Báo cáo khoa học: Nucleotide excision repair rates in rat tissues pdf

Nucleotide excision repair rates in rat tissues Anastas Gospodinov, Rumen Ivanov, Boyka Anachkova and George Russev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nuc- leotide excision repair capacity than slowly proliferating tis- sues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepa- tectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates. Keywords: DNA repair; DNA replication; protein extract; rat tissues; UV irradiation. DNA repair and particularly nucleotide excision repair (NER) is one of the primary pathways by which mamma- lian cells remove a wide variety of DNA lesions. In the process of repair, the products of more than a dozen genes are involved in damage recognition, incision, elongation and ligation of DNA to collectively restore its original structure [1–3]. Many other cellular factors, normally participating in transcription and replication, also play a role in the process of DNA repair [4–6]. The NER pathway can recognize a broad spectrum of helix-distorting lesions, such as UV- induced pyrimidine dimers, bulky adducts, cross-links, etc. and remove them by excision of oligonucleotide fragments containing the damaged site [7]. In many cases the capacity for DNA repair changes as a result of mutation of some of the genes related to the repair process. The cases of severe reductions of NER capacity due to mutations in some genes are manifested phenotypically and are diagnosed as diseases or syndromes such as xeroderma pigmentosum, Cockain syndrome, trichothiodistrophy, etc. All of them are charac- terized by decreased genetic stability and increased cancer incidence [8]. The linkage between perpetual DNA damage and oncogenic activity suggests that such persistence of DNA damage is a reflection of the diminished involvement of DNA repair activities in tumorigenesis. This suggests that DNA repair proficiency could exist as an aetiologic correlate for the general population at risk for cancer [9,10]. This problem has not been addressed in a systematic way and there are only few such investigations with contradictory results. In most of these studies, DNA repair was measured in peripheral human lymphocytes as they were easy to collect and handle [11–15]. However, this approach has at least two major drawbacks: (a) lympho- cytes are not capable of NER unless stimulated to proliferation [12], and (b) lymphocytes could have different repair capability than target tissues. Several studies tried to avoid these drawbacks by directly determining expression rates of major NER genes in tissues obtained by biopsy or other means [16,17]. These results showed considerable differences between the expression of the same genes in different tissues as well as between the expression of different genes in the same tissue. However, the results obtained could not be directly related to repair capacity as it is difficult to assess the contribution of the different genes in the overall lesion removing process. In this study our aim was to determine the NER rates of different rat tissues and to determine whether these rates correlated with the proliferative status of the tissues. To this end repair-competent cell extracts were prepared from the target tissues and UV-damaged plasmid DNA was incuba- ted in them. We used UV-damaged DNA molecules to avoid mixing up the base excision repair (BER) and NER pathways as UV-induced lesions are repaired only by NER, while many other lesions such as single-strand breaks, abasic sites, oxidative damages, etc., could be repaired by both BER and NER pathways [18]. The repair rates were calculated by comparison of the transformation efficiency of the damaged plasmids before and after incubation in the extracts. Our results indicated that rapidly proliferating tissues exhibited higher NER rates than slowly proliferating tissues. Upon stimulation to proliferation the repair rates of the tissues increased. Materials and methods Cells K562 cells were obtained from ATCC and the XPA lymphoblastoid cell line was obtained from NIGMS Human Genetic Cell Repository. They were grown in Correspondence to G. Russev, Institute of Molecular Biology, Bulgarian Academy of Sciences, Academic G. Bonchev Street, block 21, 1113 Sofia, Bulgaria. Fax/Tel.: + 359 2 72 35 07, E-mail: grs@obzor.bio21.bas.bg Abbreviations: BER, base excision repair; NER, nucleotide excision repair; TdR, thymine desoxyriboside. Note: A web site is available at http://www.bio21.bas.bg/imb/ (Received 19 November 2002, revised 15 January 2003, accepted 21 January 2003) Eur. J. Biochem. 270, 1000–1005 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03473.x suspension in RPMI 1640 medium, containing 4.5 mgÆmL )1 glucose in the case of K562. The media were supplemented with 10% foetal bovine serum and antibiotics and cells were grown in an atmosphere of 95% air/5% CO 2 at 37 °C. Splenocytes were prepared from freshly obtained rat spleens by squeezing the tissue through a metal mesh under sterile conditions. The cell suspension was overlayered onto 3 mL of Lymphoprep (Sigma) and centrifuged at 180 g for 10 min. Splenocytes were collected from the interphase, washed with NaCl/P i , suspended in RPMI 1640 supple- mented with 10% fetal bovine serum and stimulated to proliferation with 5 lgÆmL )1 each phytohemagglutinin, concanavalin A and lipopolysaccharide (Sigma-Aldrich). [ 3 H]Thymine desoxyriboside (TdR) was added at different intervals after stimulation and 1 h later splenocytes were used to prepare repair-competent extracts. DNA was isolated from the high-speed pellet obtained in the course of cell extract preparations and aliquots were counted in Beckman scintillation counter. Tissues Wistar albino rats (200 g) were killed under ether anaes- thesia by dislocation of the cervical cord and the tissues were immediately removed and rinsed in ice cold NaCl/P i .Partial hepatectomy was performed under ether anaesthesia by removing two-thirds of the liver. At 6-h intervals after the operation animals were injected intraperitoneally with 200 lCi [ 3 H]TdR and 1 h later were sacrificed and livers were used for preparation of repair-competent extracts and DNA for counting. Protein extracts Protein extracts were prepared as described in [19] with modifications. Cells were rinsed twice in ice-cold NaCl/P i , resuspended in hypotonic buffer (10 m M Tris/HCl, pH 8.0; 1 m M EDTA and a tablet of Complete TM protease inhibitor cocktail per 50 mL solution, Roche Diagnostics GmbH) and left on ice for 20 min to swell. Cells were then homogenized by 20–30 strokes in a Dounce homogenizer. When solid tissues were used 0.4–0.5 g of the material were finely minced, twice rinsed in ice cold NaCl/P i and 4 mL of hypotonic buffer added to the pellet. This crude suspension was immediately homogenized in a Teflon pestle homogenizer. To each cell lysate 4 mL of an ice cold solution containing 50 m M Tris/HCl pH 8.0, 10 m M MgCl 2 ,2m M dithiothreitol, 25% sucrose, 50% glycerol were added slowly with stirring. One ml neutralized saturated ammo- nium sulfate was then added with gentle mixing. The mixture was kept on ice with occasional stirring for 30 min, and then centrifuged at 25 000 r.p.m. in a Beckman SW41 rotor at 2 °C for 3 h. The upper two- thirds of the supernatant were withdrawn and the protein was precipitated by the addition of 0.33 gÆmL )1 neutral- ized ammonium sulfate. The precipitate was collected by centrifugation, resuspended in a minimum volume of buffer containing 25 m M Hepes/KOH pH 7.9, 0.1 M KCl, 12 m M MgCl 2 ,1m M EDTA, 2 m M dithiothreitol, 17% glycerol. The extract was desalted using a Sephadex G-25 coarse column and the total protein concentration deter- mined by the Bradford reaction. Plasmids and irradiation Plasmids pBlueScript SK+ (Stratagene) and pEGFP-N1 (Clontech) were propagated in Escherichia coli strain XL-1 Blue. The UV irradiation of pBlueScript and pEGFP-N1 was carried out with a germicidal mercury lamp as described by Rougev and Russev [20]. Plasmid DNA was dissolved in Tris/EDTA buffer to a final concentration of 100 lgÆmL )1 . It was poured into Petri dishes to form 1–2-mm thick layer, placed on ice and irradiated from a 10-cm distance for 1, 2, 4 and 10 min. With these irradiation conditions the exposure rate was 8.3 JÆm )2 Æs )1 as measured with a UV power energy meter (Scientech 362) and plasmid DNA received doses of  0.5, 1, 2 and 5 kJÆm )2 , respectively. Plasmid pEGFP-N1 irradiated with different doses of UV light was linearized by EcoRI digestion and used as a template to amplify a 1.05-kb fragment (nt 440–1490) using the primers: 5¢-CCATTGACGTCAATGGGA-3¢ and 5¢-AGGTTCAGGGGGAGGT-3¢. Twelve PCR cycles were run using the following parameters: denaturation at 95 °C for 60 s, annealing at 59 °C for 45 s and extension at 72 °C for 60 s. Amplified products were run on a 1% agarose gel, stained with ethidium bromide and quantified with Gel Pro Analyzer v.3 software for Windows (Media Cybernetics). DNA repair reactions A50-lL reaction containing 300 ng UV irradiated pBlue- Script repair substrate, 300 ng unirradiated pEGFP internal control, 45 m M Hepes/KOH pH 7.8, 70 m M KCl, 7.4 m M MgCl 2 ,0.9m M dithiothreitol, 0.4 m M EDTA, 2 m M ATP, 25 l M each of dGTP, dATP, dTTP and dCTP, 40 m M phosphocreatine, 2.5 lg creatine phosphokinase, 18 lg bovine serum albumin, 100 lg cell-extract protein were incubated at 30 °C for the specified intervals. Repair was stopped by the addition of equal volume of 40 m M EDTA, 100 m M Tris/HCl pH 8.0. Following incubation with 100 lgÆmL )1 RNaseAfor15min,SDSwasaddedto 0.5% and proteinase K to 200 lgÆmL )1 and proteins were digested for 1 h at 37 °C. The mixture was extracted with phenol/chloroform (1 : 1) once, and DNA from the aque- ous phase precipitated with 0.8 vols isopropanol and 0.1 vol. 3.3 M ammonium acetate. Plasmids were dissolved in Tris/EDTA, electrophoresed in a 1% agarose gel, stained with 0.5 lgÆmL )1 ethidium bromide and their concentration was adjusted. Bacteria and transformation E. coli strains XL-1 Blue and the UV-sensitive strain SOLR-UvrC were obtained from the Bulgarian Cell Type Collection. Transformation was carried out by adding 20 ng plasmid DNA to 200 lL competent E. coli. After 10 min on ice, bacteria were heat-shocked at 42 °C for 30 s and placed on ice for additional 10 min. One mL unselective medium was added to each transformation and incubated for 30 min at 37 °C. Bacteria were plated on Petri dishes with solid Luria–Bertani medium (10 g Bacto tryptone, 5 g Ó FEBS 2003 Nucleotide excision repair rates in rat tissues (Eur. J. Biochem. 270) 1001 yeast extract, 10 g NaCl, 15 g Bacto agar per litre) containing 100 lgÆmL )1 ampicillin or 50 lgÆmL )1 kana- mycin and grown for 12 h at 37 °C. Results and discussion Validation of the repair assay To assess the repair capacity of cells and tissues we prepared repair-competent extracts and monitored the repair of UV- irradiated BlueScript plasmid DNA incubated in them. We normalized the repair rates against the total amount of protein in the extracts rather than against any specific housekeeping protein as the expression of individual proteins varies in different tissues or in the same tissue in different physiological states. The main products of UV irradiation are pyrimidine dimers and pyrimidine (6–4)- photoproducts. Both types of lesions are effective blocks of Taq polymerase in PCR [21,22] and both are repaired by the NER pathway [23–25]. To calibrate the lesions, pEGFP- N1 was irradiated with 0.5, 1, 2 and 5 kJÆm )2 and after linearization with EcoRI was used as template for quanti- tative PCR to amplify the 1-kb fragment containing the EGFP gene. By using the Poisson formulae which gives the relationship between the average number of lesions and the proportion of the undamaged fragment, we were able to calculate that under our conditions irradiation with 1kJÆm )2 produces, on average, 0.5–1 lesions per kb in the plasmid DNA. This figure was in agreement with other recent data showing that the rate of cyclobutan pyrimidine dimer and pyrimidine (6–4) pyrimidone product [(6–4)PP] formation upon irradiation with UV light was within the range 0.3–1.2 lesions per kb per 1 kJÆm )2 [26,27]. There are data showing that plasmids carrying UV lesions are seldom replicated in the absence of SOS induction of E. coli, and do not support colony formation on selective medium [28,29]. This permits the establishment of a direct link between the number of colonies obtained after transformation of E. coli cells with irradiated plasmid and the amount of native plasmid molecules in the sample, and opens the possibility to calculate the repair capacity of the extracts. We used irradiated BlueScript and control EGFP-N1 plasmids with and without incubation in K562 extracts to transform competent E. coli cells (Fig. 1). The transformation efficiency of the UV irradiated pBlueScript was expressed relative to the transformation efficiency of the control undamaged pEGFP assuming the ratio of pBlue- Script (ampicillin resistant) colonies to pEGFP (kanamycin resistant) colonies in the case when cells were transformed with mixture of undamaged pEGFP and undamaged pBlueScript in a ratio of 1 : 1 (w/w) was 100%. As shown in Fig. 1 the K562 extract repaired plasmid DNA at an almost constant rate up to the third hour, after which it slowed considerably. To demonstrate the direct relationship between colony formation potential of the irradiated plasmids and the number of DNA lesions, we carried out two control experiments. First we incubated the irradiated pBlueScript in an extract from repair-deficient XPA cells and then used the plasmid DNA for transformation of E. coli. In this case the pBlueScript irradiated with 1 kJÆm )2 showed 5–10% transformation efficiency in comparison with the undamaged plasmid and this did not change with the time of the incubation (Fig. 1). Using the Poisson equation we were able to calculate that this plasmid contained  