Academic literature on the topic 'Zinc; genomic stability; DNA damage'

Salinity is an edaphic stress that dramatically restricts worldwide crop production. Nanomaterials and plant growth-promoting bacteria (PGPB) are currently used to alleviate the negative effects of various stresses on plant growth and development. This study investigates the protective effects of different levels of zinc oxide nanoparticles (ZnO-NPs) (0, 20, and 40 mg L−1) and PGPBs (no bacteria, Bacillus subtilis, Lactobacillus casei, Bacillus pumilus) on DNA damage and cytosine methylation changes in the tomato (Solanum lycopersicum L. ‘Linda’) seedlings under salinity stress (250 mM NaCl). Coupled Restriction Enzyme Digestion-Random Amplification (CRED-RA) and Randomly Amplified Polymorphic DNA (RAPD) approaches were used to analyze changes in cytosine methylation and to determine how genotoxic effects influence genomic stability. Salinity stress increased the polymorphism rate assessed by RAPD, while PGPB and ZnO-NPs reduced the adverse effects of salinity stress. Genomic template stability was increased by the PGPBs and ZnO-NPs application; this increase was significant when Lactobacillus casei and 40 mg L−1 of ZnO-NPs were used.A decreased level of DNA methylation was observed in all treatments. Taken together, the use of PGPB and ZnO-NPs had a general positive effect under salinity stress reducing genetic impairment in tomato seedlings.

The cytotoxic effects of metallic nanoparticles (MNPs) might be revealed in genomic and histopathological defects. Therefore current study aimed to assess the bio-persistence and adverse effects potency of zinc oxide nanoparticles (ZnONPs) in the gastropod, Monacha cartusiana. Gastropods were exposed to 74 μg/mL for 14 d then the DNA adduct and histopathological defect profiles were assessed. The results demonstrated significant decline in the estimated genomic template stability (GTS%) in haemolymph and digestive gland ranging from 10.0 to 42.9% in treated animals compared to controls. In the treated and recovered snails, randomly amplified polymorphic (RAPD)-DNA showed the appearance and/or disappearance of DNA bands, indicating DNA damage due to the cytotoxicity of ZnONPs on gastropods. Significant defects in microvilli (MV), nucleus (N), mitochondria (M), and execratory glands (EXG) were noticed in the treated individuals with respect to controls. The remaining live animals were subjected to a recovery period (14 d, without treatment) and slight recovery profiles were reported for both measures compared to the control group. The recovery pattern was recognized in the nucleus/cytoplasm ratio with 0.186 and 0.428 in the treated and recovered groups concerning their control (0.176). The studied parameters reported herein might be reliable tools to assess accumulation and bio-persistence patterns of NPs in the organisms for short-term exposure indicating the cytotoxic and genotoxic effects. Also, gastropods may provide simple models for evaluating the ecotoxicological effects of nanomaterials.

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.

rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.

The expanded uses of zinc oxide nanoparticles (ZnO NPs) have grown rapidly in the field of nanotechnology. Thus, rising production of nanoparticles (NPs) increases the possible risks to the environment and occupationally exposed humans. Hence, it is indispensable to appraise the safety toxicity including genotoxicity for these NPs. In the present study, we have evaluated the genotoxic effect of ZnO NPs after oral administration to Swiss mice at dose levels of 300 and 2000 mg/kg body weight. These doses were administered for 2 days at 24 h apart. Chromosomal aberration (CA) and micronucleus tests were conducted following Organization for Economic Co-operation and Development guidelines. DNA damage was evaluated at 0, 24, 48, and 72 h posttreatment using a randomly amplified polymorphic DNA (RAPD) assay; additionally, semen analyses were also performed at 34.5 days post oral exposure. The reactive oxygen species (ROS), 8-oxo-2′-deoxyguanosine and CAs were increased ( p < 0.05) at the highest dosage (2000 mg/kg) of ZnO NPs compared to controls. Aberrant sperm morphology with reduced sperm count and motility were also present ( p < 0.05) in the high-dose group. Based on the RAPD assay, the genomic template stability within the high-dose group (<90%) was less than the controls (100%). The results suggested that ZnO NPs are mildly genotoxic in a dose-related manner and this toxicity were induced by generation of ROS.

