Human translesion DNA polymerases ι and κ mediate tolerance to temozolomide in MGMT-deficient glioblastoma cells.

Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.

The role of chaperone-mediated autophagy in drug resistance.

In the search for alternatives to overcome the challenge imposed by drug resistance development in cancer treatment, the modulation of autophagy has emerged as a promising alternative that has achieved good results in clinical trials. Nevertheless, most of these studies have overlooked a novel and selective type of autophagy: chaperone-mediated autophagy (CMA). Following its discovery, research into CMA's contribution to tumor progression has accelerated rapidly. Therefore, we now understand that stress conditions are the primary signal responsible for modulating CMA in cancer cells. In turn, the degradation of proteins by CMA can offer important advantages for tumorigenesis, since tumor suppressor proteins are CMA targets. Such mutual interaction between the tumor microenvironment and CMA also plays a crucial part in establishing therapy resistance, making this discussion the focus of the present review. Thus, we highlight how suppression of LAMP2A can enhance the sensitivity of cancer cells to several drugs, just as downregulation of CMA activity can lead to resistance in certain cases. Given this panorama, it is important to identify selective modulators of CMA to enhance the therapeutic response.

A Novel Interaction Between RAD23A/B and Y-family DNA Polymerases.

The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.

Exploring pradimicin-IRD antineoplastic mechanisms and related DNA repair pathways.

DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent - pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.

Mutagenicity Profile Induced by UVB Light in Human Xeroderma Pigmentosum Group C Cells.

Nucleotide excision repair (NER) is one of the main pathways for genome protection against structural DNA damage caused by sunlight, which in turn is extensively related to skin cancer development. The mutation spectra induced by UVB were investigated by whole-exome sequencing of randomly selected clones of NER-proficient and XP-C-deficient human skin fibroblasts. As a model, a cell line unable to recognize and remove lesions (XP-C) was used and compared to the complemented isogenic control (COMP). As expected, a significant increase of mutagenesis was observed in irradiated XP-C cells, mainly C>T transitions, but also CC>TT and C>A base substitutions. Remarkably, the C>T mutations occur mainly at the second base of dipyrimidine sites in pyrimidine-rich sequence contexts, with 5'TC sequence the most mutated. Although T>N mutations were also significantly increased, they were not directly related to pyrimidine dimers. Moreover, the large-scale study of a single UVB irradiation on XP-C cells allowed recovering the typical mutation spectrum found in human skin cancer tumors. Eventually, the data may be used for comparison with the mutational profiles of skin tumors obtained from XP-C patients and may help to understand the mutational process in nonaffected individuals.

Reversine exerts cytotoxic effects through multiple cell death mechanisms in acute lymphoblastic leukemia.

PURPOSE: Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited therapeutic options for adult patients. Aurora kinases have drawn attention as potential targets in hematological neoplasms due to their high expression and biological functions. Aurora kinase A (AURKA) and AURKB are essential for a successful mitosis, acting in spindle mitotic organization and cytokinesis. Reversine is a synthetic purine analog that acts as a multi-kinase inhibitor with anti-neoplastic activity by targeting AURKA and AURKB. METHODS: ALL patient gene expression data were retrieved from the Amazonia! DATABASE: For functional assays, Jurkat (T-ALL) and Namalwa (B-ALL) cells were exposed to increasing concentrations of reversine and submitted to various cellular and molecular assays. RESULTS: We found that AURKB expression was higher in ALL patient samples compared to normal lymphocytes (p < 0.0001). The ALL cell lines tested displayed aberrant AURKA and AURKB expression. In Jurkat and Namalwa cells, reversine reduced cell viability in a dose- and time-dependent manner (p < 0.05). Reversine also significantly reduced the viability of primary ALL cells. Reversine induced apoptosis and autophagy, and reduced cell proliferation in both cell lines (p < 0.05). Mitotic catastrophe markers, including cell cycle arrest at G2/M, increased cell size and DNA damage, were observed upon reversine exposure. Short- and long-term treatment with reversine inhibited autonomous clonogenicity (p < 0.05). At the molecular level, reversine reduced AURKB activity, induced SQSTM1/p62 consumption, and increased LC3BII and γ-H2AX levels. In Namalwa cells, reversine modulated 25 out of 84 autophagy-related genes, including BCL2, BAD, ULK1, ATG10, IRGM and MAP1LC3B, which indicates that reversine acts by initiating and sustaining autophagy signals in ALL cells. CONCLUSIONS: From our data we conclude that reversine reduces the viability of ALL cells by triggering multiple cell death mechanisms, including apoptosis, mitotic catastrophe, and autophagy. Our findings highlight reversine as a potential anticancer agent for ALL.

