The major contribution of the DNA damage-triggered reactive oxygen species production to cell death: implications for antimicrobial and cancer therapy.

Genotoxic agents damage DNA, block DNA replication and provoke cell death. However, there is growing evidence that an important part of their cytotoxicity results from metabolic disturbances induced by treatment. This review article describes how increased production of the reactive oxygen species (ROS) induced by different genotoxic agents contribute to death of prokaryotic and eukaryotic cells. ROS are byproducts of normal cellular functioning. Because ROS are damaging cellular macromolecules, they are constantly eliminated by protective antioxidant mechanisms. However, even a small increase in ROS production may have deleterious consequences because cells possess just enough defensive mechanisms to protect themselves against endogenously produced ROS. Therefore, it may be possible to enhance cytotoxic potential of antimicrobial and anticancer drugs by increasing ROS production or by inhibiting cellular antioxidant systems.

Significance of Wild-Type p53 Signaling in Suppressing Apoptosis in Response to Chemical Genotoxic Agents: Impact on Chemotherapy Outcome.

Our genomes are subject to potentially deleterious alterations resulting from endogenous sources (e.g., cellular metabolism, routine errors in DNA replication and recombination), exogenous sources (e.g., radiation, chemical agents), and medical diagnostic and treatment applications. Genome integrity and cellular homeostasis are maintained through an intricate network of pathways that serve to recognize the DNA damage, activate cell cycle checkpoints and facilitate DNA repair, or eliminate highly injured cells from the proliferating population. The wild-type p53 tumor suppressor and its downstream effector p21WAF1 (p21) are key regulators of these responses. Although extensively studied for its ability to control cell cycle progression, p21 has emerged as a multifunctional protein capable of downregulating p53, suppressing apoptosis, and orchestrating prolonged growth arrest through stress-induced premature senescence. Studies with solid tumors and solid tumor-derived cell lines have revealed that such growth-arrested cancer cells remain viable, secrete growth-promoting factors, and can give rise to progeny with stem-cell-like properties. This article provides an overview of the mechanisms by which p53 signaling suppresses apoptosis following genotoxic stress, facilitating repair of genomic injury under physiological conditions but having the potential to promote tumor regrowth in response to cancer chemotherapy.

Bone marrow transplantation in Schimke immuno-osseous dysplasia.

Schimke immuno-osseous dysplasia (SIOD, OMIM 242900) is a rare autosomal recessive multisystem childhood disorder characterized by short stature, renal failure, T-cell immunodeficiency, and hypersensitivity to genotoxic agents. SIOD is associated with biallelic mutations in SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response enzyme with annealing helicase activity. Two features of SIOD causing much morbidity and mortality are bone marrow failure and T-cell deficiency with the consequent opportunistic infections. To address the safety and efficacy of bone marrow transplantation (BMT) in SIOD, we reviewed the outcomes of the only five SIOD patients known to us in whom bone marrow or hematopoietic stem cell transplantation has been attempted. We find that only one patient survived the transplantation procedure and that the existing indicators of a good prognosis for bone marrow transplantation were not predictive in this small cohort. Given these observations, we also discuss some considerations for the poor outcomes.

The oncoprotein BCL6 enables solid tumor cells to evade genotoxic stress.

Genotoxic agents remain the mainstay of cancer treatment. Unfortunately, the clinical benefits are often countered by a rapid tumor adaptive response. Here, we report that the oncoprotein B cell lymphoma 6 (BCL6) is a core component that confers solid tumor adaptive resistance to genotoxic stress. Multiple genotoxic agents promoted BCL6 transactivation, which was positively correlated with a weakened therapeutic efficacy and a worse clinical outcome. Mechanistically, we discovered that treatment with the genotoxic agent etoposide led to the transcriptional reprogramming of multiple pro-inflammatory cytokines, among which the interferon-α and interferon-γ responses were substantially enriched in resistant cells. Our results further revealed that the activation of interferon/signal transducer and activator of transcription 1 axis directly upregulated BCL6 expression. The increased expression of BCL6 further repressed the tumor suppressor PTEN and consequently enabled resistant cancer cell survival. Accordingly, targeted inhibition of BCL6 remarkably enhanced etoposide-triggered DNA damage and apoptosis both in vitro and in vivo. Our findings highlight the importance of BCL6 signaling in conquering solid tumor tolerance to genotoxic stress, further establishing a rationale for a combined approach with genotoxic agents and BCL6-targeted therapy.

Chromosomal Aberrations in Cattle.

