ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22phox that produces H2O2. Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H2O2, we detected an increase in H2O2 in the nucleus correlated with NOX4-p22phox perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence

Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases.

International audienceTo understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-transformed and untransformed fibroblasts representative of all genetic TTD complementation groups. Results showed that all cell lines from photosensitive TTD patients efficiently express Cat or luciferase genes in transfected plasmids carrying non-CPD lesions, including 6-4PP, and display wild-type or near-wild-type (50-70% in 3 cell lines) 6-4PP repair in the overall genome after immunoblot analysis. However, CPD lesions (the repair of which is defective in the overall genome) also block the expression of the reporter gene in transfected plasmids. Two cell lines from nonphotosensitive TTD patients showed wild-type levels of repair for both photoproducts in overall genome. A model on the lesion-specific repair in the context of the molecular defect in TTD is proposed. The implication of the defective CPD repair and efficient 6-4PP repair subpathways in cancer prevention in TTD patients is discussed

A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and nails, mental retardation, impaired sexual development, and ichthyosis. Photosensitivity has been reported in approximately 50% of the cases, but no skin cancer is associated with TTD. Virtually all photosensitive TTD patients have a deficiency in the nucleotide excision repair (NER) of UV-induced DNA damage that is indistinguishable from that of xeroderma pigmentosum (XP) complementation group D (XP-D) patients. DNA repair defects in XP-D are associated with two additional, quite different diseases; XP, a sun-sensitive and cancer-prone repair disorder, and Cockayne syndrome (CS), a photosensitive condition characterized by physical and mental retardation and wizened facial appearance. One photosensitive TTD case constitutes a new repair-deficient complementation group, TTD-A. Remarkably, both TTD-A and XP-D defects are associated with subunits of TFIIH, a basal transcription factor with a second function in DNA repair. Thus, mutations in TFIIH components may, on top of a repair defect, also cause transcriptional insufficiency, which may explain part of the non-XP clinical features of TTD. Besides XPD and TTDA, the XPB gene product is also part of TFIIH. To date, three patients with the remarkable conjunction of XP and CS but not TTD have been assigned to XP complementation group B (XP-B). Here we present the characterization of the NER defect in two mild TTD patients (TTD6VI and TTD4VI) and confirm the assignment to X-PB. The causative mutation was found to be a single base substitution resulting in a missense mutation (T119P) in a region of the XPB protein completely conserved in yeast, Drosophila, mouse, and man. These findings define a third TTD complementation group, extend the clinical heterogeneity associated with XP-B, stress the exclusive relationship between TTD and mutations in subunits of repair/transcription factor TFIIH, and strongly support the concept of "transcription syndromes.

Stable SV40-transformation and characterisation of some DNA repair properties of fibroblasts from a trichothiodystrophy patient

International audienceTo characterize nucleotide excision repair properties of cells from trichothiodystrophy (TTD) patients genetically-related to the xeroderma pigmentosum (XP) group D, TTD skin fibroblasts from two unrelated patients (TTD1VI and TTD2VI) belonging to the TTD/XPD group were transformed with a plasmid containing SV40 large T antigen-coding sequences and some DNA repair properties, such as unscheduled DNA synthesis (UDS), UV-survival, in vitro repair synthesis of cell extracts and reactivation of UV-irradiated reporter plasmid were studied. Results showed that: a) both untransformed and transformed TTD cells present a reduced UV-survival, compared to wild-type cells, but at significantly less reduced levels than XP-D cells; b) reduced repair activities were detected in both TTD and XP-D transformed cells by using in vitro cell free extract repair and reactivation of UV-irradiated plasmid procedures, and these relative reduced extents correlated with respective UV-survival; c) surprisingly, near wild-type UDS levels were detected in TTD2VILas transformed cells at different passages after the crisis, suggesting a phenotypic reversion of this transformed cell line; d) fluoro-cytometric analysis of TTD2VILas cells revealed a strong increase of a cell population containing a DNA amount more than twice as high than that of untransformed cells; finally, e) when UDS data were normalized to the DNA content in TTD2VILas cells, it appeared that the repair efficiency was only slightly higher than in untransformed cells. This implies that in transformed cells DNA repair properties should be evaluated, taking into account additional parameters. We obtained an immortalized TTD cell line which maintains DNA repair properties similar to those of parental untransformed cells and may be used to characterize the TTD defect at genetic, molecular and biochemical levels

Heterozygous mutations in the tumor suppressor gene PATCHED provoke basal cell carcinoma-like features in human organotypic skin cultures

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