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DNA-END BINDING ACTIVITY OF KU IN SYNCHRONIZED CELLS
Cell Biology International 1999, Vol. 23, No. 10, 663–670 Article No. cbir.1999.0432, available online at http://www.idealibrary.com on
DNA-END BINDING ACTIVITY OF KU IN SYNCHRONIZED CELLS LI-FEN CHOU and WEN-GANG CHOU* Department of Life Science, National Tsin-Hua University, Hsinchu, Taiwan, R.O.C. Received 9 March 1999; accepted 16 July 1999
Three different types of cells were synchronized by various methods and DNA-end binding (DEB) activities of Ku were compared with asynchronous controls. In CHO K1 cells synchronized in G1 phase by serum starvation and in S phase by serum refeeding, DEB activity was reduced in S cells but remained unchanged in G1 cells. However, the same type of cells synchronized in G1/S phase by double thymidine block and in S phase by releasing the blockage, have the same DEB activity as asynchronous controls. A similar result was found in RKO and HeLa cells synchronized by the latter method. Arresting cells in mitosis with nocodazole also generated different cell cycle effects. Ku activity was reduced in CHO K1 and RKO cells, but not in HeLa cells after treatment with nocodazole. In phase-enriched cells separated by centrifugal elutriation, DEB activities were similar at different stages of the cell cycle in all three types of cells. Thus, different synchronization procedures can give very different values of Ku activity in a cell type-dependent manner. Results from elutriated cells are consistent, and suggest DEB 1999 Academic Press activity of Ku does not change with the cell cycle. K: Ku protein; DNA-end binding; cell cycle.
INTRODUCTION The Ku protein was originally described as an autoantigen in patients with sclerodermapolymyositis overlap syndrome (Mimori et al., 1981). It is an abundant nuclear protein consisting of two polypeptides with molecular mass of 70- and 86 kDa (Reeves, 1985; Yanvea et al., 1985; Mimori et al., 1986). The heterodimeric Ku was shown to have an unique activity of binding to doublestranded DNA with free ends, but not to circular or single stranded DNAs (Mimori et al., 1986; Mimori and Hardin, 1986). In addition to this DNA-end binding (DEB) activity, Ku was later shown to bind DNA with nicks, gaps and other specialized structures such as stem-loop, dumbbellshaped and ‘bubble’ structures (Paillard and Struss, 1991; Blier et al., 1993; Falzon et al., 1993). Ku is the DNA-binding component of the DNAdependent protein kinase, DNA-PK. The kinase activity of the catalytic subunit, p460 or DNA*To whom correspondence should be addressed: Wen-Gang Chou, Department of Life Science, National Tsin-Hua University, Hsinchu, Taiwan, R.O.C. 1065–6995/99/100663+08 $30.00/0
PKcs is stimulated by the binding of Ku to DNA ends (Gottlieb and Jackson, 1993; Suwa et al., 1994). DNA-PK can phosphorylate a variety of proteins involved in signal transduction, DNA repair and replication in vitro (Anderson, 1993). Indeed, DEB activity of Ku was found to be deficient in the X-ray-sensitive (xrs) mutants derived from the CHO K1 cell line (Getts and Stamato, 1994; Rathmell and Chu, 1994). The xrs mutants are defective in repairing DNA doublestrand breaks (dsb) and V(D)J recombination (Kemp et al., 1984; Finnie et al., 1995). The role of Ku in dsb repair was confirmed by the finding that introducing the gene or cDNA of Ku86 into xrs mutants restored X-ray resistance, dsb repair and V(D)J recombination (Smider et al., 1994; Taccioli et al., 1994; Boubnov et al., 1995; Finnie et al., 1995). In addition, Ku70-deficient embryonic stem cells also have increased X-ray sensitivity and defective V(D)J recombination (Gu et al., 1997). The role of DNA-PK in DNA end-joining was also established by the discovery that SCID mice, which are unable to carry out V(D)J recombination and are sensitive to ionizing radiation, are defective 1999 Academic Press
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in p460 (Blunt et al., 1995; Kirchgessner et al., 1995). Besides an important function in DNA repair, Ku plays a role in regulating transcription of transferrin receptor gene (Roberts et al., 1994), long terminal repeat of mouse mammary tumor virus (Giffin et al., 1996), mouse ribosomal gene (Kuhn et al., 1993) and metallothionein-I (Goshal et al., 1998). Two groups have generated homozygous null mutants for mouse Ku86 gene (Nussenzweig et al., 1996; Zhu et al., 1996). These Ku86 knock-out mice were found to have impaired V(D)J recombination, and therefore both B and T cells were in low numbers and were developmentally arrested as progenitors. Furthermore, the mutant mice have a dwarf phenotype and only growing to 40-60% of the control wild type size (Zhu et al., 1996). Because many of these processes Ku seems to be engaged in are growth related, it is of interest to investigate whether Ku activity shows cell cycle dependency. MATERIALS AND METHODS Cell cultures The Chinese hamster ovary cell line, CHO K1, was cultured in F10 medium as described previously (Zhu et al., 1992). The human cervical carcinoma cell line, HeLa, and the colorectal carcinoma cell line, RKO, were cultured in DMEM with 45 m sodium bicarbonate and 10% fetal calf serum. Cells were maintained as monolayers by seeding 1105 cells in 25-cm2 flasks in a 37C, 5% CO2 and 95% air incubator. Exponentially growing cells were maintained by subculturing when confluence reached 50–70%. Cell synchronization Exponentially growing CHO K1 cells were synchronized to G1 and S phase by serum starvation and replenishing. Cells were maintained in F10 medium with 0.1% serum for 3 days and were refed with complete F10 medium. Cell cycle profiles were analyzed every 2 h after refeeding by flow cytometry and representative samples were chosen. G1 and S phase cells were those obtained 4 h and 17 h after refeeding, respectively. CHO K1, HeLa, and RKO cells were synchronized to G1/S boundary or S phase by double thymidine block. CHO K1 cells were sequentially incubated with medium containing 2 m thymidine for 12 h, normal medium for 6 h, 2 m thymidine
