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Molecular Cancer Research 3:277-285 (2005)
© 2005 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

Cisplatin Sensitizes Cancer Cells to Ionizing Radiation via Inhibition of Nonhomologous End Joining

Heather J. Boeckman, Kelly S. Trego and John J. Turchi

Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio

Requests for reprints: John J. Turchi, Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435. Phone: 937-775-3595; Fax: 937-775-3730. E-mail: john.turchi{at}wright.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The combination of cisplatin and ionizing radiation (IR) treatment represents a common modality for treating a variety of cancers. These two agents provide considerable synergy during treatment, although the mechanism of this synergy remains largely undefined. We have investigated the mechanism of cisplatin sensitization to IR using a combination of in vitro and in vivo experiments. A clear synergistic interaction between cisplatin and IR is observed in cells proficient in nonhomologous end joining (NHEJ) catalyzed repair of DNA double-strand breaks (DSB). In contrast, no interaction between cisplatin and IR is observed in NHEJ-deficient cells. Reconstituted in vitro NHEJ assays revealed that a site-specific cisplatin-DNA lesion near the terminus results in complete abrogation of NHEJ catalyzed repair of the DSB. These data show that the cisplatin-IR synergistic interaction requires the DNA-dependent protein kinase–dependent NHEJ pathway for joining of DNA DSBs, and the presence of a cisplatin lesion on the DNA blocks this pathway. In the absence of a functional NHEJ pathway, although the cells are hypersensitive to IR, there is no synergistic interaction with cisplatin.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The role of the nonhomologous end joining (NHEJ) pathway in repairing ionizing radiation (IR)–induced DNA double-strand breaks (DSB) is well established (1). However, the mechanism by which the NHEJ pathway responds to compound DNA lesions, including those induced by cis-diamminedichloroplatinum(II) (cisplatin), is less well defined. Cisplatin is a chemotherapeutic drug used in conjunction with IR to treat various types of cancer, including cervical carcinoma as well as head and neck cancers (2, 3). Although the mechanism of cisplatin sensitization to IR remains largely undefined, evidence suggests that the NHEJ pathway is involved (4, 5).

The chemotherapeutic efficacy of cisplatin is afforded by the ability to form covalent DNA adducts, including intrastrand 1,2d (GpG) and 1,3d (GpXpG) adducts and interstrand G-G cross-links. These adducts distort the DNA structure, thereby inhibiting replication and transcription, and, if not repaired, lead to cell death via the apoptotic pathway (6-8). Repair of cisplatin-DNA intrastrand lesions is largely catalyzed by the nucleotide excision repair pathway (9-11). The molecular mechanism of cisplatin interstrand cross-link repair has not been elucidated, and even less is known about the pathways required for the repair of compound lesions, such as those generated by IR treatment of cisplatin-damaged DNA.

Exposure to IR induces ssDNA and dsDNA breaks as well as various types of nucleobase damage (12). Repair of IR-induced DNA DSBs is catalyzed predominantly by the NHEJ pathway; however, the homologous recombination (HR) pathway has also been implicated in their repair (13-16). Repair of DNA DSBs via the NHEJ pathway requires the concerted action of a series of protein complexes. The DNA end-binding complex DNA-dependent protein kinase (DNA-PK) consists of the Ku heterodimer, which facilitates binding of the catalytic subunit of the DNA-PK (DNA-PKcs; refs. 17, 18). The crystal structure of the Ku heterodimer complexed with a dsDNA terminus has been solved and reveals a unique organization where a base, two pillars, and a bridge form a tunnel through which a duplex DNA can thread (19). To observe maximal kinase activity, the Ku subunits are first threaded onto the DNA substrate (20). The Ku dimer then slides inward on the DNA by a single helical turn in an ATP-independent reaction. This structure then results in the association of DNA-PKcs with Ku and the DNA termini (20-22). We have determined that Ku translocation on duplex DNA is significantly inhibited by the presence of cisplatin-DNA lesions and that DNA-PK activity is inhibited by the presence of cisplatin on the DNA termini (23). Following the association of DNA-PKcs, processing of the termini is likely to be required. Another protein, Artemis, has also been shown to form a complex with and serve as a substrate for phosphorylation by DNA-PKcs (24). Artemis has an associated 5'-3' exonuclease activity and, following phosphorylation by DNA-PKcs, acquires a single-stranded flap-like endonuclease activity (24) similar to the endonuclease activity of the Fen-1 class of enzymes (25, 26). The Mre11/Rad50/Nbs1 complex is also required for NHEJ catalyzed DSB repair and may also play an important role in processing the DNA termini (27-29). The Mre11/Rad50/Nbs1 complex exhibits ATPase, DNA binding, and DNA strand annealing activity in addition to a single-stranded exonuclease activity, all of which are potentially involved in processing DNA termini (29, 30). The processing events may facilitate the search for regions of microhomology near the termini or simply generate blunt DNA ends, which can then be ligated. In vitro analyses show that blunt-ended DNA fragments can be joined by the NHEJ pathway, although the efficiency of the reaction is increased if the DNA contains cohesive ends (31). Following processing of the DNA termini, the formation of the phosphodiester bonds completes joining of the DSB and is catalyzed by the DNA ligase IV/XRCC4 complex (30, 32, 33). In addition, the XRCC4 subunit is a substrate for DNA-PKcs phosphorylation, although mutational analysis revealed that phosphorylation of XRCC4 is not required for NHEJ (34).