Homologous recombination is involved in repairing DNA damage, contributing to maintaining the integrity and stability of viral and cellular genomes. In bacteria, the recombination mediator proteins RecO and RecR are required to load the RecA recombinase on ssDNA for homologous recombination. To structurally and functionally characterize RecO, we determined the crystal structure of RecO from Campylobacter jejuni (cjRecO) at a 1.8 Å resolution and biochemically assessed its capacity to interact with DNA and a metal ion. cjRecO folds into a curved rod-like structure that consists of an N-terminal domain (NTD), C-terminal domain (CTD), and Zn2+-binding domain (ZnD). The ZnD at the end of the rod-like structure coordinates three cysteine residues and one histidine residue to accommodate a Zn2+ ion. Based on an extensive comparative analysis of RecO structures and sequences, we propose that the Zn2+-binding consensus sequence of RecO is CxxC…C/HxxC/H/D. The interaction with Zn2+ is indispensable for the protein stability of cjRecO but does not seem to be required for the recombination mediator function. cjRecO also interacts with ssDNA as part of its biological function, potentially using the positively charged patch in the NTD and CTD. However, cjRecO displays a low ssDNA-binding affinity, suggesting that cjRecO requires RecR to efficiently recognize ssDNA for homologous recombination.

Abstract Acute promyelocytic leukemia (APL) is driven by the oncogene PML-RARA which is generated by fusion of the promyelocytic leukemia (PML) and retinoic acid receptor alpha (RARA) genes, and which strongly interferes with downstream signalling and the architecture of multiprotein structures known as PML nuclear bodies (NBs). NB disruption is a diagnostic hallmark of APL, yet the significance of this phenomenon to disease pathogenesis and treatment response remains poorly understood. The majority of APL patients can now be cured with combination therapy with arsenic trioxide (ATO) and ATRA (All Trans-Retinoic Acid), which synergize promoting re-formation of disrupted Pml NBs. To date, the importance of NB disruption has only been studied in vitro. To address this, we generated a knock-in mouse model with targeted NB disruption achieved through mutation of key zinc-binding cysteine residues (C62A/C65A) in the RING domain of Pml. Homozygous PmlC62A/C65A mice are viable, and developmentally normal. At a cellular level, Pml NB disruption was confirmed and treatment with ATO was associated with defective Pml SUMOylation and degradation. A key feature of APL fusion proteins is the capacity to homodimerise (mediated by the fusion partner e.g. PML), which is not a feature of wild-type RARα. This forced homodimerisation of RARα has been shown to be critical for APL pathogenesis. We investigated whether Pml NB disruption could cooperate in vivo with forced RARα homodimerisation (mediated artificially by linking RARα to the dimerisation domain of the NFκB p50 subunit). While no leukemias arose in PmlC62A/C65A mice, p50-RARα mice expressing PmlC62A/C65A presented a doubling in the rate of leukemia development (p<0.0001) compared to PmlWT-p50-RARα, leading to a penetrance comparable to that observed in previously published PML-RARα transgenic models. Moreover, the latency period to onset of leukemia was significantly reduced in the context of NB disruption (p=0.008). ATRA treatment significantly improved the survival of mice transplanted with PmlWT-p50-RARα or Pml-RARα leukemic blasts, but not with PmlC62A/C65A-p50-RARα. These data reveal not only the key role of PML-RARα expression-induced NB disruption in APL development, but also the importance of re-formation of NBs for an effective response to differentiating drug. While formation of the PML-RARA fusion is considered an initiating event in APL pathogenesis, it is insufficient for the full leukemic phenotype. Exome sequencing studies have consistently identified presence of cooperating mutations. Since Pml and Pml NB have established roles in DNA repair and in the maintenance of genomic stability, we speculated that loss of NB integrity could affect these functions. Whole exome sequencing revealed a pattern of higher genomic instability in PmlC62A/C65A-p50-RARα leukemia as compared to PmlWT-p50-RARα, with detection of mutations found in human APL, including Kras, Ptpn11 and Usp9y. Using DNA repair reporter assays, we demonstrated that DNA repair via both non-homologous end joining (NHEJ; p=0.01) and homologous recombination (HR; p=0.006) pathways was less efficient in PmlC62A/C65A primary cells than in PmlWT cells. Importantly, using a PML-RARα-inducible cell line, comparable defects in the NHEJ and HR pathways, which were PML-RARα dependent, were identified. These data were also supported by an increase in sister-chromatid exchange (p<0.0001) and chromosome abnormality (p=0.0002) in the context of PmlC62A/C65A versus PmlWT. Interestingly, the kinetic of repair of ionising radiation (IR)-induced DNA double-strand breaks, assessed by analysis of γH2AX foci formation and clearance, was not affected. None of the DNA repair players analysed (e.g. Blm, Rad51 and 53BP1) failed to form foci in response to IR. However, their basal levels of foci were significantly greater in the presence of PmlC62A/C65A (p<0.04; quantified using Amnis ImageStreamX Mk II imaging flow cytometer). Additionally, we found that Rad51 foci showed a defect in localisation post-IR when PmlC62A/C65A was expressed, with impairment of Rad51 co-localisation and interaction with γH2AX. Altogether, our data therefore highlight the significant contribution of Pml NB to the effectiveness of DNA damage repair processes, and the manner in which their disruption mediated by the PML-RARα oncoprotein can assist APL pathogenesis. Disclosures Hills: TEVA: Honoraria. Grimwade:TEVA: Research Funding.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida.
The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga
The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