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for 12 h and with normal medium. G1/S cells were obtained at 1 h and S phase cells were obtained at 7 h after growing in normal medium. Similarly, HeLa and RKO cells were treated with thymidine for 17 h, normal medium for 9 h, and thymidine for 15 h before being incubated in normal medium. G1/S cells were obtained at 1 h and S phase cells were obtained at 3 h after growing in normal medium. In the above experiments, cell cycle profiles were determined by flow cytometry every hour after releasing the blockage, and representative samples were chosen at the time-points indicated above. Separation of phase-specific cells by centrifugal elutriation was performed according to Murray and Meyn (1986) with minor modifications. Exponentially growing cells (2108) were harvested, suspended in 20 ml of complete medium and loaded into a Beckman JE-6B rotor. In the separation of K1 cells, the rotor speed was maintained at 1700 rpm and cells were introduced into the chamber at 12 ml/min. The flow rate was then increased from 12 to 30 ml/min in 1.5-ml/min increments. At each flow rate, cells in 80 ml were collected for determination of cell cycle stage by flow cytometry and for preparation of whole cell extract to assay DEB activity. For HeLa cells, the rotor speed was 2000 rpm and the initial flow rate was 14 ml/min. The flow rate was increased to 46 ml/min in 2-ml/min increments. For RKO cells, the rotor speed was 2000 rpm and the initial flow rate was 10 ml/min. The flow rate was increased to 34 ml/min in 2 ml/min increments. All three cell lines were arrested at M phase by treatment with 100 ng/ml nocodazole for 24 h. Cell cycle analysis For flow cytometric analysis of DNA content, cells (1–2106) were fixed in 70% ethanol at 4C for 16–24 h. The fixed cells were treated with 1 mg/ml RNAse A in PBS for 30 min at room temperature. The cells were stained with 10 g/ml propidium iodide for at least 15 min and less than 6 h. The samples were analyzed by FACScan (Becton Dickinson). For each sample, 10,000 cells in the gated region (the distribution region of cycling cells) were collected for data analysis. Assay for DEB activity Cells were harvested with a rubber policeman and by centrifugation. Whole cell extracts were prepared by the method of Jiang and Eberhardt (1995). Cell pellets were resuspended in three
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packed cell volumes of ice-cold extraction buffer (20 m HEPES–HCl, pH 7.9; 0.5 KCl; 0.2 m EDTA; 0.5 m DTT; 0.5 m PMSF and 20% glycerol). The samples were subjected to three cycles of freezing and thawing, centrifuged at 12,000g for 10 min at 4C. The supernatant was stored at 70C. Aliquot of sample was diluted with the extraction buffer without KCl to 0.1– 0.2 g/ml for assay of DEB activity. The protein concentration was determined by the Bio-Rad protein assay kit (Bradford method), with bovine serum albumin as the standard. DEB activity was measured by gel electrophoretic mobility shift assay (EMSA) described previously (Rathmell and Chu, 1994). The standard 10-l reaction contained 0.2–0.6 g of extract, 2 ng of 32P-5 -labeled linear DNA probe (approximately 105 cpm), and 300 ng of circular pGEM1 plasmid DNA. The linear DNA probe was the gel-purified 222 base-pair fragment of pGEM1 plasmid digested with restriction enzyme AvaII. The reaction mixture was separated by electrophoresis in 6% non-denaturing polyacrylamide gel (acrylamide/bis-acrylamide: 29/1). The gel was dried for autoradiography. Western blot analysis Proteins in the extract was separated with 6% SDS-PAGE, electroblotted to PVDF membrane and incubated with antibodies (diluted 2000-fold) to human Ku70 or Ku86 (Serotec, Oxford, U.K.). The signal was detected by ECL (Amersham) according to manufacturer’s suggestions. RESULTS Effect of serum on DEB activity in CHO K1 cells The interaction of Ku with DNA was analyzed by EMSA. Retardation of migration of the DNA prob when incubated with the cell extract indicated binding of proteins to the DNA. Binding to DNA ends was shown by the facts that large amounts of circular plasmid DNA containing the identical sequence as the probe did not decrease the binding activity, and that small amounts of linear salmon sperm DNA with different sequences could effectively compete for binding (Fig. 1). The level of DEB activity depends on the amount of extracts and DNA probe. In the standard reaction, binding activity was linear over a range of 0.2 to 0.6 g of the extract in the presence of 2 ng of the probe. Under these conditions, the probe is in large excess
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Fig. 1. Assay of DEB activity by EMSA. Extract (0.6 g) of CHO K1 cells were incubated with 2 ng of the 32P-labeled DNA probe in the binding reaction containing either circular plasmid DNA (2.7 kb) or sonicated salmon sperm DNA (1.4 kb) with the amount in nanograms indicated on the top. Lane 1 is the control without extract. The positions of KuDNA complex (B) and free DNA (F) are shown with arrows.
and no DNA-protein ladder due to binding of multiple Ku to the same DNA molecule was observed (data not shown). These conditions were used in order to facilitate the quantitation of DEB activities. In order to study DEB activities in cells at different stages of the cell cycle, CHO K1 cells were synchronized in G1 phase by serum starvation, in S phase by serum replenishment, or in M phase by treatment with nocodazole. Synchronization was analyzed by flow cytometry, and extracts were made from the synchronized cells to determine DEB activities. Results of cell cycle analyses show CHO K1 cells were synchronized successfully to G1, S, and M by these methods (Fig. 2A). EMSA indicated that DEB activity in G1 cells was similar to that of the asynchronous control, but was reduced 10-fold in S and M cells (Fig. 2B). These results were reproducible and the average decrease was 6.5-fold with a variation from 5 to 10-fold in four independent experiments. A mixing experiment using a fixed amount of the G1 extract and increasing amounts of S or M extract indicated that
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Fig. 2. DEB activities of CHO K1 cells treated with serum or nocodazole. A: Cell cycle analyses. Exponentially growing (C), serum starved (G1), serum refed (S) or nocodazole treated (M) cells were stained with PI to determine the cell cycle profile by flow cytometry. Data are shown as DNA histograms. Details of the treatments are described in Materials and Methods. B: DEB activities of the treated cells. Extracts were prepared from cells treated the same way as in panel A, and DEB activity of each extract (0.4 g) was determined under standard conditions.
the reduced DEB activity in cells in S and M was not due to the presence of an inhibitor (Fig. 3). In order to confirm these results and to see whether other cell lines have the same phenomenon, we used another method to synchronize cells, and two more cell lines were included for comparison. Effect of thymidine block and nocodazole on DEB activities in different cell lines CHO K1, RKO and HeLa cells were synchronized in G1/early S and S phases by double thymidine block and releasing the blockage, and in M phase by treatment with nocodazole. Flow cytometry analyses indicate that cells highly enriched in particular phases of the cell cycle were obtained (Fig. 4A). In cells arrested at G1/early S, all 3 cell types were found to have similar DEB activity as their respective asynchronous controls. In contrast
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to previous results, DEB activity in S cells of CHO K1 did not significantly differ from that of the asynchronous control. This was also true for the other two cell lines RKO and HeLa (Fig. 4B). Treatment with nocodazole decreased the DEB activities in CHO K1 and RKO cells. The average from 4 experiments was 6.5-fold for CHO K1 and 8-fold for RKO cells. In contrast, M phase HeLa cells obtained by nocodazole treatment had the same level of DEB activity as the asynchronous control (Fig. 4B). Western blot analyses showed the levels of both Ku86 and Ku70 protein were the same in asynchronous control, G1/S, S and M RKO cells (Fig. 4C). So the reduced DEB activity in M phase RKO cells was not due to a decrease of the Ku protein. As in the case of CHO K1 cells, results of a mixing experiment indicated that the reduced activity was not due to the presence of an inhibitor (data not shown). We failed to detect Ku protein in CHO K1 extract, and therefore it is not known whether decreased DEB activity in CHO K1 M phase cells was due to a decrease in the Ku protein. Regardless of the cause of the decrease, the above results indicate that different methods of synchronization give different cell cycle effects on DEB activity (S cells of CHO K1), and even the same method can cause cell type-specific variation (M cells of HeLa). We then used a further cell separation method to see whether consistent results could be obtained in the three cell types. DEB activities in cells separated by elutriation Exponentially growing cells were subjected to counter-flow centrifugal elutriation and collected fractions were analyzed for their DNA content to determine their cell cycle stage. Results in Fig. 5 show that cells in particular fractions have DNA content characteristic of G1, S and G2/M of the cell cycle (Fig. 5A). Whole cell extracts were prepared from the collected cells to determine DEB activity and protein level of Ku. In all 3 cell types, G1, S and G2/M cells were found to have similar levels of DEB activity as their respective asynchronous controls. Western analyses of RKO and HeLa extracts indicate that the levels of Ku86 and Ku70 were the same in asynchronous control, G1, S, and G2/M cells (Fig. 5C). The results in Fig. 5 are from representative fractions of the elutriation experiment. Full spectrum analyses using cells from consecutive fractions after elutriation also led to the same conclusion, and the result was reproducible in three experiments.
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Fig. 3. Absence of inhibitor of DEB activity in CHO K1 cells treated with serum or nocodazole. Extracts prepared from serumor nocodazole-treated cells as described in Fig. 2 are designed as G1, S or M extract. DEB activities were first determined using different amount of G1 extract (0 to 1 g), 0.6 g of S or M extract. To show the absence of an inhibitor in S and M extracts, a fixed amount of G1 extract (0.6 g) was mixed with increasing amounts of S or M extract (0.1 to 0.6 g), and DEB activities after mixing were determined under standard conditions.
DISCUSSION This study shows that different method of synchronization can give differed cell cycle effect on DEB activity of Ku with the same cell type. The result in CHO K1 S cells acquired by serum starvation and thymidine block is an example. In addition, the same method of synchronization can give different results with different types of cells. Treatment with nocodazole is a very common and effective method to block cells at M phase. However, the effect on DEB activities after the treatment varied among different cell types, being reduced in CHO K1 and RKO cells but having no effect in HeLa cells. The reason for these discrepancies is unknown. But there are examples of cells arrested artificially in a particular phase of the cell cycle showing abnormal protein expression. Cyclin A in nocodazole-treated HeLa cells was unstable and therefore showed a reduced level (Pines and Hunter, 1990). In early S hamster cells arrested by hydroxyurea, the level of cyclin B was abnormally high due to continuous accumulation during treatment. Interestingly, this type of abnormality was not observed in HeLa or other human cell lines (Steinmann et al., 1991). When using cells enriched at G1, S and G2/M by elutriation, DEB activities were similar in different phase of the cell cycle in all three cell types. We consider enrichment by centrifugal elutriation produces cells with less perturbation than treatment
with chemicals or serum withdrawal, and conclude that Ku activity does not change during cell cycle. This suggestion is supported by a previous report showing the protein levels of both Ku70 and Ku86 were the same in G1, S and G2/M of HeLa cells synchronized by hydroxyurea blockage and release (Lee et al., 1997). Several important points can be made from this study. First, the results clearly indicate that commonly used methods of cell synchronization give inconsistent results of cell cycle effects on DEB activity of Ku. We do not know whether the inconsistency described here exists in other types of cells. Nevertheless, it seems inadequate to depend on a single method in dealing with cell cycle effect on Ku. Confirming the results by an alternative procedure is recommended, and centrifugal elutriation seems to be a good choice. Second, it is well known that radiation sensitivity of mammalian cells changes during cell cycle, being most sensitive in G2/M, intermediate in G1, and most resistant at the S phase. Our present findings suggest that cell cycle-dependent variation of radiation sensitivity is probably not due to changes of DEB activity of Ku, although Ku plays an important role in repairing DNA double strand breaks and mutants of Ku-defective cells are hypersensitive to ionizing radiation. Third, if DEB activity and protein level of Ku are the same in different phases of the cell cycle and Ku is involved in growth regulation, the
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Fig. 4. DEB activities in different cell lines synchronized by thymidine block or nocodazole treatment. A: Cell cycle analyses. Exponentially growing CHO K1, RKO and HeLa cells (C), and those cells synchronized to G1/early S phase (G1), to S phase (S) by double thymidine block or to M phase (M) by treatment with nocodazole were stained with PI and subjected to cell cycle analyses by flow cytometry. B: DEB activities of the synchronized cells. Extracts were prepared from cells treated with thymidine (Thy) or nocodazole (Noc) as in panel A described above. DEB activities (0.4 g of K1 extract and 0.2 g of RKO or HeLa extract) were determined under standard conditions. C: Protein (10 g) of RKO extracts in panel B were analyzed by Western blot with antibodies to Ku86 and Ku70. The positions of the two subunits are shown by arrows.
relationship betweeen Ku and growth must be at another level. For example, Ku might have functions other than DNA binding and stimulation of DNA-PK activity, or different cellular location during growth. In fact, a recent report suggested that Ku might have a novel function that was not related to DNA-PK-dependent DNA repair in G2/M cells (Munoz et al., 1998). Also, a previous report showed that the Ku86 subunit in mitoticphase-arrested cells was completely away from
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Fig. 5. DEB activities in cells at different stages of the cell cycle. A: Cell cycle analyses. Exponentially growing CHO K1, RKO and HeLa cells, C=contrast, and those cells enriched at the G1, S, or G2/M phase (M) of the cell cycle by centrifugal elutriation were stained with PI and subjected to cell cycle analyses by flow cytometry. B: Determination of DEB activities. Extracts were prepared from phase-enriched cells of the three cell lines described in panel A. DEB activities (0.4 g of K1 extract and 0.2 g of RKO or HeLa extract) were determined under standard conditions. C: Protein (20 g) of RKO or HeLa extract in panel B were analyzed by Western blot with antibodies to Ku86 and Ku70. The positions of the two subunits are shown by arrows. Results of the phase-enriched cells were from representative fractions after elutriation.
chromosomes (Li and Yeh, 1992), although another result indicated that Ku70 was concentrated at the nuclear periphery in interphase cells and was associated with metaphase chromosomes (Higashiura et al., 1992). Taken together, it appears that other DNA dsb repair system which is independent of DEB activity of Ku exists, and the function or mechanism of action of Ku are more complex than it was originally thought.
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ACKNOWLEDGEMENTS We thank Ms Chia-Lian Han for preparing this manuscript. This work was supported by grants NSC87-2311-B007-015 and NSC87-2312-B007005-NU from National Science Council, Republic of China. REFERENCES A CW, 1993. DNA damage and the DNA-activated protein kinase. Trends Biochem Sci 18: 433–437. B PR, G AJ, C J, H JA, 1993. Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. J Biol Chem 268: 7594–7601. B T, F NJ, T GE, S GC, D J, G TM, M R, V AJ, A FW, J PA, J SP, 1995. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80: 813–823. B NV, H KT, W Z, L SE, H DM, B DM, P CR, B H, R W, H EA, W DT, 1995. Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen. Proc Natl Acad Sci USA 92: 890–894. F M, F JW, K EL, 1993. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J Biol Chem 268: 10,546–10,552. F NJ, G TM, B T, J PA, J SP, 1995. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc Natl Acad Sci USA 92: 320–324. G RC, S TD, 1994. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem 269: 15,981–15,984. G W, T H, R DJ, P GG, P L, H RJ, 1996. Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380: 265–268. G K, L Z, J ST, 1998. Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals. Proc Natl Acad Sci USA 95: 10,390–10,395. G TM, J SP, 1993. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72: 131–142. G Y, J S, G Y, W DT, A FW, 1997. Ku70deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc Natl Acad Sci USA 94: 8076–8081. H M, S Y, T M, M T, Y T, 1992. Immunolocalization of Ku-proteins (p80/ p70)—localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosome. Exp Cell Res 201: 444–451.
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J SW, E NL, 1995. A micro-scale method to isolate DNA-binding proteins suitable for quantitative comparison of expression levels from transfected cells. Nucleic Acids Res 23: 3607–3608. K LM, S SG, J PA, 1984. X-ray sensitive mutants of Chinese hamster ovary cells defective in doublestrand break rejoining. Mutat Res 132: 189–196. K CU, P CK, E JW, C CA, F LM, C T, O MA, B JM, 1995. DNAdependent kinase (p350) as a candidate gene for the murine SCID defect. Science 267: 1178–1183. K A, S V, G I, 1993. The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription. Nucleic Acids Res 21: 2057–2063. L SE, M RA, C A, H EA, 1997. Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle. Mol Cell Biol 17: 1425–1433. L LL, Y NH, 1992. Cell-cycle-dependent migration of DNA-binding protein Ku80 into nucleoli. Exp Cell Res 199: 262–268. M T, H JA, 1986. Mechanism of interaction between Ku protein and DNA. J Biol Chem 261: 10,375–10,379. M T, A M, Y H, I S, Y S, H M, 1981. Characterization of a high molecular weight acidic nuclear protein recognized by autoantibodies in sera from patients with polymyositis-scleroderma overlap. J Clin Invest 68: 611–620. M T, H JA, S JA, 1986. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem 261: 2274–2278. M P, Z MZ, B J-M, P J, 1998. Hypersensitivity of Ku-deficient cells toward the DNA topoisomerase II inhibitor ICRF-193 suggests a novel role for Ku antigen during the G2 and M phases of the cell cycle. Mol Cell Biol 18: 5797–5808. M D, M RE, 1986. Cell cycle-dependent cytotoxicity of alkylating agents: determination of nitrogen mustardinduced DNA cross-links and their repair in Chinese hamster ovary cells synchronized by centrifugal elutriation. Cancer Res 46: 2324–2329. N A, C C, C S V, S M, S K, N MC, L GC, 1996. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382: 551–555. P S, S F, 1991. Analysis of the mechanism of interaction of simian Ku protein with DNA. Nucleic Acids Res 19: 5619–5624. P J, H H, 1990. Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346: 760–763. R WK, C G, 1994. A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol Cell Biol 14: 4741–4748. R WH, 1985. Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera. J Exp Med 161: 18–39. R MR, H Y, F A, H L, R FH, 1994. A DNA-binding activity, TRAC, specific for the TRA element of the transferrin receptor gene copurifies with the Ku autoantigen. Proc Natl Acad Sci USA 91: 6354– 6358.
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SP, J PA, 1994. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265: 1442–1445. Y M, O R, MR DK, Z S, B H, 1985. Purification of an 86-70 kDa nuclear DNA-associated protein complex. Biochim Biophys Acta 841: 22–29. Z W, K PC, C WG, 1992. Differential gene expression in wild-type and X-ray-sensitive mutants of Chinese hamster ovary cell lines. Mutat Res 274: 237– 245. Z C, B MA, L DS, H P, R DB, 1996. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86: 379–389.
DNA-END BINDING ACTIVITY OF KU IN SYNCHRONIZED CELLS LI-FEN CHOU and WEN-GANG CHOU* Department of Life Science, National Tsin-Hua University, Hsinchu, Taiwan, R.O.C. Received 9 March 1999; accepted 16 July 1999
Three different types of cells were synchronized by various methods and DNA-end binding (DEB) activities of Ku were compared with asynchronous controls. In CHO K1 cells synchronized in G1 phase by serum starvation and in S phase by serum refeeding, DEB activity was reduced in S cells but remained unchanged in G1 cells. However, the same type of cells synchronized in G1/S phase by double thymidine block and in S phase by releasing the blockage, have the same DEB activity as asynchronous controls. A similar result was found in RKO and HeLa cells synchronized by the latter method. Arresting cells in mitosis with nocodazole also generated different cell cycle effects. Ku activity was reduced in CHO K1 and RKO cells, but not in HeLa cells after treatment with nocodazole. In phase-enriched cells separated by centrifugal elutriation, DEB activities were similar at different stages of the cell cycle in all three types of cells. Thus, different synchronization procedures can give very different values of Ku activity in a cell type-dependent manner. Results from elutriated cells are consistent, and suggest DEB 1999 Academic Press activity of Ku does not change with the cell cycle. K: Ku protein; DNA-end binding; cell cycle.
INTRODUCTION The Ku protein was originally described as an autoantigen in patients with sclerodermapolymyositis overlap syndrome (Mimori et al., 1981). It is an abundant nuclear protein consisting of two polypeptides with molecular mass of 70- and 86 kDa (Reeves, 1985; Yanvea et al., 1985; Mimori et al., 1986). The heterodimeric Ku was shown to have an unique activity of binding to doublestranded DNA with free ends, but not to circular or single stranded DNAs (Mimori et al., 1986; Mimori and Hardin, 1986). In addition to this DNA-end binding (DEB) activity, Ku was later shown to bind DNA with nicks, gaps and other specialized structures such as stem-loop, dumbbellshaped and ‘bubble’ structures (Paillard and Struss, 1991; Blier et al., 1993; Falzon et al., 1993). Ku is the DNA-binding component of the DNAdependent protein kinase, DNA-PK. The kinase activity of the catalytic subunit, p460 or DNA*To whom correspondence should be addressed: Wen-Gang Chou, Department of Life Science, National Tsin-Hua University, Hsinchu, Taiwan, R.O.C. 1065–6995/99/100663+08 $30.00/0
PKcs is stimulated by the binding of Ku to DNA ends (Gottlieb and Jackson, 1993; Suwa et al., 1994). DNA-PK can phosphorylate a variety of proteins involved in signal transduction, DNA repair and replication in vitro (Anderson, 1993). Indeed, DEB activity of Ku was found to be deficient in the X-ray-sensitive (xrs) mutants derived from the CHO K1 cell line (Getts and Stamato, 1994; Rathmell and Chu, 1994). The xrs mutants are defective in repairing DNA doublestrand breaks (dsb) and V(D)J recombination (Kemp et al., 1984; Finnie et al., 1995). The role of Ku in dsb repair was confirmed by the finding that introducing the gene or cDNA of Ku86 into xrs mutants restored X-ray resistance, dsb repair and V(D)J recombination (Smider et al., 1994; Taccioli et al., 1994; Boubnov et al., 1995; Finnie et al., 1995). In addition, Ku70-deficient embryonic stem cells also have increased X-ray sensitivity and defective V(D)J recombination (Gu et al., 1997). The role of DNA-PK in DNA end-joining was also established by the discovery that SCID mice, which are unable to carry out V(D)J recombination and are sensitive to ionizing radiation, are defective 1999 Academic Press
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in p460 (Blunt et al., 1995; Kirchgessner et al., 1995). Besides an important function in DNA repair, Ku plays a role in regulating transcription of transferrin receptor gene (Roberts et al., 1994), long terminal repeat of mouse mammary tumor virus (Giffin et al., 1996), mouse ribosomal gene (Kuhn et al., 1993) and metallothionein-I (Goshal et al., 1998). Two groups have generated homozygous null mutants for mouse Ku86 gene (Nussenzweig et al., 1996; Zhu et al., 1996). These Ku86 knock-out mice were found to have impaired V(D)J recombination, and therefore both B and T cells were in low numbers and were developmentally arrested as progenitors. Furthermore, the mutant mice have a dwarf phenotype and only growing to 40-60% of the control wild type size (Zhu et al., 1996). Because many of these processes Ku seems to be engaged in are growth related, it is of interest to investigate whether Ku activity shows cell cycle dependency. MATERIALS AND METHODS Cell cultures The Chinese hamster ovary cell line, CHO K1, was cultured in F10 medium as described previously (Zhu et al., 1992). The human cervical carcinoma cell line, HeLa, and the colorectal carcinoma cell line, RKO, were cultured in DMEM with 45 m sodium bicarbonate and 10% fetal calf serum. Cells were maintained as monolayers by seeding 1105 cells in 25-cm2 flasks in a 37C, 5% CO2 and 95% air incubator. Exponentially growing cells were maintained by subculturing when confluence reached 50–70%. Cell synchronization Exponentially growing CHO K1 cells were synchronized to G1 and S phase by serum starvation and replenishing. Cells were maintained in F10 medium with 0.1% serum for 3 days and were refed with complete F10 medium. Cell cycle profiles were analyzed every 2 h after refeeding by flow cytometry and representative samples were chosen. G1 and S phase cells were those obtained 4 h and 17 h after refeeding, respectively. CHO K1, HeLa, and RKO cells were synchronized to G1/S boundary or S phase by double thymidine block. CHO K1 cells were sequentially incubated with medium containing 2 m thymidine for 12 h, normal medium for 6 h, 2 m thymidine
