protein that impacts on expression of the viral genome. These results also point to the ability of HIV-1 to recruit proteins involved in DNA repair that are necessary for retroviral DNA integration, efficient replication and prevention of viral-induced cell death.

Estrogen receptor β‐mediated nuclear interaction between IRS‐1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or gamma-irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERbeta) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERbeta was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERbeta was also present in human medulloblastoma clinical samples. Expression of ERbeta coincided with nuclear translocation of insulin receptor substrate 1 (IRS-1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERbeta and IRS-1 bind each other, and the interaction involves C-terminal domain of IRS-1 (aa 931-1233). Following cisplatin-induced DNA damage, nuclear IRS-1 localized at the sites of damaged DNA, and interacted with Rad51--an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR-DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931-1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERbeta-mediated IRS-1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas.

Protective role of Purα to cisplatin

BACKGROUND: The nucleic acid-binding protein Purα is involved at stalled DNA replication forks, in double-strand break (DSB) DNA repair and the cellular response to DNA replication stress. Purα also regulates homologous recombination-directed DNA repair (HRR). METHODS: We investigated the effects of the DNA damage-inducing cancer chemotherapeutic agent cisplatin on mouse embryo fibroblasts (MEFs) from PURA-/- knockout mice that lack Purα. RESULTS: Cells lacking Purα showed enhanced sensitivity to cisplatin as evaluated by assays for cell viability and cell clonogenicity. This was seen both in Purα-negative MEFs and in human glioblastoma cells treated with siRNA directed against Purα. MEFs lacking Purα also showed enhanced H2AX phosphorylation in response to cisplatin. Repair of a reporter plasmid that had been treated with cisplatin was decreased in a reactivation assay using Purα-negative MEFs and the capacity of nuclear extracts from Purα-negative MEFs to perform non-homologous end-joining in vitro was also impaired. CONCLUSIONS: Purα has a role in the cellular response to cisplatin-induced DNA damage and may provide new therapeutic modalities for cisplatin-resistant tumors.

IRS‐1–Rad51 nuclear interaction sensitizes JCV T‐antigen positive medulloblastoma cells to genotoxic treatment

The large T-antigen from human polyomavirus JC (JCV T-antigen) is suspected to play a role in malignant transformation. Previously, we reported that JCV T-antigen requires the presence of a functional insulin-like growth factor I receptor (IGF-IR) for transformation of fibroblasts and for survival of medulloblastoma cell lines; that IGF-IR is phosphorylated in medulloblastoma biopsies and that JCV T-antigen inhibits homologous recombination-directed DNA repair, causing accumulation of mutations. Here we are evaluating whether JCV T-antigen positive and negative mouse medulloblastoma cell lines, which significantly differ in their tumorigenic properties, are also different in their abilities to repair double strand breaks of DNA (DSBs). Our results show that despite much stronger tumorigenic potential, JCV T-antigen positive medulloblastoma cells are more sensitive to genotoxic agents (cisplatin and gamma-irradiation). Subsequent analysis of DNA repair of DSBs indicated that homologous recombination-directed DNA repair (HRR) was selectively attenuated in JCV T-antigen positive medulloblastoma cells. JCV T-antigen did not affect HRR directly. In the presence of JCV T-antigen, insulin receptor substrate 1 (IRS-1) translocated to the nucleus where it co-localized with Rad51, possibly attenuating HRR.

JC virus large T‐antigen and IGF‐I signaling system merge to affect DNA repair and genomic integrity

The progression of cancer is often associated with genomic instability, which may develop as a result of compromised defense mechanisms responsible for the maintenance of chromosomal integrity. These include defects in telomere preservation, chromosomal segregation, and DNA repair. In this review, we discuss molecular interactions between viral and cellular signaling components, which interfere with DNA repair mechanisms, and possibly contribute to the development of a mutagenic phenotype. Our studies indicate that large T-antigen from the human polyomavirus JC (JCV T-antigen) inhibits homologous recombination directed DNA repair (HRR)-causing accumulation of mutations in the affected cells (JCP 2005, in press). Surprisingly, T-antigen does not operate directly, but utilizes insulin receptor substrate 1 (IRS-1), which is the major signaling molecule for insulin-like growth factor I receptor (IGF-IR). Following T-antigen-mediated nuclear translocation, IRS-1 binds Rad51 at the site of damaged DNA. This T-antigen-mediated inhibition of HRR does not function in cells lacking IRS-1, and can be reproduced in the absence of T-antigen by IRS-1 with an artificial nuclear localization signal. The interplay described between the IGF-IR signaling system and JCV T-antigen in the process of DNA repair could be relevant, since nearly 90% of the human population is seropositive for JC virus, JCV T-antigen transforms cells in vitro, is tumorigenic in experimental animals, and the presence of JC virus has been shown in an increasing number of biopsies of human cancer.

