Cell Biology and Toxicology

Off-target challenge for base editor-mediated genome editing

Off-target genome editing: a new discipline of gene science and a new of class medicine Abstract With an increasing growth of genome editing, off-target effects such as non-specific genetic modifications resulting from the designed process of genome editing become a new discipline of gene science and new class medicine. The degree of short-term and long-term side effects and toxicity or dynamics of the primary and secondary off-target genome editing varies with the application of different methodologies of gene editing and measuring, readouts of genetic modifications, or comparison reference. Measurements of dynamic off-target effects caused directly or indirectly by genome editing are critical in clinical application of gene editing. The quality of genome editing methods is one of the decisive factors in the occurrence of off-target effects. Mechanisms by which off-target effects of genome editing occurs are more complex and comprehensive than we expected. The heterogeneity of off-target effects of gene-edited cells at single-cell levels should be defined during the development and formation of cell clusters. In addition to off-target effects on gene-edited cells per se, alterations of gene sequence, structure, dimension, and function of related regulators caused by off-target effects may also influence intercellular communications and interactions between gene-edited cells, between gene-edited cells and non-edited cells, or between non-edited cells. Thus, controlling, measuring, defining, categorizing, and predicting off-target genome editing need to be standardized and prioritized before clinical application of gene editing.

Resveratrol inhibits the proliferation of estrogen receptor-positive breast cancer cells by suppressing EZH2 through the modulation of ERK1/2 signaling Abstract Enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in breast cancer and plays an important role in maintaining the cell proliferative capacity. However, the mechanisms underlying the transcriptional regulation of EZH2 in estrogen receptor (ER)-positive breast cancer cells remain unclear. The antitumor effects of resveratrol have been reported. However, whether EZH2 was involved in these effects needs further exploration. Here, we showed that EZH2 is required for estrogen-induced cell proliferation in ER-positive breast cancer. Exposure to 17β-estradiol (E2) upregulated EZH2 via ERα signaling, and this effect was blocked by U0126, a MEK inhibiter. Resveratrol inhibited the proliferation and colony formation in ER-positive breast cancer cells and downregulated EZH2 through inhibition of phospho-ERK1/2. These findings indicated that ERK1/2 and ER signaling–mediated EZH2 upregulation is crucial for the proliferation of ER-positive breast cancer cells. The suppression of EZH2 expression by ERK1/2 dephosphorylation is important for the antiproliferative activities of resveratrol against ER-positive breast cancer cells.

Olive leaf extract counteracts epithelial to mesenchymal transition process induced by peritoneal dialysis, through the inhibition of TGFβ1 signaling Abstract The mesothelial cells (MCs) play an important role in the morpho-functional alterations of the peritoneal membrane (PM) undergoing peritoneal dialysis (PD). MCs, through the epithelial-mesenchymal transition process (EMT), progressively acquire a myofibroblast-like phenotype, promoting peritoneal fibrosis (PF) and failure of peritoneal membrane function. Transforming growth factor β1 (TGFβ1), through canonical and non-canonical pathways, promotes the epithelial-mesenchymal transition (EMT) process leading to PF. To investigate the therapeutic potential of an olive leaf extract (OLE) on preserving peritoneal membrane function, we evaluated the effect of OLE on the TGFβ1-induced EMT in mesothelial cells, Met5A, and elucidated the underlying molecular mechanisms. As assessed by changes in the expression of epithelial, mesenchymal, and fibrotic cell markers (such as E-cadherin, N-cadherin, α-SMA, fibronectin, vimentin), levels of matrix metalloproteinases (MMP2 and MMP9), and cell migration, OLE inhibited the TGFβ1-induced EMT. Importantly, the beneficial effect of OLE was mediated by reduction of the TGFβ1-induced activation of Smad2/3 signaling and the mitigation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Smad/non-Smad signaling pathways, activated by TGFβ1, both reduce expression of epithelial marker E-cadherin which has a crucial role in EMT initiation. Interestingly, we observed that in presence of OLE activity of the E-cadherin, promoter was increased and concomitantly OLE reduced the nuclear content of its co-repressor SNAIL. Our results suggest the potential therapeutic of OLE to counteract fibrotic process in peritoneal dialysis patients.

