Добірка наукової літератури з теми "Ligase IV Inhibitor SCR7"

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency.

Abstract Multiple Myeloma (MM) is characterized by the growth of malignant plasma cells harboring numerous genomic aberrations. The molecular basis driving MM genomic instability is still largely unknown. The ability to repair DNA damages is essential for the maintenance of its integrity, especially the double-strand breaks (DSBs) which are mainly repaired by Non Homologous End Joining (NHEJ). We have investigated NHEJ pathway in myeloma and observed a significant association between up-regulated NHEJ pathway-related gene expression and poor overall survival in two large datasets (IFM and Arkansas) in myeloma. We have also observed a higher end joining (EJ) activity in MM cell lines compared to normal cells using a dual gene plasmid-based assay utilizing Luciferase (LUC) as a test gene to measures end joining, and Alkaline Phosphatase (SEAP) as a reporter gene to control for transfection efficiency. Moreover, we confirmed an increased NHEJ activity in several primary patient myeloma cells at different disease stage. Based on this rationale, since an altered NHEJ has been linked to genomic instability and its inhibition leading to eventual cell death, we hypothesized that the aberrant NHEJ can be used as a potential therapeutic target in MM. To address the relevance of NHEJ inhibition in MM cell proliferation and survival, we used SCR7, an inhibitor of Ligase IV (Lig-IV) which is essential for ligation of the double strand breaks following their recognition by the KU70/KU80 heterodimer and the recruitment of DNA-PKcs. We tested 4 different MM cell lines (U266, R8226, MM1s and Dox40), however, except for some level of inhibition in Dox40 (IC50, between 50 and 100 uM at 72 hours), the other cell line growth was not significantly affected (R8226 - IC30 at the concentration; and U266 and MM1s did not reach IC30). The same data were confirmed by Annexin V/7AAD staining and Caspase assay. Interestingly, expression of Lig-IV estimated by western blot analysis, inversely correlated with MM cells sensitivity suggesting that higher protein concentration may require higher drug levels for inhibition. Consistent with this result, we observed a strong inhibition of the NHEJ pathway by ku86-directed shRNAs, which was able to induce cell death in the more resistant MM cell line u266. Subsequently we used the dual gene plasmid-based assay to evaluate the effect of sub-lethal dose (20 uM) of SCR7 on NHEJ in 3 MM cell lines (u266, R8226 and MM1s) and observed an increased recombination activity in 2 of them. We also confirmed these data with another NHEJ inhibitor, NU7441, which target DNA-PK; and by using ku86-shRNA in U266 cell line. Moreover we observed an accumulation of unrepaired DSBs at the genome level as demonstrated by an increased γ-H2AX by western blotting. These results suggested the possibility that the inhibition of the NHEJ by blocking Lig-IV could activate the alternative NHEJ pathway (a-NHEJ), which is more error-prone compared to the classical NHEJ (c-NHEJ). To confirm this hypothesis further, we treated MM cell lines with sub-lethal dose of NU7441 (2.5 uM), Benzamide (2.5 uM), an inhibitor of PARP, which is one of the main protein involved in the a-NHEJ, or both. The different modulation observed with single and combination treatments, along with the ability of NU7441 to revert sensitivity to Benzamide in R8226 cells, suggested that inhibition of the classical pathway could switch on the a-NHEJ and indicated its basal activity at least in this cell line. Ongoing study is assessing the influence of such compounds on NHEJ in primary MM cells and their impact on acquisition of new genomic changes. In conclusion, our data confirm the aberrant activation of NHEJ in MM, and suggest the potential role for both classical and more error-prone a-NHEJ pathways in inducing genomic instability, which may require a dual inhibition to trigger myeloma cell death. Disclosures No relevant conflicts of interest to declare.

