Natália Moreno | Universidade de São Paulo (original) (raw)

Papers by Natália Moreno

Mutagenesis, 2019

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal o... more Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell re...

Carcinogenesis, 2021

XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 product... more XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in Xeroderma pigmentosum complementation group C (XP-C) (XPC-null) cells compared with an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, whereas AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine increased glutathione conce...

OncoImmunology

ABSTRACT Although active immunotherapies are effective strategies to induce activation of CD8+ T ... more ABSTRACT Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation.

Experimental and Molecular Therapeutics

Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, perf... more Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, performing translesion synthesis (TLS), a mechanism known by DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and therefore this may be a strategy that those cells use to resist treatments. Moreover, this process is error-prone and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in in tumor therapy with Temozolomide (TMZ). This drug is an alkylating agent that damage DNA. Our aim is to investigate how TMZ affects TLS mutated cells, to understand how these polymerases are related to tumor cells resistance to this chemotherapeutic agent. Hence, we firstly treated POLH mutated cells and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT) and for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, POLH mutated cells a...

Photochemistry and Photobiology

Nucleotide excision repair (NER) is one of the main pathways for genome protection against struct... more 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 non-affected individuals.

Environment International

Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan ar... more Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1β, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

Cellular Oncology

PURPOSE Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited the... more 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.

Experimental and Molecular Therapeutics

Nucleic Acids Research

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-ex... more 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 ...

Free Radical Biology and Medicine

The UVA component of sunlight induces DNA damage, which are basically responsible for skin cancer... more 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.

Photochemistry and Photobiology

The crucial role of DNA polymerase eta in protecting against sunlight-induced tumors is evidenced... more 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.

Free radical biology & medicine, Jul 18, 2017

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of... more 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,...

Free radical biology & medicine, Jun 18, 2017

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of th... more 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 in...

Environmental Toxicology and Pharmacology, 2014

Comprehensive Series in Photochemical & Photobiological Sciences

Genetics and Molecular Biology

Base and nucleotide excision repair (BER and NER) pathways are normally associated with removal o... more Base and nucleotide excision repair (BER and NER) pathways are normally associated with removal of specific types of DNA damage: small base modifications (such as those induced by DNA oxidation) and bulky DNA lesions (such as those induced by ultraviolet or chemical carcinogens), respectively. However, growing evidence indicates that this scenario is much more complex and these pathways exchange proteins and cooperate with each other in the repair of specific lesions. In this review, we highlight studies discussing the involvement of NER in the repair of DNA damage induced by oxidative stress, and BER participating in the removal of bulky adducts on DNA. Adding to this complexity, UVA light experiments revealed that oxidative stress also causes protein oxidation, directly affecting proteins involved in both NER and BER. This reduces the cell's ability to repair DNA damage with deleterious implications to the cells, such as mutagenesis and cell death, and to the organisms, such as cancer and aging. Finally, an interactome of NER and BER proteins is presented, showing the strong connection between these pathways, indicating that further investigation may reveal new functions shared by them, and their cooperation in maintaining genome stability.

Mutagenesis, 2019

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal o... more Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell re...

Carcinogenesis, 2021

XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 product... more XPC deficiency is associated with mitochondrial dysfunction, increased mitochondrial H2O2 production and sensitivity to the Complex III inhibitor antimycin A (AA), through a yet unclear mechanism. We found an imbalanced expression of several proteins that participate in important mitochondrial function and increased expression and phosphorylation of the tumor suppressor p53 in Xeroderma pigmentosum complementation group C (XP-C) (XPC-null) cells compared with an isogenic line corrected in locus with wild-type XPC (XPC-wt). Interestingly, inhibition of p53 nuclear import reversed the overexpression of mitochondrial proteins, whereas AA treatment increased p53 expression more strongly in the XP-C cells. However, inhibition of p53 substantially increased XP-C cellular sensitivity to AA treatment, suggesting that p53 is a critical factor mediating the cellular response to mitochondrial stress. On the other hand, treatment with the antioxidant N-acetylcysteine increased glutathione conce...

OncoImmunology

ABSTRACT Although active immunotherapies are effective strategies to induce activation of CD8+ T ... more ABSTRACT Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation.

Experimental and Molecular Therapeutics

Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, perf... more Translesion DNA polymerases are capable of replicating damaged DNA without removing lesions, performing translesion synthesis (TLS), a mechanism known by DNA damage tolerance. Tumor cells use this mechanism in order to survive lesions caused by chemotherapy and therefore this may be a strategy that those cells use to resist treatments. Moreover, this process is error-prone and can lead to mutagenesis increasing resistance potential of tumor cells. Little is known about the role of TLS in in tumor therapy with Temozolomide (TMZ). This drug is an alkylating agent that damage DNA. Our aim is to investigate how TMZ affects TLS mutated cells, to understand how these polymerases are related to tumor cells resistance to this chemotherapeutic agent. Hence, we firstly treated POLH mutated cells and analyzed cell proliferation by flow cytometry, cell viability by a colorimetric assay (XTT) and for survival by apoptotic markers (such sub-G1) and clonogenic assays. In fact, POLH mutated cells a...

Photochemistry and Photobiology

Nucleotide excision repair (NER) is one of the main pathways for genome protection against struct... more 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 non-affected individuals.

Environment International

Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan ar... more Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1β, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

Cellular Oncology

PURPOSE Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer with limited the... more 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.

Experimental and Molecular Therapeutics

Nucleic Acids Research

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-ex... more 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 ...

Free Radical Biology and Medicine

The UVA component of sunlight induces DNA damage, which are basically responsible for skin cancer... more 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.

Photochemistry and Photobiology

The crucial role of DNA polymerase eta in protecting against sunlight-induced tumors is evidenced... more 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.

Free radical biology & medicine, Jul 18, 2017

UVA light is hardly absorbed by the DNA molecule, but recent works point to a direct mechanism of... more 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,...

Free radical biology & medicine, Jun 18, 2017

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of th... more 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 in...

Environmental Toxicology and Pharmacology, 2014

Comprehensive Series in Photochemical & Photobiological Sciences

Genetics and Molecular Biology

Base and nucleotide excision repair (BER and NER) pathways are normally associated with removal o... more Base and nucleotide excision repair (BER and NER) pathways are normally associated with removal of specific types of DNA damage: small base modifications (such as those induced by DNA oxidation) and bulky DNA lesions (such as those induced by ultraviolet or chemical carcinogens), respectively. However, growing evidence indicates that this scenario is much more complex and these pathways exchange proteins and cooperate with each other in the repair of specific lesions. In this review, we highlight studies discussing the involvement of NER in the repair of DNA damage induced by oxidative stress, and BER participating in the removal of bulky adducts on DNA. Adding to this complexity, UVA light experiments revealed that oxidative stress also causes protein oxidation, directly affecting proteins involved in both NER and BER. This reduces the cell's ability to repair DNA damage with deleterious implications to the cells, such as mutagenesis and cell death, and to the organisms, such as cancer and aging. Finally, an interactome of NER and BER proteins is presented, showing the strong connection between these pathways, indicating that further investigation may reveal new functions shared by them, and their cooperation in maintaining genome stability.