Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy

Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents

Superhydrophobic Paper from Nanostructured Fluorinated Cellulose Esters

The development of economically and ecologically viable strategies for sup erhydrophobization offers a vast variety of interesting applications in self-cleaning surfaces. Examples include packaging materials, textiles, outdoor clothing, and microfluidic devices. In this work, we produced superhydrophobic paper by spin-coating a dispersion of nanostructured fluorinated cellulose esters. Modification of cellulose nanocrystals was accomplished using 2H,2H,3H,3H-perfluorononanoyl chloride and 2H,2H,3H,3H-perfluoroundecanoyl chloride, which are well-known for their ability to reduce surface energy. A stable dispersion of nanospherical fluorinated cellulose ester was obtained by using the nanoprecipitation technique. The hydrophobized fluorinated cellulose esters were characterized by both solid- and liquid-state nuclear magnetic resonance, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and contact angle measurements. Further, we investigated the size, shape, and structure morphology of nanostructured fluorinated cellulose esters by dynamic light scattering, scanning electron microscopy, and X-ray diffraction measurements.Peer reviewe

A Click Chemistry-Based Artificial Metallo-Nuclease

Artificial metallo-nucleases (AMNs) are promising DNA damaging drug candidates. Here, we demonstrate how the 1,2,3-triazole linker produced by the Cu-catalysed azide-alkyne cycloaddition (CuAAC) reaction can be directed to build Cu-binding AMN scaffolds. We selected biologically inert reaction partners tris(azidomethyl)mesitylene and ethynyl-thiophene to develop TC-Thio, a bioactive C3-symmetric ligand in which three thiophene-triazole moieties are positioned around a central mesitylene core. The ligand was characterised by X-ray crystallography and forms multinuclear CuII and CuI complexes identified by mass spectrometry and rationalised by density functional theory (DFT). Upon Cu coordination, CuII-TC-Thio becomes a potent DNA binding and cleaving agent. Mechanistic studies reveal DNA recognition occurs exclusively at the minor groove with subsequent oxidative damage promoted through a superoxide- and peroxide-dependent pathway. Single molecule imaging of DNA isolated from peripheral blood mononuclear cells shows that the complex has comparable activity to the clinical drug temozolomide, causing DNA damage that is recognised by a combination of base excision repair (BER) enzymes

Fusion transcript analysis reveals slower response kinetics than multiparameter flow cytometry in childhood acute myeloid leukaemia

Funding Information: We thank the employees at the Department of Clinical Chemistry at Sahlgrenska University Hospital, Haemodiagnostic Laboratory at the Aarhus University Hospital, and Department of Clinical Immunology, Copenhagen University Hospital Rigshospitalet for sample collection, processing and analyses. Publisher Copyright: © 2022 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.Introduction: Analysis of measurable residual disease (MRD) is increasingly being implemented in the clinical care of children and adults with acute myeloid leukaemia (AML). However, MRD methodologies differ and discordances in results lead to difficulties in interpretation and clinical decision-making. The aim of this study was to compare results from reverse transcription quantitative polymerase chain reaction (RT-qPCR) and multiparameter flow cytometry (MFC) in childhood AML and describe the kinetics of residual leukaemic burden during induction treatment. Methods: In 15 children who were treated in the NOPHO-AML 2004 trial and had fusion transcripts quantified by RT-qPCR, we compared MFC with RT-qPCR for analysis of MRD during (day 15) and after induction therapy. Eight children had RUNX1::RUNX1T1, one CBFB::MYH11 and six KMT2A::MLLT3. Results: When ≥0.1% was used as cut-off for positivity, 10 of 22 samples were discordant. The majority (9/10) were MRD positive with RT-qPCR but MRD negative with MFC, and several such cases showed the presence of mature myeloid cells. Fusion transcript expression was verified in mature cells as well as in CD34 expressing cells sorted from diagnostic samples. Conclusions: Measurement with RT-qPCR suggests slower response kinetics than indicated from MFC, presumably due to the presence of mature cells expressing fusion transcript. The prognostic impact of early measurements with RT-qPCR remains to be determined.Peer reviewe

