- Email: [email protected]
Bleomycin enhances random integration of transfected DNA into a human genome
Mutation Research 409 Ž1998. 1–10
Bleomycin enhances random integration of transfected DNA into a human genome Chikako Nakayama, Noritaka Adachi, Hideki Koyama
)
Kihara Institute for Biological Research, Yokohama City UniÕersity, Maioka-cho 641-12, Totsuka-ku, Yokohama 244, Japan Received 9 October 1997; revised 17 June 1998; accepted 23 June 1998
Abstract In mammalian cells, nonhomologous Žillegitimate. recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin ŽBLM., on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake andror accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Bleomycin; Transfection; Random integration; Nonhomologous recombination; DNA strand break
1. Introduction Mammalian cells possess an activity of nonhomologous Žillegitimate. recombination that requires little or no sequence homology between DNA substrates w1x. Nonhomologous recombination presumably prevents the cells from death by repairing DNA double-strand breaks, which may result from endoge) Corresponding author. Tel.: q81-45-820-1907; Fax: q81-45820-1901; E-mail: [email protected]
nous mechanisms or occasional exposure to endogenous or exogenous agents w2–4x. In contrast, it may cause deleterious genomic rearrangements, such as translocation w5x, deletion w6x, inversion w7x, or gene amplification w8x, leading to a variety of genetic diseases and cancer. When foreign DNA is transfected into mammalian cells, a nonhomologous recombination event results in random integration of the DNA into the host genome w9–11x. The practical application of the recombination is to generate transfectants that stably
0921-8777r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 8 . 0 0 0 3 6 - 6
2
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
express a gene of interest. For gene therapy, improvements in the frequency of the integration would be necessary. On the other hand, if the foreign DNA includes sequences homologous to chromosomal sequences, a homologous recombination event results in gene targeting in a small minority of transfected cells. Highly efficient gene targeting would provide the ideal protocol of gene therapy, but at present the efficiency of homologous recombination is several orders magnitude too low for this to be feasible. To this end, improvements in the frequency of homologous recombination and reductions in the proportion of random integration would be required. Thus, it will be important to understand the mechanisms of nonhomologous as well as homologous recombination, and to make attempts to control the process of genomic integration events. However, the mechanism of such integration events is poorly understood, although it appears to involve transient strand breaks of chromosomal DNA, followed by ligation of the breaks and both ends of transfected, linear DNA w4,11,12x. We recently reported that, in hamster and human cells, treatment with DNA topoisomerase Žtopo. II inhibitors after transfection with plasmid DNA significantly enhance random integration by nonhomologous recombination w13,14x. Topo II is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes, through a transient double-strand break and subsequent religation of the break w15,16x. The enzyme has been implicated in many cellular processes such as transcription, replication, or chromosome condensation and segregation w17–23x. Also, extensive studies have indicated the involvement of topo II in various nonhomologous recombination reactions in vivo w24–26x as well as in vitro w27–30x. Many of topo II inhibitors, such as VP-16 Žetoposide., VM-26 Žteniposide. or m-AMSA, are able to generate and stabilize double-strand breaks by preventing the subsequent ligation step w16,31x. In the present report, we have examined the effect of a radiomimetic drug, bleomycin ŽBLM., on nonhomologous recombination in human cells. Since many of topo II inhibitors cause double-strand breaks of DNA w16,31x, we expected that BLM would also be effective in enhancing random integration. BLM is an antitumor antibiotic that exerts its cytotoxic effect by generating single- and double-strand breaks
of DNA in the presence of FeŽII. and O 2 w32–39x. The cutting reaction occurs in the minor groove of DNA predominantly at GpC and GpT sequences w37–39x. Here we show that BLM greatly enhances random integration of transfected plasmid DNA into human chromosomes. We also suggest that this enhancement results from a direct action of the drug in a nonhomologous recombination reaction within the nucleus.
2. Materials and methods 2.1. Cells and culture methods The human cell lines PA1, HeLa and EJ-1 used here were described previously w14x. Cells were maintained in ES medium ŽNissui. supplemented with 5% fetal bovine serum Žgrowth medium. at 378C in a humidified atmosphere of 5% CO 2 in air w14x. For all experiments, logarithmically growing cells were used. For transfection, a plasmid vector, pSV2neo, carrying the bacterial neomycin-resistant gene was purified by double CsCl gradient centrifugation; prior to transfection, it was linearized, unless otherwise stated, by digestion with EcoRI which recognizes the unique site outside the neomycin-resistant gene w40x. 2.2. Recombination assay by transfection The method reported by Boussif et al. w41x with a cationic polymer polyethyleneimine ŽPEI. was modified for stable transfection by Koyama et al. Žin preparation.. Briefly, cells were plated at 2 = 10 5 cellsrwell in a 12-well plate ŽCostar. containing 1 ml of growth medium, grown for 20 h, and transfected by adding the complex consisting of 1.0 mg of pSV2neo and 3 ml of 10 mM PEI Ž50 kDa, Sigma. in 0.4 ml of serum-free ES medium. After a 5-h incubation, the medium was replaced with growth medium, and the cells were grown for a further 20 h in the presence or absence of BLM ŽBleomycin–HCl, Wako.. BLM was dissolved into water and filtered for sterilization. The cells were harvested by trypsinization, diluted appropriately, and replated at 5 = 10 4 –10 5 cellsr60-mm dish in 5 ml of growth medium containing 0.25 mgrml of active Geneticin
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
ŽG418, Gibco BRL.; the cells were subsequently cultured for 13-14 days to select for G418-resistant ŽG418 r . colonies, as described previously w13,14x. To estimate cell survival, aliquots of the BLM-treated cells were plated at 300 cellsr60-mm dish in growth medium without added G418 and cultured for 11–12 days. The resulting colonies were fixed with 10% formaldehyde in saline, and stained with 0.1% crystal violet and counted. The frequency of G418 r colonies Ži.e., recombinant frequency. was calculated as a function of surviving cells. The value of the recombinant frequency in BLM-treated cells relative to that in untreated cells was calculated and expressed as relative recombinant frequency as described w13,14x. 2.3. Luciferase assay PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, transfected with 1.0 mg of NotI-linearized pGL3-Promoter ŽPromega. as mentioned above and then cultured in growth medium in the presence or absence of BLM. After 20 h, the cells were assayed for luciferase activity as described previously w42x. The luciferase activity of untreated cells was taken as 1, and the relative activity of the drug-treated cells was calculated.
3
ŽCa2q and Mg 2q-free phosphate-buffered saline., and subjected to the comet assay essentially as described by Klaude et al. w44x. Microscope slides were pretreated by spreading 20 ml of 0.5% agarose ŽSeaKem GTG, FMC Bioproducts. in PBSy evenly over the surface, and dried. The cell suspension Ž10 3 –10 4 cellsrml. was mixed with an equal volume of 1% low melting agarose ŽSeaPlaque GTG, FMC Bioproducts.. Fifty microliters of the mixture was immediately pipetted onto the pretreated slide, covered with a coverslip and left on ice. When the gel had set, the coverslip was removed and the slides were immersed in a lysis buffer Ž2.5 M NaCl, 100 mM EDTA, 10% DMSO, 1% Triton X-100 and 10 mM Tris–HCl, pH 10. and kept on ice for 2 h. After cell lysis, the slides were placed on a horizontal slab of an electrophoretic unit containing 0.3 M NaOHr1 mM EDTA ŽpH 13.. After equilibration for 20 min at 118C, electrophoresis was carried out at 25 V Ž300 mA. for 25 min. The samples were then neutralized in 0.4 M Tris–HCl ŽpH 8., rinsed, fixed with methanol, and stained with SYBR GREEN I ŽWako.. Comet-positive cells were detected and photographed with an Olympus AX70 fluorescence microscope ŽOlympus.. Their percentage was calculated by counting more than 400 cells. 2.6. Flow cytometry
2.4. b-galactosidase assay 5
PA1 cells were plated at 2 = 10 cellsrwell in a 12-well plate, grown for 20 h, transfected with 1.0 mg of ScaI-linearized pCAG-lacZ w43x as mentioned above and then cultured in growth medium in the presence or absence of BLM. After 20 h, the cells were washed, fixed, and stained with X-gal, as described w14x. The percentage of b-galactosidase Žbgal.-positive cells was calculated by counting more than 500 cells. The percentage in untreated cells was taken as 1, and the relative percentage in the drugtreated cells was calculated.
PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, and treated with BLM at various concentrations for 20 h. The cells were then harvested, fixed with ethanol, treated with RNase, stained with propidium iodide, and analyzed on a Coulter EPICS XL flow cytometer ŽCoulter. as described w42x.