Chromosomal aberrations and their mechanisms have been studied for many years in livestock. In cattle, chromosomal abnormalities are often associated with serious reproduction-related problems, such as infertility of carriers and early mortality of embryos. In the present work, we review the mechanisms and consequences of the most important bovine chromosomal aberrations: Robertsonian translocations and reciprocal translocations. We also discuss the application of bovine cell cultures in genotoxicity studies.

Measurement of DNA Damage Using the Neutral Comet Assay in Cultured Cells.

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair for disease pathogenesis, measuring them is of considerable interest, and the comet assay is a widely used method for this. Cells treated with DNA damaging agents are embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates towards the anode depending on its degree of fragmentation, creating shapes resembling comets, which can be visualized and analysed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, and both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the neutral comet assay to assess double-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by ionizing radiation or present endogenously in the cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and cost-effective.

Measuring DNA Damage Using the Alkaline Comet Assay in Cultured Cells.

Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA-damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair in disease, measuring the amount of both aspects is of considerable interest. The comet assay is a widely used method that allows the measurement of both DNA damage and its repair in cells. For this, cells are treated with DNA-damaging agents and embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave so-called 'nucleoids' consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates toward the anode depending on its degree of fragmentation and creates shapes resembling comets, which can be subsequently visualized and analyzed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, in addition to both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the alkaline comet assay to assess single-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by agents such as hydrogen peroxide (H2O2) and methyl-methanesulfonate (MMS) or present endogenously in cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and allows the cost-effective assessment of single-strand breaks and their repair kinetics in cultured cells.

Development of a co-culture model of mouse primary hepatocytes and splenocytes to evaluate xenobiotic genotoxicity using the medium-throughput Comet assay.

To date, only a limited number of toxicological studies have focused on the establishment and validation of in vitro genotoxicity screening systems using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop an effective co-culture model of mouse-derived primary hepatocytes and splenocytes for screening chemicals for genotoxicity using the medium-throughput Comet assay. This cocultured model was constructed and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase methods. DNA damage was detected using both alkaline and formamidopyrimidine DNA glycosylase (FPG) Comet assays. Compared with the controls, DNA strand breaks and FPG-sensitive sites showed significant concentration-dependent increases in genotoxic-agent-treated groups. In contrast, DNA damage remained unchanged in non-genotoxic-agent-treated groups. In addition, different types of genotoxic agents resulted in different time-dependent DNA lesions. Our results indicated that the % tail DNA indicating both DNA strand breaks and FPG-sensitive sites might be effective markers for predicting chemical-induced DNA damage and oxidative DNA damage using the cocultured model of hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.

Comparative evaluation of micronuclei in exfoliated oral epithelial cells in potentially malignant disorders and malignant lesions using special stains.

INTRODUCTION: Micronuclei (MNs) are extranuclear cytoplasmic DNA bodies which are induced in cells by numerous genotoxic agents that damage chromosome. The MN assay in exfoliated buccal cells is a useful and minimally invasive method for monitoring genetic damage. AIM: The aim of present study was to detect and assess MNs in oral exfoliated cells in patients diagnosed with leukoplakia with dysplasia, oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC) using special stains and to determine the most appropriate staining technique for the evaluation of MNs along with a comparative evaluation of MNs with histological grading. MATERIALS AND METHODS: The study was conducted in the Department of Oral Pathology and Microbiology, CDCRI, Rajnandgaon, and a total of 45 subjects were included in the study who were subsequently divided into three groups (15 each). Four smears were obtained from each subject which were taken from the lesional tissue and stained simultaneously. ANALYSIS: The results were analyzed via Statistical Package for the Social Sciences, version 23.0 (SPSS). RESULTS: The results confirmed the association of MNs with genotoxic agents and showed an elevated number in OSCC followed by OSMF and leukoplakia. The frequency also increased with the severity of the lesion. Besides this, Papanicolaou (PAP) stain was found to be the most suitable stain for detection of MNs. CONCLUSION: Based on the above pretext, we can conclude that PAP stain was the most suitable stain for valuation of MNs and that the MN assay holds promise as a specific biomarker of genotoxicity, for screening of oral cancer and can be used as a prognostic indicator.

Identification of pharmacodynamic biomarkers and common molecular mechanisms of response to genotoxic agents in cancer cell lines.