Abstract The oncogenic BCR-ABL in CML produces increased reactive oxygen species (ROS) leading to DSB and aberrant repair. We have previously shown that CML cells demonstrate an increased frequency of errors of non homologous end-joining (NHEJ). DSB are repaired by two major pathways, homologous recombination (HR) and NHEJ, the dominant pathway in eukaryotic cells, also known as DNA-PK dependent NHEJ (D-NHEJ). Recent reports have identified alternative or “back-up” NHEJ pathways (B-NHEJ) that are highly error-prone, and may explain the altered DSB repair reported in CML. To determine the mechanism for the aberrant NHEJ repair in CML, we examined steady state levels of D-NHEJ proteins, including Ku70/86, DNA-PKcs, Artemis and DNA Ligase IV/XRCC4 in four different BCR-ABL positive CML cell lines compared with three lymphoblastoid cell lines established from normal individuals and one BCR-ABL negative CML cell line. We find that two key components of D-NHEJ, Artemis (4–7 fold) and DNA Ligase IV (2–3 fold) are down-regulated, compared with controls. These data suggest that D-NHEJ repair is compromised in CML. To determine whether alternative NHEJ repair plays a role in the aberrant repair of DSB in CML cells, we next examined expression levels of DNA Ligase IIIα/XRCC1, PARP and other proteins known to be associated with NHEJ repair, such as the protein found to be deleted in Werner’s syndrome, WRN. We find that WRN and DNA Ligase IIIα are increased (3–6 fold) in BCR-ABL-positive CML compared with control cell lines. Importantly, DNA Ligase IIIα/XRCC1 forms a complex with WRN, suggesting that it may be a new member of the alternative repair pathway. To confirm that up-regulation of DNA Ligase IIIα and WRN are elicited by BCR-ABL, we examined the levels of these proteins in primary samples (N=4) from patients with different levels of BCR-ABL, following treatment with the tyrosine kinase inhibitor Gleevec. WRN and DNA Ligase IIIα are down regulated in patient samples where BCR-ABL levels are significantly decreased. Furthermore, we confirmed that these up-regulated proteins are involved in DSB repair in CML cells because they co-localize to induced DSB in BCR-ABL-positive cell lines stably transfected with DSB-containing DRneo plasmid, using fluorescence in situ hybridization (FISH) co-immunostaining. Importantly we show that siRNA down-regulation of WRN and DNA Ligase IIIα leads to elevated levels of unrepaired DSB and a decreased frequency of DSB repair efficiency in CML cells. In addition siRNA down-regulation of WRN leads to large deletions at the site of repair, while siRNA down-regulation of DNA Ligase IIIα results in an increased frequency of misrepair. Finally, we determined whether “correction” of main NHEJ pathway proteins in CML can lead to a decrease in the frequency of errors of end-joining repair. Over-expression of Artemis using pcDNA constructs in CML cells leads to more correct end-joining, compared with vector transfected controls. We conclude that down-regulation of Artemis and DNA Ligase IV leads to compensatory up-regulation of alternative repair pathways in BCR-ABL-positive CML cells, and suggest a role for a new protein complex in CML, in protecting and joining DNA ends, thus ensuring the survival of CML cells. Inhibition of alternative NHEJ repair may be explored in combination with other agents as a therapeutic strategy in CML.

The vines and leaves of Momordica charantia L. are used as herbal medicines to treat inflammation-related disorders. However, their safety profile remains uncharacterized, and the constituents in their extracts that exert anti-inflammatory and adverse effects remain unclear. This study isolated the characteristic cucurbitane-type triterpenoid species in the vines and leaves of M. charantia L. and analyzed their cytotoxicity, anti-inflammatory effects, and underlying mechanisms. Four structurally related triterpenoids—momordicines I, II, IV, and (23E) 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al (TCD)—were isolated from the triterpenoid-rich fractions of extracts from the vines and leaves of M. charantia. Momordicine I was cytotoxic on normal cells, momordicine II exerted milder cytotoxicity, and momordicine IV and TCD had no obvious adverse effects on cell growth. TCD had anti-inflammatory activity both in vivo and in vitro. In lipopolysaccharide-stimulated RAW 264.7 cells, TCD inhibited the inhibitor kappa B kinase/nuclear factor-κB pathway and enhanced the expression of nuclear factor erythroid 2-related factor 2, heme oxygenase-1, and glutamate-cysteine ligase modifier subunit through the extracellular signal-regulated kinase1/2 and p38. Thus, the vines and leaves of M. charantia should be used with caution. An extraction protocol that can enrich TCD but remove momordicine I would likely enhance the safety of the extract.

