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 mechanism in mammalian cells. This system entails multiple steps 1Departamento de Microbiologia, Instituto de Cieˆncias Biome´dicas, Universidade de Saõ Paulo, SP, Brazil, 2 Instituto de Pesquisas Energe´ticas e that employ a number of proteins to eliminate a broad Nucleares (IPEN), Comissaõ Nacional de Energia Nuclear (CNEN/SP), spectrum of structurally unrelated lesions such as UV-induced Supervisaõ de Radiobiologia, Saõ Paulo, SP, Brazil,3 Departamento de photoproducts, mainly cyclobutane pyrimidine dimers (CPDs), Anatomia, Instituto de Cieˆncias Biome´dicas, Universidade de Saõ Paulo, SP, and (6–4) pyrimidine-pyrimidone photoproducts (3). Group A Brazil and 4Departamento de Imunologia, Instituto de Cieˆncias Biome´dicas, Universidade de Saõ Paulo e Instituto de Investigaçaõ 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 Saõ protein, possibly in combination with replication protein A Paulo, Av. Prof. Lineu Prestes, 1374, Saõ 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).

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). The isolated clones INXA15 and 0 and 4 were divided and assigned to easily distinguishable categories 1, 2 µ XPIND were always kept in culture in the presence of zeocin (150 g/ml) and 3. The number of comets in each category was counted, and an average and neomycin (300 µg/ml) antibiotics for plasmid selection. DNA damage (DD) was calculated as follows: DD ϭ (n1 ϩ 2n2 ϩ 3n3 ϩ 4n4)/ Protein analysis (Σ/100), n1 – n4 ϭ number of comets in category 1 – 4, and Σ ϭ sum of all The proteins from cellular extracts were analyzed by electrophoresis in a 12% counted comets, including category 0. Repair of DNA damage (DR) was ϭ ϫ ϭ acrylamide-SDS gel electrophoresis. For western blot, total protein samples calculated as follows: DR DDi – DD24/(DDi – DD0) 100%, DDi DNA ϭ (30 µg/lane) were transferred to a Hybond-C membrane (Amersham Pharmacia damage after exposure at time 0; DD24 DNA damage after exposure at ϭ Biotech, USA) and probed with specific antibody, anti-XPA polyclonal (Santa repair time 24 h and DD0 DNA damage without irradiation. Cruz Biotechnology, CA, USA,). Secondary antibody anti-rabbit peroxidase- Determination of endonuclease sensitive sites (ESS) conjugate IgG was obtained from Sigma-Aldrich, USA. Band intensity was Cells were grown in a complete medium containing [3H]-thymidine determined by densitometry using a GS700 densitometer and the Molecular (0.5 µCi/ml) for 48 h and then UV irradiated with 2.5 J/m2. The cells were Analysis software (Bio-Rad Laboratories, CA, USA). cultivated for a period of time after UV-irradiation for DNA repair and then Immunocytochemistry for XPA protein detection harvested. Nuclei were prepared with 0.5% Triton X-100, 0.1 M NaCl and XPA protein presence in the cells was detected following the methodology 10 mM EDTA, washed twice with PBSA, and incubated in NET buffer ϭ described by Lotfi and Armelin (15). Briefly, cells were seeded on circular (100 mM NaCl, 10 mM Tris–HCl and 10 mM EDTA, pH 7.5) with cover slips and 24 h later fixed with 3.7% formaldehyde in phosphate buffered T4-endonuclease V for 30 min at 37°C. Molecular weights of untreated and treated DNA were determined by alkaline sucrose gradient sedimentation, as saline (PBSA), for 20 min. XPA protein was visualized by immunoperoxidase 7 staining using Vectastain Elite ABC kit and DAB (Vector Laboratories, CA, described before (19). The number of ESSs expressed by 10 daltons was USA). Nuclei positive for immune complex are heavily stained brown, calculated in relation to non-irradiated cells. ESSs estimated in nuclei whereas negative nuclei appear bluish stained with hematoxylin of Harris and correspond to approximately half of the CPDs, which are accessible to the UV-endonuclease within the chromatin. differentiated with a saturated solution of Li2CO3. Cover slips were randomly coded and 100–200 nuclei per cover slip were double-blinded counted. Flow cytometry analysis of individual cells by annexin V staining Cell survival determination by colony forming ability The apoptosis was detected based on the method described in Amarante- 2 Approximately 1500 cells were plated in 60 mm Petri dishes 14–16 h before Mendes et al. (20). After the UV treatment (10 J/m ), the supernatant and ϫ UV irradiation. Cells were washed twice with prewarmed PBSA and irradiated tripsinized adherent cells were collected and centrifuged 200 g for 5 min at ϭ with a low-pressure germicidal lamp (254 nm). After this, cells were maintained room temperature. The appropriate buffer (10 mM HEPES, pH 7.4, 150 mM in culture for 15 days, then being fixed with 10% formaldehyde and stained NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) was added to the cells with 1% violet crystal. Colonies with the minimal number of 15 cells were and centrifuged again under the same conditions. The pellet was then exposed scored. Survival values were obtained as the ratio of the number of colonies to the annexin-FITC solution (BD PharMingen, CA, USA) in the dark for µ µ from irradiated cells to non-irradiated cells. 