PURPOSE: Genotoxic agents (GAs) including cisplatin, doxorubicin, gemcitabine, and topotecan are often used in cancer treatment. However, the response to GAs is variable among patients and predictive biomarkers are inadequate to select patients for treatment. Accurate and rapid pharmacodynamics measures of response can, thus, be useful for monitoring therapy and improve clinical outcomes. METHODS: This study focuses on integrating a database of genome-wide response to treatment (The NCI Transcriptional Pharmacodynamics Workbench) with a database of baseline gene expression (GSE32474) for the NCI-60 cell lines to identify mechanisms of response and pharmacodynamic (PD) biomarkers. RESULTS AND CONCLUSIONS: Our analysis suggests that GA-induced endoplasmic reticulum (ER) stress may signal for GA-induced cell death. Reducing the uptake of GA, activating DNA repair, and blocking ER-stress induction cooperate to prevent GA-induced cell death in the GA-resistant cells. ATF3, DDIT3, CARS, and PPP1R15A appear as possible candidate PD biomarkers for monitoring the progress of GA treatment. Further validation studies on the proposed intrinsic drug-resistant mechanism and candidate genes are needed using in vivo data from either patient-derived xenograft models or clinical chemotherapy trials.

Treatment May Be Harmful: Mechanisms/Prediction/Prevention of Drug-Induced DNA Damage and Repair in Multiple Myeloma.

Multiple myeloma (MM) is a malignancy characterized by accumulation of malignant plasma cells within the bone marrow (BM). MM is considered mostly without definitive treatment because of the inability of standard of care therapies to overcome drug-resistant relapse. Genotoxic agents are used in the treatment of MM and exploit the fact that DNA double-strand breaks are highly cytotoxic for cancer cells. However, their mutagenic effects are well-established and described. According to these effects, chemotherapy could cause harmful DNA damage associated with new driver genomic abnormalities providing selective advantage, drug resistance, and higher relapse risk. Several mechanisms associated with MM cell (MMC) resistance to genotoxic agents have been described, underlining MM heterogeneity. The understanding of these mechanisms provides several therapeutic strategies to overcome drug resistance and limit mutagenic effects of treatment in MM. According to this heterogeneity, adopting precision medicine into clinical practice, with the development of biomarkers, has the potential to improve MM disease management and treatment.

Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage.

BACKGROUND: Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. METHODS: Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coli and DR. shp knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. RESULTS: DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm+ genomic DNA, and to cytosine unmethylated DR and E. coli dcm- genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced >100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors. CONCLUSIONS: The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage. GENERAL SIGNIFICANCE: Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.

Recent advances in understanding/assessing toxicity to the epigenome.

The ability of non-genotoxic agents to induce cancer has been documented and clearly requires a reassessment of testing for environmental and human safety. Drug safety testing has historically relied on test batteries designed to detect DNA damage leading to mutation and cancer. The standard genetic toxicology testing battery has been a reliable tool set to identify small molecules/chemicals as hazards that could lead to genetic changes in organisms and induction of cancer. While pharmaceutical companies and regulatory agencies have extensively used the standard battery, it is not suitable for compounds that may induce epigenetic changes. Additionally, many pharmaceutical companies have changed their product portfolios to include peptides and/or other biological molecules, which are not expected to be genotoxic in their own right. If we are to best use our growing knowledge regarding chemicals and biomolecules that induce heritable changes via epigenetic mechanisms, then we must ask what changes may be needed in our testing paradigm to predict long-term downstream effects through epigenetic mechanisms.

Functional Analysis of Arabidopsis ARGONAUTEs in Meiosis and DNA Repair.

Plant ARGONAUTE (AGO) proteins regulate a wide range of cellular and developmental functions. Recent findings highlight their role during homologous recombination, a basic mechanism to repair double-strand DNA lesions (in somatic cells) and programmed DNA breaks (in meiocytes). This chapter contains an exhaustive description of procedures applied to analyze meiotic chromosome behavior (cytogenetic techniques) and DNA repair capacity (genotoxicity assays) in AGO-deficient Arabidopsis thaliana mutants.

Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines.

The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance.

Evaluation of micronuclei in tobacco users: A study in Punjabi population.

INTRODUCTION: The assessment of micronuclei in exfoliated cells is a promising tool for the study of epithelial carcinogens and can be used to detect chromosome breakage or mitotic interference, thought to be relevant to carcinogenesis. AIM: The present study aimed to detect micronuclei in exfoliated oral mucosal cells in individuals using various tobacco forms from the last 5 years. MATERIALS AND METHODS: A total of 75 healthy male subjects (25 smokeless tobacco users, 25 smokers, and 25 non-tobacco users) were selected for the study. Smears were obtained with moistened wooden spatula from buccal mucosa and fixed with 95% alcohol. All the cytologic smears were stained by Papanicolaou technique. From each slide, ~1000 cells were examined under the 400× magnification and where micronucleated (MN) cells were located, they were examined under the 1000× magnification. RESULT: MN cells were found to be significantly higher in smokeless tobacco users than in smokers and controls. CONCLUSION: A positive correlation is found between increased micronucleus frequency and tobacco-using habits. So micronucleus assay can be used as a biomarker of genotoxicity.