The mechanistic role of oxidative stress in cigarette smoke-induced cardiac stem cell dysfunction and prevention by ascorbic acid Abstract Cigarette smoking causes a vast array of diseases including cardiovascular diseases. Our laboratory focuses on investigating cigarette smoke (CS)-induced cardiovascular malfunction and the responsible mechanisms utilizing the model, c-kit-positive cardiac stem cells (CSCs). The main objective of our study is to investigate whether CS extracts (CSEs) cause impairment of CSC functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of CSC proteins and antioxidant enzymes, can modulate CSC functions and these modifications can be attenuated by ascorbate treatment. Our specific aims are (1) to investigate CSE-induced oxidative modification of CSC proteins via carbonylation, and prevention by ascorbic acid; (2) to investigate CSE-induced oxidative modification of antioxidant enzymes and ascorbic acid-mediated modulations; and (3) to investigate CSE-induced changes in CSC functions and protection by ascorbic acid. CSCs were cultured, and the aqueous extracts of CSE were prepared. CSE-induced modulations of CSC viability, oxidative modification of proteins, and antioxidant enzyme activities were detected using standard assays including Apostain, bromodeoxyuridine, and Oxiblot. CSE caused oxidative modification of CSC proteins, changed antioxidant enzyme levels, attenuated CSC proliferation, and accelerated CSC apoptosis. Ascorbic acid prevented CSE-induced CSC malfunctions, and ascorbic acid therapy might be useful in smoker CSC recipients and to condition CSCs prior to the transplant in the future. Cardiac stem cell therapy is currently undergoing in clinical trials.

Identification of cancer-type specific expression patterns for active aldehyde dehydrogenase (ALDH) isoforms in ALDEFLUOR assay Abstract Aldehyde dehydrogenases (ALDHs) defend intracellular homeostasis by catalyzing the conversion of toxic aldehydes into non-toxic carboxylic acids, which is of particular importance to the self-renewal of stem cells and cancer stem cells. The widely used ALDEFLUOR assay was initially designed to indicate the activity of ALDH1A1 in leukemia and has been demonstrated to detect the enzyme activity of several other ALDH isoforms in various cancer types in recent years. However, it is still elusive which isoforms, among the 19 ALDH isoforms in human genome, are the potential contributors in catalyzing ALDEFLUOR assay in different cancers. In the current study, we performed a screening via overexpressing each ALDH isoform to assess their ability of catalyzing ALDEFLUOR assay. Our results demonstrate that nine isoforms are active in ALDEFLUOR assay, whose overexpression significantly increases ALDH-positive (ALDH+) population. Further analysis of the expression of these active isoforms in various cancers reveals cancer-type specific expression patterns, suggesting that different cancer types may exhibit ALDEFLUOR activity through expression of specific active ALDH isoforms. This study strongly indicates that a detailed elucidation of the functions for each active ALDH isoform in CSCs is necessary and important for a profound understanding of the underlying mechanisms of ALDH-associated stemness.