3. Results 3.1. BLM enhances random integration in human cells
2.5. Comet assay PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, and treated with BLM at various concentrations for 20 h. The cells were harvested by trypsinization, suspended in PBSy
We examined the effect of BLM on random integration of transfected plasmid DNA into human chromosomes. As described in Section 2, PA1 cells were transfected with EcoRI-linearized pSV2neo and then treated with varying concentrations of BLM for
4
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
20 h, followed by cultivation to determine the number of G418-resistant ŽG418 r . colonies and surviving cells. The resulting colonies were scored, and recombinant frequency was calculated. Fig. 1A shows the relative recombinant frequency plotted against BLM concentration Žleft panel.. In untreated, control cells, the basal level of recombination frequency was 4.2 = 10y4 on average Ž9 inde-
pendent experiments.. Relative recombinant frequencies in cells treated with 0.05, 0.1, or 0.15 mM BLM were 6.4, 9.3 or 6.2, respectively, indicating that at 0.1 mM the drug caused a maximal enhancement. Above this dose, however, the effect was saturated or rather reduced. This may partly be due to a decrease in the number of surviving cells, because cell survival was gradually decreased with increasing
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
doses of the drug Žright panel.. It should be noted that the maximal enhancement was observed at the concentration that reduced the survival fraction to ; 50%. This result is reminiscent of the stimulation seen with a variety of topo II inhibitors w13,14x. A closed-circular pSV2neo plasmid was used in place of its linear form, and similar recombination enhancement by BLM was observed Ždata not shown.. We examined the effect of BLM with varying amounts of plasmid DNA ŽFig. 1B.. As expected, the basal levels of recombinant frequency in untreated cells increased with the amount of pSV2neo used and, at every dose, the relative recombinant frequency was elevated to a similar extent Ž8-fold. by treatment of PA1 cells with 0.1 mM BLM. In addition, a 20-h treatment with 0.1 mM BLM following transfection was enough to produce a maximal stimulation of the integration Ždata not shown.. The BLM enhancement was not restricted to PA1 cells. As shown in Fig. 1C, essentially the same effect was observed with HeLa cells, and with EJ-1 cells Ždata not shown.. In addition, the BLM effect was found to be independent of a transfection method. We transfected PA1 cells with pSV2neo by lipofection or electroporation as described previously w14x and treated for 20 h with BLM. Stimulatory effects of BLM were also found which were comparable to that found by the PEI method used here Ždata not shown.. Therefore, the BLM-induced recombination is a general phenomenon that does not depend on the cell type, the molecular form of transfected plasmid DNA, or the transfection method. We purified genomic DNAs of individual G418 r colonies from BLM-treated and untreated cultures, analyzed them by PCR with primers specific for the neomycin-resistant gene and confirmed the existence
5
of integrated pSV2neo DNA in their genome Ždata not shown.. 3.2. BLM does not enhance transient expression To address whether BLM influences the transient expression of introduced DNA, PA1 cells were transfected with the pGL3-Promoter plasmid carrying the luciferase reporter gene w42x and then treated with 0.1 mM BLM for 20 h. Crude extracts were prepared from the drug-treated and untreated cells, and assayed for luciferase activity. As shown in Fig. 2A, it should be emphasized that the BLM treatment did not enhance the transient expression; rather, the treated cells revealed a reduced level Ž0.63. of the enzyme activity compared to the untreated cells. Similarly, PA1 cells were transfected with the pCAG-lacZ plasmid carrying the bacterial lacZ reporter gene w43x, treated with 0.1 mM BLM for 20 h and stained for b-galactosidase Žb-gal. activity. As shown in Fig. 2B, the relative percentage of b-galpositive cells was reduced to 0.82 in the drug-treated cells. These results clearly indicate that no stimulation of transient expression occurred by the BLM treatment after transfection. Given that the levels of such transient expression reflect the number of transfected DNA molecules that are present in the cell nucleus, we conclude that BLM has no stimulatory effect on the uptake andror accumulation of input DNA within the nucleus.
4. BLM causes DNA strand breaks Transient expression analyses suggested that recombination enhancement by BLM was due to a
Fig. 1. BLM enhances random integration of transfected DNA in human cells. ŽA. PA1 cells were transfected with an EcoRI-linearized pSV2neo and then treated with BLM at the indicated concentrations for 20 h, followed by cultivation to determine the number of G418 r colonies and surviving cells. The resulting colonies were scored, and the recombinant frequency was determined as a function of surviving cells at each concentration of BLM Žright panel shows the survival curve.. The value of recombinant frequency in the drug-treated cells relative to that in untreated cells was calculated and expressed as relative recombinant frequency Žleft panel.. Error bars represent the standard deviation of 9 experiments. ŽB. Effect of BLM with varying amounts of transfected DNA. PA1 cells were transfected with an EcoRI-lnearized pSV2neo at the indicated amounts and treated with 0.1 mM BLM. The hatched and open boxes show the relative recombinant frequency in BLM-treated and untreated cells, respectively. The relative values to the recombinant frequency found in BLM-untreated cells transfected with 0.5 mgrwell of pSV2neo are shown. ŽC. HeLa cells were transfected with linearized pSV2neo and treated with BLM at the indicated concentrations. The relative recombinant frequency was calculated and expressed as in ŽA.. Error bars represent the standard deviation of 3 experiments.
6
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
Fig. 2. BLM does not enhance transient expression of two reporter genes. ŽA. PA1 cells were transfected with the pGL3-Promoter plasmid and then treated with 0.1 mM BLM for 20 h. Crude extracts were prepared from untreated or BLM-treated cells, and assayed for luciferase activity. The luciferase activity in untreated cells Žopen box. was taken as 1, and the relative activity in the drug-treated cells Žhatched box. was calculated. Error bars represent the standard deviation of 5 experiments. ŽB. PA1 cells were transfected with the pCAG-lacZ plasmid w43x and processed as above. The b-galactosidase Žb-gal.-positive cells were detected by staining with X-gal. Their percentage in untreated cells Žopen box. was taken as 1, and the relative percentage in the BLM-treated cells Žhatched box. was calculated. Error bars represent the standard deviation of five experiments.
direct involvement of this drug in a nonhomologous recombination reaction. It has been demonstrated that BLM induces DNA strand breaks w33–39x. Accordingly, one possible explanation is that such BLM-induced breaks are causative of enhanced recombination. However, it remained unclear whether BLM at the dose of 0.1 mM effective for yielding the maximal enhancement Žsee Fig. 1A. is sufficient to cause strand breaks, since this dose is considerably low compared with those at which others have observed DNA strand breaks w33–39x. To clarify this point, we carried out the comet assay which allows us to detect DNA strand breaks with high sensitivity at the single cell level w44,45x. As summarized in Table 1, BLM treatment caused DNA strand breaks in a dose-dependent manner, as judged by the number of comet-positive PA1 cells. Importantly, BLM at the concentration as low as 0.1 mM was sufficient to induce comet-positive cells: the percentage was quite low but significantly higher than that of untreated cells. Thus, it is evident that BLM, at the concentration for the maximal enhancement ŽFig. 1A., does cause DNA strand breaks. The G2rM block is known to be a hallmark of DNA damage such as strand breaks w16,31x. We therefore performed flow cytometric analysis to examine whether BLM at 0.1 mM is capable of arresting cells at G2rM phase. When untreated cells were
subjected to the FACS analysis as described in Section 2, the cell population in G2rM phase was 20% ŽFig. 3., reflecting an active cycling of cells. In contrast, in cells treated with 0.1, 0.3, or 1 mM BLM for 20 h, the G2rM population was elevated to 27%, 34%, or 55%, respectively, with a concomitant decrease in the population of G1- and S-phase cells. It should be noted that treatment with BLM at the concentration of 0.1 mM indeed increased the G2rM population but scarcely affected the S population. These results clearly indicate that, in a dose-depen-
Table 1 BLM causes DNA strand breaks in a dose-dependent manner BLM ŽmM.
Comet-positive cells
0 0.1 0.3 1
2.3"0.6% 4.7"0.8 10.8"4.3 26.3"16.6
PA1 cells were treated with BLM at the indicated concentrations for 20 h, collected by trypsinization and subjected to the comet assay as described in Section 2. Comet-positive cells were stained with SYBR GREEN I, photographed with an Olympus AX70 fluorescence microscope and counted. Their percentages were calculated by counting more than 400 cells. Data are presented as mean"S.D. of three experiments.
