The relative roles of DNA damage induced by UVA irradiation in human cells.

UVA light (320-400 nm) represents approximately 95% of the total solar UV radiation that reaches the Earth's surface. UVA light induces oxidative stress and the formation of DNA photoproducts in skin cells. These photoproducts such as pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs, and pyrimidine (6-4) pyrimidone photoproducts, 6-4PPs) are removed by nucleotide excision repair (NER). In this repair pathway, the XPA protein is recruited to the damage removal site; therefore, cells deficient in this protein are unable to repair the photoproducts. The aim of this study was to investigate the involvement of oxidative stress and the formation of DNA photoproducts in UVA-induced cell death. In fact, similar levels of oxidative stress and oxidised bases were detected in XP-A and NER-proficient cells exposed to UVA light. Interestingly, CPDs were detected in both cell lines; however, 6-4PPs were detected only in DNA repair-deficient cells. XP-A cells were also observed to be significantly more sensitive to UVA light compared to NER-proficient cells, with an increased induction of apoptosis, while necrosis was similarly observed in both cell lines. The induction of apoptosis and necrosis in XP-A cells using adenovirus-mediated transduction of specific photolyases was investigated and we confirm that both types of photoproducts are the primary lesions responsible for inducing cell death in XP-A cells and may trigger the skin-damaging effects of UVA light, particularly skin ageing and carcinogenesis.

Protective effect of a Phyllanthus orbicularis aqueous extract against UVB light in human cells.

CONTEXT: One approach to protect human skin against the dangerous effects of solar ultraviolet (UV) irradiation is the use of natural products, such as photoprotectors. Phyllanthus orbicularis Kunth (Euphorbiaceae) is a Cuban endemic plant used in popular medicine. Its antigenotoxicity effect against some harmful agents has been investigated. However, the effect in ultraviolet B (UVB)-irradiated human cells has not been previously assessed. OBJECTIVE: The protective effect of a P. orbicularis extract against UVB light-induced damage in human cells was evaluated. MATERIALS AND METHODS: DNA repair proficient (MRC5-SV) and deficient (XP4PA, complementation group XPC) cell-lines were used. Damaging effects of UVB light were evaluated by clonogenic assay and apoptosis induction by flow cytometry techniques. The extent of DNA repair itself was determined by the removal of cyclobutane pyrimidine dimers (CPDs). The CPDs were detected and quantified by slot-blot assay. RESULTS: Treatment of UVB-irradiated MRC5-SV cells with P. orbicularis extract increased the percentage of colony-forming cells from 36.03 ± 3.59 and 4.42 ± 1.45 to 53.14 ± 8.8 and 14.52 ± 1.97, for 400 and 600 J/m(2), respectively. A decrease in apoptotic cell population was observed in cells maintained within the extract. The P. orbicularis extract enhanced the removal of CPD from genomic DNA. The CPDs remaining were found to be about 27.7 and 1.1%, while with plant extract, treatment these values decreased to 16.1 and 0.2%, for 3 and 24 h, respectively. DISCUSSION AND CONCLUSION: P. orbicularis aqueous extract protects human cells against UVB damage. This protective effect is through the modulation of DNA repair effectiveness.

Development of a DNA-dosimeter system for monitoring the effects of solar-ultraviolet radiation.

Solar radiation sustains and affects all life forms on Earth. In recent years, the increase in environmental levels of solar-UV radiation due to depletion of the stratospheric ozone layer, as a result of anthropogenic emission of destructive chemicals, has highlighted serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions, where the intensity of solar radiation is higher. To better understand the impact of the harmful effects of solar-UV radiation on the DNA molecule, we developed a reliable biological monitoring system based on the exposure of plasmid DNA to artificial UV lamps and sunlight. The determination and quantification of different types of UV photoproducts were performed through the use of specific DNA repair enzymes and antibodies. As expected, a significant number of CPDs and 6-4PPs was observed when the DNA-dosimeter system was exposed to increasing doses of UVB radiation. Moreover, CPDs could also be clearly detected in plasmid DNA when this system was exposed to either UVA or directly to sunlight. Interestingly, although less abundant, 6-4PPs and oxidative DNA damage were also generated after exposure to both UVA and sunlight. These results confirm the genotoxic potential of sunlight, reveal that UVA may also produce CPDs and 6-4PPs directly in naked DNA and demonstrate the applicability of a DNA-dosimeter system for monitoring the biological effects of solar-UV radiation.

CPDs and 6-4PPs play different roles in UV-induced cell death in normal and NER-deficient human cells.

Ultraviolet (UV) light generates two major DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs), but the specific participation of these two lesions in the deleterious effects of UV is a longstanding question. In order to discriminate the precise role of unrepaired CPDs and 6-4PPs in UV-induced responses triggering cell death, human fibroblasts were transduced by recombinant adenoviruses carrying the CPD-photolyase or 6-4PP-photolyase cDNAs. Both photolyases were able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair (NER) to a similar extent, while in NER-proficient cells UV-induced apoptosis was prevented only by CPD-photolyase, with no effects observed when 6-4PPs were removed by the specific photolyase. These results strongly suggest that both CPDs and 6-4PPs contribute to UV-induced apoptosis in NER-deficient cells, while in NER-proficient cells, CPDs are the only lesions responsible for UV-killing, probably due to the rapid repair of 6-4PPs by NER. As a consequence, the difference in skin photosensitivity, including carcinogenesis, of most of the xeroderma pigmentosum patients and of normal people is probably not only a quantitative aspect, but depends on the type of DNA damage induced by sunlight and its rate of repair.

Adenovirus mediated transduction of the human DNA polymerase eta cDNA.

Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.

Restoring DNA repair capacity of cells from three distinct diseases by XPD gene-recombinant adenovirus.

The nucleotide excision repair (NER) is one of the major human DNA repair pathways. Defects in one of the proteins that act in this system result in three distinct autosomal recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). TFIIH is a nine-protein complex essential for NER activity, initiation of RNA polymerase II transcription and with a possible role in cell cycle regulation. XPD is part of the TFIIH complex and has a helicase function, unwinding the DNA in the 5' --> 3' direction. Mutations in the XPD gene are found in XP, TTD and XP/CS patients, the latter exhibiting both XP and CS symptoms. Correction of DNA repair defects of these cells by transducing the complementing wild-type gene is one potential strategy for helping these patients. Over the last years, adenovirus vectors have been largely used in gene delivering because of their efficient transduction, high titer, and stability. In this work, we present the construction of a recombinant adenovirus carrying the XPD gene, which is coexpressed with the EGFP reporter gene by an IRES sequence, making it easier to follow cell infection. Infection by this recombinant adenovirus grants full correction of SV40-transformed and primary skin fibroblasts obtained from XP-D, TTD and XP/CS patients.