Local implant-based delivery of rationally selected combination of chemotherapeutics has some major advantages for the treatment of glioblastoma such as: (a) 100 % bio-availability locally in brain can be achieved at the tumor site (b) avoid systemic leakage and associated toxicity, and (c) simultaneous inhibition of multiple, mutually exclusive cancer mechanisms is possible. Here, we report a polymeric brain implant capable of delivering two different drugs in recur-rent glioma cells. We have selected a combination of clinically used DNA alkylating agent, Te-mozolomide, and a DNA mismatch repair protein (Ligase IV) inhibitor, SCR-7, and delivered simultaneously into tumor spheroids formed by rat glioma cells, C6. The dual-drug loaded polymeric wafer, prepared by lyophilization method, could deliver both the drugs in a controlled fashion. To test the efficacy of this system, we have optimized an in vitro recurrent model of glioma spheroids wherein, the implant released both the drugs in a sustained fashion, thereby continuously exposing the cells to DNA methylation while inhibiting the DNA repair pathways. This leads to synergistic toxicity and inhibition of tumor recurrence for extended duration compared to free drug combination.

In CRISPR–Cas9 genome editing, double-strand DNA breaks (DSBs) primarily undergo repair through nonhomologous end joining (NHEJ), which produces insertion or deletion of random nucleotides within the targeted region (indels). As a result, frameshift mutation-mediated loss-of-function mutants are frequently produced. An alternative repair mechanism, homology-directed repair (HDR), can be used to fix DSBs at relatively low frequency. By injecting a DNA-homology repair construct with the CRISPR–Cas components, specific nucleotide sequences can be introduced within the target region by HDR. We have taken advantage of the fact that Xenopus oocytes have much higher levels of HDR than eggs to increase the effectiveness of creating precise mutations. We introduced the oocyte host transfer technique, well established for knockdown of maternal mRNA for loss-of-function experiments, to CRISPR–Cas9-mediated genome editing. The host-transfer technique is based on the ability of Xenopus oocytes to be isolated, injected with CRISPR–Cas components, and cultured in vitro for up to 5 d before fertilization. During these 5 d, CRISPR–Cas components degrade, preventing further alterations to the paternal or maternal genomes after fertilization and resulting in heterozygous, nonmosaic embryos. Treatment of oocytes with a DNA ligase IV inhibitor, which blocks the NHEJ repair pathway, before fertilization further improves the efficiency of HDR. This method allows straightforward generation of either nonmosaic F0 heterozygous indel mutant Xenopus or Xenopus with efficient, targeted insertion of small DNA fragments (73–104 nt). The germline transmission of mutations in these animals allows homozygous mutants to be obtained one generation (F1) sooner than previously reported.

