DNA Damage in Nijmegen Breakage Syndrome Cells Leads to PARP Hyperactivation and Increased Oxidative Stress

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency

Abstract Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals

Raising cellular NAD+ levels in human cells by nicotinic acid supplementation : biological consequences related to PARP-1 mediated reactions

The cellular response to DNA damage includes a number of mechanisms to detect and repair various types of DNA damage in order to preserve the integrity and stability of the genome. One reaction involved is the poly(ADP-ribosyl)ation (PARylation) of proteins, a modification performed by nuclear poly(ADP-ribose)polymerase-1 or -2 (PARP-1 and PARP-2) immediately after DNA damage infliction. PARPs covalently attach ADP-ribose units in a sequential fashion to target proteins including themselves, synthesizing a negatively charged polymer by using nicotinamide adenine dinucleotide (NAD+) as substrate. According to the level of DNA damage and intracellular NAD+ status, the most active enzyme PARP-1 and its product PAR mediate the recruitment of DNA repair factors to sites of lesions, facilitate DNA repair and thus maintain genomic integrity under conditions of moderate stress. In this scenario a tolerable amount of total cellular NAD+ is used for polymer synthesis. In contrast, the critical expenditure of NAD+ due to massive activation of PARP-1 under severe stress conditions can lead to cell death thus influencing deleterious or health-enhancing processes, as is apparent in inflammatory diseases or neurodegenerative disorders.One important parameter determining the cellular response to stresses is the level of available NAD+, which is crucial for adequate PAR synthesis and other NAD+ dependent processes, as the energy metabolism or sirtuin functions. Sirtuins can act as deacetylases in terms of response to cellular damage and to metabolic imbalances thus regulating fundamental processes as well.In order to analyse the biological consequences of elevated NAD+ levels in respect to PARP-1 mediated reactions and to address the question, if modulated NAD+ and/or PAR levels contribute to physiological or pathophysiological outcomes, distinct end points were investigated.Human blood peripheral mononuclear cells (NAD+) were ex vivo supplemented with the NAD+ precursor nicotinic aid (NA), which significantly raised intracellular nucleotide pools and led to an intensified PAR formation in response to genotoxic stimuli.It was observed that NA supplementation reduces cell death after genotoxic stress and shifts the residual fraction from necrosis to apoptosis, which is less harmful in regard to tissue integrity. To investigate, if this is a result of improved DNA repair, strand break formation and subsequent repair within the first 40 minutes was accessed. Interestingly, we observed that strand break rejoining is tightly regulated but was positively affected in NA supplemented cells under massive DNA damaging conditions and to a minor extent also under mild genotoxic stress. Furthermore, enhanced NAD+ levels seem to be beneficial in context of genomic integrity, as supplemented cells displayed reduced frequencies of micronucleus formation. In addition to the impact on already mentioned cellular functions, it was monitored if higher NAD+ levels favour also sirtuin activities. The SIRT-1 target p53 is acetylated in response to DNA damage. Preliminary results give evidence that the DNA damaged induced p53 acetylation is reduced in nicotinic acid supplemented cells, potentially due to increased deacetylation rate.In summary, increased NAD+ levels by NA supplementation can be beneficial for a cellular system, most notably in context of massive damage, but also in situations of minor damage. So far no adverse effects were observed for the investigated parameters. Based on the current results, supplying nicotinic acid seems to be a valuable approach to augment NAD+ levels especially in the case of acute damage or to correct suboptimal NAD+ levels to prevent negative consequences, often linked with excessive or elevated PARP-1 activation

The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation

NAD+ is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD+ consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD+ levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes.We observed that nicotinic acid supplementation increased NAD+ levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD+ concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD+ levels may be limiting under conditions of genotoxic stress and that boosting the NAD+ system with nicotinic acid can improve genomic stability

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD+ as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD+ depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD+ precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD+ levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N′-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD+ levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death

Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells

Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within protocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing. Technologies for small non-coding RNA sequencing at the single-cell level are less mature than for sequencing mRNAs. Here the authors evaluate available protocols for analysis of circulating lung cancer tumour cells

Microfluidic enrichment, isolation and characterization of disseminated melanoma cells from lymph node samples

For the first time in melanoma, novel therapies have recently shown efficacy in the adjuvant therapy setting, which makes companion diagnostics to guide treatment decisions a desideratum. Early spread of disseminated cancer cells (DCC) to sentinel lymph nodes (SLN) is indicative of poor prognosis in melanoma and early DCCs could therefore provide important information about the malignant seed. Here, we present a strategy for enrichment of DCCs from SLN suspensions using a microfluidic device (Parsortix (TM), Angle plc). This approach enables the detection and isolation of viable DCCs, followed by molecular analysis and identification of genetic changes. By optimizing the workflow, the established protocol allows a high recovery of DCC from melanoma patient-derived lymph node (LN) suspensions with harvest rates above 60%. We then assessed the integrity of the transcriptome and genome of individual, isolated DCCs. In LNs of melanoma patients, we detected the expression of melanoma-associated transcripts including MLANA (encoding for MelanA protein), analyzed the BRAF and NRAS mutational status and confirmed the malignant origin of isolated melanoma DCCs by comparative genomic hybridization. We demonstrate the feasibility of epitope-independent isolation of LN DCCs using Parsortix (TM) for subsequent molecular characterization of isolated single DCCs with ample application fields including the use for companion diagnostics or subsequent cellular studies in personalized medicine

Rapid NAD⁺ depletion in NBS patient fibroblasts after DNA damage.

<p>Relative levels of NAD⁺ in NBS-1LBI NBS patient fibroblasts (•) and LN9 control fibroblasts (▪) after DNA damage are shown. NAD⁺ levels in untreated cells were set at 100%.</p

High levels of ROS in <i>Nbn</i> null mutant murine fibroblasts and NBS patient cells after DNA damage.

<p>(A) FACS profiles of ROS measurements in murine cells with the indicated genotypes with or without treatment with bleomycin. Cells were stained with CM-H₂DCFDA 12 hours after treatment with bleomycin. Fluorescence intensity is proportional to ROS. The experiment was repeated six times and the same profiles were obtained. (B) Western-blot demonstration of conditional <i>Nbn</i> null mutation in murine fibroblasts. Lysates from <i>Nbn</i>^Ins-6/lox-6 fibroblasts with and without treatment with HTNC were probed on immunoblots with anti-nibrin and anti-actin antibodies. (C) Representative FACS profiles of ROS measurements in LN9 wild type and GM7166VA7 NBS patient fibroblasts with or without treatment with bleomycin. Cells were stained with CM-H₂DCFDA 12 hours after treatment with bleomycin. Fluorescence intensity is proportional to ROS. The experiment was repeated more than five times and essentially the same profiles were obtained.</p

Increased PARP activity in <i>Nbn^−/−</i> murine fibroblasts and NBS patient fibroblasts after DNA damage.

<p>Lysates from mouse (A) and human LN9 and GM166VA7 fibroblasts (B) with the given genotypes were harvested at the indicated timepoints (minutes) after a bleomycin treatment and probed on immunoblots with antibodies directed against poly(ADP-ribose) and ß-actin.</p