ABSTRACT
The extent to which the three-dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We generated a high-resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and used high-throughput genome-wide translocation sequencing to map translocations from target DNA double-strand breaks (DSBs) within it. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high-frequency DSBs at these loci and their colocalization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and subchromosomal domains in a manner directly related to pre-existing spatial proximity. By combining two high-throughput genomic methods in a genetically tractable system, we provide a new lens for viewing cancer genomes.
Subject(s)
Genome , Neoplasms/genetics , Translocation, Genetic , Animals , DNA Breaks, Double-Stranded/radiation effects , G1 Phase , High-Throughput Nucleotide Sequencing , Mice , Mice, 129 Strain , Mice, Inbred BALB C , Neoplasms/drug therapy , Neoplasms/pathology , Precursor Cells, B-Lymphoid/cytology , Receptors, Antigen/geneticsABSTRACT
Macrophages constitute a first line of pathogen defense by triggering a number of inflammatory responses and the secretion of various pro-inflammatory cytokines. Recently, we and others found that IκBζ, an atypical IκB family member and transcriptional coactivator of selected NF-κB target genes, is essential for macrophage expression of a subset of pro-inflammatory cytokines, such as IL-6, IL-12, and CCL2. Despite defective pro-inflammatory cytokine expression, however, IκBζ-deficient mice develop symptoms of chronic inflammation. To elucidate this discrepancy, we analyzed a regulatory role of IκBζ for the expression of anti-inflammatory cytokines and identified IκBζ as an essential activator of IL-10 expression. LPS-challenged peritoneal and bone marrow-derived macrophages from IκBζ-deficient mice revealed strongly decreased transcription and secretion of IL-10 compared with wild-type mice. Moreover, ectopic expression of IκBζ was sufficient to stimulate Il10 transcription. On the molecular level, IκBζ directly activated the Il10 promoter at a proximal κB site and was required for the transcription-enhancing trimethylation of histone 3 at lysine 4. Together, our findings show for the first time the IκBζ-dependent expression of an anti-inflammatory cytokine that is crucial in controlling immune responses.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Interleukin-10/metabolism , Macrophages/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line , Cells, Cultured , Embryo, Mammalian/cytology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Immunoblotting , Inflammation/genetics , Inflammation/metabolism , Inflammation Mediators/metabolism , Interleukin-10/genetics , Macrophages/cytology , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/metabolism , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/antagonists & inhibitors , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.
Subject(s)
Cell Nucleus/genetics , DNA Damage , Genome, Mitochondrial , Real-Time Polymerase Chain Reaction/methods , Cells, Cultured , Comet Assay , DNA, Mitochondrial/chemistry , Humans , Jurkat CellsABSTRACT
Cellular senescence, a state of sustained cell cycle arrest, has been identified as an important anti-tumor barrier. Senescent cells secrete various growth factors and cytokines, such as IL6 and IL8, which collectively constitute the senescence-associated secretory phenotype (SASP). The SASP can signal to the tumor environment and elicit the immune-mediated clearance of tumor cells or, depending on the context, could potentially promote tumor progression. Despite the importance of the SASP to tumor biology, its regulation remains relatively unknown. Here, we show that IκBζ, an atypical member of the inhibitor of NFκB proteins and selective coactivator of particular NFκB target genes, is an important regulator of SASP expression. Several models of DNA damage- and oncogene-induced senescence revealed a robust induction of IκBζ expression. RNAi-mediated knockdown of IκBζ impaired IL6 and IL8 expression, whereas transgenic IκBζ expression resulted in enhanced SASP cytokine expression. Importantly, during senescence of IκBζ knockout cells induction of IL6 and IL8, but not of the cell cycle inhibitor p21(WAF/CIP1), was completely abolished. Thus, we propose an important and hitherto unappreciated role of IκBζ in SASP formation in both DNA damage- and oncogene-induced senescence.