10 min on ice. Next, 400 lofa100 g/ml of propidium iodide (PI) solution was added and immediately centrifuged. The PI excess was removed by Unscheduled DNA synthesis (UDS) washing the cells three times with the buffer. The final pellet was kept in the Analysis of DNA repair synthesis was carried out as previously described dark, on ice, until analysis, for a maximum of 1 h. Samples were transferred with modifications (16). Briefly, 104 cells were grown on glass cover slips to microtubes, and annexin V fluorescence was read by flow cytometry for 24 h. After 24 h of culture in a serum-deprived medium (0.5% FCS), (FACScalibur, Becton Dickinson), 10 000 events each. Results were obtained 1 µCi/ml of [3H]-methyl thymidine (86.0 Ci/mmol, Amersham Pharmacia as a percentage of M1, which represents apoptotic cells. Biotech, USA) was added to the medium for 1 h. The cells were washed with PBSA and then UV irradiated with 10 J/m2. After 3 h in the presence of 3H-thymidine, followed by a chase of 1 hour with cold thymidine (100 µM), Results the cells were fixed with methanol–acetate (3:1) mounted onto glass slides, and washed three times with 5% TCA for 15 min each, then being rinsed two Characterization of the clone INXA15 times with 70% ethanol and once with absolute ethanol. The slides were In order to modulate the xpa gene expression, inducible genetic dipped in EM-1 (Amersham Pharmacia Biotech, Inc., USA) emulsion and vectors were constructed, carrying the cDNA xpa. The system exposed for 1 week at 4°C. After development, the mean number of grains per nucleus was obtained by counting at least 30 non-S-phase nuclei. that best fitted into our experimental conditions was the muristerone A inducible vector described by No et al. (11), Alkaline single cell gel test (the comet assay) even though some leakiness is still present. The comet assay was conducted in a modified version of the method described by Collins et al. (17). Basically, a 1% normal agarose layer (90 µl) was The vectors pINXA or pIND and pVgRXR were cotrans- prepared on frosted microscope slides using 22 ϫ 22 mm cover slips. After fected into XP12RO, and cells were selected in a medium UV treatment (2.5 J/m2) and DNA repair time (0 or 24 h), cells were gently supplied with zeocin and neomycin antibiotics. Thirty clones 1040 Low amounts of XPA protein are enough for DNA repair

was only possible as an overexpressed product in INXA15 cells inducible by muristerone A (0.1 µM/48 h, data not shown). Even in control cells, carrying the intact xpa gene, the detection of XPA transcript was not possible under our conditions. INXA15 survival after UV irradiation Since the XPA protein levels can be monitored in INXA15 cells, experiments were performed to check biological functions of this protein. Cell survival after UV irradiation was deter- Fig. 1. XPA expression from INXAs clones. Protein extracts from the cells mined in different cells and conditions. The results are shown were analyzed by western blot, using anti-XPA as a probe: MRC5-V1, DNA in Figure 4. As expected, XP12RO and XPIND cell lines are repair proficient cells (positive control); XPIND, XP12RO cells with the extremely sensitive to UV-irradiation. DNA repair proficient empty vectors (negative control); clones INXA15, INXA16, INXA17 and ϩ µ cells (MRC5-V1) present a higher UV resistance. The INXA15 INXA18, induced ( ) or not (–) with 0.1 M/72 h of muristerone A (Mu). clone is able to recover UV resistance in both reduced and overexpressed levels of the XPA protein, their survival curves were obtained and the expression of the XPA protein in these being indistinguishable. Moreover, the overexpression of xpa cells was analyzed in western blots, under conditions of does not promote any toxic effect to the cells, plating efficiency induction or without muristerone A. The clone INXA15 showed in all situations being similar (~40%). the highest XPA expression when 0.1 µM of muristerone A Unscheduled DNA synthesis activity in the INXA15 clone was added to the cell medium, with very low basal levels To correlate the recovery of UV survival with the ability to under non-induction conditions. As a negative control, we perform DNA repair synthesis, the incorporation of established a cell population by the cotransfection of the empty [3H]-thymidine was measured following UV irradiation using vector pIND and the pVgRXR plasmid. In Figure 1, an classical UDS method. The results are shown in Figure 5. example of such western blots is shown when a protein band XPIND cells exhibit a reduced UDS level after UV exposition with ~42 kDa, corresponding to the XPA protein, is observed. (4.4 Ϯ 1.9 grains per nucleus and 3.8 Ϯ 1.7 in non-irradiated Clearly, the XPA expression in the INXA15 cells is dependent cells). Control cells MRC5-V1 exhibit a high UDS level, on the presence of muristerone A in the medium. The inducible 32.5 Ϯ 1.4 after UV treatment, and 3.7 Ϯ 1.7 grains per response is better for these cells than for any other clone. No nucleus in non-irradiated cells. For INXA15 cells, the UDS XPA protein was detected in XPIND cell extracts, and the level was restored to almost normal levels under induced and levels of XPA induction in INXA15 cells were consistently non-induced conditions after UV exposition, at 38.9 Ϯ 27.3 higher than in MRC5-V1 control cells. and 33.7 Ϯ 24.4 grains per nucleus, respectively. However, it In situ immunocytochemistry was also employed to check is clear that the distribution of DNA repair activity in the for XPA expression. This technique allows for the detection INXA15 clone is very heterogeneous when compared to control of XPA expression in single cells, thus providing more accurate cells. This heterogeneity in the number of grains per nucleus data regarding the efficiency of the cloning process and the may reflect differences in XPA protein expression in single homogeneity of protein expression within a cell population. cells, as observed in immunocytochemistry. Results are presented in Figure 2 and it can be observed that some INXA15 cells were expressing enough XPA protein to Removal of CPDs after UV irradiation be detected by this technique (2.7%) even in the absence of To define the events associated with DNA damage and repair muristerone A, this possibly reflects the basal expression that could be correlated with survival, the removal of associated with this inducible system. After induction, the photoproducts by global genomic repair was assayed by T4 percentage of cells expressing XPA rises to 83.9%. Despite endonuclease DNA cleavage detection by the ‘comet assay’ the fact that the majority of cells are expressing XPA after and sedimentation on alkaline sucrose gradients. In both induction, this expression was not completely homogeneous. methodologies, CPD frequencies in cells carrying the cDNA Heavy and light brown stained cells could be detected which xpa with or without muristerone A induction were compared might be a signal of differential XPA expression rates. In order with DNA repair proficient and deficient cells. Figure 6 shows to better evaluate the ability of the INXA15 clone to induce typical results obtained with the ‘comet assay’. In preparations XPA expression, 0.1 µM of muristerone A was added to the from control cells, the undamaged DNA remains within a culture medium for different periods of time. brightly fluorescent nuclei core. However, when the DNA Figure 3 summarizes the kinetics of XPA expression induc- contains breaks, it moves from this core towards the anode, tion. The increased expression starts to be detected after 4 h, forming an image aptly described as a ‘comet’. The size of and 48 h after hormone addition the levels of XPA protein the comet, and the distribution intensity of fluorescence, has detected had increased ~20 times in relation to non-induced been correlated quantitatively with the amount of DNA breaks cells. Comparing XPA amounts in different human cells by (21). In this work, DNA breaks were introduced by cleavage the western blot, the basal levels of XPA protein expression with T4 endonuclease V, which nicks specifically at CPD sites. in INXA15 cells were found to be 20 (Ϯ8)% (average of five The DNA damage repair (DR) rate was used to compare different experiments) of what is normally obtained in human different cell lines after UV irradiation (2.5 J/m2). The XPA fibroblast cell lines, normal for the NER process (HeLa and deficient cell line (XPIND) was not able to reduce the ‘comet MRC5-V1). The induction with 0.1 µM of muristerone A in tail’ and even a slight increase of tail size was observed in 24 and 48 h can increase the XPA protein up to two and four the 24 h following UV irradiation (DR ϭ –3.5%). For MRC5- times, respectively, compared to control cells. Interestingly, V1 cells and INXA15 cells, induced or not by muristerone A, the detection of the XPA transcript by Northern blot (~1.2 kb) there is a strong decrease of the comet tail if they are allowed 1041 A.R.Muotri et al.

Fig. 2. Immunocytochemistry for XPA protein detection in human cell lines. Cells were prepared as described in Material and methods and anti-XPA was used as a probe. Nuclei positive for XPA are heavily stained brown, negative nuclei appears bluish. Mu, muristerone A induction 0.1 µM/48 h before. Scale bar corresponds to 5 µm.

Fig. 3. Kinetics of XPA induction by muristerone A. (A) Protein extracts from cells harvested at different periods after Mu (0.1 µM) addition were analyzed by western blot, using anti-XPA as a probe. (B) Values obtained from quantiﬁcation by densitometry of ‘A’, in relation to the non-induced situation.

Fig. 4. UV survival of INXA15 cells is similar to DNA repair proficient to repair for 24 h of culture after UV irradiation. The cell lines. Cells were UV-irradiated at the indicated doses and the survival is results indicated that the DR for MRC5-V1, INXA15 and determined as a colony-forming ability. INXA15 ϩ Mu were 52.9, 51.4 and 50.0%, respectively. The number of ESSs remaining in the genome 24 and 48 h after UV irradiation was also determined by alkaline For the cell lines proficient in XPA protein, the removal of sucrose gradients. As shown in Figure 7, the XPA deficient ESS seems to be similar, as observed in the comet assay cells are unable to remove CPDs. In fact, there is a reproducible methodology. These results indicated that even in cells increase in the number of breaks in DNA (independent of T4 expressing low levels of XPA protein, INXA15 without a endonuclease V, data not shown) 48 h after UV irradiation. hormone, a normal DNA repair activity is detected. 1042 Low amounts of XPA protein are enough for DNA repair

Fig. 6. CPDs removal detected by the ‘comet assay’. Nuclei from non- irradiated (A) and UV-irradiated cells (2.5 J/m2) harvested immediately (B) or 24 h after UV (C) were treated with T4 endonuclease V in order to generate the breaks that are detected as tails in this assay. Cell lines are indicated. Scale bar corresponds to 5 µm.

Fig. 7. CPDs removal kinetics by sucrose gradients in different human cell lines. Remaining SSE was determined as described in Materials and methods. Cells lines are indicated.

in clear contrast with the DNA repair complementation results reported above.

Fig. 5. DNA repair activity (UDS) in different human cell lines. UDS was determined as described in Material and methods. The UDS activity is Discussion expressed by grains per nucleus. (A) XPIND; (B) MRC5-V1; (C) INXA15; XPA protein plays an important role in the initial steps of and (D) INXA15 ϩ Mu. Black bars, non-irradiated cells and gray bars, UV- irradiated cells (10 J/m2). nucleotide excision repair pathways. After DNA damage recognition by either the XPC-hHR23B complex (for global genomic repair – GGR) or the RNA transcription machinery (for transcription coupled repair – TCR), XPA binds to damaged UV-induced apoptosis DNA, interacting with several other DNA repair proteins. It UV-induced apoptosis was also determined in these INXA15 may act by correctly positioning these proteins in relation to cells, under conditions of XPA induction or not. This was the damage (4,23). Thus, this protein is an essential part of done by FACs analysis of annexin V positive cells. Figure 8A the preincision complex of nucleotide excision repair. This illustrates the kind of results obtained and Figure 8B presents crucial role implies that cells deficient in this protein are the average of four experiments performed. As can be observed, completely unable to repair certain types of DNA damage, the early events of UV-induced apoptosis are highly increased including those induced by UV irradiation, resulting in severe in XPA deficient cells (XP12RO and XPIND), when compared anomalies in patients that are genetically XPA deficient (2). with SV40-transformed control cells, confirming results Modulation of XPA protein levels within the cells could, in observed by others (22). Cells overexpressing XPA protein principal, directly affect the first steps of nucleotide excision (INXA15 cells in the presence of muristerone A) behave as repair, thus limiting final performance. This modulation was control cells, with lower levels of annexin V positive events. achieved by constructing a cell line (INXA15) that expresses However, INXA15 cells expressing low amounts of XPA the xpa gene under the control of an inducible promoter. The protein (without inductor) also increased UV-induced results, however, indicate that although we were able to apoptosis, at least at early times (2 and 8 h after UV). This is adjust the amount of XPA protein by just adding hormone 1043 A.R.Muotri et al.

