Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance
Alysson R. Muotri1,
Maria C.N. Marchetto1,
Miriam F. Suzuki2,
Kayo Okazaki2,
Claudimara F.P. Lotfi3,
Gabriela Brumatti4,
Gustavo P. Amarante-Mendes4 and
Carlos F.M. Menck1,5
1 Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil,
2 Instituto de Pesquisas Energéticas e Nucleares (IPEN), Comissão Nacional de Energia Nuclear (CNEN/SP), Supervisão de Radiobiologia, São Paulo, SP, Brazil,
3 Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil and
4 Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo e Instituto de Investigacião em Imunologia, Instituto do Milênio, SP, Brazil
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Abstract
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DNA integrity is threatened by the damaging effects of physical and chemical agents that can affect its function. Nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove damages causing DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (64) photoproducts, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). The modulation of xpa expression may modify the DNA repair rate in the cell genome, providing a valuable contribution to an understanding of the NER process. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15 clone shows good induction of the XPA protein and total complementation of XP12RO cell deficiency. Overexpression of this protein resulted in UV cell survival comparable to normal control human cells. Moreover, low expression of the XPA protein in these cells is sufficient for total complementation in cellular UV sensitivity and DNA repair activity. These data demonstrate that XPA protein concentration is not a limiting factor for DNA repair.
Abbreviations: CPDs, cyclobutane pyrimidine dimers; ESS, endonuclease-sensitive site; GGR, global genome repair; Mu, muristerone A hormone; NER, nucleotide excision repair; TCR, transcription-coupled repair; UDS, unscheduled DNA repair; UV, ultraviolet; XPA, xeroderma pigmentosum group A.
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Introduction
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Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease clinically characterized by hypersensitivity to UV rays, high predisposition for developing skin cancers on sunlight exposed areas, and in some cases, neurological disorders (1,2). XP cells have impaired nucleotide excision repair (NER). NER is considered a major DNA repair mechanism in mammalian cells. This system entails multiple steps that employ a number of proteins to eliminate a broad spectrum of structurally unrelated lesions such as UV-induced photoproducts, mainly cyclobutane pyrimidine dimers (CPDs), and (64) pyrimidine-pyrimidone photoproducts (3). Group A XP cells are defective in the XPA protein essential for NER acting together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage (4). The XPA protein, possibly in combination with replication protein A (RPA), is involved in the pre-incision step of NER, in both global and transcription coupled repair (TCR) (5,6). Tissue-specific variations in the levels of XPA mRNAs were demonstrated, suggesting that XPA expression may be transcriptionally regulated in a cell-type-specific manner (7).
Because DNA damage recognition is certainly the most challenging and rate-limiting step of any repair process, and is particularly intriguing in the case of NER, as the process can repair so many different kinds of DNA lesions, the XPA protein has been elected as one possible candidate for NER modulation. In fact, it has been shown that modest increases in xpa expression can have dramatic effects on UV resistance by the selective repair of DNA damage (8). It has also been shown that age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process, such as XPA (9). Moreover, low levels of XPA protein have been proposed to be the principal cause of cisplatin sensitivity in testis tumours (10). These reports could lead to the simple conclusion that intracellular concentration of XPA is responsible for NER modulation in mammalian cells. The consequences of this interpretation are (i) the XPA protein is the key factor for NER and TCR modulation; (ii) the natural cell resistance against DNA damage is limited by XPA concentration and (iii) cell resistance to DNA lesions could be improved by increasing xpa expression.
Contrary to this idea, we herein describe an efficient system for xpa modulation in human cells. Based on experimental results, we conclude that xpa modulation in human cells does not interfere with DNA repair rates measured by UDS and 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, low levels of XPA protein in the cells led to an increased number of apoptotic cells a short time after UV-irradiation.
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Materials and methods
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Construction of the pINXA muristerone A-inducible vector
Xpa cDNA was obtained from Dr Alain Sarasin (Laboratory of Genetic Instability and Cancer, CNRS, Institut André Lwoff, Villejuif, France) and was cloned into the Eco RI restriction site of the pIND plasmid in the sense position related to the promoter, generating the pINXA inducible vector (11). These vectors are normally cotransfected with the pVgRXR vector. The ecdysone-inducible expression system utilizes a heterodimer of the ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), that binds a hybrid ecdysone response element (E/GRE) in the presence of the synthetic analog of ecdysone, muristerone A (11). The ecdysone receptor (VgEcR) is derived from the natural Drosophila receptor and modified to contain the VP16 transaction domain (12). Binding of the heterodimer to the modified ecdysone response element (EcRE), present in the Drosophila minimal heat shock promoter in the pIND vector, activates transcription (11).
Cell culture and clonal selection
Cells were routinely grown at 37°C in 5% CO2 humidified atmosphere in a Dulbecco Modified Essential Medium (DMEM, Life Technologies, USA) supplemented with heat inactivated 10% fetal calf serum (FCS, Cultilab, Brazil) and antibiotics at 1 µg/ml each of penicillin and streptomycin and 2.5 µg/ml of fungizone. The MRC5-V1 cell line is derived from the normal lung tissue of a 14-week-old male fetus. The HeLa cell line is derived from the cervical adenocarcinoma of a 31-year-old Negro female (ATCC CCL2). XP12RO fibroblast cells carry a C
T transition at nucleotide 619 of exon 5 in the xpa gene. This mutation alters the Arg-207 codon (CGA) to a nonsense codon (TGA) in both alleles (13). MRC5-V1 and HeLa cells are normal for DNA excision repair. MRC5-V1 and XP12RO cells are transformed by SV40 and were kindly provided by Dr Alain Sarasin (Laboratory of Genetic Instability and Cancer, CNRS, Institut André Lwoff, Villejuif, France). The XP12RO cells were co-transfected with 5 µg of pINXA or pIND and pVgRXR plasmids, with 1 mg/ml of lipofectin (14). The isolated clones INXA15 and XPIND were always kept in culture in the presence of zeocin (150 µg/ml) and neomycin (300 µg/ml) antibiotics for plasmid selection.
Protein analysis
The proteins from cellular extracts were analyzed by electrophoresis in a 12% acrylamide-SDS gel electrophoresis. For western blot, total protein samples (30 µg/lane) were transferred to a Hybond-C membrane (Amersham Pharmacia Biotech, USA) and probed with specific antibody, anti-XPA polyclonal (Santa Cruz Biotechnology, CA, USA,). Secondary antibody anti-rabbit peroxidase-conjugate IgG was obtained from Sigma-Aldrich, USA. Band intensity was determined by densitometry using a GS700 densitometer and the Molecular Analysis software (Bio-Rad Laboratories, CA, USA).
Immunocytochemistry for XPA protein detection
XPA protein presence in the cells was detected following the methodology described by Lotfi and Armelin (15). Briefly, cells were seeded on circular cover slips and 24 h later fixed with 3.7% formaldehyde in phosphate buffered saline (PBSA), for 20 min. XPA protein was visualized by immunoperoxidase staining using Vectastain Elite ABC kit and DAB (Vector Laboratories, CA, USA). Nuclei positive for immune complex are heavily stained brown, whereas negative nuclei appear bluish stained with hematoxylin of Harris and differentiated with a saturated solution of Li2CO3. Cover slips were randomly coded and 100200 nuclei per cover slip were double-blinded counted.
Cell survival determination by colony forming ability
Approximately 1500 cells were plated in 60 mm Petri dishes 1416 h before UV irradiation. Cells were washed twice with prewarmed PBSA and irradiated with a low-pressure germicidal lamp (254 nm). After this, cells were maintained in culture for 15 days, then being fixed with 10% formaldehyde and stained with 1% violet crystal. Colonies with the minimal number of 15 cells were scored. Survival values were obtained as the ratio of the number of colonies from irradiated cells to non-irradiated cells.
Unscheduled DNA synthesis (UDS)
Analysis of DNA repair synthesis was carried out as previously described with modifications (16). Briefly, 104 cells were grown on glass cover slips for 24 h. After 24 h of culture in a serum-deprived medium (0.5% FCS), 1 µCi/ml of [3H]-methyl thymidine (86.0 Ci/mmol, Amersham Pharmacia 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), the cells were fixed with methanolacetate (3:1) mounted onto glass slides, and washed three times with 5% TCA for 15 min each, then being rinsed two times with 70% ethanol and once with absolute ethanol. The slides were dipped in EM-1 (Amersham Pharmacia Biotech, Inc., USA) emulsion and 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.
Alkaline single cell gel test (the comet assay)
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 prepared on frosted microscope slides using 22 x 22 mm cover slips. After UV treatment (2.5 J/m2) and DNA repair time (0 or 24 h), cells were gently trypsinized and resuspended at 1 x 106 cells/ml with 0.7% low melting point agarose. A second layer was added to each slide using 45 µl of the cell suspension. Slides were maintained on ice and in the dark. After this, the slides were immersed in a lysis solution (2.5 M NaCl, 200 mM NaOH, 10% dimethyl sulphoxide and 1% Triton X-100, pH = 10.0) at 4°C for at least 1 h and then washed (3 x 160 ml for 5 min) with T4 endonuclease V buffer (10 mM, TrisHCl pH = 8.0, 10 mM EDTA and 75 mM NaCl). Cells embedded in agarose were overlaid with 50 µl of T4 endonuclease V (20 µg/ml). Cover slips were added and the slides incubated for 30 min at 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 buffer (300 mM NaOH, 1 mM EDTA) for 25 min, and 25 V 300 mA were applied for 25 min more. Following electrophoresis, slides were washed three times with neutralizing Tris-buffer (400 mM Tris base, pH = 7.5) for 5 min each and stained with 30 µl of ethidium bromide solution (20 mg/ml). The extent of DNA migration was evaluated by visual scoring under a fluorescent microscope equipped with a 515560 nm filter, 250 x (LEICA DMLB). The number of comets counted on each slide ranged from 100 to 200, depending on cell density. The extent of DNA migration was evaluated by visual scoring. Quantitative DNA damage repair (DR) was estimated according to the method firstly described by Jaloszynski et al. (18). Comets were classified and assigned to five categories (04) according to the extent of DNA-tail migration. Cells with bright heads and no apparent tails were assigned to category 0, comets with very small heads and long diffused tails to category 4. Comets displaying features intermediary between category 0 and 4 were divided and assigned to easily distinguishable categories 1, 2 and 3. The number of comets in each category was counted, and an average DNA damage (DD) was calculated as follows: DD = (n1 + 2n2 + 3n3 + 4n4)/(
/100), n1 n4 = number of comets in category 1 4, and
= sum of all counted comets, including category 0. Repair of DNA damage (DR) was calculated as follows: DR = DDi DD24/(DDi DD0) x 100%, DDi =DNA damage after exposure at time 0; DD24 = DNA damage after exposure at repair time 24 h and DD0 = DNA damage without irradiation.
Determination of endonuclease sensitive sites (ESS)
Cells were grown in a complete medium containing [3H]-thymidine (0.5 µCi/ml) for 48 h and then UV irradiated with 2.5 J/m2. The cells were cultivated for a period of time after UV-irradiation for DNA repair and then harvested. Nuclei were prepared with 0.5% Triton X-100, 0.1 M NaCl and 10 mM EDTA, washed twice with PBSA, and incubated in NET buffer (100 mM NaCl, 10 mM TrisHCl and 10 mM EDTA, pH = 7.5) with T4-endonuclease V for 30 min at 37°C. Molecular weights of untreated and treated DNA were determined by alkaline sucrose gradient sedimentation, as described before (19). The number of ESSs expressed by 107 daltons was calculated in relation to non-irradiated cells. ESSs estimated in nuclei correspond to approximately half of the CPDs, which are accessible to the UV-endonuclease within the chromatin.
Flow cytometry analysis of individual cells by annexin V staining
The apoptosis was detected based on the method described in Amarante-Mendes et al. (20). After the UV treatment (10 J/m2), the supernatant and tripsinized adherent cells were collected and centrifuged 200x g for 5 min at room temperature. The appropriate buffer (10 mM HEPES, pH = 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) was added to the cells and centrifuged again under the same conditions. The pellet was then exposed to the annexin-FITC solution (BD PharMingen, CA, USA) in the dark for 10 min on ice. Next, 400 µl of a 100 µg/ml of propidium iodide (PI) solution was added and immediately centrifuged. The PI excess was removed by washing the cells three times with the buffer. The final pellet was kept in the dark, on ice, until analysis, for a maximum of 1 h. Samples were transferred to microtubes, and annexin V fluorescence was read by flow cytometry (FACScalibur, Becton Dickinson), 10 000 events each. Results were obtained as a percentage of M1, which represents apoptotic cells.
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Results
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Characterization of the clone INXA15
In order to modulate the xpa gene expression, inducible genetic vectors were constructed, carrying the cDNA xpa. The system that best fitted into our experimental conditions was the muristerone A inducible vector described by No et al. (11), even though some leakiness is still present.
The vectors pINXA or pIND and pVgRXR were cotransfected into XP12RO, and cells were selected in a medium supplied with zeocin and neomycin antibiotics. Thirty clones were obtained and the expression of the XPA protein in these cells was analyzed in western blots, under conditions of induction or without muristerone A. The clone INXA15 showed the highest XPA expression when 0.1 µM of muristerone A was added to the cell medium, with very low basal levels under non-induction conditions. As a negative control, we established a cell population by the cotransfection of the empty vector pIND and the pVgRXR plasmid. In Figure 1
, an example of such western blots is shown when a protein band with ~42 kDa, corresponding to the XPA protein, is observed. Clearly, the XPA expression in the INXA15 cells is dependent on the presence of muristerone A in the medium. The inducible response is better for these cells than for any other clone. No XPA protein was detected in XPIND cell extracts, and the levels of XPA induction in INXA15 cells were consistently higher than in MRC5-V1 control cells.

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Fig. 1. XPA expression from INXAs clones. Protein extracts from the cells were analyzed by western blot, using anti-XPA as a probe: MRC5-V1, DNA repair proficient cells (positive control); XPIND, XP12RO cells with the empty vectors (negative control); clones INXA15, INXA16, INXA17 and INXA18, induced (+) or not () with 0.1 µM/72 h of muristerone A (Mu).
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In situ immunocytochemistry was also employed to check for XPA expression. This technique allows for the detection of XPA expression in single cells, thus providing more accurate data regarding the efficiency of the cloning process and the homogeneity of protein expression within a cell population. Results are presented in Figure 2
and it can be observed that some INXA15 cells were expressing enough XPA protein to be detected by this technique (2.7%) even in the absence of muristerone A, this possibly reflects the basal expression associated with this inducible system. After induction, the percentage of cells expressing XPA rises to 83.9%. Despite the fact that the majority of cells are expressing XPA after induction, this expression was not completely homogeneous. Heavy and light brown stained cells could be detected which might be a signal of differential XPA expression rates. In order to better evaluate the ability of the INXA15 clone to induce XPA expression, 0.1 µM of muristerone A was added to the culture medium for different periods of time.

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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.
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Figure 3
summarizes the kinetics of XPA expression induction. The increased expression starts to be detected after 4 h, and 48 h after hormone addition the levels of XPA protein detected had increased ~20 times in relation to non-induced cells. Comparing XPA amounts in different human cells by the western blot, the basal levels of XPA protein expression in INXA15 cells were found to be 20 (±8)% (average of five different experiments) of what is normally obtained in human fibroblast cell lines, normal for the NER process (HeLa and MRC5-V1). The induction with 0.1 µM of muristerone A in 24 and 48 h can increase the XPA protein up to two and four times, respectively, compared to control cells. Interestingly, the detection of the XPA transcript by Northern blot (~1.2 kb) 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.

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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.
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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 determined in different cells and conditions. The results are shown in Figure 4
. As expected, XP12RO and XPIND cell lines are extremely sensitive to UV-irradiation. DNA repair proficient cells (MRC5-V1) present a higher UV resistance. The INXA15 clone is able to recover UV resistance in both reduced and overexpressed levels of the XPA protein, their survival curves being indistinguishable. Moreover, the overexpression of xpa does not promote any toxic effect to the cells, plating efficiency in all situations being similar (~40%).

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Fig. 4. UV survival of INXA15 cells is similar to DNA repair proficient cell lines. Cells were UV-irradiated at the indicated doses and the survival is determined as a colony-forming ability.