Abstract The Pml gene is the target of t(15;17) chromosome translocation in acute promyelocytic leukemia. PML protein is known to localize in discrete nuclear speckles, named PML nuclear bodies (NBs). In NBs, PML interacts with several transcription factors, such as p53 and AML1, and their co-activators, such as HIPK2 and p300. PML activates transcription of their target genes. PML is thought to stabilize transcription factor complex and function as a mediator in transcription activation, but little is known about the molecular mechanism by which PML activates transcription. To clarify the role of PML in transcription regulation, we purified the PML complex and identified a novel F-box protein (FBP), Skp1, and Cullin1 (Cul1) in the PML complex by LC/MS/MS analysis. FBPs form SCF ubiquitin ligase complexes with Skp1, Cul1 and ROC1 and mediate recognition of specific substrates for ubiquitination. We found that the FBP that we identified here also forms a SCF complex with Skp1, Cul1 and ROC1. To identify substrates for the SCF complex, we tested several proteins that could bind to PML, and found that the FBP promotes degradation of HIPK2 and p300. These degradations were inhibited in the presence of a proteasome inhibitor, MG132. The FBP stimulated ubiquitination of HIPK2. These results suggest that the SCF promotes degradation of these proteins by the ubiquitin-proteasome pathway. The fact that the SCF is a part of the PML complex suggests that PML plays a role in the SCF-mediated degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. In order to clarify the role of PML in degradation of HIPK2 and p300, we tested effects of PML on the degradation and found that PML inhibited the SCF-mediated degradation of HIPK2 and p300 without inhibition of ubiquitination. To clarify roles of HIPK2, PML IV and the FBP in p53-dependent transcription, we performed reporter analysis using the MDM2 promoter in H1299 cells. Since the FBP promotes degradation of HIPK2, we initially thought that the FBP might inhibit activation of p53-dependent transcription by HIPK2 and PML IV. However, the FBP, HIPK2 and PML synergistically stimulated the p53-dependent transcriptional activation. Taken together our data suggest that the SCF-induced ubiquitination of transcription co-activators HIPK2 and p300 plays a critical role in transcriptional regulation, and that PML stimulates transcription by protecting HIPK2 and p300 from ubiquitin-dependent degradation.

En cancérologie, la radiothérapie est une des armes essentielles pour éradiquer les cellules tumorales. Les cassures des deux brins de l'ADN dites "double-brin" qu'elle induit sont particulièrement toxiques et constituent la principale cause de mort cellulaire. La NHEJ (Jonction d'Extrémités Non-Homologues) est la voie métabolique majeure de réparation de ces cassures double-brin de l'ADN et par ce mécanisme, les cellules humaines adoptent une résistance à la radiothérapie. Ce mécanisme de réparation constitue donc une cible de choix pour un traitement anticancéreux combiné en vue d'augmenter la sensibilité des cellules cancéreuses aux rayons ionisants (radiosensibilisation). Au cours du mécanisme NHEJ, la ligature finale des extrémités d'ADN est assurée par le complexe protéique tripartite: XRCC4/ADN Ligase IV/Cernunnos-XLF. Les interfaces protéiques concernées représentent toutes des cibles potentielles dans une stratégie rationnelle d'isolement de molécules inhibitrices, guidée par les structures tridimensionnelles de chaque protéine. A travers des expériences de criblage virtuel et de validation à la fois biophysique et biochimique, nous avons isolé les premières molécules capable de prévenir in vitro les interactions protéine-protéine pour les complexes XRCC4/Lig4 et XRCC4/Cer-XLF, respectivement. Ces composés sont des points de départ pour l'élaboration d'inhibiteurs potentiels de plus haute affinité grâce à l'apport de la biologie structurale, en vue d'un effet radiosensibilisant cellulaire
Radiotherapy is a major weapon used against cancer. Radio-induced DNA double strand breaks (DSB) are the main lesions responsible for cell death. Non-homologous end-joining (NHEJ) is a predominant DSB repair mechanism which contributes to cancer cells resistance to radiotherapy. NHEJ is thus a good target for strategies which aim at increasing the radio-sensitivity of tumors. Through in silico screening and biophysical and biochemical assays, our objective was to find specific ligands for the XRCC4/Lig4 and XRCC4/Cer-XLF protein-protein interactions involved in NHEJ. Here, we isolated the first compounds able to prevent their interaction in vitro. These early stage inhibitors are promising tools for cancer therapy with the hope to develop more specific compounds for cellular assays through the 3D structure of the protein/inhibitor complexes