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for 12 h and with normal medium. G1/S cells were obtained at 1 h and S phase cells were obtained at 7 h after growing in normal medium. Similarly, HeLa and RKO cells were treated with thymidine for 17 h, normal medium for 9 h, and thymidine for 15 h before being incubated in normal medium. G1/S cells were obtained at 1 h and S phase cells were obtained at 3 h after growing in normal medium. In the above experiments, cell cycle profiles were determined by flow cytometry every hour after releasing the blockage, and representative samples were chosen at the time-points indicated above. Separation of phase-specific cells by centrifugal elutriation was performed according to Murray and Meyn (1986) with minor modifications. Exponentially growing cells (2108) were harvested, suspended in 20 ml of complete medium and loaded into a Beckman JE-6B rotor. In the separation of K1 cells, the rotor speed was maintained at 1700 rpm and cells were introduced into the chamber at 12 ml/min. The flow rate was then increased from 12 to 30 ml/min in 1.5-ml/min increments. At each flow rate, cells in 80 ml were collected for determination of cell cycle stage by flow cytometry and for preparation of whole cell extract to assay DEB activity. For HeLa cells, the rotor speed was 2000 rpm and the initial flow rate was 14 ml/min. The flow rate was increased to 46 ml/min in 2-ml/min increments. For RKO cells, the rotor speed was 2000 rpm and the initial flow rate was 10 ml/min. The flow rate was increased to 34 ml/min in 2 ml/min increments. All three cell lines were arrested at M phase by treatment with 100 ng/ml nocodazole for 24 h. Cell cycle analysis For flow cytometric analysis of DNA content, cells (1–2106) were fixed in 70% ethanol at 4C for 16–24 h. The fixed cells were treated with 1 mg/ml RNAse A in PBS for 30 min at room temperature. The cells were stained with 10 g/ml propidium iodide for at least 15 min and less than 6 h. The samples were analyzed by FACScan (Becton Dickinson). For each sample, 10,000 cells in the gated region (the distribution region of cycling cells) were collected for data analysis. Assay for DEB activity Cells were harvested with a rubber policeman and by centrifugation. Whole cell extracts were prepared by the method of Jiang and Eberhardt (1995). Cell pellets were resuspended in three
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packed cell volumes of ice-cold extraction buffer (20 m HEPES–HCl, pH 7.9; 0.5 KCl; 0.2 m EDTA; 0.5 m DTT; 0.5 m PMSF and 20% glycerol). The samples were subjected to three cycles of freezing and thawing, centrifuged at 12,000g for 10 min at 4C. The supernatant was stored at 70C. Aliquot of sample was diluted with the extraction buffer without KCl to 0.1– 0.2 g/ml for assay of DEB activity. The protein concentration was determined by the Bio-Rad protein assay kit (Bradford method), with bovine serum albumin as the standard. DEB activity was measured by gel electrophoretic mobility shift assay (EMSA) described previously (Rathmell and Chu, 1994). The standard 10-l reaction contained 0.2–0.6 g of extract, 2 ng of 32P-5 -labeled linear DNA probe (approximately 105 cpm), and 300 ng of circular pGEM1 plasmid DNA. The linear DNA probe was the gel-purified 222 base-pair fragment of pGEM1 plasmid digested with restriction enzyme AvaII. The reaction mixture was separated by electrophoresis in 6% non-denaturing polyacrylamide gel (acrylamide/bis-acrylamide: 29/1). The gel was dried for autoradiography. Western blot analysis Proteins in the extract was separated with 6% SDS-PAGE, electroblotted to PVDF membrane and incubated with antibodies (diluted 2000-fold) to human Ku70 or Ku86 (Serotec, Oxford, U.K.). The signal was detected by ECL (Amersham) according to manufacturer’s suggestions. RESULTS Effect of serum on DEB activity in CHO K1 cells The interaction of Ku with DNA was analyzed by EMSA. Retardation of migration of the DNA prob when incubated with the cell extract indicated binding of proteins to the DNA. Binding to DNA ends was shown by the facts that large amounts of circular plasmid DNA containing the identical sequence as the probe did not decrease the binding activity, and that small amounts of linear salmon sperm DNA with different sequences could effectively compete for binding (Fig. 1). The level of DEB activity depends on the amount of extracts and DNA probe. In the standard reaction, binding activity was linear over a range of 0.2 to 0.6 g of the extract in the presence of 2 ng of the probe. Under these conditions, the probe is in large excess
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Fig. 1. Assay of DEB activity by EMSA. Extract (0.6 g) of CHO K1 cells were incubated with 2 ng of the 32P-labeled DNA probe in the binding reaction containing either circular plasmid DNA (2.7 kb) or sonicated salmon sperm DNA (1.4 kb) with the amount in nanograms indicated on the top. Lane 1 is the control without extract. The positions of KuDNA complex (B) and free DNA (F) are shown with arrows.
