RESUMEN
Although correlations between RNA polymerase II (RNAPII) transcription stress, R-loops, and genome instability have been established, the mechanisms underlying these connections remain poorly understood. Here, we used a mutant version of the transcription elongation factor TFIIS (TFIISmut), aiming to specifically induce increased levels of RNAPII pausing, arrest, and/or backtracking in human cells. Indeed, TFIISmut expression results in slower elongation rates, relative depletion of polymerases from the end of genes, and increased levels of stopped RNAPII; it affects mRNA splicing and termination as well. Remarkably, TFIISmut expression also dramatically increases R-loops, which may form at the anterior end of backtracked RNAPII and trigger genome instability, including DNA strand breaks. These results shed light on the relationship between transcription stress and R-loops and suggest that different classes of R-loops may exist, potentially with distinct consequences for genome stability.
Asunto(s)
Inestabilidad Genómica , Estructuras R-Loop , ARN Mensajero/genética , Estrés Fisiológico , Transcripción Genética , Factores de Elongación Transcripcional/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Mutación , ARN Polimerasa II/metabolismo , Empalme del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/genéticaRESUMEN
UV-induced photoproducts are responsible for the pathological effects of sunlight. Mutations in nucleotide excision repair (NER) cause severe pathologies characterized by sunlight sensitivity, coupled to elevated predisposition to cancer and/or neurological dysfunctions. We have previously shown that in UV-irradiated non-cycling cells, only a particular subset of lesions activates the DNA damage response (DDR), and this requires NER and EXO1 activities. To define the molecular mechanism acting at these lesions, we demonstrate that Y family TLS polymerases are recruited at NER- and EXO1-positive lesion sites in non-S phase cells. The coordinated action of EXO1 and Y family TLS polymerases promotes checkpoint activation, leads to lesion repair, and is crucial to prevent cytotoxic double-strand break (DSB) formation.
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Puntos de Control del Ciclo Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Rayos Ultravioleta/efectos adversos , Muerte Celular/efectos de la radiación , Línea Celular , Enzimas Reparadoras del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Exodesoxirribonucleasas/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transporte de Proteínas , ADN Polimerasa iotaRESUMEN
Pancreatic cancer is a very aggressive disease with a dismal prognosis. The tumor microenvironment exerts immunosuppressive activities through the secretion of several cytokines, including interleukin (IL)-1. The IL-1/IL-1 receptor (IL-1R) axis is a key regulator in tumor-promoting T helper (Th)2- and Th17-type inflammation. Th2 cells are differentiated by dendritic cells endowed with Th2-polarizing capability by the thymic stromal lymphopoietin (TSLP) that is secreted by IL-1-activated cancer-associated fibroblasts (CAFs). Th17 cells are differentiated in the presence of IL-1 and other IL-1-regulated cytokines. In pancreatic cancer, the use of a recombinant IL-1R antagonist (IL1RA, anakinra, ANK) in in vitro and in vivo models has shown efficacy in targeting the IL-1/IL-1R pathway. In this study, we have developed sphingomyelin nanosystems (SNs) loaded with ANK (ANK-SNs) to compare their ability to inhibit Th2- and Th17-type inflammation with that of the free drug in vitro. We found that ANK-SNs inhibited TSLP and other pro-tumor cytokines released by CAFs at levels similar to ANK. Importantly, inhibition of IL-17 secretion by Th17 cells, but not of interferon-γ, was significantly higher, and at lower concentrations, with ANK-SNs compared to ANK. Collectively, the use of ANK-SNs might be beneficial in reducing the effective dose of the drug and its toxic effects.