Repair of DNA breaks is essential for maintenance of genomic integrity. DNA double-strand breaks (DSBs) are considered as the most harmful DNA lesions within the cells which if left unrepaired or are misrepaired, can lead to a wide variety of genetic alterations, chromosomal rearrangements either culminating in oncogenic transformation or apoptosis. Therefore, the cell adopts two major pathways, Homologous recombination (HR) and nonhomologous end joining (NHEJ), in order to repair these DSBs thereby maintaining genome stability. HR mediated DSB repair is error-free and is functional during the S and G2 phases of cell cycle and requires the participation of a sister chromatid to act as a template in the repair process. However, in higher eukaryotes, DSBs are primarily repaired by nonhomologous DNA end joining (NHEJ), which is an error-prone DNA repair mechanism and is active throughout the cell cycle. During NHEJ, KU70/KU80 heterodimer binds to the broken DNA ends and recruits DNA-PKcs, Artemis, and Pol µ or ι to the repair site, resulting in processing of broken DNA ends. Following the end processing, ligation occurs with the help of Ligase IV, XRCC4, XLF and PAXX complex. In general, NHEJ is considered as a DSB repair pathway that does not have a sequence preference and hence thought to be random. Using an oligomer based, cell-free assay system, we evaluated the role of DNA sequence during the end-to-end joining of broken DNA, both at the overhangs and at its flanking regions. Using radiolabeled double-stranded oligomeric DNA substrates, possessing DSBs with different overhang sequences, we observed that there is a distinct difference in joining efficiency of mammalian extracts (rat testis, rat lungs, rat brain and rat heart) when overhangs with G:C rich sequences were studied. In contrast, we observed an enhanced efficiency in DNA end joining when A:T-rich flanking sequences were examined. Specifically, when flanking sequences were homopolymers of A:T or sequences enriched with A:T (adenines and/or thymines), joining efficiency was higher compared to that of random or G:C-rich flanking sequences. In order to investigate the factors that contribute towards the observed sequence specificity of NHEJ, we have overexpressed and purified one of the essential factors involved during NHEJ, the Ligase IV/XRCC4 complex. Ligase IV has a multidomain architecture, consisting of a conserved DNA binding domain at N-terminus and a tandem BRCT domain at C-terminus. The central catalytic domain comprises of adenylation (AD) and oligo-binding (OB-fold) domains. The N-terminal DNA binding domain (DBD) of Ligase IV is crucial for its interaction with DNA. Two hypomorphic mutations (A3V and T9I) in the DBD are observed in Ligase IV syndrome patients. Previous studies suggested that recruitment of Ligase IV/XRCC4 complex is dependent on KU protein complex. Interestingly, we observed that binding of Ligase IV/XRCC4 complex can occur independent of KU70/80, however, in a sequence dependent manner. In the present study, we uncover the sequence preference of Ligase IV/XRCC4 complex for the first time and show that it can bind directly to DNA containing DSBs, when flanked with A:T rich sequence in a KU independent manner irrespective of the sequence of the overhangs. All DNA substrates possessing A:T rich flank sequences investigated in the current study, including the one derived from human genome showed preferential binding to Ligase IV/XRCC4, with a binding constant that was ~10 fold higher than that of G:C rich substrates. Gel mobility shift assays, in conjunction with shift-western blotting assay using various radiolabeled double-stranded oligomers and purified Ligase IV/XRCC4 complex revealed stable DNA-protein complex formation in the absence of KU70/80 heterodimer. Importantly, SCR7, a well-known Ligase IV inhibitor, inhibited the recruitment of Ligase IV/XRCC4 in a concentration dependent manner. The DNA:protein complex formed was resistant to DNase I digestion. In addition, biolayer interferometry (BLI) studies using biotinylated double-stranded oligomers possessing A:T or GC rich sequences flanking the DSBs and purified proteins demonstrated that both Ligase IV/XRCC4 complex and Ligase IV could bind strongly to the immobilized double-stranded oligomer, in a sequence dependent manner (KD values were as low as in nanomolar range for AT rich flanking DNA substrates), unlike XRCC4 (KD= 4.12 ± 0.924 µM). Furthermore, BLI results also revealed that the binding of DNA binding domain (DBD) of Ligase IV was restricted to biotinylated polynucleotide of thymines or AT rich flanking ds DNA substrates. This binding got abrogated when point mutations seen in Ligase IV syndrome patients were introduced to DBD, thereby