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
7
Fig. 3. BLM arrests PA1 cells at G2rM phase. PA1 cells were treated with BLM at the concentrations indicated for 20 h and subjected to flow cytometric analysis as described in Section 2. The percentage of the population of G1 -, S-, and G2rM-cells was determined on a Coulter EPICS XL flow cytometer. Data are presented as mean " S.D. of three determinations.
dent manner, BLM arrests PA1 cells at G2rM phase, supporting the authenticity of DNA strand breaks detected in the above comet assay. Taken together, our results strongly suggest that BLM acts directly at an illegitimate recombination reaction through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes.
5. Discussion We have shown that BLM enhances random integration of transfected plasmid DNA through nonhomologous recombination into human cells. Clearly, this enhancement is a general phenomenon, since the effect is independent of the molecular form of the plasmid, the cell type, or even the transfection method. We analyzed the enhancement mechanism and suggested that BLM acts directly at a nonhomologous recombination reaction through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. In support of this, transient expression analyses revealed no stimulation of the expression of two reporter genes by BLM treatment, indicating that recombination enhancement by this drug is not attributable to increased uptake andror accumulation of transfected DNA in the drug-treated cell nuclei. These results are analogous to our recent findings that a variety of topo II inhibitors, such as VP-16,
VM-26, m-AMSA or ICRF-193, enhance random integration of transfected plasmids in hamster and human cells w13,14x. Among these drugs, VP-16, VM-26, and m-AMSA cause DNA double-strand breaks by stabilizing a cleavable, ternary complex comprising the drug, topo II, and DNA w16,24,31x. Thus, including BLM, four kinds of DNA-damaging agents possess the ability to stimulate nonhomologous recombination. Moreover, there are several papers in which treatment of mouse or hamster cells with agents such as X-rays w46x, UV w46–49x, or FUdR w49x resulted in increased transfection efficiency, although in these studies, no evidence for DNA strand breaks was shown. Therefore, it is likely that chromosomal DNA strand breaks produced by the drugs we used trigger the integration of transfected plasmids. Such breaks may be mended by a direct action of recombination andror repair enzymes, or may be converted into certain DNA lesions, leading to enhanced recombination events including random integration w13,14,50–54x. However, the above explanation is not still exclusive. For example, ICRF-193, unlike other topo II inhibitors, has been reported to be incapable of producing DNA strand breaks w55–58x. We have confirmed the finding with PA1 cells under the present conditions ŽFujimaki et al., in preparation.. Therefore, recombination enhancement by this drug argues against the mechanism based on strand breaks as mentioned above, although it might be possible that topo II inhibition by this drug generates an
8
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
undetectable level of DNA lesions. Rather, direct inhibition of topo II activity by ICRF-193 Žand possibly by the other drugs. may render the chromosomal DNA recombinogenic through changes in the higher order structure of chromatin w13,14x. We examined whether BLM treatment affected the expression of topo II and observed no detectable change in the enzyme content in PA1 cells Žunpublished observations.. Given that BLM has no direct, inhibitory effect on topo II activity, these two drugs may stimulate recombination through a distinct pathway: i.e., ICRF-193 enhances the recombination via topo II inhibition, while BLM does so via formation of DNA strand breaks. Alternatively, since all the drugs capable of enhancing recombination have been reported to bring about cell cycle arrest at G2rM phase w57x, abnormal cell cycle progression, i.e., elongation of the G2rM phase, may lead to enhanced recombination events. It is also possible that treatment of cells with these drugs generates unstable regions in the genome, into which transfected DNA molecules would preferentially integrate w54x. BLM produces both single- and double-strand breaks of DNA w33,36x. Recently, Dar and Jorgensen w59x have reported that deletions are characteristic mutations for BLM-induced double-strand breaks, while single-strand breaks mostly lead to base substitutions. Together with the fact that double-strand breaks have been implicated in nonhomologous as well as homologous recombination w60–64x, we prefer, at present, an interpretation that BLM stimulates random integration mainly via double-strand breaks of the host genome. This idea is also compatible with our previous observations that a topo I inhibitor camptothecin, which causes single-strand breaks but not double-strand breaks, fails to enhance random integration w13,14x. The application of random integration is to generate transfectants that stably express a gene of interest, to study its function in a biological process, or to utilize the transfectants with a new function. It is obvious that, especially for gene therapy, further improvements in the frequency of the integration should be crucial. Our findings will facilitate the development of transfection protocols with higher efficiency, as well as the understanding of genomic integration events via nonhomologous recombination in mammalian cells.
Acknowledgements We thank Ms. A. Onozuka for her excellent technical assistance. The pCAG-lacZ plasmid was kindly supplied by Dr. J. Miyazaki, Osaka University. This work was supported by grants from Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, and the Ministry of Education, Science, Sports and Culture of Japan.
References w1x D.B. Roth, J.H. Wilson, Illegitimate recombination in mammalian cells, in: R. Kucherlapati, G.R. Smith ŽEds.., Genetic Recombination, American Society for Microbiology, WA, 1988, pp. 621–653. w2x C.T. Wake, F. Vernaleone, J.H. Wilson, Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells, Mol. Cell. Biol. 5 Ž1985. 2080–2089. w3x M.K. Derbyshire, L.H. Epstein, C.S.H. Young, P.L. Munz, R. Fishel, Nonhomologous recombination in human cells, Mol. Cell. Biol. 14 Ž1994. 156–169. w4x A.L. Nicolas, ´ C.S.H. Young, Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific, Mol. Cell. Biol. 14 Ž1994. 170–180. w5x T.H. Rabbitts, Chromosomal translocations in human cancer, Nature 372 Ž1994. 143–149. w6x D. Lasko, W. Cavenee, Loss of constitutional heterozygosity in human cancer, Annu. Rev. Genet. 25 Ž1991. 281–314. w7x C.T. Denny, G.F. Hollis, F. Hecht, R. Morgan, M.P. Link, S.D. Smith, I.R. Kirsch, Common mechanism of chromosome inversion in B- and T-cell tumors: relevance to lymphoid development, Science 234 Ž1986. 197–200. w8x G.R. Strark, M. Debatisse, E. Giulotto, G.M. Wahl, Recent progress in understanding mechanisms of mammalian DNA amplification, Cell 57 Ž1989. 901–908. w9x D.B. Roth, J.H. Wilson, Relative rates of homologous and nonhomologous recombination in transfected DNA, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 3355–3359. w10x M.R. Capecchi, Altering the genome by homologous recombination, Science 244 Ž1989. 1288–1292. w11x J.P. Murnane, M.J. Yezzi, B.R. Young, Recombination events during integration of transfected DNA into normal human cells, Nucleic Acids Res. 18 Ž1990. 2733–2738. w12x D.M. Robins, S. Ripley, A.S. Henderson, R. Axel, Transforming DNA integrates into the host chromosome, Cell 23 Ž1981. 29–39. w13x Y. Aratani, T. Andoh, H. Koyama, Effects of DNA topoisomerase inhibitors on nonhomologous and homologous recombination in mammalian cells, Mutat. Res. 362 Ž1996. 181–191.
C. Nakayama et al.r Mutation Research 409 (1998) 1–10 w14x F. Fujimaki, Y. Aratani, S. Fujisawa, S. Motomura, T. Okubo, H. Koyama, DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes, Somat. Cell Mol. Genet. 22 Ž1996. 279–290. w15x J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 54 Ž1985. 665–697. w16x L.F. Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem. 58 Ž1989. 351–375. w17x S. DiNardo, K. Voelkel-Meiman, R. Sternglanz, DNA topoisomerase II mutant of Saccharomyces cereÕisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication, Proc. Natl. Acad. Sci. U.S.A. 81 Ž1984. 2616–2620. w18x C. Holm, T. Goto, J.C. Wang, D. Botstein, DNA topoisomerase II is required at the time of mitosis in yeast, Cell 41 Ž1985. 553–563. w19x U. Uemura, H. Ohkura, Y. Adachi, K. Morino, K. Shiozaki, M. Yanagida, DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe, Cell 50 Ž1987. 917–925. w20x S.J. Brill, R. Sternglanz, Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants, Cell 54 Ž1988. 403–411. w21x M.F. Christman, F.S. Dietrich, G.R. Fink, Mitotic recombination in the rDNA of S. cereÕisiae is suppressed by the combined action of DNA topoisomerases I and II, Cell 55 Ž1988. 413–425. w22x R.A. Kim, J.C. Wang, A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings, Cell 57 Ž1989. 975–985. w23x Y. Adachi, M. Luke, U.K. Laemmli, Chromosome assembly in vitro: topoisomerase II is required for condensation, Cell 64 Ž1991. 137–148. w24x H. Ikeda, DNA topoisomerase-mediated illegitimate recombination, in: N. Cozzarelli, J.C. Wang ŽEds.., DNA Topoisomerase, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1990, pp. 341–359. w25x J.C. Wang, P.R. Caron, R.A. Kim, The role of DNA topoisomerases in recombination and genome stability: A doubleedged sword?, Cell 62 Ž1990. 403–406. w26x A. Miura-Masuda, H. Ikeda, The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo, Mol. Gen. Genet. 220 Ž1990. 345–352. w27x H. Ikeda, Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro, Proc. Natl. Acad. Sci. U.S.A. 83 Ž1986. 922–926. w28x H. Ikeda, K. Aoki, A. Naito, illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules, Proc. Natl. Acad. Sci. U.S.A. 79 Ž1982. 3724–3728. w29x Y.-S. Bae, I. Kawasaki, H. Ikeda, L.F. Liu, Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 2076– 2080. w30x K.C. Gale, N. Osheroff, Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase: II. Potential roles in recombination, J. Biol. Chem. 267 Ž1992. 12090–12097.
