UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil–DNA glycosylase UNG is the major route for FU–DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil–DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU–DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd

Interlaboratory evaluation of a digital holographic microscopy–based assay for label-free in vitro cytotoxicity testing of polymeric nanocarriers

State-of-the-art in vitro test systems for nanomaterial toxicity assessment are based on dyes and several staining steps which can be affected by nanomaterial interference. Digital holographic microscopy (DHM), an interferometry-based variant of quantitative phase imaging (QPI), facilitates reliable proliferation quantification of native cell populations and the extraction of morphological features in a fast and label- and interference-free manner by biophysical parameters. DHM therefore has been identified as versatile tool for cytotoxicity testing in biomedical nanotechnology. In a comparative study performed at two collaborating laboratories, we investigated the interlaboratory variability and performance of DHM in nanomaterial toxicity testing, utilizing complementary standard operating procedures (SOPs). Two identical custom-built off-axis DHM systems, developed for usage in biomedical laboratories, equipped with stage-top incubation chambers were applied at different locations in Europe. Temporal dry mass development, 12-h dry mass increments and morphology changes of A549 human lung epithelial cell populations upon incubation with two variants of poly(alkyl cyanoacrylate) (PACA) nanoparticles were observed in comparison to digitonin and cell culture medium controls. Digitonin as cytotoxicity control, as well as empty and cabazitaxel-loaded PACA nanocarriers, similarly impacted 12-h dry mass development and increments as well as morphology of A549 cells at both participating laboratories. The obtained DHM data reflected the cytotoxic potential of the tested nanomaterials and are in agreement with corresponding literature on biophysical and chemical assays. Our results confirm DHM as label-free cytotoxicity assay for polymeric nanocarriers as well as the repeatability and reproducibility of the technology. In summary, the evaluated DHM assay could be efficiently implemented at different locations and facilitates interlaboratory in vitro toxicity testing of nanoparticles with prospects for application in regulatory science.publishedVersio

The PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions

Decitabine (5-aza-2′-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1–DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML

A children’s health perspective on nano- and microplastics

BACKGROUND : Pregnancy, infancy, and childhood are sensitive windows for environmental exposures. Yet the health effects of exposure to nano- and microplastics (NMPs) remain largely uninvestigated or unknown. Although plastic chemicals are a well-established research topic, the impacts of plastic particles are unexplored, especially with regard to early life exposures. OBJECTIVES : This commentary aims to summarize the knowns and unknowns around child- and pregnancy-relevant exposures to NMPs via inhalation, placental transfer, ingestion and breastmilk, and dermal absorption. METHODS : A comprehensive literature search to map the state of the science on NMPs found 37 primary research articles on the health relevance of NMPs during early life and revealed major knowledge gaps in the field. We discuss opportunities and challenges for quantifying child-specific exposures (e.g., NMPs in breastmilk or infant formula) and health effects, in light of global inequalities in baby bottle use, consumption of packaged foods, air pollution, hazardous plastic disposal, and regulatory safeguards. We also summarize research needs for linking child health and NMP exposures and address the unknowns in the context of public health action. DISCUSSION : Few studies have addressed child-specific sources of exposure, and exposure estimates currently rely on generic assumptions rather than empirical measurements. Furthermore, toxicological research on NMPs has not specifically focused on child health, yet children’s immature defense mechanisms make them particularly vulnerable. Apart from few studies investigating the placental transfer of NMPs, the physicochemical properties (e.g., polymer, size, shape, charge) driving the absorption, biodistribution, and elimination in early life have yet to be benchmarked. Accordingly, the evidence base regarding the potential health impacts of NMPs in early life remains sparse. Based on the evidence to date, we provide recommendations to fill research gaps, stimulate policymakers and industry to address the safety of NMPs, and point to opportunities for families to reduce early life exposures to plastic.The European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant.https://ehp.niehs.nih.govdm2022School of Health Systems and Public Health (SHSPH

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques. The characterization strategy and the analytical measurements described here will have an important role during formulation development and in determining robust quality attributes ultimately supporting the quality assessment of these innovative RNA therapeutics.publishedVersio

Small molecule inhibitor of OGG1 blocks oxidative DNA damage repair at telomeres and potentiates methotrexate anticancer effects

