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Metabolic regulation by p53 prevents R-loop-associated genomic instability

Mar 2, 2023 | 2022, Cell Reports, Journal publications

Abstract

Gene-environment interactions can perturb the epigenome, triggering network alterations that participate in cancer pathogenesis. Integrating epigenomics, transcriptomics, and metabolic analyses with functional perturbation, we show that the tumor suppressor p53 preserves genomic integrity by empowering adequate levels of the universal methyl donor S-adenosylmethionine (SAM). In p53-deficient cells, perturbation of DNA methylation promotes derepression of heterochromatin, massive loss of histone H3-lysine 9 methylation, and consequent upregulation of satellite RNAs that triggers R-loop-associated replication stress and chromosomal aberrations. In p53-deficient cells, the inadequate SAM level underlies the inability to respond to perturbation because exogenous reintroduction of SAM represses satellite elements and restores the ability to cope with stress. Mechanistically, p53 transcriptionally controls genes involved in one-carbon metabolism, including Slc43a2, the methionine uptake transporter that is critical for SAM synthesis. Supported by clinical data, our findings shed light on the role of p53-mediated metabolism in preventing unscheduled R-loop-associated genomic instability.

Mapping the cellular response to electron transport chain inhibitors reveals selective signaling networks triggered by mitochondrial perturbation

Mar 2, 2023 | 2022, Archives of Toxicology, Journal publications

Abstract

Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes.

On the Usefulness of Animals as a Model System (Part II): Considering Benefits within Distinct Use Domains

Mar 2, 2023 | 2022, Altex, Journal publications

Abstract

In many countries, animal experiments can only be performed when their necessity has been demonstrated in a legal document. As the usefulness of animals in research is also a significant societal and political issue, criteria to structure debates and evaluations are needed. Here, background information is given on laboratory animal studies. Moreover, parameters that may be considered in judging their usefulness are suggested. The discussion is strictly focused on animals used as tools/test systems/models to provide information on humans. In this context, general features and performance characteristics of models are discussed. Examples are given for well-recognized criteria (e.g., robustness, relevance, predictivity) to judge the usefulness of predictive models. The main hypothesis put forward here is that a benefits evaluation (usefulness metrics) is only possible within sharply circumscribed “use domains”. Examples are given for the research fields of drug and vaccine research, toxicology, disease pathogenesis, and basic biological research. Efficacy, safety, and quality studies are highlighted as “use domains” within the field of drug discovery and production. A further separation into individual diseases, drug targets or symptoms is suggested for, e.g., efficacy studies or pathophysiology. Finally, an outlook is given on the evaluation of model advantages and disadvantages to arrive at their “net benefit”. Moreover, the need to compare the net benefits of animal models versus that of their alternatives is highlighted.

Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions

Mar 2, 2023 | 2022, Journal publications, Stem Cells

Abstract

In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the “fraction of reactive cells” in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Distinct and Dynamic Transcriptome Adaptations of iPSC-Generated Astrocytes after Cytokine Stimulation

Mar 2, 2023 | 2022, Cells, Journal publications

Abstract

Astrocytes (ACs) do not only play a role in normal neurogenesis and brain homeostasis, but also in inflammatory and neurodevelopmental disorders. We studied here the different patterns of inflammatory activation triggered by cytokines in human induced pluripotent stem cell (iPSC)-derived ACs. An optimized differentiation protocol provided non-inflamed ACs. These cells reacted to TNFα with a rapid translocation of NFκB, while AC precursors showed little response. Transcriptome changes were quantified at seven time points (2-72 h) after stimulation with TNFα, IFNγ or TNFα plus IFNγ. TNFα triggered a strong response within 2 h. It peaked from 12-24 h and reverted towards the ground state after 72 h. Activation by IFNγ was also rapid, but the response pattern differed from that of TNFα. For instance, several chemokines up-regulated by TNFα were not affected by IFNγ. Instead, MHC-II-related antigen presentation was drastically enhanced. The combination of the two cytokines led to a stronger and more persistent response. For instance, TRIB3 up-regulation by the combination of TNFα plus IFNγ may have slowed NFκB inactivation. Additionally, highly synergistic regulation was observed for inflammation modifiers, such as CASP4, and for STAT1-controlled genes. The combination of the cytokines also increased oxidative stress markers (e.g., CHAC1), led to phenotypic changes in ACs and triggered markers related to cell death. In summary, these data demonstrate that there is a large bandwidth of pro-inflammatory AC states, and that single markers are not suitable to describe AC activation or their modulation in disease, development and therapy.

Classification of Developmental Toxicants in a Human iPSC Transcriptomics-Based Test

Mar 2, 2023 | 2022, Chemical Research in Toxicology, Journal publications

Abstract

Despite the progress made in developmental toxicology, there is a great need for in vitro tests that identify developmental toxicants in relation to human oral doses and blood concentrations. In the present study, we established the hiPSC-based UKK2 in vitro test and analyzed genome-wide expression profiles of 23 known teratogens and 16 non-teratogens. Compounds were analyzed at the maximal plasma concentration (Cmax) and at 20-fold Cmax for a 24 h incubation period in three independent experiments. Based on the 1000 probe sets with the highest variance and including information on cytotoxicity, penalized logistic regression with leave-one-out cross-validation was used to classify the compounds as test-positive or test-negative, reaching an area under the curve (AUC), accuracy, sensitivity, and specificity of 0.96, 0.92, 0.96, and 0.88, respectively. Omitting the cytotoxicity information reduced the test performance to an AUC of 0.94, an accuracy of 0.79, and a sensitivity of 0.74. A second method, which used the number of significantly deregulated probe sets to classify the compounds, resulted in a specificity of 1; however, the AUC (0.90), accuracy (0.90), and sensitivity (0.83) were inferior compared to those of the logistic regression-based procedure. Finally, no increased performance was achieved when the high test concentrations (20-fold Cmax) were used, in comparison to testing within the realistic clinical range (1-fold Cmax). In conclusion, although further optimization is required, for example, by including additional readouts and cell systems that model different developmental processes, the UKK2-test in its present form can support the early discovery-phase detection of human developmental toxicants.