9
w31x E.M. Nelson, K.M. Tewey, L.F. Liu, Mechanism of antitumor drug action: poisoning of mammalian DNA topoisoX merase II on DNA by 4 -Ž9-acridinylamino.-methanesulfonm-anisidide, Proc. Natl. Acad. Sci. U.S.A. 81 Ž1984. 1361– 1365. w32x L.F. Povirk, W. Wubker, W. Kohnlein, F. Hutchinson, DNA ¨ ¨ double-strand breaks and alkali-labile bonds produced by bleomycin, Nucleic Acids Res. 4 Ž1977. 3573–3580. w33x R.M. Burger, S.J. Projan, S.B. Horwitz, J. Peisach, The DNA cleavage mechanism of iron-bleomycin: kinetic resolution of strand scission from base propenal release, J. Biol. Chem. 261 Ž1986. 15955–15959. w34x L.F. Povirk, Y.-H. Han, R.J. Steighner, Structure of bleomycin-induced DNA double-strand breaks: predomiX nance of blunt ends and single-base 5 extensions, Biochemistry 28 Ž1989. 5808–5814. w35x R.J. Steighner, L.F. Povirk, Bleomycin-induced DNA lesions at mutational hot spots: Implications for the mechanism of double-strand cleavage, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 8350–8354. w36x J.W. Sam, X.-J. Tang, J. Peisach, Electrospray mass spectrometry of iron bleomycin: Demonstration that activated bleomycin is a ferric peroxide complex, J. Am. Chem. Soc. 116 Ž1994. 5250–5256. w37x S.A. Kane, S.M. Hecht, Polynucleotide recognition and degradation by bleomycin, Prog. Nucl. Acid Res. Mol. Biol. 49 Ž1994. 313–352. w38x A. Natrajan, S.M. Hecht, Bleomycins: Mechanism of polynucleotide recognition and oxidative degradation, in: S. Neidle, M.J. Waring ŽEds.., Molecular Aspects of Anticancer DrugDNA Interactions, Vol. 2, Macmillan, Basingstoke and London, 1994, pp. 197–242. w39x J. Stubbe, J.W. Kozarich, W. Wu, D.E. Vanderwall, Bleomycins: A structural model for specificity, binding, and double strand cleavage, Acc. Chem. Res. 29 Ž1996. 322–330. w40x P.J. Southern, P. Berg, Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter, J. Mol. Appl. Genet. 1 Ž1982. 327–341. w41x O. Boussif, F. Lezoualc’h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.-P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 7297–7301. w42x N. Adachi, M. Kobayashi, H. Koyama, Cell cycle-dependent regulation of the mouse DNA topoisomerase IIa gene promoter, Biochem. Biophys. Res. Commun. 230 Ž1997. 105– 109. w43x H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene 108 Ž1991. 193–200. w44x M. Klaude, S. Eriksson, J. Nygren, G. Ahnstrom, ¨ The comet assay: mechanisms and technical considerations, Mutat. Res. 363 Ž1996. 89–96. w45x K. Kasamatsu, K. Kohda, Y. Kawazoe, Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay, Mutat. Res. 369 Ž1996. 1–6. w46x P.G. Debenham, M.B.T. Webb, The effect of X-rays and
10
w47x
w48x
w49x
w50x
w51x
w52x
w53x
w54x
w55x
w56x
C. Nakayama et al.r Mutation Research 409 (1998) 1–10 ultraviolet light on DNA-mediated gene transfer in mammalian cells, Int. J. Radiat. Biol. 46 Ž1984. 555–568. Y. Nagata, H. Takagi, T. Morita, M. Oishi, Stimulation of DNA-mediated transformation by UV irradiation of recipient Žmouse FM3A. cells, J. Cell. Physiol. 121 Ž1984. 453–457. M. Van Duin, A. Westerveld, J.H.J. Hoeijmaker, UV stimulation of DNA-mediated transformation of human cells, Mol. Cell. Biol. 5 Ž1985. 734–741. E.H. Postel, Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA, Mol. Gen. Genet. 201 Ž1985. 136–139. N.A. Berger, S. Chatterjee, J.A. Schmotzer, S.R. Helms, ŽVP-16-213.-induced gene alterations: potential contribution to cell death, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 8740–8743. Y.-S. Bae, M. Chiba, M. Ohira, H. Ikeda, A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency, Gene 101 Ž1991. 285–289. A.L. Bodley, H.-C. Huang, C. Yu, L.F. Liu, Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 Žteniposide., Mol. Cell. Biol. 13 Ž1993. 6190–6200. D.W. Russell, I.E. Alexander, A.D. Miller, DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 5719–5723. R.V. Merrihew, K. Marburger, S.L. Pennington, D.B. Roth, J.H. Wilson, High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome, Mol. Cell. Biol. 16 Ž1996. 10–18. K. Tanabe, Y. Ikegami, R. Ishida, T. Andoh, Inhibition of topoisomerase II by antitumor agents bisŽ2,6-dioxopiperazine. derivatives, Cancer Res. 51 Ž1991. 4903–4908. R. Ishida, T. Miki, T. Narita, R. Yui, M. Sato, K.R. Utsumi, K. Tanabe, T. Andoh, Inhibition of intracellular topoisomerase II by antitumor bisŽ2,6-dioxopiperazine. derivatives:
w57x
w58x
w59x
w60x
w61x
w62x
w63x
w64x
mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors, Cancer Res. 51 Ž1991. 4909–4916. C.S. Downes, D.J. Clarke, A.M. Mullinger, J.F. Gimenez´ Abian, ´ A.M. Creighton, R.T. Johnson, A topoisomerase IIdependent G2 cycle checkpoint in mammalian cells, Nature 372 Ž1994. 467–470. R. Ishida, M. Sato, T. Narita, K.R. Utsumi, T. Nishimoto, T. Morita, H. Nagata, T. Andoh, Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events, J. Cell Biol. 26 Ž1994. 1341–1351. M.D. Dar, T.J. Jorgensen, Deletion at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system, Nucleic Acids Res. 23 Ž1995. 3224–3230. J.W. Phillips, W.F. Morgan, Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome, Mol. Cell. Biol. 14 Ž1994. 5794–5803. P. Rouet, F. Smih, M. Jasin, Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 6064– 6068. A.R. Godwin, R.J. Bollag, D.M. Christie, R.M. Liskay, Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 12554–12558. A. Choulika, A. Perrin, B. Dujon, J.-F. Nicolas, Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cereÕisiae, Mol. Cell. Biol. 15 Ž1995. 1968–1973. R.G. Sargent, M.A. Brenneman, J.H. Wilson, Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination, Mol. Cell. Biol. 16 Ž1997. 267–277.
