RESUMO
Xeroderma pigmentosum (XP) variant cells are deficient in the translesion synthesis (TLS) DNA polymerase Polη (eta). This protein contributes to DNA damage tolerance, bypassing unrepaired UV photoproducts and allowing S-phase progression with minimal delay. In the absence of Polη, backup polymerases perform TLS of UV lesions. However, which polymerase plays this role in human cells remains an open question. Here, we investigated the potential role of Polι (iota) in bypassing ultraviolet (UV) induced photoproducts in the absence of Polη, using NER-deficient (XP-C) cells knocked down for Polι and/or Polη genes. Our results indicate that cells lacking either Polι or Polη have increased sensitivity to UVC radiation. The lack of both TLS polymerases led to increased cell death and defects in proliferation and migration. Loss of both polymerases induces a significant replication fork arrest and G1/S-phase blockage, compared to the lack of Polη alone. In conclusion, we propose that Polι acts as a bona fide backup for Polη in the TLS of UV-photoproducts.
Assuntos
DNA Polimerase iota , Xeroderma Pigmentoso , Humanos , Dano ao DNA , Síntese de DNA Translesão , Replicação do DNA , Xeroderma Pigmentoso/genética , Raios Ultravioleta , Reparo do DNARESUMO
BACKGROUND AND AIMS: Telomere attrition is a major risk factor for end-stage liver disease. Due to a lack of adequate models and intrinsic difficulties in studying telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver disease in patients with telomere syndromes remain elusive. To circumvent that, we used genome editing to generate isogenic human embryonic stem cells (hESCs) harboring clinically relevant mutations in telomerase and subjected them to an in vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte development in vivo. APPROACH AND RESULTS: Using this platform, we observed that while telomerase is highly expressed in hESCs, it is quickly silenced, specifically due to telomerase reverse transcriptase component (TERT) down-regulation, immediately after endoderm differentiation and completely absent in in vitro-derived hepatocytes, similar to what is observed in human primary hepatocytes. While endoderm derivation is not impacted by telomere shortening, progressive telomere dysfunction impaired hepatic endoderm formation. Consequently, hepatocyte derivation, as measured by expression of specific hepatic markers as well by albumin expression and secretion, is severely compromised in telomerase mutant cells with short telomeres. Interestingly, this phenotype was not caused by cell death induction or senescence. Rather, telomere shortening prevents the up-regulation and activation of human hepatocyte nuclear factor 4 alpha (HNF4α) in a p53-dependent manner. Both reactivation of telomerase and silencing of p53 rescued hepatocyte formation in telomerase mutants. Likewise, the conditional expression (doxycycline-controlled) of HNF4α, even in cells that retained short telomeres, accrued DNA damage, and exhibited p53 stabilization, successfully restored hepatocyte formation from hESCS. CONCLUSIONS: Our data show that telomere dysfunction acts as a major regulator of HNF4α during hepatocyte development, pointing to a target in the treatment of liver disease in telomere-syndrome patients.
Assuntos
Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Telômero/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias , Hepatócitos/citologia , Humanos , Telomerase/genéticaRESUMO
The striking and complex phenotype of Cockayne syndrome (CS) patients combines progeria-like features with developmental deficits. Since the establishment of the in vitro culture of skin fibroblasts derived from patients with CS in the 1970s, significant progress has been made in the understanding of the genetic alterations associated with the disease and their impact on molecular, cellular, and organismal functions. In this review, we provide a historic perspective on the research into CS by revisiting seminal papers in this field. We highlighted the great contributions of several researchers in the last decades, ranging from the cloning and characterization of CS genes to the molecular dissection of their roles in DNA repair, transcription, redox processes and metabolism control. We also provide a detailed description of all pathological mutations in genes ERCC6 and ERCC8 reported to date and their impact on CS-related proteins. Finally, we review the contributions (and limitations) of many genetic animal models to the study of CS and how cutting-edge technologies, such as cell reprogramming and state-of-the-art genome editing, are helping us to address unanswered questions.
RESUMO
Tumorigenesis is associated with the development of a highly variable pattern of cellular diversity, consequence of genetic and epigenetic diversification, followed by clonal selection and expansion. This process is shaped by the microenvironment and leads to intratumoral heterogeneity, which is characterized by differences between cancer cells in terms of gene expression, phenotypic markers, growth dynamics, and resistance to treatment. Another relevant aspect in intratumor heterogeneity is cell plasticity-the ability of a cell to switch to new identities. In this review, we focus on the mechanisms that regulate cancer cell plasticity within a tumor, and explore the concept of tumor propagating cells, or TPCs, a cancer cell able to propagate/phenocopy the parental tumor and recapitulate tumor heterogeneity. We discuss the influence of the microenvironment and driver mutations on TPCs formation and function, the existence of phenotypically distinct TPC clones within a tumor, the evolution of TPCs with disease progression, and their implications for therapy.