Zinc (Zn) is an essential trace element required for both optimal human health and maintaining genomic stability. The main aim of this thesis was to address important knowledge gaps regarding the possible impact of Zn status on genomic stability events in both lymphocytes and epithelial cells using both in vitro and in vivo models. The project also aimed to study the differential impact of Zn Carnosine (ZnC) and Zn Sulphate (ZnSO₄) on genome stability as the former is a newly emerging commercially available supplement renown for its antioxidant capacity. The in vitro studies investigated the effects of ZnSO₄ and ZnC on cell proliferation via MTT assay and DNA damage rates and was measured using both the comet assay and the Cytokinesis-block micronucleus cytome (CBMN-Cyt) assay in the WIL2-NS human lymphoblastoid cell line and HOK cell line. This study also investigated the impact of Zn status on both telomere length and telomere base damage in vitro. An in vivo study was designed to further investigate the effect of Zn supplementation in minimising genome instability events in lymphocytes. An increased intake of Zn may reduce the risk of degenerative diseases but may be toxic if taken in excess. This study aimed to investigate whether taking daily supplements of 20 mg of Zn as Zn Carnosine can improve Zn status, genome stability events and Zn transporter genes in an elderly South Australian cohort characterised by having low plasma Zn levels. In conclusion, the in vitro studies suggest that 1) Zn deficiency (0 μM) and high Zn concentrations increase DNA damage; 2) Zn at 4-16 μM is optimal in maintaining genome stability events; 3) Zn at 16-32 μM is optimal in protecting the cell against DNA damage induced by irradiation and hydrogen peroxide challenges; and 4) Zn may play an important role in telomere maintenances. The in vivo study suggests that Zn supplementation may be beneficial in an elderly population with marginal lowered Zn status by raising plasma Zn levels, lowering DNA damage events and modifies Zn transporter gene expression.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2012