Impaired homologous recombination DNA repair and enhanced sensitivity to DNA damage in prostate cancer cells exposed to anchorage-independence

During metastases, cancer cells are temporarily exposed to the condition in which interactions with extracellular environment can be restricted (anchorage-independence). We demonstrate that the sensitivity of prostate cancer cell lines, DU145 and PC-3, to genotoxic treatment (cisplatin and gamma-irradiation) increased several folds when cells were forced to grow in anchorage-independence. This enhanced drug sensitivity was associated with a severe impairment of homologous recombination-directed DNA repair (HRR). The mechanism involves Rad51, which is the major enzymatic component of HRR. The protein level of Rad51 and its recruitment to DNA double-strand breaks (DSBs) were both attenuated. Rad51 deficiency in anchorage-independence was not associated with Rad51 promoter activity, and was not compensated by a constitutive overexpression of Rad51 cDNA. Instead, Rad51 protein level and its ability to colocalize with DSBs were restored in the presence of proteosome inhibitors, or when cells from the suspension cultures were allowed reattachment. Presented results indicate that anchorage-independence sensitizes prostate cancer cells to genotoxic agents; however, it also attenuates faithful component of DNA repair by targeting stability of Rad51. This temporal attenuation of HRR may contribute to the accumulation mutations after DNA damage, and possibly the selection of new adaptations in cells, which survived genotoxic treatment.

Impairment of homologous recombination directed DNA repair in prostate cancer cells exposed to anchorage independence

9638 Background: The majority of patients with androgen dependant prostate cancer develop a transition to highly invasive metastatic disease, which is refractory to hormonal therapy. This complication relies on the acquisition of new properties, which allow cancer cells to invade other tissues. One mechanism via which rapid genetic changes could occur is a failure of systems controlling stability of the genome, including DNA repair. In parallel, metastatic cells are temporarily exposed to anchorage-independence -a condition in which cellular interactions with extracellular matrix proteins are restricted. The purpose of this study was to evaluate whether and how anchorage-independence could predispose prostate cancer cells to develop genomic instability. Methods: DNA repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination directed DNA repair (HRR) were evaluated in human prostate cancer cell lines: DU145, LNCaP and PC-3. Cell survival was evaluated by trypan blue exclusion, cell cycle distribution by flow-cytometry, and DNA repair proteins by western blotting and immunocytofluorescence. Results: The sensitivity of prostate cancer cells to irradiation, cisplatin and mitomycin increased several fold in anchorage-independence. This enhanced drug sensitivity was associated with a severe impairment of HRR. The mechanism involved Rad51, which is the major enzymatic component of HRR. The protein level of Rad51 and its recruitment to DNA breaks were both attenuated. Rad51 deficiency in anchorage-independence was not associated with Rad51 promoter activity, and was not compensated by a constitutive overexpression of Rad51 cDNA. However, Rad51 protein was efficiently restored in the presence of the proteosom inhibitor epoxomycin, or when cells from suspension cultures were allowed to reattach. Conclusions: Anchorage-independence sensitizes prostate cancer cells to genotoxic agents; however, it also attenuates a faithful component of DNA repair by targeting the stability of Rad51. This may contribute to the development of the metastatic phenotype in cells, which survive genotoxic treatment. No significant financial relationships to disclose.

Impairment of homologous recombination directed DNA repair in prostate cancer cells exposed to anchorage independence

9638 Background: The majority of patients with androgen dependant prostate cancer develop a transition to highly invasive metastatic disease, which is refractory to hormonal therapy. This complication relies on the acquisition of new properties, which allow cancer cells to invade other tissues. One mechanism via which rapid genetic changes could occur is a failure of systems controlling stability of the genome, including DNA repair. In parallel, metastatic cells are temporarily exposed to anchorage-independence -a condition in which cellular interactions with extracellular matrix proteins are restricted. The purpose of this study was to evaluate whether and how anchorage-independence could predispose prostate cancer cells to develop genomic instability. Methods: DNA repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination directed DNA repair (HRR) were evaluated in human prostate cancer cell lines: DU145, LNCaP and PC-3. Cell survival was evaluated by trypan blue exclusion, cell cycle distribution by flow-cytometry, and DNA repair proteins by western blotting and immunocytofluorescence. Results: The sensitivity of prostate cancer cells to irradiation, cisplatin and mitomycin increased several fold in anchorage-independence. This enhanced drug sensitivity was associated with a severe impairment of HRR. The mechanism involved Rad51, which is the major enzymatic component of HRR. The protein level of Rad51 and its recruitment to DNA breaks were both attenuated. Rad51 deficiency in anchorage-independence was not associated with Rad51 promoter activity, and was not compensated by a constitutive overexpression of Rad51 cDNA. However, Rad51 protein was efficiently restored in the presence of the proteosom inhibitor epoxomycin, or when cells from suspension cultures were allowed to reattach. Conclusions: Anchorage-independence sensitizes prostate cancer cells to genotoxic agents; however, it also attenuates a faithful component of DNA repair by targeting the stability of Rad51. This may contribute to the development of the metastatic phenotype in cells, which survive genotoxic treatment. No significant financial relationships to disclose.

Role of the insulin-like growth factor I/insulin receptor substrate 1 axis in Rad51 trafficking and DNA repair by homologous recombination.

The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome.