Effect Of XPA Protein In DNA Damage And Repair After UVB Irradiation And Intervention Study Of EGCG

BackgroundUltraviolet irradiation of sunlight including UVA and UVB is the most powerful wavelength which causes sunburn,photoaging and skin tumors.The photo-damage effect of UVB is 800～1000 times stronger than that of UVA under the same dosage irradiation.UVB irradiation can cause the generation of free radicals and relevant reactive oxygen species (ROS),the cytokine production and secretion,the local and systemic immune suppression,DNA damage and mutation,and contribute to photo-aging and photo-carcinogenesis.The skin is the first protective layer to UV irradiation and keratinocytes of the skin are the important target cells of UV irradiation.UVB radiation leads directly production of photoproducts,the most prevalent products cyclobutane pyrimidine dimmers(CPDs)and n-(6-4)-photoproducts(6-4PPs).These lesions are repaired by nucleotide excision repair(NER),including recognition and processing of DNA damage,unwinding of DNA by helicases,excision of the damage-containing fragment and re-synthesis by DNA polymerase. More than 30 kinds of genes and proteins such as XPA,ERCC1,PCNA, RPA,p53 are involved in the procedures of DNA repair.The XPA protein specifically recognizes the UV damaged DNA lesion,and triggers the NER process.XPA binds to RPA or ERCC1.The ERCC1 polypeptide is required for NER of damaged DNA.In mammalian cells XPG cleaves 3' of the DNA lesion while the ERCC1-XPF complex makes the 5' incision. If UVB-induced lesions are not repaired,DNA mutations representing initial events of multi-step carcinogenesis could occur.Xeroderma pigmentosum(XP)is an autosomal recessive photosensitive disorder with an extremely high incidence of UV-related skin cancers associated with impaired ability to repair UV-induced DNA damage.There are seven NER complementation groups(A through G) and a NER proficient form(XP variant).DNA damage is not repaired by the NER system equally throughout the genome.There are two DNA repair pathways:1)transcription-coupled repair,and 2)global genome repair.Many factors involved in these pathways are related to the pathogenesis of XP.There is no effective therapy for XP,therefore recovering of NER of DNA damage and tumor immune will be a new way to cure XP.Clinical management consists of early diagnosis followed by a rigorous program of sun protection including avoidance of unnecessary UV exposure,wearing UV blocking clothing,and use of sunblocks on the skin.Although there is no cure for XP,the efficacy of oral retinoids for the prevention of new skin cancers and the external use of a prokaryotic DNA repair enzyme have been reported. Curing hereditary human diseases by providing functional copies of relevant genes is an active area of ongoing research.The XPA genetic complementation group(defective in the XPA gene)comprises one of the largest groups among XP patients.We assume that a recombinant lentivirus as a vehicle for delivery of human XPA cDNA to XPA-mutant human skin cell may be a new therapy for XP.EGCG,which is the major and most effective component of TP extracted from green tea,has been proved to be able to improve antioxidation, anti-inflammation and anti-carcinogenesis.We focused on the production and removal of CPDs in primary keratinocyte cells after UVB irradiation and the intervention of EGCG;effects of XPA and ERCC1 protein in DNA damage and repair after UVB irradiation and the intervention of EGCG,and then the protein expressions of XPA in primary kerationcyte cells infected by XPA lentivirus vector under UVB irradiation and EGCG intervention.What we try is to find a new possibility for XP treatment by gene therapy,meanwhile it will contribute to the development and application of the natural sunscreens.ObjectiveTo study the photo-damage of UVB on HaCaT cells and photo-protection of EGCG and to choose the experimental concentration of EGCG;to study the production and removal of CPDs in primary KC after UVB irradiation and the intervention of EGCG;to observe the protein expressions of XPA and ERCC1 in HaCaT cells and primary KC and intervention of EGCG;to observe the protein expressions of XPA in XPA lentivirus vector infected primary kerationcyte cells under UVB irradiation and EGCG intervention.Materials and methods1.Selection of EGCG concentration:HaCaT cells were treated with different concentrations of EGCG and irritated by UVB.MTT assay was used to measure cell viability and concentration of EGCG was choosed.2.Cell culture:HaCaT cells were cultured in RMPI-1640 medium with 10%calf serum and cells were plated in 3.5 cm dishes and 96-well plate with an equal number of cells.Primary kerationcyte cells were cultured in K-SFM medium and cells were plated in 6-well plate with an equal number of cells.3.Ultraviolet irradiation:The time-effect and dosage-effect of UVB irradiation were conducted according to the experiment design.EGCG was added into the medium before or after UVB irradiation.4.Photoproducts assay:The production and removal of CPDs in primary KC after UVB irradiation were dectected by immunohistochemical method.Positive stained cells were calculated by microscope.5.XPA lentivirus vector infected primary kerationcyte cells:XPA lentivirus vector was constructed and packaged and tite was determined. Primary kerationcyte cells were infected by XPA lentivirus vector. 6 Western blotting:Total protein of cells was harvested and extracted. The protein expressions of XPA and ERCC1 of different conditioned samples were measured by Western blotting.7 Statistical analysis:The experimental data were analyzed with SPSS.P values less than 0.05 were considered to be statistically significant.Results1.EGCG of 25-200μg/mL had no cytotoxicity on viability of HaCaT cells.UVB could decrease cell viability and the photo-damage was dose-dependent.EGCG could reduce the damage caused by UVB irradiation and the concentration of 100,50μg/mL were optimal selection.2.Naturally,CPDs could be removed rapidly from primary keratinocyte cells during the first 4 h and then removed slowly and almost cleared out up to 24 h after UVB irradiation.The production of thymine dimers was decreased when primary keratinocyte cells were preincubated with EGCG before UVB irradiation.The removal of thymine dimers was accelerated when primary keratinocyte cells were coincubated with EGCG immediately right after UVB irradiation.3.UVB irradiation could increase the protein expressions of XPA and ERCC1 in cultured HaCaT cells and primary KC and protein levels of XPA and ERCC1 were decreased after EGCG intervention in UVB irradiated group to some degree.4.XPA lentivirus vector could infect primary kerationcyte cells successfully and XPA protein was expressed.XPA protein expression was increased after UVB irradiation and EGCG could down-regulate the expression of XPA protein in lentivirus vector.ConclusionsThe photodamage effect of UVB irradiation on cultured HaCaT cells is dose-dependent and EGCG can relieve cell damage caused by UVB irradiation partly and efficiently.There seems to be two phases in the removal of CPDs:a rapid phase occurring within the first 4h and a slower phase thereafter up to 24h.EGCG added into medium before UVB irradiation can decrease the production of CPDs,EGCG added into medium after UVB irradiation can accelerate the removal of CPDs. EGCG has the potential photo-protective efficacy on HaCaT cells and primary keratinocyte cells damaged from UVB irradiation.Decreasing the expressions of proteins of XPA and ERCC1 genes may be part of the mechanisms related to reducing the photo-damage effects of UVB irradiation.XPA lentivirus vector could infect primary kerationcyte cells successfully and XPA protein was expressed.It would be a new way for XP therapy.