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
A integridade genómica é assegurada por várias moléculas que trabalham conjuntamente para erradicar ou minimizar a lesão do DNA. Estas constituem a resposta à lesão do DNA (DDR). Vários micronutrientes atuam em reações essenciais da DDR, sendo a adequada biodisponibilidade destes fatores dietéticos crucial para o seu ótimo funcionamento. O zinco (Zn) é particularmente relevante por estar envolvido em várias funções fundamentais à célula desde a resposta ao stress oxidativo, apoptose, regulação do ciclo celular, do metabolismo, síntese e reparação do DNA. Notavelmente, vários mecanismos de reparação do DNA envolvem o Zn. Apesar do papel do Zn na prevenção da doença ser bem definido, as suas funções na carcinogénese são menos conhecidas. No contexto do sistema hematopoiético, uma adequada DDR é essencial à manutenção das células estaminais hematopoiéticas, evitando a acumulação de lesões nestes precursores. Alterações da DDR têm sido reportadas na leucemia mielóide aguda (LMA) e relacionadas com a origem da doença. Outra observação comum nos doentes com leucemia é o decréscimo sérico de Zn. Apesar da frequência deste achado, o significado biológico do Zn na LMA e a relação com a DDR nas células leucémicas não são bem compreendidos. Este trabalho pretendeu estudar o papel do Zn na modulação da DDR na LMA e explorar o seu potencial como coadjuvante da terapia anticancerígena. Para tal, um modelo celular de LMA (HEL) foi incubado em condições normais de cultura (standard) com o teor basal de zinco presente no soro fetal bovino, bem como em depleção de Zn e em suplementação com ZnSO4 por 2, 7 e 15 dias. As respostas basais e induzidas por exposição de 30 minutos a 10µM de H2O2 e a 60 segundos de radiação ultravioleta (Ee=2.9841 W.cm-2) foram avaliadas a cada momento temporal. Para compreender se os papéis do Zn variam no contexto fisiológico e patológico, uma linha celular de linfócitos humanos normais (IMC) foi submetida às mesmas experiências. A lesão cromossómica, morte celular e níveis de divisão celular foram avaliados pelo teste do micronúcleo com bloqueio da citocinese. A monitorização da yH2AX, biomarcador da lesão e da cinética de reparação, foi feita a cada momento temporal antes da exposição genotóxica, após indução da lesão e 1 hora e 24 horas após exposição. A expressão de genes da DDR (PARP1, XRCC1, MSH2, MSH6, MLH1, XPA, ERCC1, RAD23B, RAD51, PRKDC, XRCC6, PALB2, FANCD2 e MGMT) foi avaliada por qRT-PCR. Para compreender o papel do Zn na modulação de inibidores da DDR e de compostos quimioterapêuticos utilizados na LMA, as células HEL foram incubadas com olaparib e citarabina em monoterapia e combinação com Zn. Os resultados foram analisados estatisticamente considerando um nível de significância de 95% (p <0,05). Comparativamente a standard, a suplementação com Zn diminuiu a lesão basal e a morte celular nos linfócitos normais e aumentou ligeiramente a proliferação. Pelo contrário, nas células de LMA verificou-se aumento da lesão basal ao longo do tempo de suplementação, ligeiro aumento da morte celular e redução dos níveis de divisão celular. Após estímulo genotóxico, os linfócitos suplementados apresentaram uma resposta mais eficiente à lesão do que os da condição standard, manifestado pelo decréscimo da lesão cromossómica, particularmente ao 7º e 15º dia de suplementação, aumento da morte celular e diminuição dos níveis de divisão celular. Por oposição, os linfócitos em depleção de Zn revelaram uma resposta menos eficiente e maior lesão cromossómica. Nas células de LMA suplementadas houve aumento da lesão cromossómica comparativamente às da condição standard, enquanto que os menores níveis de lesão se verificaram na ausência de Zn. A monitorização da yH2AX pareceu corroborar que nos linfócitos suplementados poderá haver reparação, que não é atingida na ausência de Zn, e que na linha de LMA a lesão persiste após exposição genotóxica. A análise da expressão génica não revelou diferenças significativas entre condições, com exceção da diminuição significativa (1.02 vezes inferior, p=0.0315) da expressão basal de XRCC6 nas células HEL suplementadas. Os resultados da monoterapia e da terapia combinada revelaram redução da proliferação e viabilidade celular de forma dependente da dose e do tempo, tendo a terapia combinada apresentado efeitos mais eficazes. Estes foram claramente demonstrados pelo efeito sinérgico do Zn com o olaparib e citarabina na maioria das doses testadas e pelo marcado decréscimo do IC50 do olaparib e da citarabina em combinação com o Zn comparativamente à monoterapia (doses 2,8 vezes e 5,4 vezes inferiores, respetivamente). Em suma, os dados corroboram a importância do Zn na manutenção de respostas adequadas à lesão do DNA no contexto preventivo da doença e a sua possível aplicabilidade como coadjuvante potenciador de terapias genotóxicas ou das que têm como alvo a DDR, revelando um duplo papel do zinco no contexto preventivo e terapêutico do cancro, em particular da LMA.
