RESUMO
BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.
Assuntos
Leucócitos Mononucleares , RecQ Helicases , Adulto , Feminino , Humanos , Masculino , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Leucócitos Mononucleares/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/metabolismo , Telômero/genética , Homeostase do Telômero/genéticaRESUMO
PURPOSE: To assess Meibomian gland dysfunction using meibography in patients with xeroderma pigmentosum and correlate with ocular surface changes. METHODS: This cross-sectional study evaluated patients with xeroderma pigmentosum. All patients underwent a comprehensive and standardized interview. The best-corrected visual acuity of each eye was determined. Detailed ophthalmic examination was conducted, including biomicroscopy examination of the ocular surface, Schirmer test type I, and meibography, and fundus examination was also performed when possible. Meibomian gland dysfunction was assessed by non-contact meibography using Oculus Keratograph® 5M (OCULUS Inc., Arlington, WA, USA). Saliva samples were collected using the Oragene DNA Self-collection kit (DNA Genotek Inc., Ottawa, Canada), and DNA was extracted as recommended by the manufacturer. Factors associated with abnormal meiboscores were assessed using generalized estimating equation models. RESULTS: A total of 42 participants were enrolled, and 27 patients underwent meibography. The meiboscore was abnormal in the upper eyelid in 8 (29.6%) patients and in the lower eyelid in 17 (62.9%). The likelihood of having abnormal meiboscores in the lower eyelid was 16.3 times greater than that in the upper eyelid. In the final multivariate model, age (p=0.001), mutation profile (p=0.006), and presence of ocular surface malignant tumor (OSMT) (p=0.014) remained significant for abnormal meiboscores. For a 1-year increase in age, the likelihood of abnormal meiboscores increased by 12%. Eyes with OSMT were 58.8 times more likely to have abnormal meiboscores than eyes without ocular surface malignant tumor. CONCLUSION: In the final model, age, xeroderma pigmentosum profile, previous cancer, and clinical alterations on the eyelid correlated with a meiboscore of ≥2. Meibomian gland dysfunction was common in patients with xeroderma pigmentosum, mainly in the lower eyelid. The severity of Meibomian gland dysfunction increases with age and is associated with severe eyelid changes.
Assuntos
Neoplasias Oculares , Disfunção da Glândula Tarsal , Xeroderma Pigmentoso , Humanos , Estudos Transversais , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/diagnóstico por imagem , Pálpebras , DNARESUMO
ABSTRACT Purpose: To assess Meibomian gland dysfunction using meibography in patients with xeroderma pigmentosum and correlate with ocular surface changes. Methods: This cross-sectional study evaluated patients with xeroderma pigmentosum. All patients underwent a comprehensive and standardized interview. The best-corrected visual acuity of each eye was determined. Detailed ophthalmic examination was conducted, including biomicroscopy examination of the ocular surface, Schirmer test type I, and meibography, and fundus examination was also performed when possible. Meibomian gland dysfunction was assessed by non-contact meibography using Oculus Keratograph® 5M (OCULUS Inc., Arlington, WA, USA). Saliva samples were collected using the Oragene DNA Self-collection kit (DNA Genotek Inc., Ottawa, Canada), and DNA was extracted as recommended by the manufacturer. Factors associated with abnormal meiboscores were assessed using generalized estimating equation models. Results: A total of 42 participants were enrolled, and 27 patients underwent meibography. The meiboscore was abnormal in the upper eyelid in 8 (29.6%) patients and in the lower eyelid in 17 (62.9%). The likelihood of having abnormal meiboscores in the lower eyelid was 16.3 times greater than that in the upper eyelid. In the final multivariate model, age (p=0.001), mutation profile (p=0.006), and presence of ocular surface malignant tumor (OSMT) (p=0.014) remained significant for abnormal meiboscores. For a 1-year increase in age, the likelihood of abnormal meiboscores increased by 12%. Eyes with OSMT were 58.8 times more likely to have abnormal meiboscores than eyes without ocular surface malignant tumor. Conclusion: In the final model, age, xeroderma pigmentosum profile, previous cancer, and clinical alterations on the eyelid correlated with a meiboscore of ≥2. Meibomian gland dysfunction was common in patients with xeroderma pigmentosum, mainly in the lower eyelid. The severity of Meibomian gland dysfunction increases with age and is associated with severe eyelid changes.
RESUMO
Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.