reflect the differential expression of the xpa gene in different tissues (7). In fact, NER requires the activity of an intricate combination of protein factors, and possibly some of them may be limiting in the process. But these limitations may differ depending on the cells employed and on the kind of damage being studied. In our observations not only global genomic repair was fully complemented by low quantities of XPA protein, but also cell survival after UV irradiation was indistinguishable between control cells and XP cells expressing low or high levels of XPA protein. These results are consistent with the work of Kobayashi et al. (27), who have also found that cells overproducing XPA protein have a UV resistance similar to control cells (Hela and WI38VA13 cell lines). Thus, our results point to the fact that low amounts of XPA (~20% of the protein found in normal human cells) are sufficient for carrying out excision repair, and this protein is not a limiting factor for global genomic repair or cell resistance to UV irradiation. Cleaver et al. (8) have employed a different inducible promoter Fig. 8. UV-induced apoptosis in different human cell lines. (A) Histogram analysis immediately, and 8 h, after 10 J/m2 of UV irradiation. Data system to modulate the expression of the xpa gene in the obtained from FACs analysis. M1 represents the cell percentage positive for XP12RO cell line, with some of their cell clones overexpressing annexin V. Mu: 0.1 µM/48 h of muristerone A before UV exposition. (B) the xpa cDNA presented an increased resistance to UV light Percentage of apoptotic cells (M1) after UV irradiation (10 J/m2)in when compared to control cells (GM637). Although the authors µ different periods of time. Mu: 0.1 M/48 h of muristerone A before UV interpret these results as due to a possible rate-limiting role exposition. of XPA protein, our data completely contrast with their observations, as expression of four times more XPA protein, (muristerone A) into the cell medium, the effects on DNA relative to normal control levels, did not produce cells more repair synthesis, CPD removal or cell resistance to UV could resistant to UV. The different cell survival observed by these not be detected, as even low concentrations of XPA were authors could be explained by a different general genetic sufficient for full complementation of these endpoints. background between XP12RO and GM637 cell lines, and not UDS analysis directly detects DNA synthesis by the damage to the expression of high levels of XPA protein alone. Curiously, repair apparatus (24). INXA15 cells are able to complement Cleaver et al. (8) also did not detect any removal of CPDs by DNA repair synthesis deficiency for XPA cells in both reduced immunological methods, in cells overexpressing xpa cDNA, and overexpressed conditions, with no sign of cell toxicity. in complete discrepancy to the normal global genome repair Cells overexpressing XPA does not have an increase in UDS of these lesions detected in INXA15 cells. Since a different activity. Curiously, in both cases there is a clear heterogeneity technology was employed, it is difficult to compare the two in the distribution of cells capable or not of performing UDS, sets of data, although normal CPD removal is the expected which gives a good correlation with the heterogeneity of XPA result for UV resistance recovery in cells overproducing XPA protein expression detected by immunocytochemistry. The protein. It is also important to note, however, that DNA repair, removal of CPDs was also analyzed by detecting these lesions especially GGR of CPDs, may be reduced in SV40-transformed with T4 endonuclease V (ESSs), employing the comet assay cell lines. This may occur due to the fact that LT-antigen of and sedimentation in alkaline sucrose gradients. These are SV40 binds to the p53 tumor suppressor protein (28). This direct measurements of global genome nucleotide excision could also interfere in the expression of p48 protein (deficient repair for CPDs. In both analyses, there was a full DNA repair in XPE individuals), necessary for GGR activity in mammalian complementation in INXA15 cells, even in the absence of the cells (29,30). In this work, the use of both control and XP-A inducer. These results are consistent with classical findings cell lines, transformed with SV40, was performed in order to that human heterozygous for the xpa gene and, more recently, minimize possible differences due to p53 inhibition, as GGR heterozygous knockout mice, do not present any clinical activity is probably equally affected. symptoms. Cells from human or mouse heterozygous support During analysis of CPD removal in these cells, we found normal levels of DNA repair (25). Giannelli and Pawsey (26) that XPA deficient cell lines have an increased number of found a similar conclusion when analyzing heterokaryons of breaks measured by alkaline sucrose gradients or comet assay. XP and normal cells concluding that small quantities of xpa A part of these breaks is independent of T4 endonuclease V mRNA is enough to complement DNA repair synthesis. These cleavage, that is, they occur as a product of cell metabolism results indicate that the modulation of xpa expression does not in these cells (31,32). These breaks could be a consequence interfere with the rate of CPD removal, at least in global of UV induced apoptosis, which is normally increased in these genomic repair. cells (22). This prompted us to analyze the patterns of apoptosis However, Koberle et al. (10) reached different conclusions. in these SV40-transformed cells. We were unable to obtain a These authors found that testicular germ tumor cells are clear pattern of internucleosomal cleavage (also known as particularly sensitive to cisplatin, this explaining the success DNA ladder) in any of the cell lines employed, except for of cisplatin-based chemotherapy. In vitro experiments indicated HeLa cells (33). However, detection of the externalization of that extracts from these cells have a reduced ability to carry phosphatidil serine by annexin V, an early signal of apoptosis, on repair of cisplatin-induced DNA damage, this probably was observed in these cells in a short period after UV being due to reduced levels of XPA protein. This may also irradiation. This phenomenon is detected much faster in 1044 Low amounts of XPA protein are enough for DNA repair

XP12RO cells, when compared with MRC5-V1 cells: 2 h after Acknowledgements UV ~20% of the XP cells present these signals, this frequency This work was supported by the Fundaçaõ de Amparo a` Pesquisa do Estado still going up 8 h and later, while control cells have a much de Saõ Paulo – FAPESP (proc. # 98/11119-7, Saõ Paulo, Brazil) and Conselho delayed entry into apoptosis. However, cells that have only a Nacional de Desenvolvimento Cient´ıfico e Tecnolo´gico (CNPq, Bras´ılia, low amount of XPA protein behave like deficient cells, while Brazil). M.C.N.M. and G.B. have MS Fellowships and A.R.M. has a PhD cells that overexpress this protein behave like control cells. fellowship from FAPESP, respectively. We thank Dr Roge´rio Meneghini (IQ- USP, Brazil) for providing the T4 endonuclease V and Dr Alain Sarasin for These results are distinct from what have been observed for the cell lines XP12RO and MRC5-V1 (Laboratory of Genetic Instability and other endpoints such as DNA repair measurements and UV Cancer, CNRS, Institut Andre´ Lwoff, Villejuif, France). resistance, where low levels of XPA protein induce full complementation. Yamaizumi and Sugano (34) have proposed References that apoptotic responses to UV can be related to the repair 1.Machado,C.R. and Menck,C.F.M. (1997) Mini-review: Human DNA capacity of actively transcribed genes. Moreover, human cell repair diseases: from genome instability to cancer. Braz. J. Gen., 20, lines deficient in TCR, such as those deficient in the xpa gene, 755–762. have shown to present a strong correlation between inhibition of 2.Moriwaki,S. and Kraemer,K.H. (2001) Xeroderma pigmentosum: bridging RNA transcription and apoptosis induction after UV irradiation a gap between clinic and laboratory. Photodermatol. Photoimmunol. Photomed., 2,47–54. (35,36). Removal of CPDs by photoreactivation prevents the 3.De Boer,J. and Hoeijmakers,J.H.J. (2000) Nucleotide excision repair and early steps of UV induced apoptosis, which is coherent with human syndromes. Carcinogenesis, 21, 453–460. the hypothesis that DNA damage may block RNA transcription 4.De Laat,W., Jaspers,N.G.J. and Hoeijmakers,J.H.J. (1999) Molecular by RNA polymerase, signaling for apoptosis in the UV mechanism of nucleotide excision repair. Genes Develop., 13, 768–785. irradiated cells (33,37). Cells that have inefficient systems for 5.Sugasawa,K., Ng,J.M., Matsutani,C., et al. (1998) Two human homologs of Rad23 are functionally interchangeable in complex formation and the removal of these lesions have increased signals for stimulation of XPC repair activity. Mol. Cell Biol., 17, 6924–6931. apoptosis. Indeed, Van Oosten et al. (38) have demonstrated 6.Batty,D.P. and Wood,R.D. (2000) Damage recognition in nucleotide that TCR is responsible for apoptosis prevention