Clarissa Ribeiro Reily Rocha

The role of DNA repair in the pluripotency and differentiation of human stem cells.

All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.

Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη(KD) cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype.

Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

Nucleotide excision repair (NER) is the most flexible of all known DNA‐repair mechanisms, and XPG is a 3′‐endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP‐G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP‐G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV‐irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV‐ but not oxidative‐stress‐induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.

NRF2 and glutathione are key resistance mediators to temozolomide in glioma and melanoma cells

Cancer is a leading cause of death worldwide, and while great advances have been made particularly in chemotherapy, many types of cancer still present a dismal prognosis. In the case of glioma, temozolomide (TMZ) is the main option for treatment, but it has limited success due to drug resistance. While this resistance is usually associated to DNA repair mechanisms, in this work we demonstrate that oxidative stress plays an important role. We showed that upon TMZ treatment there is an induction of the nuclear factor erythroid 2-related factor 2 (NRF2), which is the main antioxidant transcription factor regulator in human cells. This is accompanied by an enhancement of glutathione (GSH) concentration in the tumor cells. The effectiveness of this pathway was proven by silencing NFR2, which greatly enhanced cell death upon TMZ treatment both in vitro and in vivo. Also, higher DNA damage and induced cell death was observed by combining BSO - a GSH inhibitor - with TMZ. Similar effects were also observed using in vitro and in vivo models of melanoma, thus possibly indicating that GSH has a decisive role in TMZ resistance in a wider range of tumors. Thus, a combined regimen of BSO and TMZ configures an interesting therapeutic alternative for fighting both glioma and melanoma.

Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.

The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.

DNA Damage and Its Cellular Response in Mother and Fetus Exposed to Hyperglycemic Environment

The increased production of reactive oxygen species (ROS) plays a key role in pathogenesis of diabetic complications. ROS are generated by exogenous and endogenous factors such as during hyperglycemia. When ROS production exceeds the detoxification and scavenging capacity of the cell, oxidative stress ensues. Oxidative stress induces DNA damage and when DNA damage exceeds the cellular capacity to repair it, the accumulation of errors can overwhelm the cell resulting in cell death or fixation of genome mutations that can be transmitted to future cell generations. These mutations can lead to and/or play a role in cancer development. This review aims at (i) understanding the types and consequences of DNA damage during hyperglycemic pregnancy; (ii) identifying the biological role of DNA repair during pregnancy, and (iii) proposing clinical interventions to maintain genome integrity. While hyperglycemia can damage the maternal genetic material, the impact of hyperglycemia on fetal cells is still unclear. DNA repair mechanisms may be important to prevent the deleterious effects of hyperglycemia both in mother and in fetus DNA and, as such, prevent the development of diseases in adulthood. Hence, in clinical practice, maternal glycemic control may represent an important point of intervention to prevent the deleterious effects of maternal hyperglycemia to DNA.

Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells

Abstract Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria.

Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease.

PNKP (polynucleotide kinase 3′-phosphatase, OMIM #605610) product is involved in the repair of strand breaks and base damage in the DNA molecule mainly caused by radical oxygen species. Deleterious variants affecting this gene have been previously associated with microcephaly, epilepsy, and developmental delay.1 According to a previous report, homozygous loss-of-function substitution in PNKP was associated with cerebellar atrophy, neuropathy, microcephaly, epilepsy, and intellectual disability.2 Recently, whole-exome sequencing (WES) performed in a cohort of Portuguese families with ataxia with oculomotor apraxia (AOA) disclosed pathogenic variants in PNKP in 11 individuals. Other clinical features in that study included neuropathy, dystonia, cognitive impairment, decreased vibration sense, pyramidal signs, mild elevation in α-fetoprotein, and low levels of albumin. This condition was named AOA type 4 (OMIM #616267), as the phenotype of AOA has been previously associated with 3 other genes: APTX, SETX, and PIK3R5.3 Altogether, these reports demonstrate the great phenotypic diversity associated with PNKP mutations. In this article, we further enlarge this variability by demonstrating that early-onset axonal sensory-motor neuropathy (or axonal Charcot-Marie-Tooth (CMT) disease) followed years later by ataxia without oculomotor apraxia can be caused by deleterious variants in PNKP. Full consent was obtained from the patient and his parents for this publication. This study was approved by institutional ethics committees.

Overexpression of KLC2 due to a homozygous deletion in the non-coding region causes SPOAN syndrome

SPOAN syndrome is a neurodegenerative disorder mainly characterized by spastic paraplegia, optic atrophy and neuropathy (SPOAN). Affected patients are wheelchair bound after 15 years old, with progressive joint contractures and spine deformities. SPOAN patients also have sub normal vision secondary to apparently non-progressive congenital optic atrophy. A potential causative gene was mapped at 11q13 ten years ago. Here we performed next-generation sequencing in SPOAN-derived samples. While whole-exome sequencing failed to identify the causative mutation, whole-genome sequencing allowed to detect a homozygous 216-bp deletion (chr11.hg19:g.66,024,557_66,024,773del) located at the non-coding upstream region of the KLC2 gene. Expression assays performed with patients fibroblasts and motor neurons derived from SPOAN patients showed KLC2 overexpression. Luciferase assay in constructs with 216-bp deletion confirmed the overexpression of gene reporter, varying from 48 to 74%, as compared with wild-type. Knockdown and overexpression of klc2 in Danio rerio revealed mild to severe curly-tail phenotype, which is suggestive of a neuromuscular disorder. Overexpression of a gene caused by a small deletion in the non-coding region is a novel mechanism, which to the best of our knowledge, was never reported before in a recessive condition. Although the molecular mechanism of KLC2 up-regulation still remains to be uncovered, such example adds to the importance of non-coding regions in human pathology.

Chloroquine-induced glioma cells death is associated with mitochondrial membrane potential loss, but not oxidative stress.

Chloroquine (CQ), a quinolone derivative widely used to treat and prevent malaria, has been shown to exert a potent adjuvant effect when combined with conventional glioblastoma therapy. Despite inducing lysosome destabilization and activating p53 in human glioma cells, the mechanisms underlying cell death induced by this drug are poorly understood. Here, we analyzed in a time- and dose-dependent manner, the effects of CQ upon mitochondria integrity, autophagy regulation and redox processes in four human glioma cell lines that differ in their resistance to this drug. NAC-containing media protected cells against CQ-induced loss of mitochondrial membrane potential (MMP), autophagic vacuoles (LC3II) accumulation and loss of cell viability induced by CQ. However, we noticed that part of this protection was due to media acidification in NAC preparations, alerting for problems in experimental procedures using NAC. The results indicate that although CQ induces accumulation of LC3II, mitochondria, and oxidative stress, neither of these events is clearly correlated to cell death induced by this drug. The only event elicited in all cell lines at equitoxic doses of CQ was the loss of MMP, indicating that mitochondrial stability is important for cells resistance to this drug. Finally, the data indicate that higher steady-state MMP values can predict cell resistance to CQ treatment.