Bleomycin enhances random integration of transfected DNA into a human genome Chikako Nakayama, Noritaka Adachi, Hideki Koyama
)
Kihara Institute for Biological Research, Yokohama City UniÕersity, Maioka-cho 641-12, Totsuka-ku, Yokohama 244, Japan Received 9 October 1997; revised 17 June 1998; accepted 23 June 1998
Abstract In mammalian cells, nonhomologous Žillegitimate. recombination is a predominant pathway to repair DNA double-strand breaks. We have shown that DNA topoisomerase II inhibitors are capable of enhancing random integration of foreign DNA via nonhomologous recombination. Since this enhancement is likely due to stabilized DNA strand breaks, we examined the effect of a radiomimetic antitumor drug, bleomycin ŽBLM., on nonhomologous recombination. We found that BLM greatly enhances the random integration of transfected plasmids into human cells. Importantly, this enhancement was independent of the molecular form of the plasmid, the cell type or the transfection method, suggesting that the BLM effect is intrinsically general. Transient expression analysis revealed no stimulation of reporter gene expression by the drug, suggesting that the effect is not attributable to increased uptake andror accumulation of transfected DNA in the drug-treated cell nuclei. In addition, the comet assay and flow cytometric analyses revealed the occurrence of low but significant strand breaks in cells treated with the BLM concentration which maximally enhanced the integration. These results strongly suggest that BLM acts directly at a nonhomologous recombination reaction that is initiated through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. Our findings will facilitate the understanding of DNA integration events through nonhomologous recombination and the development of transfection protocols with higher efficiencies. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Bleomycin; Transfection; Random integration; Nonhomologous recombination; DNA strand break
1. Introduction Mammalian cells possess an activity of nonhomologous Žillegitimate. recombination that requires little or no sequence homology between DNA substrates w1x. Nonhomologous recombination presumably prevents the cells from death by repairing DNA double-strand breaks, which may result from endoge) Corresponding author. Tel.: q81-45-820-1907; Fax: q81-45820-1901; E-mail: [email protected]
nous mechanisms or occasional exposure to endogenous or exogenous agents w2–4x. In contrast, it may cause deleterious genomic rearrangements, such as translocation w5x, deletion w6x, inversion w7x, or gene amplification w8x, leading to a variety of genetic diseases and cancer. When foreign DNA is transfected into mammalian cells, a nonhomologous recombination event results in random integration of the DNA into the host genome w9–11x. The practical application of the recombination is to generate transfectants that stably
0921-8777r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 9 8 . 0 0 0 3 6 - 6
2
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
express a gene of interest. For gene therapy, improvements in the frequency of the integration would be necessary. On the other hand, if the foreign DNA includes sequences homologous to chromosomal sequences, a homologous recombination event results in gene targeting in a small minority of transfected cells. Highly efficient gene targeting would provide the ideal protocol of gene therapy, but at present the efficiency of homologous recombination is several orders magnitude too low for this to be feasible. To this end, improvements in the frequency of homologous recombination and reductions in the proportion of random integration would be required. Thus, it will be important to understand the mechanisms of nonhomologous as well as homologous recombination, and to make attempts to control the process of genomic integration events. However, the mechanism of such integration events is poorly understood, although it appears to involve transient strand breaks of chromosomal DNA, followed by ligation of the breaks and both ends of transfected, linear DNA w4,11,12x. We recently reported that, in hamster and human cells, treatment with DNA topoisomerase Žtopo. II inhibitors after transfection with plasmid DNA significantly enhance random integration by nonhomologous recombination w13,14x. Topo II is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes, through a transient double-strand break and subsequent religation of the break w15,16x. The enzyme has been implicated in many cellular processes such as transcription, replication, or chromosome condensation and segregation w17–23x. Also, extensive studies have indicated the involvement of topo II in various nonhomologous recombination reactions in vivo w24–26x as well as in vitro w27–30x. Many of topo II inhibitors, such as VP-16 Žetoposide., VM-26 Žteniposide. or m-AMSA, are able to generate and stabilize double-strand breaks by preventing the subsequent ligation step w16,31x. In the present report, we have examined the effect of a radiomimetic drug, bleomycin ŽBLM., on nonhomologous recombination in human cells. Since many of topo II inhibitors cause double-strand breaks of DNA w16,31x, we expected that BLM would also be effective in enhancing random integration. BLM is an antitumor antibiotic that exerts its cytotoxic effect by generating single- and double-strand breaks
of DNA in the presence of FeŽII. and O 2 w32–39x. The cutting reaction occurs in the minor groove of DNA predominantly at GpC and GpT sequences w37–39x. Here we show that BLM greatly enhances random integration of transfected plasmid DNA into human chromosomes. We also suggest that this enhancement results from a direct action of the drug in a nonhomologous recombination reaction within the nucleus.
2. Materials and methods 2.1. Cells and culture methods The human cell lines PA1, HeLa and EJ-1 used here were described previously w14x. Cells were maintained in ES medium ŽNissui. supplemented with 5% fetal bovine serum Žgrowth medium. at 378C in a humidified atmosphere of 5% CO 2 in air w14x. For all experiments, logarithmically growing cells were used. For transfection, a plasmid vector, pSV2neo, carrying the bacterial neomycin-resistant gene was purified by double CsCl gradient centrifugation; prior to transfection, it was linearized, unless otherwise stated, by digestion with EcoRI which recognizes the unique site outside the neomycin-resistant gene w40x. 2.2. Recombination assay by transfection The method reported by Boussif et al. w41x with a cationic polymer polyethyleneimine ŽPEI. was modified for stable transfection by Koyama et al. Žin preparation.. Briefly, cells were plated at 2 = 10 5 cellsrwell in a 12-well plate ŽCostar. containing 1 ml of growth medium, grown for 20 h, and transfected by adding the complex consisting of 1.0 mg of pSV2neo and 3 ml of 10 mM PEI Ž50 kDa, Sigma. in 0.4 ml of serum-free ES medium. After a 5-h incubation, the medium was replaced with growth medium, and the cells were grown for a further 20 h in the presence or absence of BLM ŽBleomycin–HCl, Wako.. BLM was dissolved into water and filtered for sterilization. The cells were harvested by trypsinization, diluted appropriately, and replated at 5 = 10 4 –10 5 cellsr60-mm dish in 5 ml of growth medium containing 0.25 mgrml of active Geneticin
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
ŽG418, Gibco BRL.; the cells were subsequently cultured for 13-14 days to select for G418-resistant ŽG418 r . colonies, as described previously w13,14x. To estimate cell survival, aliquots of the BLM-treated cells were plated at 300 cellsr60-mm dish in growth medium without added G418 and cultured for 11–12 days. The resulting colonies were fixed with 10% formaldehyde in saline, and stained with 0.1% crystal violet and counted. The frequency of G418 r colonies Ži.e., recombinant frequency. was calculated as a function of surviving cells. The value of the recombinant frequency in BLM-treated cells relative to that in untreated cells was calculated and expressed as relative recombinant frequency as described w13,14x. 2.3. Luciferase assay PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, transfected with 1.0 mg of NotI-linearized pGL3-Promoter ŽPromega. as mentioned above and then cultured in growth medium in the presence or absence of BLM. After 20 h, the cells were assayed for luciferase activity as described previously w42x. The luciferase activity of untreated cells was taken as 1, and the relative activity of the drug-treated cells was calculated.
3
ŽCa2q and Mg 2q-free phosphate-buffered saline., and subjected to the comet assay essentially as described by Klaude et al. w44x. Microscope slides were pretreated by spreading 20 ml of 0.5% agarose ŽSeaKem GTG, FMC Bioproducts. in PBSy evenly over the surface, and dried. The cell suspension Ž10 3 –10 4 cellsrml. was mixed with an equal volume of 1% low melting agarose ŽSeaPlaque GTG, FMC Bioproducts.. Fifty microliters of the mixture was immediately pipetted onto the pretreated slide, covered with a coverslip and left on ice. When the gel had set, the coverslip was removed and the slides were immersed in a lysis buffer Ž2.5 M NaCl, 100 mM EDTA, 10% DMSO, 1% Triton X-100 and 10 mM Tris–HCl, pH 10. and kept on ice for 2 h. After cell lysis, the slides were placed on a horizontal slab of an electrophoretic unit containing 0.3 M NaOHr1 mM EDTA ŽpH 13.. After equilibration for 20 min at 118C, electrophoresis was carried out at 25 V Ž300 mA. for 25 min. The samples were then neutralized in 0.4 M Tris–HCl ŽpH 8., rinsed, fixed with methanol, and stained with SYBR GREEN I ŽWako.. Comet-positive cells were detected and photographed with an Olympus AX70 fluorescence microscope ŽOlympus.. Their percentage was calculated by counting more than 400 cells. 2.6. Flow cytometry
2.4. b-galactosidase assay 5
PA1 cells were plated at 2 = 10 cellsrwell in a 12-well plate, grown for 20 h, transfected with 1.0 mg of ScaI-linearized pCAG-lacZ w43x as mentioned above and then cultured in growth medium in the presence or absence of BLM. After 20 h, the cells were washed, fixed, and stained with X-gal, as described w14x. The percentage of b-galactosidase Žbgal.-positive cells was calculated by counting more than 500 cells. The percentage in untreated cells was taken as 1, and the relative percentage in the drugtreated cells was calculated.
PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, and treated with BLM at various concentrations for 20 h. The cells were then harvested, fixed with ethanol, treated with RNase, stained with propidium iodide, and analyzed on a Coulter EPICS XL flow cytometer ŽCoulter. as described w42x.
3. Results 3.1. BLM enhances random integration in human cells
2.5. Comet assay PA1 cells were plated at 2 = 10 5 cellsrwell in a 12-well plate, grown for 20 h, and treated with BLM at various concentrations for 20 h. The cells were harvested by trypsinization, suspended in PBSy
We examined the effect of BLM on random integration of transfected plasmid DNA into human chromosomes. As described in Section 2, PA1 cells were transfected with EcoRI-linearized pSV2neo and then treated with varying concentrations of BLM for
4
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
20 h, followed by cultivation to determine the number of G418-resistant ŽG418 r . colonies and surviving cells. The resulting colonies were scored, and recombinant frequency was calculated. Fig. 1A shows the relative recombinant frequency plotted against BLM concentration Žleft panel.. In untreated, control cells, the basal level of recombination frequency was 4.2 = 10y4 on average Ž9 inde-
pendent experiments.. Relative recombinant frequencies in cells treated with 0.05, 0.1, or 0.15 mM BLM were 6.4, 9.3 or 6.2, respectively, indicating that at 0.1 mM the drug caused a maximal enhancement. Above this dose, however, the effect was saturated or rather reduced. This may partly be due to a decrease in the number of surviving cells, because cell survival was gradually decreased with increasing
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
doses of the drug Žright panel.. It should be noted that the maximal enhancement was observed at the concentration that reduced the survival fraction to ; 50%. This result is reminiscent of the stimulation seen with a variety of topo II inhibitors w13,14x. A closed-circular pSV2neo plasmid was used in place of its linear form, and similar recombination enhancement by BLM was observed Ždata not shown.. We examined the effect of BLM with varying amounts of plasmid DNA ŽFig. 1B.. As expected, the basal levels of recombinant frequency in untreated cells increased with the amount of pSV2neo used and, at every dose, the relative recombinant frequency was elevated to a similar extent Ž8-fold. by treatment of PA1 cells with 0.1 mM BLM. In addition, a 20-h treatment with 0.1 mM BLM following transfection was enough to produce a maximal stimulation of the integration Ždata not shown.. The BLM enhancement was not restricted to PA1 cells. As shown in Fig. 1C, essentially the same effect was observed with HeLa cells, and with EJ-1 cells Ždata not shown.. In addition, the BLM effect was found to be independent of a transfection method. We transfected PA1 cells with pSV2neo by lipofection or electroporation as described previously w14x and treated for 20 h with BLM. Stimulatory effects of BLM were also found which were comparable to that found by the PEI method used here Ždata not shown.. Therefore, the BLM-induced recombination is a general phenomenon that does not depend on the cell type, the molecular form of transfected plasmid DNA, or the transfection method. We purified genomic DNAs of individual G418 r colonies from BLM-treated and untreated cultures, analyzed them by PCR with primers specific for the neomycin-resistant gene and confirmed the existence
5
of integrated pSV2neo DNA in their genome Ždata not shown.. 3.2. BLM does not enhance transient expression To address whether BLM influences the transient expression of introduced DNA, PA1 cells were transfected with the pGL3-Promoter plasmid carrying the luciferase reporter gene w42x and then treated with 0.1 mM BLM for 20 h. Crude extracts were prepared from the drug-treated and untreated cells, and assayed for luciferase activity. As shown in Fig. 2A, it should be emphasized that the BLM treatment did not enhance the transient expression; rather, the treated cells revealed a reduced level Ž0.63. of the enzyme activity compared to the untreated cells. Similarly, PA1 cells were transfected with the pCAG-lacZ plasmid carrying the bacterial lacZ reporter gene w43x, treated with 0.1 mM BLM for 20 h and stained for b-galactosidase Žb-gal. activity. As shown in Fig. 2B, the relative percentage of b-galpositive cells was reduced to 0.82 in the drug-treated cells. These results clearly indicate that no stimulation of transient expression occurred by the BLM treatment after transfection. Given that the levels of such transient expression reflect the number of transfected DNA molecules that are present in the cell nucleus, we conclude that BLM has no stimulatory effect on the uptake andror accumulation of input DNA within the nucleus.
4. BLM causes DNA strand breaks Transient expression analyses suggested that recombination enhancement by BLM was due to a
Fig. 1. BLM enhances random integration of transfected DNA in human cells. ŽA. PA1 cells were transfected with an EcoRI-linearized pSV2neo and then treated with BLM at the indicated concentrations for 20 h, followed by cultivation to determine the number of G418 r colonies and surviving cells. The resulting colonies were scored, and the recombinant frequency was determined as a function of surviving cells at each concentration of BLM Žright panel shows the survival curve.. The value of recombinant frequency in the drug-treated cells relative to that in untreated cells was calculated and expressed as relative recombinant frequency Žleft panel.. Error bars represent the standard deviation of 9 experiments. ŽB. Effect of BLM with varying amounts of transfected DNA. PA1 cells were transfected with an EcoRI-lnearized pSV2neo at the indicated amounts and treated with 0.1 mM BLM. The hatched and open boxes show the relative recombinant frequency in BLM-treated and untreated cells, respectively. The relative values to the recombinant frequency found in BLM-untreated cells transfected with 0.5 mgrwell of pSV2neo are shown. ŽC. HeLa cells were transfected with linearized pSV2neo and treated with BLM at the indicated concentrations. The relative recombinant frequency was calculated and expressed as in ŽA.. Error bars represent the standard deviation of 3 experiments.
6
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
Fig. 2. BLM does not enhance transient expression of two reporter genes. ŽA. PA1 cells were transfected with the pGL3-Promoter plasmid and then treated with 0.1 mM BLM for 20 h. Crude extracts were prepared from untreated or BLM-treated cells, and assayed for luciferase activity. The luciferase activity in untreated cells Žopen box. was taken as 1, and the relative activity in the drug-treated cells Žhatched box. was calculated. Error bars represent the standard deviation of 5 experiments. ŽB. PA1 cells were transfected with the pCAG-lacZ plasmid w43x and processed as above. The b-galactosidase Žb-gal.-positive cells were detected by staining with X-gal. Their percentage in untreated cells Žopen box. was taken as 1, and the relative percentage in the BLM-treated cells Žhatched box. was calculated. Error bars represent the standard deviation of five experiments.
direct involvement of this drug in a nonhomologous recombination reaction. It has been demonstrated that BLM induces DNA strand breaks w33–39x. Accordingly, one possible explanation is that such BLM-induced breaks are causative of enhanced recombination. However, it remained unclear whether BLM at the dose of 0.1 mM effective for yielding the maximal enhancement Žsee Fig. 1A. is sufficient to cause strand breaks, since this dose is considerably low compared with those at which others have observed DNA strand breaks w33–39x. To clarify this point, we carried out the comet assay which allows us to detect DNA strand breaks with high sensitivity at the single cell level w44,45x. As summarized in Table 1, BLM treatment caused DNA strand breaks in a dose-dependent manner, as judged by the number of comet-positive PA1 cells. Importantly, BLM at the concentration as low as 0.1 mM was sufficient to induce comet-positive cells: the percentage was quite low but significantly higher than that of untreated cells. Thus, it is evident that BLM, at the concentration for the maximal enhancement ŽFig. 1A., does cause DNA strand breaks. The G2rM block is known to be a hallmark of DNA damage such as strand breaks w16,31x. We therefore performed flow cytometric analysis to examine whether BLM at 0.1 mM is capable of arresting cells at G2rM phase. When untreated cells were
subjected to the FACS analysis as described in Section 2, the cell population in G2rM phase was 20% ŽFig. 3., reflecting an active cycling of cells. In contrast, in cells treated with 0.1, 0.3, or 1 mM BLM for 20 h, the G2rM population was elevated to 27%, 34%, or 55%, respectively, with a concomitant decrease in the population of G1- and S-phase cells. It should be noted that treatment with BLM at the concentration of 0.1 mM indeed increased the G2rM population but scarcely affected the S population. These results clearly indicate that, in a dose-depen-
Table 1 BLM causes DNA strand breaks in a dose-dependent manner BLM ŽmM.
Comet-positive cells
0 0.1 0.3 1
2.3"0.6% 4.7"0.8 10.8"4.3 26.3"16.6
PA1 cells were treated with BLM at the indicated concentrations for 20 h, collected by trypsinization and subjected to the comet assay as described in Section 2. Comet-positive cells were stained with SYBR GREEN I, photographed with an Olympus AX70 fluorescence microscope and counted. Their percentages were calculated by counting more than 400 cells. Data are presented as mean"S.D. of three experiments.