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Unscheduled DNA synthesis activity in the INXA15 clone
To correlate the recovery of UV survival with the ability to perform DNA repair synthesis, the incorporation of [3H]-thymidine was measured following UV irradiation using classical UDS method. The results are shown in Figure 5
. XPIND cells exhibit a reduced UDS level after UV exposition (4.4 ± 1.9 grains per nucleus and 3.8 ± 1.7 in non-irradiated cells). Control cells MRC5-V1 exhibit a high UDS level, 32.5 ± 1.4 after UV treatment, and 3.7 ± 1.7 grains per nucleus in non-irradiated cells. For INXA15 cells, the UDS level was restored to almost normal levels under induced and non-induced conditions after UV exposition, at 38.9 ± 27.3 and 33.7 ± 24.4 grains per nucleus, respectively. However, it is clear that the distribution of DNA repair activity in the INXA15 clone is very heterogeneous when compared to control cells. This heterogeneity in the number of grains per nucleus may reflect differences in XPA protein expression in single cells, as observed in immunocytochemistry.

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Fig. 5. DNA repair activity (UDS) in different human cell lines. UDS was determined as described in Material and methods. The UDS activity is expressed by grains per nucleus. (A) XPIND; (B) MRC5-V1; (C) INXA15; and (D) INXA15 + Mu. Black bars, non-irradiated cells and gray bars, UV-irradiated cells (10 J/m2).
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Removal of CPDs after UV irradiation
To define the events associated with DNA damage and repair that could be correlated with survival, the removal of photoproducts by global genomic repair was assayed by T4 endonuclease DNA cleavage detection by the `comet assay' and sedimentation on alkaline sucrose gradients. In both methodologies, CPD frequencies in cells carrying the cDNA xpa with or without muristerone A induction were compared with DNA repair proficient and deficient cells. Figure 6
shows typical results obtained with the `comet assay'. In preparations from control cells, the undamaged DNA remains within a brightly fluorescent nuclei core. However, when the DNA contains breaks, it moves from this core towards the anode, forming an image aptly described as a `comet'. The size of the comet, and the distribution intensity of fluorescence, has been correlated quantitatively with the amount of DNA breaks (21). In this work, DNA breaks were introduced by cleavage with T4 endonuclease V, which nicks specifically at CPD sites. The DNA damage repair (DR) rate was used to compare different cell lines after UV irradiation (2.5 J/m2). The XPA deficient cell line (XPIND) was not able to reduce the `comet tail' and even a slight increase of tail size was observed in the 24 h following UV irradiation (DR = 3.5%). For MRC5-V1 cells and INXA15 cells, induced or not by muristerone A, there is a strong decrease of the comet tail if they are allowed to repair for 24 h of culture after UV irradiation. The results indicated that the DR for MRC5-V1, INXA15 and INXA15 + Mu were 52.9, 51.4 and 50.0%, respectively.

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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.
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The number of ESSs remaining in the genome 24 and 48 h after UV irradiation was also determined by alkaline sucrose gradients. As shown in Figure 7
, the XPA deficient cells are unable to remove CPDs. In fact, there is a reproducible increase in the number of breaks in DNA (independent of T4 endonuclease V, data not shown) 48 h after UV irradiation. For the cell lines proficient in XPA protein, the removal of ESS seems to be similar, as observed in the comet assay methodology. These results indicated that even in cells expressing low levels of XPA protein, INXA15 without a hormone, a normal DNA repair activity is detected.

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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.
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UV-induced apoptosis
UV-induced apoptosis was also determined in these INXA15 cells, under conditions of XPA induction or not. This was done by FACs analysis of annexin V positive cells. Figure 8A
illustrates the kind of results obtained and Figure 8B
presents the average of four experiments performed. As can be observed, the early events of UV-induced apoptosis are highly increased in XPA deficient cells (XP12RO and XPIND), when compared with SV40-transformed control cells, confirming results observed by others (22). Cells overexpressing XPA protein (INXA15 cells in the presence of muristerone A) behave as control cells, with lower levels of annexin V positive events. However, INXA15 cells expressing low amounts of XPA protein (without inductor) also increased UV-induced apoptosis, at least at early times (2 and 8 h after UV). This is in clear contrast with the DNA repair complementation results reported above.

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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 obtained from FACs analysis. M1 represents the cell percentage positive for annexin V. Mu: 0.1 µM/48 h of muristerone A before UV exposition. (B) Percentage of apoptotic cells (M1) after UV irradiation (10 J/m2) in different periods of time. Mu: 0.1 µM/48 h of muristerone A before UV exposition.