Spermine protects alpha-synuclein expressing dopaminergic neurons from manganese-induced degeneration Abstract Manganese exposure is among the many environmental risk factors linked to the progression of neurodegenerative diseases, such as manganese-induced parkinsonism. In animal models, chronic exposure to manganese causes loss of cell viability, neurodegeneration, and functional deficits. Polyamines, such as spermine, have been shown to rescue animals from age-induced neurodegeneration in an autophagy-dependent manner; nonetheless, it is not understood whether polyamines can prevent manganese-induced toxicity. In this study, we used two model systems, the Caenorhabditis elegans UA44 strain and SK-MEL-28 cells, both expressing the protein alpha-synuclein (α-syn) to determine whether spermine could ameliorate manganese-induced toxicity. Manganese caused a substantial reduction in the viability of SK-MEL-28 cells and hastened neurodegeneration in the UA44 strain. Spermine protected both the SK-MEL-28 cells and the UA44 strain from manganese-induced toxicity. Spermine also reduced the age-associated neurodegeneration observed in the UA44 strain compared with a control strain without α-syn expression and led to improved avoidance behavior in a functional assay. Treatment with berenil, an inhibitor of polyamine catabolism, which leads to increased intracellular polyamine levels, also showed similar cellular protection against manganese toxicity. While both translation blocker cycloheximide and autophagy blocker chloroquine caused a reduction in the cytoprotective effect of spermine, transcription blocker actinomycin D had no effect. This study provides new insights on the effect of spermine in preventing manganese-induced toxicity, which is most likely via translational regulation of several candidate genes, including those of autophagy. Thus, our results indicate that polyamines positively influence neuronal health, even when exposed to high levels of manganese and α-syn, and supplementing polyamines through diet might delay the onset of diseases involving degeneration of dopaminergic neurons.

Cell–cell communication: old mystery and new opportunity

Biochemical effects of some CeO 2 , SiO 2 , and TiO 2 nanomaterials in HepG2 cells Abstract The potential mammalian hepatotoxicity of nanomaterials was explored in dose-response and structure-activity studies in human hepatic HepG2 cells exposed to between 10 and 1000 μg/ml of five different CeO2, three SiO2, and one TiO2-based particles for 3 days. Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. Few indications of cytotoxicity were observed between 10 and 30 μg/ml. In the 100 to 300 μg/ml exposure range, a moderate degree of cytotoxicity was often observed. At 1000 μg/ml exposures, all but TiO2 showed a high degree of cytotoxicity. Cytotoxicity per se did not seem to fully explain the observed patterns of biochemical parameters. Four nanomaterials (all three SiO2) decreased glucose 6-phosphate dehydrogenase activity with some significant decreases observed at 30 μg/ml. In the range of 100 to 1000 μg/ml, the activities of glutathione reductase (by all three SiO2) and glutathione peroxidase were decreased by some nanomaterials. Decreased glutathione concentration was also found after exposure to four nanomaterials (all three nano SiO2 particles). In this study, the more responsive and informative assays were glucose 6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, lactate dehydrogenase, and aspartate transaminase. In this study, there were six factors that contribute to oxidative stress observed in nanomaterials exposed to hepatocytes (decreased glutathione content, reduced glucose 6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and increased catalase activities). With respect to structure-activity, nanomaterials of SiO2 were more effective than CeO2in reducing glutathione content, glucose 6-phosphate dehydrogenase, glutathione reductase, and superoxide dismutase activities.

Definition of clinical gene tests Abstract Clinical tests of gene sequence, structure, and function are to predict, diagnose, monitor, and prognose human disease–specific phenomes, characters severities, durations, stages, and responses to therapy. The concept and content of gene tests for clinical application mainly include chromosome/chromatins, DNA, and RNA. Structures and functions of chromosomes and chromatins vary among various durations, phases, and conditions, with the priority consideration in clinical gene tests. Sequences and functions of DNA and associated regulators are an important partial of clinical gene test. Another large group of RNA and RNA-associated factors also contribute to gene expression, regulation, and function. DNA/RNA sequencing is used to measure tumor mutation and heterogeneity, recategorize molecular phenomes and types of cancer, or guide and predict target-based therapies. The structure and function of genome dimensions and regulations as well as various factor involvement and contributions should be seriously considered in clinical gene tests, although there are a number of challenges to be overcome, e.g., method sensitivity, specificity, stability, analysis, and clinical significance. It is also critical to have the national and international standardization, guideline, and consortium of sample handling, experimental operation, quality control, data analysis, and clinical interpretation, when clinical gene tests are developed and applied for clinical application. Thus, there is an urgent need to discover and validate those gene tests according to disease phenomes, subtypes, severity, duration, phase, progression, prognosis, and response to therapy.