Subject(s)
DNA Damage , I-kappa B Proteins/metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cellular Senescence/genetics , Cellular Senescence/physiology , Cytokines/metabolism , Humans , I-kappa B Proteins/biosynthesis , I-kappa B Proteins/genetics , MCF-7 Cells , Oncogenes , Phenotype , Signal TransductionABSTRACT
CCL2, also referred to as MCP-1, is critically involved in directing the migration of blood monocytes to sites of inflammation. Consequently, excessive CCL2 secretion has been linked to many inflammatory diseases, whereas a lack of expression severely impairs immune responsiveness. We demonstrate that IκBζ, an atypical IκB family member and transcriptional coactivator required for the selective expression of a subset of NF-κB target genes, is a key activator of the Ccl2 gene. IκBζ-deficient macrophages exhibited impaired secretion of CCL2 when challenged with diverse inflammatory stimuli, such as LPS or peptidoglycan. These findings were reflected at the level of Ccl2 gene expression, which was tightly coupled to the presence of IκBζ. Moreover, mechanistic insights acquired by chromatin immunoprecipitation demonstrate that IκBζ is directly recruited to the proximal promoter region of the Ccl2 gene and is required for transcription-enhancing histone H3 at lysine-4 trimethylation. Finally, IκBζ-deficient mice showed significantly impaired CCL2 secretion and monocyte infiltration in an experimental model of peritonitis. Together, these findings suggest a distinguished role of IκBζ in mediating the targeted recruitment of monocytes in response to local inflammatory events.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription, Genetic/genetics , Adaptor Proteins, Signal Transducing/immunology , Animals , Cells, Cultured , Chemokine CCL2/immunology , Female , Gene Expression/genetics , Gene Expression/immunology , Histones/genetics , Histones/immunology , Histones/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Lipopolysaccharides/immunology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Monocytes/immunology , Monocytes/metabolism , NF-kappa B/genetics , NF-kappa B/immunology , NF-kappa B/metabolism , Nuclear Proteins/immunology , Peritonitis/genetics , Peritonitis/immunology , Peritonitis/metabolism , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/immunology , Transcription, Genetic/immunologyABSTRACT
Targeted Radionuclide Therapies (TRT) involve the tailored combination of a therapeutic radionuclide and a targeting molecule, as for instance antibodies or fragments thereof. Despite their short shelf-life, these drug products must meet stringent regulatory standards before use. We introduce a novel, efficient method utilizing Bio-Layer Interferometry (BLI) for rapid identity testing of TRT drug products in less than five minutes. This approach not only reduces radioactive waste but also minimizes operator exposure to radiation. This label-free method has been successfully developed and validated for three different TRT products, ensuring compliance with Good Manufacturing Practices (GMP). Furthermore, we outline our strategic approach to the production and testing of custom biosensors for each product, firmly grounded in Quality-by-Design (QbD) principles.
Subject(s)
Interferometry , Interferometry/methods , Biosensing Techniques/methods , Radioisotopes/chemistry , Humans , Radiopharmaceuticals/chemistryABSTRACT
Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.
Subject(s)
Glutathione Peroxidase/metabolism , Glutathione/metabolism , Induced Pluripotent Stem Cells/metabolism , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Differentiation , Cells, Cultured , DNA Damage/drug effects , DNA Damage/radiation effects , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/metabolism , DNA, Mitochondrial/radiation effects , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Glutathione Peroxidase/antagonists & inhibitors , Glutathione Peroxidase/genetics , Humans , Hydrogen Peroxide/toxicity , Induced Pluripotent Stem Cells/cytology , Mitochondria/metabolism , Oxidative Stress/drug effects , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Ultraviolet RaysABSTRACT
Due to its role in aging and antitumor defense, cellular senescence has recently attracted increasing interest. However, there is currently no single specific marker that can unequivocally detect senescent cells. Here, we identified α-L-fucosidase (α-Fuc) as a novel sensitive biomarker for cellular senescence. Regardless of the stress stimulus and cell type, α-Fuc activity was induced in all canonical types of cellular senescence, including replicative, DNA damage- and oncogene-induced senescence. Strikingly, in most models the degree of α-Fuc upregulation was higher than the induction of senescence-associated ß-galactosidase (SA-ß-Gal), the current gold standard for senescence detection. As α-Fuc is convenient and easy to measure, we suggest its utility as a valuable marker, in particular in cells with low SA-ß-Gal activity.
Subject(s)
Biomarkers/metabolism , Cellular Senescence/physiology , alpha-L-Fucosidase/metabolism , Animals , Cells, Cultured , Cellular Senescence/genetics , HCT116 Cells , Humans , Immunohistochemistry , Mice, Inbred ICR , Real-Time Polymerase Chain Reaction , alpha-L-Fucosidase/geneticsABSTRACT
BACKGROUND: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in ß-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS: Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE: These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.
Subject(s)
Cell Nucleus/metabolism , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/antagonists & inhibitors , Insulin/metabolism , Islets of Langerhans/cytology , Protein Kinase C-delta/metabolism , Animals , Apoptosis , Blotting, Western , Humans , Insulin Secretion , Islets of Langerhans/metabolism , Mice , Microscopy, Confocal , PhosphorylationABSTRACT
Recurrent chromosomal abnormalities, especially chromosomal translocations, are strongly associated with certain subtypes of leukemia, lymphoma and solid tumors. The appearance of particular translocations or associated genomic alterations can be important indicators of disease prognosis, and in some cases, certain translocations may indicate appropriate therapy protocols. To date, most of our knowledge about chromosomal translocations has derived from characterization of the highly selected recurrent translocations found in certain cancers. Until recently, mechanisms that promote or suppress chromosomal translocations, in particular, those responsible for their initiation, have not been addressed. For translocations to occur, two distinct chromosomal loci must be broken, brought together (synapsed) and joined. Here, we discuss recent findings on processes and pathways that influence the initiation of chromosomal translocations, including the generation fo DNA double strand breaks (DSBs) by general factors or in the context of the Lymphocyte-specific V(D)J and IgH class-switch recombination processes. We also discuss the role of spatial proximity of DSBs in the interphase nucleus with respect to how DSBs on different chromosomes are justaposed for joining. In addition, we discuss the DNA DSB response and its role in recognizing and tethering chromosomal DSBs to prevent translocations, as well as potential roles of the classical and alternative DSB end-joining pathways in suppressing or promoting translocations. Finally, we discuss the potential roles of long range regulatory elements, such as the 3'IgH enhancer complex, in promoting the expression of certain translocations that are frequent in lymphomas and, thereby, contributing to their frequent appearance in tumors.