0.9 UV lesionsÆkb )1 , which is in reasonable agreement with the figures obtained with the PCR assay, and with data in the literature [26,27]. This experiment showed that the increase in the number of colonies obtained after incubation of the irradiated plasmid in repair-competent cell extracts was a result of DNA repair that had taken place in the extract. Second, we transformed in parallel the E. coli XL-1 Blue strain and the UvrC-deficient SOLR E. coli strain, which is unable to repair UV lesions, with irradiated plasmids. There were no differences in the colony formation efficiency with the repair-competent XL1 Blue and repair deficient SOLR E. coli strains (data not shown), which showed that no DNA repair was taking place in the host E. coli cells. This confirmed the finding that in the absence of SOS induction E. coli cells were not able to replicate damaged plasmid molecules [28,29]. NER rates in rat tissues We next prepared repair competent extracts from several rat tissues and determined their repair rates by using the described protocol. To this end we determined the number of lesions per kb of irradiated plasmid DNA prior to and after incubation in the respective extracts for 3 h and the Fig. 1. NER in protein extracts from K562 cells (d) and XPA cells (s). A 1 : 1 (w/w) mixture of pBlueScript UV irradiated at 1 kJÆm )2 and untreated pEGFP-N1 were incubated in K562 and XPA extracts. At different time intervals aliquots were withdrawn, plasmid DNA was isolated and used to transform competent E. coli cells. Transformed cells were plated on ampicillin- or kanamycin-containing agar and 12 h later the number of colonies was counted. The number of ampicillin- resistant colonies was expressed as percentage of the kanamycin- resistant colonies, assuming that the ratio of the ampicillin resistant to kanamycin resistant colonies when both plasmids were unirradiated was 100%. The results are means of six independent experiments and the standard deviations from the means are shown by error bars. 1002 A. Gospodinov et al. (Eur. J. Biochem. 270) Ó FEBS 2003 difference was taken as the number of lesions repaired by the extracts (Fig. 2). The results showed that different rat tissues differ in their ability to repair UV lesions. Kidney, testis and liver showed higher repair capability close to the repair capability of transformed K562 cells. Muscle, lung and brain showed somewhat lower repair capability, heart showed even lower repair capability and finally splenocytes showed practically no repair capability. This arrangement roughly corresponds to the replication activity of the respective tissues. Thus, kidney, liver and testis are consid- ered rapidly proliferative tissues, while muscle, heart, brain, lung and spleen are considered slowly proliferating tissues. Similar results have been reported by others, who also had found that the rapidly proliferative tissues such as skin, testis, ovary, breast, liver, kidney, colon, etc. show higher relative expression of five NER genes than the slowly proliferative tissues such as muscle, heart, brain, spleen, lung, etc. [16]. Cell proliferation and NER To investigate further the observed relationship between proliferation activity and NER, we carried out experiments in which we determined the repair rates in two tissues in norma and upon stimulation to proliferation. First we took advantage of the fact that rat liver is capable of intense regeneration after partial hepatectomy and followed both DNA synthesis and repair capacity of regenerating rat livers in the course of the first 36 h after a two-thirds hepatectomy. In agreement with the literature data, a sharp wave of DNA synthesis was observed between 16 and 24 h of the liver regeneration. Simultaneously an equally sharp increase of the liver’s ability to repair UV lesions was registered (Fig. 3). In a second experiment we isolated rat splenocytes and determined their repair capability prior to and after stimulation to proliferation with mixture of lectins. Here again we observed a synchronous wave of DNA synthesis about 24 h after the mitogen stimulation and a simulta- neous sharp several-fold increase of the repair rate in rat splenocytes. (Fig. 3). It is well established that DNA replication and DNA repair pathways share many common proteins including DNA polymerases. The activity of the DNA polymerases is of crucial significance in the process of DNA repair, as without the proper polymerase the end-product of most repair pathways, i.e. the repaired DNA, will not be produced, even if all other repair proteins are present and active. It is especially relevant that the gap-filling step in NER is carried out