Genome integrity is assured by a plethora of molecules that work together to eradicate or minimize repair DNA damage. These constitute the DNA damage response (DDR). Many micronutrients are crucial for key DDR reactions, meaning that adequate bioavailability of these dietary factors is critical for optimal DDR functioning. Zinc (Zn) is particularly important for playing pivotal roles in the cell, from oxidative stress responses, to apoptosis, cell cycle regulation, metabolism, DNA synthesis and repair. Notably, many DNA repair mechanisms involve Zn. Despite the role of Zn in disease prevention is well defined, its functions in carcinogenesis are far less explored. In the context of the hematopoietic system, an appropriate DDR is essential for the maintenance of the hematopoietic stem cells’ pool homeostasis, by avoiding the accumulation of DNA damage in these precursors. DDR alterations have been found in acute myeloid leukemia (AML) and related to the leukemogenesis process. Moreover, a common observation in leukemia patients is the decrease in serological Zn. Despite the frequency of this finding, the biological significance of Zn for the leukemogenesis process and relation with the DDR in AML cells is not understood. This work aimed to study the role of Zn in the modulation of the DDR in AML and explore its potential as co-adjuvant in leukemia therapy. To do so, cellular model of AML (HEL) was incubated in standard (Std) culture conditions containing the basal Zn levels presented in fetal bovine serum, in Zn depletion and in supplementation with ZnSO4 for 2, 7 and 15 days. Basal and induced cellular responses to exposure to 10µM of H2O2 for 30 minutes and 60 seconds of UV radiation (Ee=2.9841 W.cm-2) were evaluated at each time point. To understand whether Zn’s roles differ in health and disease, a cell line of normal human lymphocytes (IMC) was submitted to the same experiments. Chromosomal damage, cell death and cell division rates were assessed by the cytokinesis-block micronucleus assay. The monitorization of yH2AX, biomarker of DNA damage and repair kinetics, was performed at all evaluation times before genotoxic exposure, after initial damage induction, and 1 hour and 24 hours following exposure. The expression of DDR genes (PARP1, XRCC1, MSH2, MSH6, MLH1, XPA, ERCC1, RAD23B, RAD51, PRKDC, XRCC6, PALB2, FANCD2 and MGMT) was evaluated by two-step qRT-PCR. To acknowledge the role of Zn in the modulation of DDR inhibitors and chemotherapeutic compounds used in AML, HEL cells were submitted to treatment with olaparib and cytarabine in monotherapy and in combination with Zn. Results were statistically analyzed considering a confidence level of 95% (p<0.05). Comparatively to Std Zn conditions, supplementation reduced basal chromosomal damage and cell death in normal lymphocytes and led to slight increases in proliferation. Oppositely, in AML cells it was observed an increase of basal chromosomal damage through time of supplementation, a slight increase in basal cell death and a decrease in cell division rates. Upon genotoxic exposure, Zn-supplemented lymphocytes demonstrated a more efficient response than those from Std conditions, manifested by the decrease in chromosomal damage, particularly at the 7th and 15th days of supplementation, increase in cell death and decrease of cell division rates. Contrastingly, Zn-depleted lymphocytes shown a less efficient damage response, presenting increased chromosomal damage levels. Supplemented AML cells shown an increase in DNA damage comparatively to those from Std conditions, while the lowest chromosomal damage scores were found in Zn-depletion. The monitorization of yH2AX seemed to corroborate that in supplemented lymphocytes there may be a repair response that is not achieved in Zn absence, and that in the AML cell line damage persists following initial genotoxic exposure. Gene expression analyses did not reveal significant differences between conditions, except for a significant decrease (1.0 fold, p=0.0315) in XRCC6 basal expression in HEL supplemented cells. The results from monotherapy as well as combination therapy revealed a reduction in cellular proliferation and viability in a dose and time dependent manner, with combination therapy displaying more efficient effects. This was clearly demonstrated by the synergistical effect of Zn with olaparib and cytarabine in most of the tested concentrations and the marked decrease in olaparib and cytarabine IC50 when in combination with Zn comparatively to monotherapy (2.8-fold and 5.4-fold decreases, respectively). In sum, the results corroborate the importance of Zn in the maintenance of adequate responses to DNA damage in the context of disease prevention and a possible application of Zn as a co-adjuvant potentiator of the effects of genotoxic or DDR-targeting therapies, revealing a dual role of Zn both in cancer prevention and therapy, particularly in AML.