after UV excision repair of DNA. Gene, 241, 193–204. irradiation. Therefore, it would be interesting to investigate 7.Layer,S.K. and Cleaver,J.E. (1997) Quantification of XPA gene expression levels in human and mouse cell lines by competitive RT-PCR. Mutat. the efficiency of TCR and RNA transcription inhibition in Res., 383,9–19. non-induced INXA15 cells, in order to check for the possibility 8.Cleaver,J.E., Charles,W.C., McDowell,M.L., Sadinski,W.J. and that XPA protein may in fact be rate limiting for this kind of Mitchell,D.L. (1995) Overexpression of the xpa repair gene increases repair. Kobayashi et al. (27) have performed similar experi- resistance to ultraviolet radiation in human cells by selective repair of ments with another inducible system for this gene, but the DNA damage. Cancer Res., 55, 6152–6160. 9.Goukassian,D., Gad,F., Yaar,M., Eller,M.S., Nehal,U.S. and Gilcherest,B.A. reduction in TCR was very low and they did not analyze (2000) Mechanisms and implications of the age-associated decrease in global genomic repair to confirm whether XPA may denote a DNA repair capacity. FASEB J., 14, 1325–1334. preference to act in one or the other kind of repair. Interestingly, 10.Koberle,B., Masters,J.R.W., Hartley,A.A. and Wood,R.D. (1999) Defective the differences observed for early apoptosis signals (externaliz- repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cells tumors. Curr. Biol., 9, 273–276. ation of phosphatidil serine) and cell death (UV survival) 11.No,D., Yao,T.P. and Evas,R.M. (1996) Ecdysone-inducible gene expression indicate that these two events may bev inde- in mammalian cells and transgenic mice. Proc. Natl Acad. Sci. USA, 93, pendent. This is in agreement with recent findings with 3346–3351. Caenorhabiditis elegans cells, which can still recover cell 12.Triezenberg,S.J., LaMarco,K.L. and McKnight,S.L. (1988) Evidence of survival after a stage that resembles the early phase of apoptosis DNA:protein interaction that mediate HSV-1 immediate early gene activation by VP16. Genes Dev., 2, 730–742. (39,40). Thus, changes such as externalization of phosphatidil 13.Satokata,I., Tanaka,K., Yuba,S. and Okada,Y. (1992) Identification of serine in UV-irradiated mammalian cells are also not necessar- splicing mutations of the last nucleotides of exons, a nonsense mutation ily fatal. and a missense mutation of the XPAC gene as causes of group A xeroderma Finally, the fact that modulation of XPA protein production pigmentosum. Mutat. Res., 273, 203–212. 14.Felgner,P.L., Gadek,T.R., Holm,M., Roman,R., Chan,H.W., Wenz,M., did not allow for modulation of global nucleotide excision Northrop,J.P., Ringold,G.M. and Danielsen,M. (1987) Lipofectin: a highly repair and UV resistance indicates that this protein is not rate efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. limiting in the process. As only a small amount of protein can Sci. USA, 84, 7413–7417. cope with the global repair, it may also point to a short time 15.Lotfi,C.F. and Armelin,.HA. (2001) cfos and cjun antisense oligonucleotides as being required for its presence during repair. This can be block mitogenesis triggered by fibroblast growth factor-2 and ACTH in mouse Y1 adrenocortical cells. J. Endocrinol., 168, 381–389. possible once XPA participates in the early recognition steps 16.Friedberg,E.C. and Hanawalt,P.C. (1981) DNA Repair: A Laboratory of NER, and, once the other proteins are correctly placed for Manual of Research Procedures. Marcel Dekker, New York, Vols. 1 and 2. damage removal, XPA could be liberated to participate in a 17.Collins,A.R. (1993) Mutant rodent cell lines sensitive to ultraviolet light following event of repair. The high stability of the XPA protein ionizing radiation and cross-linking agents. A comprehensive survey of genetic and biochemical characteristics. Mutat. Res., 293, 99–118. may also contribute to these data. In fact, previous findings 18.Jaloszynski,P., Kujawski,M., Czub-Swierczek,M., Markowska,J. and indicated that DNA repair could be carried on in the absence Szyfter,K. (1997) Bleomycin-induced DNA damage and its removal in of protein synthesis in such a way that the turnover period for lymphocytes of breast cancer patients studied by comet assay. Mutat. Res., this protein in human cells must be very long (41). High 385, 223–233. protein stability is also consistent with the very low levels of 19.Menck,C.F.M. and Meneghini,R. (1982) Resistance of 3T3 mouse cells to UV light in relation to excision and transfer of dimers to daughter strands. 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