Whole-exome sequencing reveals the impact of UVA light mutagenesis in xeroderma pigmentosum variant human cells.

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.

ATR/Chk1 Pathway is Activated by Oxidative Stress in Response to UVA Light in Human Xeroderma Pigmentosum Variant Cells.

The crucial role of DNA polymerase eta in protecting against sunlight-induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP-V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XP-V cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP-V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP-V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and γH2AX signaling. Corroborating ATR participation in UVA-DDR, a significant increase in Chk1 protein phosphorylation, as well as S-phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N-acetylcysteine (NAC), which significantly protects XP-V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA-induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells.

The key role of UVA-light induced oxidative stress in human Xeroderma Pigmentosum Variant cells.

The UVA component of sunlight induces DNA damage, which are basically responsible for skin cancer formation. Xeroderma Pigmentosum Variant (XP-V) patients are defective in the DNA polymerase pol eta that promotes translesion synthesis after sunlight-induced DNA damage, implying in a clinical phenotype of increased frequency of skin cancer. However, the role of UVA-light in the carcinogenesis of these patients is not completely understood. The goal of this work was to characterize UVA-induced DNA damage and the consequences to XP-V cells, compared to complemented cells. DNA damage were induced in both cells by UVA, but lesion removal was particularly affected in XP-V cells, possibly due to the oxidation of DNA repair proteins, as indicated by the increase of carbonylated proteins. Moreover, UVA irradiation promoted replication fork stalling and cell cycle arrest in the S-phase for XP-V cells. Interestingly, when cells were treated with the antioxidant N-acetylcysteine, all these deleterious effects were consistently reverted, revealing the role of oxidative stress in these processes. Together, these results strongly indicate the crucial role of oxidative stress in UVA-induced cytotoxicity and are of interest for the protection of XP-V patients.

Direct participation of DNA in the formation of singlet oxygen and base damage under UVA irradiation.

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of DNA lesion by these wavelengths. UVA light also excite endogenous chromophores, which causes DNA damage through ROS. In this study, DNA samples were irradiated with UVA light in different conditions to investigate possible mechanisms involved in the induction of DNA damage. The different types of DNA lesions formed after irradiation were determined through the use of endonucleases, which recognize and cleave sites containing oxidized bases and cyclobutane pyrimidine dimers (CPDs), as well as through antibody recognition. The formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG) was also studied in more detail using electrochemical detection. The results show that high NaCl concentration and concentrated DNA are capable of reducing the induction of CPDs. Moreover, concerning damage caused by oxidative stress, the presence of sodium azide and metal chelators reduce their induction, while deuterated water increases the amounts of oxidized bases, confirming the involvement of singlet oxygen in the generation of these lesions. Curiously, however, high concentrations of DNA also enhanced the formation of oxidized bases, in a reaction that paralleled the increase in the formation of singlet oxygen in the solution. This was interpreted as being due to an intrinsic photosensitization mechanism, depending directly on the DNA molecule to absorb UVA and generate singlet oxygen. Therefore, the DNA molecule itself may act as a chromophore for UVA light, locally producing a damaging agent, which may lead to even greater concerns about the deleterious impact of sunlight.

Sunlight damage to cellular DNA: Focus on oxidatively generated lesions.

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells.

Genotoxic effects of the herbicide Roundup Transorb and its active ingredient glyphosate on the fish Prochilodus lineatus.

Roundup Transorb (RT) is a glyphosate-based herbicide and despite its wide use around the world there are few studies comparing the effects of the active ingredient with the formulated product. In this context the purpose of this study was to compare the genotoxicity of the active ingredient glyphosate with the formulated product RT in order to clarify whether the active ingredient and the surfactant of the RT formula may exert toxic effects on the DNA molecule in juveniles of fish Prochilodus lineatus. Erythrocytes and gill cells of fish exposed to glyphosate and to RT showed DNA damage scores significantly higher than control animals. These results revealed that both glyphosate itself and RT were genotoxic to gill cells and erythrocytes of P. lineatus, suggesting that their use should be carefully monitored considering their potential impact on tropical aquatic biota.