The relevance of preconceptional and prenatal toxicant exposures for genomic stability in offspring is difficult to analyze in human populations, because gestational exposures usually cannot be separated from preconceptional exposures. To analyze the roles of exposures during gestation and conception on genomic stability in the offspring, stability was assessed via the Comet assay and highly sensitive, semiautomated confocal laser scans of gammaH2AX foci in cord, maternal, and paternal blood as well as spermatozoa from 39 families in Crete, Greece, and the United Kingdom. With use of multivariate linear regression analysis with backward selection, preconceptional paternal smoking (% tail DNA: P>0.032; gammaH2AX foci: P>0.018) and gestational maternal (% tail DNA: P>0.033) smoking were found to statistically significantly predict DNA damage in the cord blood of F1 offspring. Maternal passive smoke exposure was not identified as a predictor of DNA damage in cord blood, indicating that the effect of paternal smoking may be transmitted via the spermatozoal genome. Taken together, these studies reveal a role for cigarette smoke in the induction of DNA alterations in human F1 offspring via exposures of the fetus in utero or the paternal germline. Moreover, the identification of transgenerational DNA alterations in the unexposed F1 offspring of smoking-exposed fathers supports the claim that cigarette smoke is a human germ cell mutagen.

De nombreuses voies de signalisation cellulaire complexes permettent de répondre à la présence de dommages à l’ADN. Cette réponse cellulaire est indispensable afin d’éviter l’accumulation de mutations pouvant éventuellement conduire à la transformation tumorale. Ces différentes voies de réponse aux dommages à l’ADN sont hautement coordonnées et sont regroupées au sein d’un mécanisme global appelé DNA damage response (DDR). Les facteurs du DDR sont régulés à plusieurs niveaux de la cascade de l’expression des gènes. De façon notable, plusieurs protéines de liaison à l’ARN (RBP) participent à la régulation de l’expression des gènes du DDR via la régulation post- transcriptionnelle de leur ARN messager. La RBP STAU2 est connue pour lier plusieurs ARNm codant pour des protéines impliquées dans le contrôle du cycle cellulaire ainsi que dans les voies du DDR. La protéine STAU2 est elle-même régulée au niveau transcriptionnel par le facteur de transcription E2F1. De récentes observations laissent penser que la kinase centrale du DDR, CHK1, pourrait être impliquée dans la régulation de la stabilité de STAU2. Par ailleurs, les conséquences cellulaires de la diminution du niveau d’expression de STAU2 sont à ce jour très peu connues. Ce mémoire a d’abord été entrepris dans le but de mieux comprendre l’implication de la voie de la kinase CHK1 dans la régulation de la protéine de liaison à l’ARN STAU2. CHK1 est une protéine centrale des voies du DDR ainsi que du contrôle de la progression du cycle cellulaire en l’absence de dommages à l’ADN. Nos résultats montrent que la diminution de CHK1 induit une dégradation rapide de STAU2 par les caspases d’une façon indépendante de l’apoptose. Nous avons également renforcé ce lien entre STAU2 et les mécanismes de réparation des dommages à l’ADN en identifiant plusieurs protéines des voies de réparation dans l’environnement immédiat de STAU2. D’autre part nos travaux visent à mettre en évidence les conséquences de la déplétion de STAU2 dans plusieurs types cellulaires. STAU2 étant une RBP, sa dérégulation impacte inévitablement le devenir de plusieurs ARNm. Afin de caractériser ces différentes conséquences, nous avons dans un premier temps réalisé la déplétion totale de STAU2 dans des cellules hTert-RPE par la technique de CRISPR/Cas9. Nos résultats montrent que ces cellules accumulent anormalement des dommages à l’ADN et prolifèrent plus rapidement que des cellules normales. En outre plusieurs gènes impliqués dans la réparation des dommages à l’ADN se retrouvent diminués dans ces cellules. Dans un second temps, afin de définir si cet effet est dépendant du type cellulaire, nous avons induit la diminution de l’expression de STAU2 dans des cellules IMR90. Nous avons montré que dans ce cas, la diminution de STAU2 induit un arrêt du cycle cellulaire et une entrée des cellules en sénescence. Ainsi, les données présentées dans ce mémoire contribuent à mieux comprendre l’implication de STAU2 dans les processus cellulaires majeurs que sont la régulation du DDR et le contrôle du cycle cellulaire.
Many complex cellular pathways are induced in response to DNA damages. This cellular response is indispensable to prevent the accumulation of mutations and to avoid malignant transformation. These different pathways are highly coordinated and are organized in a global mechanism called DNA damage response (DDR). Proteins involved in the DDR are regulated at different levels of the gene expression process. Notably, several RNA binding proteins are involved in the regulation of DDR gene expression through the post-transcriptional control of their mRNA. The RBP STAU2 is known to bind various mRNAs coding for proteins involved in the DDR or cell cycle control. STAU2 is regulated at the transcriptional levels by the major transcription factor E2F1. Recent observations suggest that CHK1 could be implicated in the control of the steady-state level of STAU2. Otherwise, the cellular consequences of STAU2 downregulation remain elusive. The purpose of this research was first to elucidate the implication of CHK1 pathway in STAU2 regulation. CHK1 is a major protein involved in the DDR regulation as well as in the control of cell cycle progression in the absence of DNA damage. Our data show that the downregulation of CHK1 rapidly leads to a caspase-dependent degradation of STAU2 independently of apoptosis. The link between STAU2 and mechanisms of DNA repair was reinforced by our BioID2 experiment that identified several proteins of the DDR in close proximity with STAU2. On the other hand, the aim of this study was to determine the consequences of STAU2 downregulation in different cell lines. Given that STAU2 is an RBP, its dysregulation will inevitably change the fate of several mRNA. In order to increase our understanding of theses consequences, we generated an hTert-RPE1 STAU2-KO cell line using the CRISPR/Cas9 technique. Our data show that these cells accumulate DNA damage and have an increased proliferation rate. Moreover, several genes involved in the DNA repair pathway are downregulated. We also downregulated STAU2 in IMR90 to determine if the previous observations are cell-type specifics. In the latter case, STAU2 diminution triggers cell cycle arrest and cellular senescence. Altogether, these results contribute to improve our knowledge of STAU2 function, especially in DNA damage response pathway and in cell cycle regulation.

