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Low Amounts of the DNA Repair XPA Protein Are Sufficient to Recover UV

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Low Amounts of the DNA Repair XPA Protein Are Sufficient to Recover UV Carcinogenesis vol.23 no.6 pp.1039–1046, 2002 Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance Alysson R.Muotri1, Maria C.N.Marchetto1, to UV rays, high predisposition for developing skin cancers Miriam F.Suzuki2, Kayo Okazaki2, Claudimara F.P.Lotfi3, on sunlight exposed areas, and in some cases, neurological Gabriela Brumatti4, Gustavo P.Amarante-Mendes4 and disorders (1,2). XP cells have impaired nucleotide excision Carlos F.M.Menck1,5 repair (NER). NER is considered a major DNA repair mechan- ism in mammalian cells. This system entails multiple steps 1Departamento de Microbiologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, SP, Brazil, 2 Instituto de Pesquisas Energe´ticas e that employ a number of proteins to eliminate a broad Nucleares (IPEN), Comissa˜o Nacional de Energia Nuclear (CNEN/SP), spectrum of structurally unrelated lesions such as UV-induced Supervisa˜o de Radiobiologia, Sa˜o Paulo, SP, Brazil,3 Departamento de photoproducts, mainly cyclobutane pyrimidine dimers (CPDs), Anatomia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, SP, and (6–4) pyrimidine-pyrimidone photoproducts (3). Group A Brazil and 4Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo e Instituto de Investigac¸a˜o em Imunologia, XP cells are defective in the XPA protein essential for NER Instituto do Mileˆnio, SP, Brazil acting together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage (4). The XPA 5To whom corrrespondence should be addressed at: Departmento de Microbiologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o protein, possibly in combination with replication protein A Paulo, Av. Prof. Lineu Prestes, 1374, Sa˜o Paulo/SP, 05508-900, Brazil (RPA), is involved in the pre-incision step of NER, in both Email: [email protected] global and transcription coupled repair (TCR) (5,6). Tissue- DNA integrity is threatened by the damaging effects of specific variations in the levels of XPA mRNAs were physical and chemical agents that can affect its function. demonstrated, suggesting that XPA expression may be tran- Nucleotide excision repair (NER) is one of the most known scriptionally regulated in a cell-type-specific manner (7). and flexible mechanisms of DNA repair. This mechanism Because DNA damage recognition is certainly the most can recognize and remove damages causing DNA double- challenging and rate-limiting step of any repair process, and helix distortion, including the cyclobutane pyrimidine is particularly intriguing in the case of NER, as the process dimers (CPDs) and the pyrimidine-pyrimidone (6–4) can repair so many different kinds of DNA lesions, the XPA photoproducts, promoted by ultraviolet light (UV). The protein has been elected as one possible candidate for NER human syndrome xeroderma pigmentosum (XP) is clinic- modulation. In fact, it has been shown that modest increases ally characterized chiefly by the early onset of severe in xpa expression can have dramatic effects on UV resistance photosensitivity of the exposed regions of the skin, a very by the selective repair of DNA damage (8). It has also been high incidence of skin cancers and frequent neurological shown that age-associated decrease in the repair of UV-induced abnormalities. The xpa gene seems to be involved during DNA damage results at least in part from decreased levels of UV damage recognition, in both global genome repair proteins that participate in the repair process, such as XPA (GGR) and transcription-coupled repair (TCR). The modu- (9). Moreover, low levels of XPA protein have been proposed lation of xpa expression may modify the DNA repair rate to be the principal cause of cisplatin sensitivity in testis in the cell genome, providing a valuable contribution to tumours (10). These reports could lead to the simple conclusion an understanding of the NER process. The controlled that intracellular concentration of XPA is responsible for NER expression of the cDNA xpa in XP12RO deficient cells modulation in mammalian cells. The consequences of this was achieved through the transfection of a muristerone-A interpretation are (i) the XPA protein is the key factor for inducible vector, pINXA. The INXA15 clone shows good NER and TCR modulation; (ii) the natural cell resistance induction of the XPA protein and total complementation against DNA damage is limited by XPA concentration and of XP12RO cell deficiency. Overexpression of this protein (iii) cell resistance to DNA lesions could be improved by resulted in UV cell survival comparable to normal control increasing xpa expression. human cells. Moreover, low expression of the XPA protein Contrary to this idea, we herein describe an efficient system in these cells is sufficient for total complementation in for xpa modulation in human cells. Based on experimental cellular UV sensitivity and DNA repair activity. These results, we conclude that xpa modulation in human cells does data demonstrate that XPA protein concentration is not a not interfere with DNA repair rates measured by UDS and limiting factor for DNA repair. CPDs elimination, and has no consequence on cellular UV-sensitivity. These results suggest that this protein may only be transiently employed in nucleotide excision repair. However, Introduction low levels of XPA protein in the cells led to an increased number of apoptotic cells a short time after UV-irradiation. Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease clinically characterized by hypersensitivity Materials and methods Abbreviations: CPDs, cyclobutane pyrimidine dimers; ESS, endonuclease- Construction of the pINXA muristerone A-inducible vector sensitive site; GGR, global genome repair; Mu, muristerone A hormone; Xpa cDNA was obtained from Dr Alain Sarasin (Laboratory of Genetic NER, nucleotide excision repair; TCR, transcription-coupled repair; UDS, Instability and Cancer, CNRS, Institut Andre´ Lwoff, Villejuif, France) and unscheduled DNA repair; UV, ultraviolet; XPA, xeroderma pigmentosum was cloned into the Eco RI restriction site of the pIND plasmid in the sense group A. position related to the promoter, generating the pINXA inducible vector (11). © Oxford University Press 1039 A.R.Muotri et al. These vectors are normally cotransfected with the pVgRXR vector. The trypsinized and resuspended at 1 ϫ 106 cells/ml with 0.7% low melting point ecdysone-inducible expression system utilizes a heterodimer of the ecdysone agarose. A second layer was added to each slide using 45 µl of the cell receptor (VgEcR) and the retinoid X receptor (RXR), that binds a hybrid suspension. Slides were maintained on ice and in the dark. After this, the ecdysone response element (E/GRE) in the presence of the synthetic analog slides were immersed in a lysis solution (2.5 M NaCl, 200 mM NaOH, 10% of ecdysone, muristerone A (11). The ecdysone receptor (VgEcR) is derived dimethyl sulphoxide and 1% Triton X-100, pH ϭ 10.0) at 4°C for at least from the natural Drosophila receptor and modified to contain the VP16 1 h and then washed (3 ϫ 160 ml for 5 min) with T4 endonuclease V buffer transaction domain (12). Binding of the heterodimer to the modified ecdysone (10 mM, Tris–HCl pH ϭ 8.0, 10 mM EDTA and 75 mM NaCl). Cells response element (EcRE), present in the Drosophila minimal heat shock embedded in agarose were overlaid with 50 µl of T4 endonuclease V promoter in the pIND vector, activates transcription (11). (20 µg/ml). Cover slips were added and the slides incubated for 30 min at Cell culture and clonal selection 37°C in a humidified atmosphere. Control slides were incubated as above with 50 µl of buffer. Slides were then placed in a chilled alkali electrophoresis Cells were routinely grown at 37°Cin5%CO2 humidified atmosphere in a buffer (300 mM NaOH, 1 mM EDTA) for 25 min, and 25 V 300 mA were Dulbecco Modified Essential Medium (DMEM, Life Technologies, USA) applied for 25 min more. Following electrophoresis, slides were washed three supplemented with heat inactivated 10% fetal calf serum (FCS, Cultilab, times with neutralizing Tris-buffer (400 mM Tris base, pH ϭ 7.5) for µ Brazil) and antibiotics at 1 g/ml each of penicillin and streptomycin and 5 min each and stained with 30 µl of ethidium bromide solution µ 2.5 g/ml of fungizone. The MRC5-V1 cell line is derived from the normal (20 mg/ml). The extent of DNA migration was evaluated by visual scoring lung tissue of a 14-week-old male fetus. The HeLa cell line is derived from under a fluorescent microscope equipped with a 515–560 nm filter, the cervical adenocarcinoma of a 31-year-old Negro female (ATCC CCL2). 250 ϫ (LEICA DMLB). The number of comets counted on each slide ranged → XP12RO fibroblast cells carry a C T transition at nucleotide 619 of exon 5 from 100 to 200, depending on cell density. The extent of DNA migration in the xpa gene. This mutation alters the Arg-207 codon (CGA) to a nonsense was evaluated by visual scoring. Quantitative DNA damage repair (DR) was codon (TGA) in both alleles (13). MRC5-V1 and HeLa cells are normal for estimated according to the method firstly described by Jaloszynski et al. (18). DNA excision repair. MRC5-V1 and XP12RO cells are transformed by SV40 Comets were classified and assigned to five categories (0–4) according to the and were kindly provided by Dr Alain Sarasin (Laboratory of Genetic extent of DNA-tail migration. Cells with bright heads and no apparent tails Instability and Cancer, CNRS, Institut Andre´ Lwoff, Villejuif, France). The were assigned to category 0, comets with very small heads and long diffused µ XP12RO cells were co-transfected with 5 g of pINXA or pIND and pVgRXR tails to category 4. Comets displaying features intermediary between category plasmids, with 1 mg/ml of lipofectin (14).
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