Regulation of the Epstein-Barr Virus Latent Membrane Protein 1 Expression

Epstein-Barr virus (EBV) is a probably the most effective and successful human virus, infecting more than 90% of the world’s adult population. As with the other members of the herpesvirus family, EBV establishes latent infection in its host and persists life-long. EBV infection is generally harmless in children but can cause infectious mononucleosis (IM) in young adults. EBV is associated with a number of human malignancies including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), nasopharyngeal carcinoma (NPC), nasal T/NK lymphoma (NL), peripheral T cell lymphoma, gastric carcinoma, and lymphoproliferative diseases in immunocompromised patients. A compromised immune system and an aberrant EBV latent gene expression are thought to be important players in the aetiology of EBV malignancies. EBV is one of the most potent transforming agents in vitro and immortalizes B cells into lymphoblastoid cell lines (LCLs). Latent membrane protein 1 (LMP1) is the main EBV oncogene, which is critically involved in immortalisation and proliferation of LCLs, and is associated with most EBV malignancies. LMP1 functions as a constitutively active tumour necrosis factor receptor (TNFR) and upregulates anti-apoptotic and pro-survival proteins through the activation of cellular signalling pathways. Thus, inappropriate expression of LMP1 is probably a central process in EBV associated tumourigenesis. The aim of this PhD project was to delineate the regulation of LMP1 gene expression in response to cellular factors. The LMP1 protein expression is regulated differently according to the expression pattern of the other EBV latent proteins as well as the cell type in which it is expressed in. In latency III infected B cells all of the EBV latent proteins are expressed, and LMP1 expression is driven by the viral transcription factor EBNA2. The EBNA2 protein lacks DNA binding ability itself, and requires cellular factors (adaptors) to be recruited to promoters. In latency II cells that represent most EBV tumours and different cell-type hosts, a more limited set of EBV latent proteins are expressed, and LMP1 expression occurs in the absence of EBNA2. Regardless of the mode of expression and cell type, LMP1 transactivation is critically dependent on cellular proteins. In the course of this investigation, a new EBNA2 adaptor was identified that bound an AP-2 site in the LMP1 promoter and mediated the relief of promoter repression and activation of the LMP1 promoter. We also report EBNA2-independent upregulation of the LMP1 promoter in response to upregulation of the p38 kinase pathway. The p38 signalling pathway activates the ATF1-CREB heterodimer that has been previously shown as an activator of LMP1 transcription. The binding of ATF1-CREB to a CRE site is a central event in LMP1 regulation both in the presence and absence of EBNA2. Additionally, we showed the presence of a mutation in the LMP1-CRE site of the P3HR1 EBV variant. This mutation led to a reduced binding efficiency of ATF1-CREB to the CRE site and a two fold reduction of LMP1 promoter activity. This finding together with reports from other groups indicate that sequence variations in the CRE site of LMP1 are evolutionary, selected probably to modulate the expression levels of the protein. Our results also indicate that the NF-κB dimers, p50-p65 and p50-p50, bind an NF¬-κB site in the LMP1 promoter and activate transcription independently of EBNA2. The EBNA2 independent activation of LMP1 transcription by NF-κB suggests that this signalling pathway may play a role in LMP1 activation in latency II infected B cells. Since the NF-κB pathway is activated by LMP1, a positive autoregulatory loop in LMP1 activation may exist. The positive autoregulation of LMP1 is supported by reports from other groups. Finally, we showed that histone acetylation and modulation of the chromatin structure of the LMP1 promoter are involved in the activation of LMP1 transcription. We hypothesise a model whereby the EBNA2 is recruited through interaction with several EBNA2 adaptors at the promoter and mediates activation. Alternatively, several transcriptional activators such as NF-κB factors and ATF1-CREB bind the promoter in the absence EBNA2 and cooperatively activate the promoter. In both cases factor-binding to the promoter leads to the recruitment of histone acetylases and chromatin remodelling enzymes to the LMP1 promoter to facilitate transcription

The p38 Signaling Pathway Upregulates Expression of the Epstein-Barr Virus LMP1 Oncogene ▿

The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation

Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression▿

The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression

Shining light on single-strand lesions caused by the chemotherapy drug bleomycin

Quantification of the DNA damage induced by chemotherapy in patient cells may aid in personalization of the dose used. However, assays to evaluate individual patient response to chemotherapy are not available today. Here, we present an assay that quantifies single-stranded lesions caused by the chemotherapeutic drug Bleomycin (BLM) in peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals. We use base excision repair (BER) enzymes to process the DNA damage induced by BLM and then extend the processed sites with fluorescent nucleotides using a DNA polymerase. The fluorescent patches are quantified on single DNA molecules using fluorescence microscopy. Using the assay, we observe a significant variation in the in vitro induced BLM damage and its repair for different individuals. Treatment of the cells with the BER inhibitor CRT0044876 leads to a lower level of repair of BLM-induced damage, indicating the ability of the assay to detect a compromised DNA repair in patients. Overall, the data suggest that our assay could be used to sensitively detect the variation in BLM-induced DNA damage and repair in patients and can potentially be able to aid in personalizing patient doses