Assuntos
Plasticidade Celular , Heterogeneidade Genética , Mutação , Neoplasias/fisiopatologia , Microambiente Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genéticaRESUMO
Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.
Assuntos
Disceratose Congênita/prevenção & controle , Células-Tronco Embrionárias/citologia , Hematopoese , Mutação , RNA Nucleotidiltransferases/antagonistas & inibidores , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Telomerase/metabolismo , Proteínas de Ciclo Celular/genética , Disceratose Congênita/metabolismo , Disceratose Congênita/patologia , Células-Tronco Embrionárias/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , Proteínas Nucleares/genética , RNA/genética , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Telomerase/genética , TelômeroRESUMO
Most of the studies on air pollution focus on emissions from fossil fuel burning in urban centers. However, approximately half of the world's population is exposed to air pollution caused by biomass burning emissions. In the Brazilian Amazon population, over 10 million people are directly exposed to high levels of pollutants resulting from deforestation and agricultural fires. This work is the first study to present an integrated view of the effects of inhalable particles present in emissions of biomass burning. Exposing human lung cells to particulate matter smaller than 10 µm (PM10), significantly increased the level of reactive oxygen species (ROS), inflammatory cytokines, autophagy, and DNA damage. Continued PM10 exposure activated apoptosis and necrosis. Interestingly, retene, a polycyclic aromatic hydrocarbon present in PM10, is a potential compound for the effects of PM10, causing DNA damage and cell death. The PM10 concentrations observed during Amazon biomass burning were sufficient to induce severe adverse effects in human lung cells. Our study provides new data that will help elucidate the mechanism of PM10-mediated lung cancer development. In addition, the results of this study support the establishment of new guidelines for human health protection in regions strongly impacted by biomass burning.
Assuntos
Poluentes Atmosféricos/toxicidade , Poluição do Ar , Morte Celular , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Exposição por Inalação , Pulmão/efeitos dos fármacos , Células A549 , Agricultura/métodos , Brasil , Conservação dos Recursos Naturais , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Modelos BiológicosRESUMO
Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase.
Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas Nucleares/genética , Nucleotidiltransferases/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Linhagem Celular Transformada , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribodipirimidina Fotoliase , Fibroblastos/citologia , Fibroblastos/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Humano , Humanos , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Dímeros de Pirimidina/metabolismo , Fase S/genética , Transdução Genética , Raios UltravioletaRESUMO
Oxidative DNA damage is considered to be a major cause of neurodegeneration and internal tumors observed in syndromes that result from nucleotide excision repair (NER) deficiencies, such as Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS). Recent evidence has shown that NER aids in removing oxidized DNA damage and may interact with base excision repair (BER) enzymes. Here, we investigated APE1 and OGG1 expression, localization and activity after oxidative stress in XPC-deficient cells. The endogenous APE1 and OGG1 mRNA levels were lower in XPC-deficient fibroblasts. However, XPC-deficient cells did not show hypersensitivity to oxidative stress compared with NER-proficient cells. To confirm the impact of an XPC deficiency in regulating APE1 and OGG1 expression and activity, we established an XPC-complemented cell line. Although the XPC complementation was only partial and transient, the transfected cells exhibited greater OGG1 expression and activity compared with XPC-deficient cells. However, the APE1 expression and activity did not significantly change. Furthermore, we observed a physical interaction between the XPC and APE1 proteins. Together, the results indicate that the responses of XPC-deficient cells under oxidative stress may not only be associated with NER deficiency per se but may also include new XPC functions in regulating BER proteins.
Assuntos
DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Cultivadas , DNA Glicosilases/genética , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoprecipitação , Oxidantes/farmacologia , Estresse Oxidativo , RNA Mensageiro/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologiaRESUMO
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.
Assuntos
Cloroquina/farmacologia , Glioma/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Acetilcisteína/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Glioma/metabolismo , Glioma/patologia , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Autophagy is a lysosome-dependent degradation pathway that allows cells to recycle damaged or superfluous cytoplasmic content, such as proteins, organelles, and lipids. As a consequence of autophagy, the cells generate metabolic precursors for macromolecular biosynthesis or ATP generation. Deficiencies in this pathway were associated to several pathological conditions, such as neurodegenerative and cardiac diseases, cancer, and aging. The aim of this review is to summarize recent discoveries showing that autophagy also plays a critical role in stem cell maintenance and in a variety of cell differentiation processes. We also discuss a possible role for autophagy during cellular reprogramming and induced pluripotent stem (iPS) cell generation by taking advantage of ATP generation for chromatin remodeling enzyme activity and mitophagy. Finally, the significance of autophagy modulation is discussed in terms of augmenting efficiency of iPS cell generation and differentiation processes.