Assuntos
Neoplasias Cutâneas , Xeroderma Pigmentoso , Humanos , Xeroderma Pigmentoso/genética , Retroelementos/genética , Mutação , Reparo do DNA , Neoplasias Cutâneas/genética , Raios Ultravioleta/efeitos adversosAssuntos
Neoplasias Cutâneas , Xeroderma Pigmentoso , Humanos , Imiquimode/uso terapêutico , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/tratamento farmacológico , Estudos Prospectivos , Neoplasias Cutâneas/prevenção & controle , Neoplasias Cutâneas/tratamento farmacológico , QuimioprevençãoRESUMO
Human DNA polymerases can bypass DNA lesions performing translesion synthesis (TLS), a mechanism of DNA damage tolerance. Tumor cells use this mechanism to survive lesions caused by specific chemotherapeutic agents, resulting in treatment relapse. Moreover, TLS polymerases are error-prone and, thus, can lead to mutagenesis, increasing the resistance potential of tumor cells. DNA polymerase eta (pol eta) - a key protein from this group - is responsible for protecting against sunlight-induced tumors. Xeroderma Pigmentosum Variant (XP-V) patients are deficient in pol eta activity, which leads to symptoms related to higher sensitivity and increased incidence of skin cancer. Temozolomide (TMZ) is a chemotherapeutic agent used in glioblastoma and melanoma treatment. TMZ damages cells' genomes, but little is known about the role of TLS in TMZ-induced DNA lesions. This work investigates the effects of TMZ treatment in human XP-V cells, which lack pol eta, and in its complemented counterpart (XP-V comp). Interestingly, TMZ reduces the viability of XP-V cells compared to TLS proficient control cells. Furthermore, XP-V cells treated with TMZ presented increased phosphorylation of H2AX, forming γH2AX, compared to control cells. However, cell cycle assays indicate that XP-V cells treated with TMZ replicate damaged DNA and pass-through S-phase, arresting in the G2/M-phase. DNA fiber assay also fails to show any specific effect of TMZ-induced DNA damage blocking DNA elongation in pol eta deficient cells. These results show that pol eta plays a role in protecting human cells from TMZ-induced DNA damage, but this can be different from its canonical TLS mechanism. The new role opens novel therapeutic possibilities of using pol eta as a target to improve the efficacy of TMZ-based therapies against cancer.
Assuntos
Antineoplásicos , Xeroderma Pigmentoso , Antineoplásicos/farmacologia , DNA , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Temozolomida/farmacologia , Xeroderma Pigmentoso/genéticaRESUMO
Ultraviolet (UV) radiation is one of the most genotoxic, universal agents present in the environment. UVB (280-315 nm) radiation directly damages DNA, producing cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs). These photolesions interfere with essential cellular processes by blocking transcription and replication polymerases, and may induce skin inflammation, hyperplasia and cell death eventually contributing to skin aging, effects mediated mainly by keratinocytes. Additionally, these lesions may also induce mutations and thereby cause skin cancer. Photolesions are repaired by the Nucleotide Excision Repair (NER) pathway, responsible for repairing bulky DNA lesions. Both types of photolesions can also be repaired by distinct (CPD- or 6-4PP-) photolyases, enzymes that specifically repair their respective photolesion by directly splitting each dimer through a light-dependent process termed photoreactivation. However, as photolyases are absent in placental mammals, these organisms depend solely on NER for the repair of DNA UV lesions. However, the individual contribution of each UV dimer in the skin effects, as well as the role of keratinocytes has remained elusive. In this study, we show that in NER-deficient mice, the transgenic expression and photorepair of CPD-photolyase in basal keratinocytes completely inhibited UVB-induced epidermal thickness and cell proliferation. On the other hand, photorepair by 6-4PP-photolyase in keratinocytes reduced but did not abrogate these UV-induced effects. The photolyase mediated removal of either CPDs or 6-4PPs from basal keratinocytes in the skin also reduced UVB-induced apoptosis, ICAM-1 expression, and myeloperoxidase activation. These findings indicate that, in NER-deficient rodents, both types of photolesions have causal roles in UVB-induced epidermal cell proliferation, hyperplasia, cell death and inflammation. Furthermore, these findings also support the notion that basal keratinocytes, instead of other skin cells, are the major cellular mediators of these UVB-induced effects.
Assuntos
Desoxirribodipirimidina Fotoliase , Animais , DNA , Reparo do DNA , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Feminino , Hiperplasia , Inflamação , Queratinócitos/metabolismo , Mamíferos/genética , Camundongos , Placenta/metabolismo , GravidezRESUMO
Trypanosoma cruzi is the etiologic agent of Chagas' disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2-related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.
Assuntos
Doença de Chagas/parasitologia , Dano ao DNA , Interações Hospedeiro-Parasita , Estresse Oxidativo , Trypanosoma cruzi/fisiologia , Antioxidantes/metabolismo , Morte Celular , Linhagem Celular , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , Regulação para Baixo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells pretreated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.
RESUMO
Somatic hypermutation of immunoglobulin genes is a highly mutagenic process that is B cell-specific and occurs during antigen-driven responses leading to antigen specificity and antibody affinity maturation. Mutations at the Ig locus are initiated by Activation-Induced cytidine Deaminase and are equally distributed at G/C and A/T bases. This requires the establishment of error-prone repair pathways involving the activity of several low fidelity DNA polymerases. In the physiological context, the G/C base pair mutations involve multiple error-prone DNA polymerases, while the generation of mutations at A/T base pairs depends exclusively on the activity of DNA polymerase η. Using two large cohorts of individuals with xeroderma pigmentosum variant (XP-V), we report that the pattern of mutations at Ig genes becomes highly enriched with large deletions. This observation is more striking for patients older than 50 years. We propose that the absence of Pol η allows the recruitment of other DNA polymerases that profoundly affect the Ig genomic landscape.