and no DNA-protein ladder due to binding of multiple Ku to the same DNA molecule was observed (data not shown). These conditions were used in order to facilitate the quantitation of DEB activities. In order to study DEB activities in cells at different stages of the cell cycle, CHO K1 cells were synchronized in G1 phase by serum starvation, in S phase by serum replenishment, or in M phase by treatment with nocodazole. Synchronization was analyzed by flow cytometry, and extracts were made from the synchronized cells to determine DEB activities. Results of cell cycle analyses show CHO K1 cells were synchronized successfully to G1, S, and M by these methods (Fig. 2A). EMSA indicated that DEB activity in G1 cells was similar to that of the asynchronous control, but was reduced 10-fold in S and M cells (Fig. 2B). These results were reproducible and the average decrease was 6.5-fold with a variation from 5 to 10-fold in four independent experiments. A mixing experiment using a fixed amount of the G1 extract and increasing amounts of S or M extract indicated that
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Fig. 2. DEB activities of CHO K1 cells treated with serum or nocodazole. A: Cell cycle analyses. Exponentially growing (C), serum starved (G1), serum refed (S) or nocodazole treated (M) cells were stained with PI to determine the cell cycle profile by flow cytometry. Data are shown as DNA histograms. Details of the treatments are described in Materials and Methods. B: DEB activities of the treated cells. Extracts were prepared from cells treated the same way as in panel A, and DEB activity of each extract (0.4 g) was determined under standard conditions.
the reduced DEB activity in cells in S and M was not due to the presence of an inhibitor (Fig. 3). In order to confirm these results and to see whether other cell lines have the same phenomenon, we used another method to synchronize cells, and two more cell lines were included for comparison. Effect of thymidine block and nocodazole on DEB activities in different cell lines CHO K1, RKO and HeLa cells were synchronized in G1/early S and S phases by double thymidine block and releasing the blockage, and in M phase by treatment with nocodazole. Flow cytometry analyses indicate that cells highly enriched in particular phases of the cell cycle were obtained (Fig. 4A). In cells arrested at G1/early S, all 3 cell types were found to have similar DEB activity as their respective asynchronous controls. In contrast
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to previous results, DEB activity in S cells of CHO K1 did not significantly differ from that of the asynchronous control. This was also true for the other two cell lines RKO and HeLa (Fig. 4B). Treatment with nocodazole decreased the DEB activities in CHO K1 and RKO cells. The average from 4 experiments was 6.5-fold for CHO K1 and 8-fold for RKO cells. In contrast, M phase HeLa cells obtained by nocodazole treatment had the same level of DEB activity as the asynchronous control (Fig. 4B). Western blot analyses showed the levels of both Ku86 and Ku70 protein were the same in asynchronous control, G1/S, S and M RKO cells (Fig. 4C). So the reduced DEB activity in M phase RKO cells was not due to a decrease of the Ku protein. As in the case of CHO K1 cells, results of a mixing experiment indicated that the reduced activity was not due to the presence of an inhibitor (data not shown). We failed to detect Ku protein in CHO K1 extract, and therefore it is not known whether decreased DEB activity in CHO K1 M phase cells was due to a decrease in the Ku protein. Regardless of the cause of the decrease, the above results indicate that different methods of synchronization give different cell cycle effects on DEB activity (S cells of CHO K1), and even the same method can cause cell type-specific variation (M cells of HeLa). We then used a further cell separation method to see whether consistent results could be obtained in the three cell types. DEB activities in cells separated by elutriation Exponentially growing cells were subjected to counter-flow centrifugal elutriation and collected fractions were analyzed for their DNA content to determine their cell cycle stage. Results in Fig. 5 show that cells in particular fractions have DNA content characteristic of G1, S and G2/M of the cell cycle (Fig. 5A). Whole cell extracts were prepared from the collected cells to determine DEB activity and protein level of Ku. In all 3 cell types, G1, S and G2/M cells were found to have similar levels of DEB activity as their respective asynchronous controls. Western analyses of RKO and HeLa extracts indicate that the levels of Ku86 and Ku70 were the same in asynchronous control, G1, S, and G2/M cells (Fig. 5C). The results in Fig. 5 are from representative fractions of the elutriation experiment. Full spectrum analyses using cells from consecutive fractions after elutriation also led to the same conclusion, and the result was reproducible in three experiments.
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Fig. 3. Absence of inhibitor of DEB activity in CHO K1 cells treated with serum or nocodazole. Extracts prepared from serumor nocodazole-treated cells as described in Fig. 2 are designed as G1, S or M extract. DEB activities were first determined using different amount of G1 extract (0 to 1 g), 0.6 g of S or M extract. To show the absence of an inhibitor in S and M extracts, a fixed amount of G1 extract (0.6 g) was mixed with increasing amounts of S or M extract (0.1 to 0.6 g), and DEB activities after mixing were determined under standard conditions.