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Discussion
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XPA protein plays an important role in the initial steps of 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 DNA, interacting with several other DNA repair proteins. It may act by correctly positioning these proteins in relation to the damage (4,23). Thus, this protein is an essential part of the preincision complex of nucleotide excision repair. This crucial role implies that cells deficient in this protein are completely unable to repair certain types of DNA damage, including those induced by UV irradiation, resulting in severe anomalies in patients that are genetically XPA deficient (2). Modulation of XPA protein levels within the cells could, in principal, directly affect the first steps of nucleotide excision repair, thus limiting final performance. This modulation was achieved by constructing a cell line (INXA15) that expresses the xpa gene under the control of an inducible promoter. The results, however, indicate that although we were able to adjust the amount of XPA protein by just adding hormone (muristerone A) into the cell medium, the effects on DNA repair synthesis, CPD removal or cell resistance to UV could not be detected, as even low concentrations of XPA were sufficient for full complementation of these endpoints.
UDS analysis directly detects DNA synthesis by the damage repair apparatus (24). INXA15 cells are able to complement DNA repair synthesis deficiency for XPA cells in both reduced and overexpressed conditions, with no sign of cell toxicity. Cells overexpressing XPA does not have an increase in UDS activity. Curiously, in both cases there is a clear heterogeneity in the distribution of cells capable or not of performing UDS, which gives a good correlation with the heterogeneity of XPA protein expression detected by immunocytochemistry. The removal of CPDs was also analyzed by detecting these lesions with T4 endonuclease V (ESSs), employing the comet assay and sedimentation in alkaline sucrose gradients. These are direct measurements of global genome nucleotide excision repair for CPDs. In both analyses, there was a full DNA repair complementation in INXA15 cells, even in the absence of the inducer. These results are consistent with classical findings that human heterozygous for the xpa gene and, more recently, heterozygous knockout mice, do not present any clinical symptoms. Cells from human or mouse heterozygous support normal levels of DNA repair (25). Giannelli and Pawsey (26) found a similar conclusion when analyzing heterokaryons of XP and normal cells concluding that small quantities of xpa mRNA is enough to complement DNA repair synthesis. These results indicate that the modulation of xpa expression does not interfere with the rate of CPD removal, at least in global genomic repair.
However, Koberle et al. (10) reached different conclusions. These authors found that testicular germ tumor cells are particularly sensitive to cisplatin, this explaining the success of cisplatin-based chemotherapy. In vitro experiments indicated that extracts from these cells have a reduced ability to carry on repair of cisplatin-induced DNA damage, this probably being due to reduced levels of XPA protein. This may also 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 system to modulate the expression of the xpa gene in the XP12RO cell line, with some of their cell clones overexpressing the xpa cDNA presented an increased resistance to UV light when compared to control cells (GM637). Although the authors interpret these results as due to a possible rate-limiting role of XPA protein, our data completely contrast with their observations, as expression of four times more XPA protein, relative to normal control levels, did not produce cells more resistant to UV. The different cell survival observed by these authors could be explained by a different general genetic background between XP12RO and GM637 cell lines, and not to the expression of high levels of XPA protein alone. Curiously, Cleaver et al. (8) also did not detect any removal of CPDs by immunological methods, in cells overexpressing xpa cDNA, in complete discrepancy to the normal global genome repair of these lesions detected in INXA15 cells. Since a different technology was employed, it is difficult to compare the two sets of data, although normal CPD removal is the expected result for UV resistance recovery in cells overproducing XPA protein. It is also important to note, however, that DNA repair, especially GGR of CPDs, may be reduced in SV40-transformed cell lines. This may occur due to the fact that LT-antigen of SV40 binds to the p53 tumor suppressor protein (28). This could also interfere in the expression of p48 protein (deficient in XPE individuals), necessary for GGR activity in mammalian cells (29,30). In this work, the use of both control and XP-A cell lines, transformed with SV40, was performed in order to minimize possible differences due to p53 inhibition, as GGR activity is probably equally affected.