Asunto(s)
Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1 , Neoplasias Pancreáticas , Esfingomielinas , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Humanos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1/metabolismo , Esfingomielinas/metabolismo , Citocinas/metabolismo , Línea Celular Tumoral , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Células Th17/inmunología , Células Th17/efectos de los fármacos , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/efectos de los fármacos , Células Th2/metabolismo , Microambiente Tumoral/efectos de los fármacos , Nanopartículas/química , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/efectos de los fármacosRESUMEN
The role and regulation of innate immune cells is poorly understood in B-cell non-Hodgkin lymphoma (NHL). As natural killer (NK) cells, helper innate lymphoid cells (ILCs) are lymphocytes endowed with either anti- or pro-tumour activity and involved in inflammatory processes. In our ex vivo analysis of NK cells and ILCs from NHL patients, we observed that, in comparison to healthy donors (HD), the frequency of the cytotoxic subset of NK cells, the CD16+ NK, decreased in patients' peripheral blood. In general, circulating NK cells showed a pro-tumorigenic phenotype, while ILCs displayed a more activated/cytotoxic phenotype. Conversely, at the tumour site, in patients' lymph nodes, ILCs showed a low expression of granzyme.In vitromixed lymphocyte-tumour cell cultures with HD PBMCs and NHL cell lines demonstrated that ILC cytotoxic potential was lowered by the presence of tumour cells but, in the absence of T regulatory cells (Tregs), their cytolytic potential was recovered. Our data shed novel light on dysfunctional innate immunity in NHL. We suggest a new mechanism of tumour immuno-escape based on the reduction of cell cytotoxicity involving ILCs and likely controlled by Tregs.
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Antineoplásicos , Linfoma no Hodgkin , Neoplasias , Humanos , Escape del Tumor , Inmunidad Innata , Linfocitos , Células Asesinas Naturales , Neoplasias/patología , Linfoma no Hodgkin/patologíaRESUMEN
The role of innate lymphoid cells (ILCs), including natural killer (NK) cells, is pivotal in inflammatory modulation and cancer. Natural killer cell activity and count have been demonstrated to be regulated by the expression of activating and inhibitory receptors together with and as a consequence of different stimuli. The great majority of NK cell populations have an anti-tumor activity due to their cytotoxicity, and for this reason have been used for cellular therapies in cancer patients. On the other hand, the recently classified helper ILCs are fundamentally involved in inflammation and they can be either helpful or harmful in cancer development and progression. Tissue niche seems to play an important role in modulating ILC function and conversion, as observed at the transcriptional level. In the past, these cell populations have been classified by the presence of specific cellular receptor markers; more recently, due to the advent of single-cell RNA sequencing (scRNA-seq), it has been possible to also explore them at the transcriptomic level. In this article we review studies on ILC (and NK cell) classification, function and their involvement in cancer. We also summarize the potential application of NK cells in cancer therapy and give an overview of the most recent studies involving ILCs and NKs at scRNA-seq, focusing on cancer. Finally, we provide a resource for those who wish to start single-cell transcriptomic analysis on the context of these innate lymphoid cell populations.
RESUMEN
The costs of developing, validating and buying new drugs are dramatically increasing. On the other hand, sobering economies have difficulties in sustaining their healthcare systems, particularly in countries with an elderly population requiring increasing welfare. This conundrum requires immediate action, and a possible option is to study the large, already present arsenal of drugs approved and to use them for innovative therapies. This possibility is particularly interesting in oncology, where the complexity of the cancer genome dictates in most patients a multistep therapeutic approach. In this review, we discuss a) Computational approaches; b) preclinical models; c) currently ongoing or already published clinical trials in the drug repurposing field in oncology; and d) drug repurposing to overcome resistance to previous therapies.
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Reposicionamiento de Medicamentos , Neoplasias , Anciano , Humanos , Neoplasias/tratamiento farmacológicoRESUMEN
The never-ending explosion in the cost of new oncology drugs is reducing in many countries the access to the most recent, effective anticancer therapies and represents a significant obstacle to the design and realization of combinatorial trials. Already approved, anticancer and nonanticancer drugs can be considered for in silico, preclinical, and clinical repurposing approaches and offer the significant advantages of a potentially cheaper, faster, and safer validation. This review discusses recent advances and challenges in the field.
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Reposicionamiento de Medicamentos/métodos , Humanos , Oncología MédicaRESUMEN
The local UV irradiation technique enables detection, kinetic measurements of recruitment, and quantification of DNA Damage Response (DDR) proteins at the site of UV-induced DNA damage.Using Isopore filters with high density pores of a broad range of sizes, it is possible to UV irradiate and damage only a very small portion of the nucleus of a cell by letting UV light pass only through the pores. Immunofluorescent analyses of modified DNA nucleotides, proteins, or fluorescently tagged versions of target factors can be used as markers to label and study UV-induced lesions and their repair.