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
7
Fig. 3. BLM arrests PA1 cells at G2rM phase. PA1 cells were treated with BLM at the concentrations indicated for 20 h and subjected to flow cytometric analysis as described in Section 2. The percentage of the population of G1 -, S-, and G2rM-cells was determined on a Coulter EPICS XL flow cytometer. Data are presented as mean " S.D. of three determinations.
dent manner, BLM arrests PA1 cells at G2rM phase, supporting the authenticity of DNA strand breaks detected in the above comet assay. Taken together, our results strongly suggest that BLM acts directly at an illegitimate recombination reaction through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes.
5. Discussion We have shown that BLM enhances random integration of transfected plasmid DNA through nonhomologous recombination into human cells. Clearly, this enhancement is a general phenomenon, since the effect is independent of the molecular form of the plasmid, the cell type, or even the transfection method. We analyzed the enhancement mechanism and suggested that BLM acts directly at a nonhomologous recombination reaction through DNA strand breaks, promoting the integration process of transfected plasmids into human chromosomes. In support of this, transient expression analyses revealed no stimulation of the expression of two reporter genes by BLM treatment, indicating that recombination enhancement by this drug is not attributable to increased uptake andror accumulation of transfected DNA in the drug-treated cell nuclei. These results are analogous to our recent findings that a variety of topo II inhibitors, such as VP-16,
VM-26, m-AMSA or ICRF-193, enhance random integration of transfected plasmids in hamster and human cells w13,14x. Among these drugs, VP-16, VM-26, and m-AMSA cause DNA double-strand breaks by stabilizing a cleavable, ternary complex comprising the drug, topo II, and DNA w16,24,31x. Thus, including BLM, four kinds of DNA-damaging agents possess the ability to stimulate nonhomologous recombination. Moreover, there are several papers in which treatment of mouse or hamster cells with agents such as X-rays w46x, UV w46–49x, or FUdR w49x resulted in increased transfection efficiency, although in these studies, no evidence for DNA strand breaks was shown. Therefore, it is likely that chromosomal DNA strand breaks produced by the drugs we used trigger the integration of transfected plasmids. Such breaks may be mended by a direct action of recombination andror repair enzymes, or may be converted into certain DNA lesions, leading to enhanced recombination events including random integration w13,14,50–54x. However, the above explanation is not still exclusive. For example, ICRF-193, unlike other topo II inhibitors, has been reported to be incapable of producing DNA strand breaks w55–58x. We have confirmed the finding with PA1 cells under the present conditions ŽFujimaki et al., in preparation.. Therefore, recombination enhancement by this drug argues against the mechanism based on strand breaks as mentioned above, although it might be possible that topo II inhibition by this drug generates an
8
C. Nakayama et al.r Mutation Research 409 (1998) 1–10
undetectable level of DNA lesions. Rather, direct inhibition of topo II activity by ICRF-193 Žand possibly by the other drugs. may render the chromosomal DNA recombinogenic through changes in the higher order structure of chromatin w13,14x. We examined whether BLM treatment affected the expression of topo II and observed no detectable change in the enzyme content in PA1 cells Žunpublished observations.. Given that BLM has no direct, inhibitory effect on topo II activity, these two drugs may stimulate recombination through a distinct pathway: i.e., ICRF-193 enhances the recombination via topo II inhibition, while BLM does so via formation of DNA strand breaks. Alternatively, since all the drugs capable of enhancing recombination have been reported to bring about cell cycle arrest at G2rM phase w57x, abnormal cell cycle progression, i.e., elongation of the G2rM phase, may lead to enhanced recombination events. It is also possible that treatment of cells with these drugs generates unstable regions in the genome, into which transfected DNA molecules would preferentially integrate w54x. BLM produces both single- and double-strand breaks of DNA w33,36x. Recently, Dar and Jorgensen w59x have reported that deletions are characteristic mutations for BLM-induced double-strand breaks, while single-strand breaks mostly lead to base substitutions. Together with the fact that double-strand breaks have been implicated in nonhomologous as well as homologous recombination w60–64x, we prefer, at present, an interpretation that BLM stimulates random integration mainly via double-strand breaks of the host genome. This idea is also compatible with our previous observations that a topo I inhibitor camptothecin, which causes single-strand breaks but not double-strand breaks, fails to enhance random integration w13,14x. The application of random integration is to generate transfectants that stably express a gene of interest, to study its function in a biological process, or to utilize the transfectants with a new function. It is obvious that, especially for gene therapy, further improvements in the frequency of the integration should be crucial. Our findings will facilitate the development of transfection protocols with higher efficiency, as well as the understanding of genomic integration events via nonhomologous recombination in mammalian cells.
Acknowledgements We thank Ms. A. Onozuka for her excellent technical assistance. The pCAG-lacZ plasmid was kindly supplied by Dr. J. Miyazaki, Osaka University. This work was supported by grants from Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, and the Ministry of Education, Science, Sports and Culture of Japan.
References w1x D.B. Roth, J.H. Wilson, Illegitimate recombination in mammalian cells, in: R. Kucherlapati, G.R. Smith ŽEds.., Genetic Recombination, American Society for Microbiology, WA, 1988, pp. 621–653. w2x C.T. Wake, F. Vernaleone, J.H. Wilson, Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells, Mol. Cell. Biol. 5 Ž1985. 2080–2089. w3x M.K. Derbyshire, L.H. Epstein, C.S.H. Young, P.L. Munz, R. Fishel, Nonhomologous recombination in human cells, Mol. Cell. Biol. 14 Ž1994. 156–169. w4x A.L. Nicolas, ´ C.S.H. Young, Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific, Mol. Cell. Biol. 14 Ž1994. 170–180. w5x T.H. Rabbitts, Chromosomal translocations in human cancer, Nature 372 Ž1994. 143–149. w6x D. Lasko, W. Cavenee, Loss of constitutional heterozygosity in human cancer, Annu. Rev. Genet. 25 Ž1991. 281–314. w7x C.T. Denny, G.F. Hollis, F. Hecht, R. Morgan, M.P. Link, S.D. Smith, I.R. Kirsch, Common mechanism of chromosome inversion in B- and T-cell tumors: relevance to lymphoid development, Science 234 Ž1986. 197–200. w8x G.R. Strark, M. Debatisse, E. Giulotto, G.M. Wahl, Recent progress in understanding mechanisms of mammalian DNA amplification, Cell 57 Ž1989. 901–908. w9x D.B. Roth, J.H. Wilson, Relative rates of homologous and nonhomologous recombination in transfected DNA, Proc. Natl. Acad. Sci. U.S.A. 82 Ž1985. 3355–3359. w10x M.R. Capecchi, Altering the genome by homologous recombination, Science 244 Ž1989. 1288–1292. w11x J.P. Murnane, M.J. Yezzi, B.R. Young, Recombination events during integration of transfected DNA into normal human cells, Nucleic Acids Res. 18 Ž1990. 2733–2738. w12x D.M. Robins, S. Ripley, A.S. Henderson, R. Axel, Transforming DNA integrates into the host chromosome, Cell 23 Ž1981. 29–39. w13x Y. Aratani, T. Andoh, H. Koyama, Effects of DNA topoisomerase inhibitors on nonhomologous and homologous recombination in mammalian cells, Mutat. Res. 362 Ž1996. 181–191.