DISCUSSION This study shows that different method of synchronization can give differed cell cycle effect on DEB activity of Ku with the same cell type. The result in CHO K1 S cells acquired by serum starvation and thymidine block is an example. In addition, the same method of synchronization can give different results with different types of cells. Treatment with nocodazole is a very common and effective method to block cells at M phase. However, the effect on DEB activities after the treatment varied among different cell types, being reduced in CHO K1 and RKO cells but having no effect in HeLa cells. The reason for these discrepancies is unknown. But there are examples of cells arrested artificially in a particular phase of the cell cycle showing abnormal protein expression. Cyclin A in nocodazole-treated HeLa cells was unstable and therefore showed a reduced level (Pines and Hunter, 1990). In early S hamster cells arrested by hydroxyurea, the level of cyclin B was abnormally high due to continuous accumulation during treatment. Interestingly, this type of abnormality was not observed in HeLa or other human cell lines (Steinmann et al., 1991). When using cells enriched at G1, S and G2/M by elutriation, DEB activities were similar in different phase of the cell cycle in all three cell types. We consider enrichment by centrifugal elutriation produces cells with less perturbation than treatment
with chemicals or serum withdrawal, and conclude that Ku activity does not change during cell cycle. This suggestion is supported by a previous report showing the protein levels of both Ku70 and Ku86 were the same in G1, S and G2/M of HeLa cells synchronized by hydroxyurea blockage and release (Lee et al., 1997). Several important points can be made from this study. First, the results clearly indicate that commonly used methods of cell synchronization give inconsistent results of cell cycle effects on DEB activity of Ku. We do not know whether the inconsistency described here exists in other types of cells. Nevertheless, it seems inadequate to depend on a single method in dealing with cell cycle effect on Ku. Confirming the results by an alternative procedure is recommended, and centrifugal elutriation seems to be a good choice. Second, it is well known that radiation sensitivity of mammalian cells changes during cell cycle, being most sensitive in G2/M, intermediate in G1, and most resistant at the S phase. Our present findings suggest that cell cycle-dependent variation of radiation sensitivity is probably not due to changes of DEB activity of Ku, although Ku plays an important role in repairing DNA double strand breaks and mutants of Ku-defective cells are hypersensitive to ionizing radiation. Third, if DEB activity and protein level of Ku are the same in different phases of the cell cycle and Ku is involved in growth regulation, the
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Fig. 4. DEB activities in different cell lines synchronized by thymidine block or nocodazole treatment. A: Cell cycle analyses. Exponentially growing CHO K1, RKO and HeLa cells (C), and those cells synchronized to G1/early S phase (G1), to S phase (S) by double thymidine block or to M phase (M) by treatment with nocodazole were stained with PI and subjected to cell cycle analyses by flow cytometry. B: DEB activities of the synchronized cells. Extracts were prepared from cells treated with thymidine (Thy) or nocodazole (Noc) as in panel A described above. DEB activities (0.4 g of K1 extract and 0.2 g of RKO or HeLa extract) were determined under standard conditions. C: Protein (10 g) of RKO extracts in panel B were analyzed by Western blot with antibodies to Ku86 and Ku70. The positions of the two subunits are shown by arrows.
relationship betweeen Ku and growth must be at another level. For example, Ku might have functions other than DNA binding and stimulation of DNA-PK activity, or different cellular location during growth. In fact, a recent report suggested that Ku might have a novel function that was not related to DNA-PK-dependent DNA repair in G2/M cells (Munoz et al., 1998). Also, a previous report showed that the Ku86 subunit in mitoticphase-arrested cells was completely away from
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Fig. 5. DEB activities in cells at different stages of the cell cycle. A: Cell cycle analyses. Exponentially growing CHO K1, RKO and HeLa cells, C=contrast, and those cells enriched at the G1, S, or G2/M phase (M) of the cell cycle by centrifugal elutriation were stained with PI and subjected to cell cycle analyses by flow cytometry. B: Determination of DEB activities. Extracts were prepared from phase-enriched cells of the three cell lines described in panel A. DEB activities (0.4 g of K1 extract and 0.2 g of RKO or HeLa extract) were determined under standard conditions. C: Protein (20 g) of RKO or HeLa extract in panel B were analyzed by Western blot with antibodies to Ku86 and Ku70. The positions of the two subunits are shown by arrows. Results of the phase-enriched cells were from representative fractions after elutriation.
chromosomes (Li and Yeh, 1992), although another result indicated that Ku70 was concentrated at the nuclear periphery in interphase cells and was associated with metaphase chromosomes (Higashiura et al., 1992). Taken together, it appears that other DNA dsb repair system which is independent of DEB activity of Ku exists, and the function or mechanism of action of Ku are more complex than it was originally thought.
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ACKNOWLEDGEMENTS We thank Ms Chia-Lian Han for preparing this manuscript. This work was supported by grants NSC87-2311-B007-015 and NSC87-2312-B007005-NU from National Science Council, Republic of China. REFERENCES A CW, 1993. DNA damage and the DNA-activated protein kinase. Trends Biochem Sci 18: 433–437. B PR, G AJ, C J, H JA, 1993. Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks. J Biol Chem 268: 7594–7601. B T, F NJ, T GE, S GC, D J, G TM, M R, V AJ, A FW, J PA, J SP, 1995. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80: 813–823. B NV, H KT, W Z, L SE, H DM, B DM, P CR, B H, R W, H EA, W DT, 1995. Complementation of the ionizing radiation sensitivity, DNA end binding, and V(D)J recombination defects of double-strand break repair mutants by the p86 Ku autoantigen. Proc Natl Acad Sci USA 92: 890–894. F M, F JW, K EL, 1993. EBP-80, a transcription factor closely resembling the human autoantigen Ku, recognizes single- to double-strand transitions in DNA. J Biol Chem 268: 10,546–10,552. F NJ, G TM, B T, J PA, J SP, 1995. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc Natl Acad Sci USA 92: 320–324. G RC, S TD, 1994. Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repair-deficient mutant. J Biol Chem 269: 15,981–15,984. G W, T H, R DJ, P GG, P L, H RJ, 1996. Sequence-specific DNA binding by Ku autoantigen and its effects on transcription. Nature 380: 265–268. G K, L Z, J ST, 1998. Overexpression of the large subunit of the protein Ku suppresses metallothionein-I induction by heavy metals. Proc Natl Acad Sci USA 95: 10,390–10,395. G TM, J SP, 1993. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 72: 131–142. G Y, J S, G Y, W DT, A FW, 1997. Ku70deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc Natl Acad Sci USA 94: 8076–8081. H M, S Y, T M, M T, Y T, 1992. Immunolocalization of Ku-proteins (p80/ p70)—localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosome. Exp Cell Res 201: 444–451.
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