During analysis of CPD removal in these cells, we found that XPA deficient cell lines have an increased number of breaks measured by alkaline sucrose gradients or comet assay. A part of these breaks is independent of T4 endonuclease V cleavage, that is, they occur as a product of cell metabolism in these cells (31,32). These breaks could be a consequence of UV induced apoptosis, which is normally increased in these cells (22). This prompted us to analyze the patterns of apoptosis in these SV40-transformed cells. We were unable to obtain a clear pattern of internucleosomal cleavage (also known as DNA ladder) in any of the cell lines employed, except for HeLa cells (33). However, detection of the externalization of phosphatidil serine by annexin V, an early signal of apoptosis, was observed in these cells in a short period after UV irradiation. This phenomenon is detected much faster in XP12RO cells, when compared with MRC5-V1 cells: 2 h after UV ~20% of the XP cells present these signals, this frequency still going up 8 h and later, while control cells have a much delayed entry into apoptosis. However, cells that have only a low amount of XPA protein behave like deficient cells, while cells that overexpress this protein behave like control cells. These results are distinct from what have been observed for other endpoints such as DNA repair measurements and UV resistance, where low levels of XPA protein induce full complementation. Yamaizumi and Sugano (34) have proposed that apoptotic responses to UV can be related to the repair capacity of actively transcribed genes. Moreover, human cell lines deficient in TCR, such as those deficient in the xpa gene, have shown to present a strong correlation between inhibition of RNA transcription and apoptosis induction after UV irradiation (35,36). Removal of CPDs by photoreactivation prevents the early steps of UV induced apoptosis, which is coherent with the hypothesis that DNA damage may block RNA transcription by RNA polymerase, signaling for apoptosis in the UV irradiated cells (33,37). Cells that have inefficient systems for the removal of these lesions have increased signals for apoptosis. Indeed, Van Oosten et al. (38) have demonstrated that TCR is responsible for apoptosis prevention after UV irradiation. Therefore, it would be interesting to investigate the efficiency of TCR and RNA transcription inhibition in non-induced INXA15 cells, in order to check for the possibility that XPA protein may in fact be rate limiting for this kind of repair. Kobayashi et al. (27) have performed similar experiments with another inducible system for this gene, but the reduction in TCR was very low and they did not analyze global genomic repair to confirm whether XPA may denote a preference to act in one or the other kind of repair. Interestingly, the differences observed for early apoptosis signals (externalization of phosphatidil serine) and cell death (UV survival) indicate that these two events may bev independent. This is in agreement with recent findings with Caenorhabiditis elegans cells, which can still recover cell survival after a stage that resembles the early phase of apoptosis (39,40). Thus, changes such as externalization of phosphatidil serine in UV-irradiated mammalian cells are also not necessarily fatal.
Finally, the fact that modulation of XPA protein production did not allow for modulation of global nucleotide excision repair and UV resistance indicates that this protein is not rate limiting in the process. As only a small amount of protein can cope with the global repair, it may also point to a short time as being required for its presence during repair. This can be possible once XPA participates in the early recognition steps of NER, and, once the other proteins are correctly placed for damage removal, XPA could be liberated to participate in a following event of repair. The high stability of the XPA protein may also contribute to these data. In fact, previous findings indicated that DNA repair could be carried on in the absence of protein synthesis in such a way that the turnover period for this protein in human cells must be very long (41). High protein stability is also consistent with the very low levels of XPA mRNA expression in human cells (estimated to be ~58 copies per cell) (7). The results in this work also indicate that high amounts of XPA protein do not accelerate repair nor help cell survival after UV irradiation. Thus, achieving an increased expression of this protein in vivo would probably not contribute to protecting against hazards caused by agents that introduce potentially carcinogenic DNA damage.
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Notes
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5 To whom corrrespondence should be addressed at: Departmento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, São Paulo/SP, 05508-900, Brazil Email: cfmmenck{at}usp.br 
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Acknowledgments
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This work was supported by the Fundacião de Amparo à Pesquisa do Estado de São Paulo FAPESP (proc. # 98/11119-7, São Paulo, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil). M.C.N.M. and G.B. have MS Fellowships and A.R.M. has a PhD fellowship from FAPESP, respectively. We thank Dr Rogério Meneghini (IQ-USP, Brazil) for providing the T4 endonuclease V and Dr Alain Sarasin for the cell lines XP12RO and MRC5-V1 (Laboratory of Genetic Instability and Cancer, CNRS, Institut André Lwoff, Villejuif, France).
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Received November 16, 2001;
revised January 7, 2002;
accepted April 1, 2002.