A number of pathways have evolved in order to repair DNA. Mismatch repair (MMR) operates when an improper nucleotide is used or when an insertion or deletion occurs during replication. Nucleotide excision repair (NER) repairs damage that distorts the DNA helix such as the presence of pyrimidine dimers induced by ultraviolet light. Base excision repair (BER) removes damaged or altered DNA bases that do not result in a conformational change in the chromatin. Single-strand break repair (SSBR) uses the same enzymatic steps as BER. Double-strand break (DSB) repair can involve either non-homologous end-joining (NHEJ) or homologous recombination (HR). In NHEJ, the broken DNA ends are joined directly. HR requires that one of the strands of the broken DNA molecule participates in the strand invasion of the sister chromatid. The site of the DSB must be modified to allow access to the repair machinery. This modification involves remodeling complexes, as well as histone-modifying enzymes.

The DNA contains the information that determines the cell phenotype. Epigenetic regulation of DNA transcription, repair of DNA damage, and tight control of the cell cycle are fundamental cell processes that determine the cellular heterogeneity, survival, plasticity, and repair in the nervous system. Epigenetics refers to heritable changes in gene expression that are independent of the DNA sequence (genetic code). Epigenetic mechanisms include: DNA methylation, histone and chromatin modifications, and effects of noncoding RNAs. Specific mutations in genes along these pathways can be associated with both neurodevelopmental and neurodegenerative disorders. To maintain genomic stability, cells activate a DNA damage response that detects and repairs the damaged DNA cycle. The elucidation of these mechanisms has led to development of novel approaches including DNA editing for treatment.