increasing the KD values many folds. Further, immunodepletion of KU70 protein from mammalian tissue extracts and ex vivo knockdown of Ku70 gene in mammalian cells reduced the joining efficiency of a random DNA substrate, while the NHEJ efficiency of DNA substrates remained unperturbed, when specific sequences were present in the flank region. However, in vitro ligation assays with Ligase IV immunodepleted cell free extracts exhibited reduced NHEJ efficiency irrespective of the substrates used. These observations suggest the unique DNA sequence dependence of Ligase IV involved during classical NHEJ repair pathway, which is KU-independent. The requirement of process of ligation catalysed by any of the three mammalian ligases (Ligase I, III or IV) is absolute, during physiological processes such as DNA replication, recombination and in almost all DNA repair pathways. This makes DNA ligases as attractive therapeutic targets to treat cancer. All three DNA ligases share a high degree of homology both at structural and functional levels (Ligase IV shares 13% and 15% sequence identity to Ligase I and Ligase III, respectively). They possess a well conserved catalytic domain but differ particularly in the DNA recognizing well conserved DNA Binding domain (DBD), that if bound by small molecules, the joining activity of ligases might get severely impaired. Based on homology modelling, and considering the anti-tumor properties of a well-known Ligase IV inhibitor, SCR7, we describe its first water soluble, auto cyclized and oxidized form, known as Sodium salt of SCR7-Pyrazine (Na-SCR7-P). Na-SCR7-P exhibited enhanced bioavailability, unlike the other DMSO-soluble Ligase inhibitors. In the present study, we found that like its parental compound (SCR7), Na-SCR7-P, also inhibited NHEJ in a Ligase IV dependent manner. However, unlike SCR7, it blocked joining catalysed by all three ligases in vitro, making it as an ideal tool for cancer therapeutic studies, as it may target multiple DNA transaction processes within the cancer cells. In depth studies revealed that Na-SCR7-P treatment resulted in reduction of mitochondrial membrane potential and activation of apoptosis culminating in cell death in various cancer cell lines. Importantly, administration of Na-SCR7-P led to significant reduction in tumor growth from 12th day of treatment and its impact was significantly higher than previously described SCR7, which predominantly targets Ligase IV within cells. Antitumor activity of Na-SCR7-P in mice resulted in enhanced lifespan, with minimal side effects. In addition, in ovo chorio-allantoic membrane assay revealed the potent anti-angiogenic property of Na-SCR7-P. Thus, we successfully identified another potent DNA ligase inhibitor, Na-SCR7-P that can potentially be used as a strategy for cancer treatment, owing to its water solubility. In addition, based on various biochemical and biophysical screening approaches, we identified two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Considering the indispensable role of DNA Ligase I during physiological processes such as DNA replication, repair and recombination, DNA Ligase I is considered as an important target for cancer therapy as it can impede proliferation of cancer cells upon treatment with specific small molecule inhibitors. Both the inhibitors blocked the ligation of nicks on DNA catalysed by cell-free extracts or purified Ligase I in a concentration-dependent manner. Docking studies in conjunction with biolayer interferometry and gel shift assays revealed that both SCR17 and SCR21 can bind to Ligase I, particularly to the DNA Binding Domain of Ligase I with KD values in nanomolar range (39 nM ± 8.88 nM and 42 nM ± 12.49 nM, respectively). The inhibitors did not show significant affinities towards DNA Ligase III/XRCC1 and DNA Ligase IV/XRCC4. Further, addition of DNA Ligase I could restore the joining, when the inhibitors were treated with testicular cell-free extracts. Ex vivo studies using multiple assays showed that even though cell death was limited in the presence of inhibitors in cancer cells, their proliferation was certainly compromised. Hence, we identify two promising DNA Ligase I inhibitors, which can be used in biochemical and cellular assays, and could be further modified and optimized to target cancer cells. In summary, the present study describes a sequence specific mechanism of recruitment of Ligase IV/XRCC4 to the broken DNA ends, which will have implications in understanding the sequence preference of NHEJ, in mammalian cells. In addition, we successfully developed novel DNA Ligase I inhibitors (SCR 17 and SCR21) and, a novel, water-soluble inhibitor (Na-SCR7-P) of NHEJ pathway which can further be improved as novel therapeutic strategies for sensitizing cancer cells to DSBs.