C. Nakayama et al.r Mutation Research 409 (1998) 1–10 w14x F. Fujimaki, Y. Aratani, S. Fujisawa, S. Motomura, T. Okubo, H. Koyama, DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes, Somat. Cell Mol. Genet. 22 Ž1996. 279–290. w15x J.C. Wang, DNA topoisomerases, Annu. Rev. Biochem. 54 Ž1985. 665–697. w16x L.F. Liu, DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem. 58 Ž1989. 351–375. w17x S. DiNardo, K. Voelkel-Meiman, R. Sternglanz, DNA topoisomerase II mutant of Saccharomyces cereÕisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication, Proc. Natl. Acad. Sci. U.S.A. 81 Ž1984. 2616–2620. w18x C. Holm, T. Goto, J.C. Wang, D. Botstein, DNA topoisomerase II is required at the time of mitosis in yeast, Cell 41 Ž1985. 553–563. w19x U. Uemura, H. Ohkura, Y. Adachi, K. Morino, K. Shiozaki, M. Yanagida, DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe, Cell 50 Ž1987. 917–925. w20x S.J. Brill, R. Sternglanz, Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants, Cell 54 Ž1988. 403–411. w21x M.F. Christman, F.S. Dietrich, G.R. Fink, Mitotic recombination in the rDNA of S. cereÕisiae is suppressed by the combined action of DNA topoisomerases I and II, Cell 55 Ž1988. 413–425. w22x R.A. Kim, J.C. Wang, A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings, Cell 57 Ž1989. 975–985. w23x Y. Adachi, M. Luke, U.K. Laemmli, Chromosome assembly in vitro: topoisomerase II is required for condensation, Cell 64 Ž1991. 137–148. w24x H. Ikeda, DNA topoisomerase-mediated illegitimate recombination, in: N. Cozzarelli, J.C. Wang ŽEds.., DNA Topoisomerase, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1990, pp. 341–359. w25x J.C. Wang, P.R. Caron, R.A. Kim, The role of DNA topoisomerases in recombination and genome stability: A doubleedged sword?, Cell 62 Ž1990. 403–406. w26x A. Miura-Masuda, H. Ikeda, The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo, Mol. Gen. Genet. 220 Ž1990. 345–352. w27x H. Ikeda, Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro, Proc. Natl. Acad. Sci. U.S.A. 83 Ž1986. 922–926. w28x H. Ikeda, K. Aoki, A. Naito, illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules, Proc. Natl. Acad. Sci. U.S.A. 79 Ž1982. 3724–3728. w29x Y.-S. Bae, I. Kawasaki, H. Ikeda, L.F. Liu, Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 2076– 2080. w30x K.C. Gale, N. Osheroff, Intrinsic intermolecular DNA ligation activity of eukaryotic topoisomerase: II. Potential roles in recombination, J. Biol. Chem. 267 Ž1992. 12090–12097.
9
w31x E.M. Nelson, K.M. Tewey, L.F. Liu, Mechanism of antitumor drug action: poisoning of mammalian DNA topoisoX merase II on DNA by 4 -Ž9-acridinylamino.-methanesulfonm-anisidide, Proc. Natl. Acad. Sci. U.S.A. 81 Ž1984. 1361– 1365. w32x L.F. Povirk, W. Wubker, W. Kohnlein, F. Hutchinson, DNA ¨ ¨ double-strand breaks and alkali-labile bonds produced by bleomycin, Nucleic Acids Res. 4 Ž1977. 3573–3580. w33x R.M. Burger, S.J. Projan, S.B. Horwitz, J. Peisach, The DNA cleavage mechanism of iron-bleomycin: kinetic resolution of strand scission from base propenal release, J. Biol. Chem. 261 Ž1986. 15955–15959. w34x L.F. Povirk, Y.-H. Han, R.J. Steighner, Structure of bleomycin-induced DNA double-strand breaks: predomiX nance of blunt ends and single-base 5 extensions, Biochemistry 28 Ž1989. 5808–5814. w35x R.J. Steighner, L.F. Povirk, Bleomycin-induced DNA lesions at mutational hot spots: Implications for the mechanism of double-strand cleavage, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 8350–8354. w36x J.W. Sam, X.-J. Tang, J. Peisach, Electrospray mass spectrometry of iron bleomycin: Demonstration that activated bleomycin is a ferric peroxide complex, J. Am. Chem. Soc. 116 Ž1994. 5250–5256. w37x S.A. Kane, S.M. Hecht, Polynucleotide recognition and degradation by bleomycin, Prog. Nucl. Acid Res. Mol. Biol. 49 Ž1994. 313–352. w38x A. Natrajan, S.M. Hecht, Bleomycins: Mechanism of polynucleotide recognition and oxidative degradation, in: S. Neidle, M.J. Waring ŽEds.., Molecular Aspects of Anticancer DrugDNA Interactions, Vol. 2, Macmillan, Basingstoke and London, 1994, pp. 197–242. w39x J. Stubbe, J.W. Kozarich, W. Wu, D.E. Vanderwall, Bleomycins: A structural model for specificity, binding, and double strand cleavage, Acc. Chem. Res. 29 Ž1996. 322–330. w40x P.J. Southern, P. Berg, Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter, J. Mol. Appl. Genet. 1 Ž1982. 327–341. w41x O. Boussif, F. Lezoualc’h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, J.-P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 7297–7301. w42x N. Adachi, M. Kobayashi, H. Koyama, Cell cycle-dependent regulation of the mouse DNA topoisomerase IIa gene promoter, Biochem. Biophys. Res. Commun. 230 Ž1997. 105– 109. w43x H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene 108 Ž1991. 193–200. w44x M. Klaude, S. Eriksson, J. Nygren, G. Ahnstrom, ¨ The comet assay: mechanisms and technical considerations, Mutat. Res. 363 Ž1996. 89–96. w45x K. Kasamatsu, K. Kohda, Y. Kawazoe, Comparison of chemically induced DNA breakage in cellular and subcellular systems using the comet assay, Mutat. Res. 369 Ž1996. 1–6. w46x P.G. Debenham, M.B.T. Webb, The effect of X-rays and
10
w47x
w48x
w49x
w50x
w51x
w52x
w53x
w54x
w55x
w56x
C. Nakayama et al.r Mutation Research 409 (1998) 1–10 ultraviolet light on DNA-mediated gene transfer in mammalian cells, Int. J. Radiat. Biol. 46 Ž1984. 555–568. Y. Nagata, H. Takagi, T. Morita, M. Oishi, Stimulation of DNA-mediated transformation by UV irradiation of recipient Žmouse FM3A. cells, J. Cell. Physiol. 121 Ž1984. 453–457. M. Van Duin, A. Westerveld, J.H.J. Hoeijmaker, UV stimulation of DNA-mediated transformation of human cells, Mol. Cell. Biol. 5 Ž1985. 734–741. E.H. Postel, Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA, Mol. Gen. Genet. 201 Ž1985. 136–139. N.A. Berger, S. Chatterjee, J.A. Schmotzer, S.R. Helms, ŽVP-16-213.-induced gene alterations: potential contribution to cell death, Proc. Natl. Acad. Sci. U.S.A. 88 Ž1991. 8740–8743. Y.-S. Bae, M. Chiba, M. Ohira, H. Ikeda, A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency, Gene 101 Ž1991. 285–289. A.L. Bodley, H.-C. Huang, C. Yu, L.F. Liu, Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 Žteniposide., Mol. Cell. Biol. 13 Ž1993. 6190–6200. D.W. Russell, I.E. Alexander, A.D. Miller, DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 5719–5723. R.V. Merrihew, K. Marburger, S.L. Pennington, D.B. Roth, J.H. Wilson, High-frequency illegitimate integration of transfected DNA at preintegrated target sites in a mammalian genome, Mol. Cell. Biol. 16 Ž1996. 10–18. K. Tanabe, Y. Ikegami, R. Ishida, T. Andoh, Inhibition of topoisomerase II by antitumor agents bisŽ2,6-dioxopiperazine. derivatives, Cancer Res. 51 Ž1991. 4903–4908. R. Ishida, T. Miki, T. Narita, R. Yui, M. Sato, K.R. Utsumi, K. Tanabe, T. Andoh, Inhibition of intracellular topoisomerase II by antitumor bisŽ2,6-dioxopiperazine. derivatives:
w57x
w58x
w59x
w60x
w61x
w62x
w63x
w64x
mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors, Cancer Res. 51 Ž1991. 4909–4916. C.S. Downes, D.J. Clarke, A.M. Mullinger, J.F. Gimenez´ Abian, ´ A.M. Creighton, R.T. Johnson, A topoisomerase IIdependent G2 cycle checkpoint in mammalian cells, Nature 372 Ž1994. 467–470. R. Ishida, M. Sato, T. Narita, K.R. Utsumi, T. Nishimoto, T. Morita, H. Nagata, T. Andoh, Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events, J. Cell Biol. 26 Ž1994. 1341–1351. M.D. Dar, T.J. Jorgensen, Deletion at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system, Nucleic Acids Res. 23 Ž1995. 3224–3230. J.W. Phillips, W.F. Morgan, Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome, Mol. Cell. Biol. 14 Ž1994. 5794–5803. P. Rouet, F. Smih, M. Jasin, Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 6064– 6068. A.R. Godwin, R.J. Bollag, D.M. Christie, R.M. Liskay, Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 12554–12558. A. Choulika, A. Perrin, B. Dujon, J.-F. Nicolas, Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cereÕisiae, Mol. Cell. Biol. 15 Ž1995. 1968–1973. R.G. Sargent, M.A. Brenneman, J.H. Wilson, Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination, Mol. Cell. Biol. 16 Ž1997. 267–277.