The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment

DNA excision repair of uracil and 5-fluorouracil in human cancercell lines

DNA er et tilsynelatende stabilt molekyl, som overføres så å si uten endringer fra unnfangelse til alderdom og fra generasjon til generasjon. Men arvestoffet vårt er ikke så uforanderlig som det kan se ut som. DNA kan endres kjemisk ved å reagere med en rekke stoffer som er påført utenfra eller som normalt finnes inne i enhver celle. DNA består av repeterende enheter av nukleotider, som igjen består av fosfat-, sukker- og basegrupper. Fosfat og sukkergruppene danner en ryggrad, mens basene parer med andre baser på en motstående DNA-tråd. Fokus for denne avhandlingen er baseskadene uracil og 5-fluorouracil. Uracil dannes ved at den normale basen cytosin reagerer med vann. Dette resulterer i et uracil:guanin base-par. Eventuelt kan både uracil og 5-fluorouracil inkorporeres i stedet for den normale basen thymin under DNA-replikasjon. Dette resulterer i uracil paret med adenin, mens 5-fluorouracil kan pare med adenin eller guanin. For å unngå at skader på DNA resulterer i mutasjoner finnes det flere mekanismer i cellen som erstatter og reparerer skadd DNA. En av de viktigste reparasjonsmekanismene er base eksisjonsreparasjon (BER). BER initieres ved at en DNA-glykosylase kutter en skadd eller unormal base fra DNA. Hos mennesker er det identifisert fire forskjellige glykosylaser som alle kan initiere reparasjon av uracil og 5-fluorouracil: UNG, SMUG1, TDG og MBD4. En APendonuklease kutter så sukker-fosfat ryggraden ved å kutte ved siden av den nå base-løse sukkergruppen, og et nytt nukleotid settes inn av en DNA polymerase. Restene av sukkergruppen kan så fjernes direkte av DNA-polymerasen, før ryggraden på DNA-tråden bindes sammen av en DNA ligase. I sum blir dermed en skadd base erstattet med en normal (ennukleotid BER). I enkelte tilfeller klarer imidlertid ikke polymerasen å fjerne det som er igjen av sukkergruppen, dette skjer gjerne når sukkergruppen har blitt redusert eller oksidert. Da vil polymerasen sette inn flere nukleotider, slik at den skadde sukkergruppen fortrenges. Dette skaper en spesiell struktur som gjenkjennes av en flap endonuklease, som kutter ut den fortrengte biten, før en DNA ligase knytter DNA-tråden sammen igjen. Dermed fører reparasjon av en skadd base til at flere nukleotider erstattes (fler-nukleotid BER). Inntil nylig har ikke fler-nukleotid BER vært observert i cellenes mitokondrier, som har sitt eget DNA å ta vare på. Hvordan ville i så fall mitokondriene håndtere skader som i cellekjernen repareres av fler-nukleotid BER? Dette har vi undersøkt i det første arbeidet, hvor vi fant at også mitokondriene kunne utføre fler-nukleotid BER. Uttrykket av glykosylasen UNG varierer mellom forskjellige mennesker, organer og cellelinjer. I det andre arbeidet viser vi at hastigheten til BER sporet som helhet kontrolleres på det første trinnet, det vil si av mengde og aktivitet av DNA-glykosylasen som initierer reparasjonen. UNG initerte all observerbar reparasjon av uracil paret med adenin, mens reapasjon av uracil paret med guanin ble initiert hovedsakelig av UNG, med et relativt stort bidrag fra TDG i en av cellelinjene. I det tredje arbeidet har vi studert hvordan 5-fluorouracil repareres i DNA og hvilken betydning DNA-reparasjon har å si for virkningsmekanismen for 5-fluoruracil. Vi fant at BER, initiert av UNG2, står for det aller meste av reparasjonen når 5-fluorouracil er paret med adenin. Når 5-fluorouracil er paret med guanin utfører BER, initert av UNG2, SMUG1 eller TDG det meste av reparasjonen, mens mismatch-reparasjon ser ut til å være av mindre betydning. Nedregulering av de nevnte glykosylasene og hemming av BER-sporet påvirket imidlertid ikke kreftcellelinjers følsomhet for 5-fluorouracil. Dermed later det til at i dette tilfellet spiller ikke inkorporering i DNA og påfølgende DNA-reparasjon noen stor rolle for celledød. I stedet ser det ut som om 5-fluorouracil heller dreper celler via inkorporering i RNA, samt ved at dannelsen av thymidin-nukleotider hemmes

Systematic review: predictive value of organoids in colorectal cancer

Abstract While chemotherapy alone or in combination with radiotherapy and surgery are important modalities in the treatment of colorectal cancer, their widespread use is not paired with an abundance of diagnostic tools to match individual patients with the most effective standard-of-care chemo- or radiotherapy regimens. Patient-derived organoids are tumour-derived structures that have been shown to retain certain aspects of the tissue of origin. We present here a systematic review of studies that have tested the performance of patient derived organoids to predict the effect of anti-cancer therapies in colorectal cancer, for chemotherapies, targeted drugs, and radiation therapy, and we found overall a positive predictive value of 68% and a negative predictive value of 78% for organoid informed treatment, which outperforms response rates observed with empirically guided treatment selection

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.We sincerely thank the patients’ family for participating in this study. This work was supported by: The Spanish Ministry of Health—Fondo de Investigación Sanitaria (FIS) (Ref: PI15/00707); the Diputación General de Aragón (Grupo Consolidado B20), European Social Fund (“Construyendo Europa desde Aragón”) CHROMATIN-Net funded by the German Federal Ministry of Education and Research (BMBF) to Frank J. Kaiser. The Spanish Ministry of Economy (Refs: IPT2011-0964-900000 and SAF2011-13156-E) to Paulino Gómez-Puertas. Swedish Research Council to Lena Ström and the Norwegian Research Council (205217) to Torkild Visnes Beatriz Puisac, María Hernández-Marcos, María-Esperanza Teresa-Rodrigo, María-Concepción Gil-Rodríguez, Feliciano J. Ramos and Juan Pié are members of CIBERER-GCV02 and ISS-Aragon at the School of Medicine, University of Zaragoza and the Hospital Clínico Universitario “Lozano Blesa”.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)