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1.
Exp Dermatol ; 31(4): 608-614, 2022 04.
Article in English | MEDLINE | ID: mdl-34758172

ABSTRACT

The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.


Subject(s)
Glutathione , NF-kappa B , Antioxidants/metabolism , Endothelial Cells/metabolism , Glutathione/metabolism , Glutathione Disulfide/metabolism , Glutathione Disulfide/pharmacology , Homeostasis , Imiquimod/pharmacology , NF-kappa B/metabolism , Oxidation-Reduction , Oxidative Stress , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolism , Toll-Like Receptor 7
2.
RNA Biol ; 15(7): 845-848, 2018.
Article in English | MEDLINE | ID: mdl-29683386

ABSTRACT

Gene expression and DNA repair are fundamental processes for life. During the last decade, accumulating experimental evidence point towards different modes of coupling between these processes. Here we discuss the molecular mechanisms by which RNAPII-dependent transcription affects repair by the Nucleotide Excision Repair system (NER) and how NER activity, through the generation of single stranded DNA intermediates and activation of the DNA damage response kinase ATR, drives gene expression in a genotoxic scenario. Since NER-dependent repair is compromised in Xeroderma Pigmentosum (XP) patients, and having in mind that these patients present a high degree of clinical heterogeneity, we speculate that some of the clinical features of XP patients can be explained by misregulation of gene expression.


Subject(s)
DNA Repair/physiology , DNA, Single-Stranded/metabolism , Gene Expression/radiation effects , Xeroderma Pigmentosum/enzymology , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cockayne Syndrome/enzymology , DNA Damage , DNA Helicases/genetics , Humans , Mutation , RNA Polymerase II/metabolism , Skin/radiation effects , Transcription, Genetic/physiology , Ultraviolet Rays/adverse effects
3.
Cell Rep ; 18(12): 2868-2879, 2017 03 21.
Article in English | MEDLINE | ID: mdl-28329680

ABSTRACT

We have previously found that UV irradiation promotes RNA polymerase II (RNAPII) hyperphosphorylation and subsequent changes in alternative splicing (AS). We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs) abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER), and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.


Subject(s)
Alternative Splicing/genetics , DNA Repair , Pyrimidine Dimers/metabolism , Ultraviolet Rays , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA/metabolism , DNA Repair/genetics , Humans , Keratinocytes/metabolism , Keratinocytes/radiation effects , Phosphorylation/radiation effects , RNA Polymerase II/metabolism , Skin/cytology , Skin/radiation effects , Transcription, Genetic/radiation effects
4.
J Mol Biol ; 428(12): 2636-2651, 2016 Jun 19.
Article in English | MEDLINE | ID: mdl-26979557

ABSTRACT

Multicellular organisms must ensure genome integrity to prevent accumulation of mutations, cell death, and cancer. The DNA damage response (DDR) is a complex network that senses, signals, and executes multiple programs including DNA repair, cell cycle arrest, senescence, and apoptosis. This entails regulation of a variety of cellular processes: DNA replication and transcription, RNA processing, mRNA translation and turnover, and post-translational modification, degradation, and relocalization of proteins. Accumulated evidence over the past decades has shown that RNAs and RNA metabolism are both regulators and regulated actors of the DDR. This review aims to present a comprehensive overview of the current knowledge on the many interactions between the DNA damage and RNA fields.


Subject(s)
DNA Damage/genetics , RNA/genetics , DNA Repair/genetics , Gene Expression/genetics , Humans
5.
FEBS Lett ; 589(22): 3370-8, 2015 Nov 14.
Article in English | MEDLINE | ID: mdl-26296319

ABSTRACT

Coupling of transcription and alternative splicing via regulation of the transcriptional elongation rate is a well-studied phenomenon. Template features that act as roadblocks for the progression of RNA polymerase II comprise histone modifications and variants, DNA-interacting proteins and chromatin compaction. These may affect alternative splicing decisions by inducing pauses or decreasing elongation rate that change the time-window for splicing regulatory sequences to be recognized. Herein we discuss the evidence supporting the influence of template structural modifications on transcription and splicing, and provide insights about possible roles of non-B DNA conformations on the regulation of alternative splicing.


Subject(s)
Alternative Splicing , Chromatin/chemistry , Chromatin/genetics , DNA/chemistry , DNA/genetics , Animals , Humans , Transcription, Genetic/genetics
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