by the replicative polymerases -d and -e, Fig. 2. NER rates in rat tissues. Mixtures of pBlueScript irradiated at 1kJÆm )2 and unirradiated pEGFP-N1 were incubated in cell extracts prepared from different rat tissues for 3 h and were used to transform XL-1 Blue E. coli cells. Repair capacity of the tissues was expressed in number of lesions per kb of DNA removed during the incubation and were determined from the number of colonies calculated as in Fig. 1 by usingthePoissonformula.Tissues:1,testis;2,kidney;3,liver;4,lung; 5,muscle;6,brain;7,heart;8,splenocytes.Theresultsaremeansfrom six animals and the standard deviations are shown by error bars. Fig. 3. NER and DNA replication rates in normal and regenerating rat liver and unstimulated and stimulated rat splenocytes. Wistar albino rats were subjected to partial hepatectomy and the excised parts of the livers were used to prepare repair competent extract. After 24 h the animals were sacrificed and the regenerating livers were removed and again used to prepare repair-competent extract. To follow DNA syn- thesis one group of animals was injected intraperitoneally with 200 lCi [ 3 H]TdR 1 h before the operation and the second group of animals was injected with the same dose 1 h before sacrifice. DNA was isolated and counted and the radioactivity was expressed as countsÆmg )1 DNA. Splenocytes were isolated from the spleens of rats and cultured in RPMI 1460. Aliquots were labelled with 1 lCiÆmL )1 [ 3 H]TdR for 1 h and used to prepare repair competent cell extract and to isolate DNA. The remaining splenocytes were stimulated with mitogens as described in Materials and methods and 24 h later were labelled with 1 lCiÆmL )1 [ 3 H]TdR for 1 h and were used to prepare repair competent extracts and DNA. pBlueScript irradiated at 1 kJÆm )2 and control pEGFP were incubated in the liver and splenocyte extracts and used to transform E. coli. Repair capacity (black columns) of liver tissue and splenocytes were determined as in Fig. 2 and the proliferative rates are shown by the 3 H counts incorporated in DNA (shadowed columns). 1, Normal liver; 2, regenerating liver; 3, normal splenocytes; 4, sti- mulated splenocytes. The results are means of six independent experiments and the standard deviations from the mean are shown with vertical error bars. Ó FEBS 2003 Nucleotide excision repair rates in rat tissues (Eur. J. Biochem. 270) 1003 which are mostly expressed in proliferating cells [6,30]. This explains why quiescent, or slowly proliferating tissues, where the levels of these enzymes are low, are less effective in NER, although they may express most of the NER specific proteins [12]. On the other hand as BER is using polymerase-b, whose expression is relatively independent of the proliferative state of the cells, this repair pathway is efficient in both proliferative and quiescent tissues and this explains why lymphocytes can remove a number of lesions repaired by it [31,32]. Apart from the DNA polymerases, products of other genes could be also involved in the relationship between DNA replication and repair. Recent reports show that the transcription of the major repair genes differ during development and follow distinct temporal patterns that are functionally linked to initiation of DNA replication and progression through S phase [17,30,33]. It has also been reported that PCNA, which is expressed only in proliferating cells, is required for normal repair activity [34,35]. Our results show that there is a considerable difference in the NER rates of rat tissues and that it is dependent on the proliferative activity of the tissues. In this respect we assume that it is not correct to deduce the NER capacity of any tissues from that of other surrogate tissues. However, when this has to be done, surrogate tissues should be chosen that have the same proliferative activity as the target tissues, which would ensure that their NER rates would be similar. Acknowledgements This work was supported by the Bulgarian National Scientific Council (grant MU-K-1002/00 to A.G) and the European Commission (grant QLK4-1999–01629 to G.R). References 1. Friedberg, E., Graham, W. & Siede, W. (1995) DNA Repair and Mutagenesis. ASM Press, Washington, USA. 2. Sancar, A. (1996) DNA excision repair. Ann. Rev. Biochem. 65, 43–81. 3. Wood, R.D. 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NER rates in rat tissues We next prepared repair competent extracts from several rat tissues and determined their repair rates by using the described. results indicated that rapidly proliferating tissues exhibited higher NER rates than slowly proliferating tissues. Upon stimulation to proliferation the repair