To further investigate the role of NHEJ in cisplatin sensitization to IR, we have employed a series of cell lines that are either wild-type (WT) or devoid of DNA-PK (35) and determined the interaction between cisplatin and IR. We have selected median effect analysis as the method to determine the dose-effect relationship between cisplatin and IR treatment (36). Median effect analysis has been used extensively to determine the interaction of two drugs in combined treatment regimens (37-39). This analysis has also been employed specifically to analyze the importance of dosing schedule in cisplatin and IR combined treatment (40). By employing a reconstituted in vitro assay using modified synthetic DNA substrates, we show that a single cisplatin-DNA lesion near a DSB is sufficient to completely block rejoining of the DNA termini via NHEJ. These results show that the molecular mechanism of cisplatin sensitization of cells to IR involves inhibition of DNA-PK catalyzed phosphorylation of target proteins and ultimately inhibition of NHEJ catalyzed DNA end joining.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
NHEJ Status Influences Cisplatin Radiosensitization
In an effort to elucidate the molecular mechanism by which cisplatin and IR synergize to afford greater cytotoxicity than either agent individually, we undertook analyzing the cisplatin-IR interaction in a series of cell lines differing in their ability to catalyze DSB repair via NHEJ. We employed median effect analysis, which allows potential interactions, both antagonistic and synergistic, to be quantitatively studied over the complete range of cells affected by the combined treatment. We have measured the end point of colony-forming ability following treatment; therefore, the fraction of cells affected is analogous to the degree of cytotoxicity. Procedurally, median effect analysis involves determination of LD50 values for the individual agents and then treatment of the cells with increasing doses of the agents together while maintaining a constant ratio of each agent based on the LD50 (36). The combination index (CI) is then calculated over a range of cells affected, which allows the measured effect of the combined treatment to be compared with the expected result if the two agents act in a simply additive fashion. A CI of 1 indicates no interaction, whereas a CI of <1 indicates synergy. Antagonistic interactions are indicated by a CI value of >1. To validate this methodology and provide a baseline of synergism, we employed the ovarian epithelial cancer cell line A2780, which displays WT levels of DNA-PK (7) and is sensitized to IR by cisplatin (41, 42). Analysis of A2780 cell sensitivity to cisplatin revealed a toxicity curve similar to that published previously (8) and a LD50 of 3.0 µmol/L was calculated (data not shown). The IR toxicity curve of A2780 cells is presented in Fig. 1A and revealed a LD50 of ~0.5 Gy. The combined treatment protocol consisted of a 4-hour pretreatment with cisplatin. The cisplatin-containing medium was removed and replaced with PBS in the absence of cisplatin, and the cells were immediately treated with the indicated doses of IR. Following these treatments, fresh medium was added and the clonogenic colony-forming survival assay was done as described in Materials and Methods. The CI was calculated and plotted versus fraction of cells affected (Fig. 1B). The results revealed that at the lower fraction of cells affected IR and cisplatin were antagonistic, indicated by CI values of >1. As the fraction of cells affected increased, a transition through an additive interaction into synergism was observed as evidenced by the CI values decreasing below a value of 1. These results show that the interaction of cisplatin and IR was clearly dependent on the total dose and differed as the fraction of cells affected increased. The treatment with low levels of cisplatin was antagonistic to IR and may be the result of a checkpoint response, which enhances repair of the DNA DSBs induced by IR treatment. Having validated the median effect analysis in A2780 cells, we further examined the effect of cisplatin and IR in a pair of human glioma cell lines. The MO59J cells are devoid of DNA-PK, deficient in NHEJ, and therefore hypersensitive to IR. A second cell line, MO59K, is established from the same cancer, displays near WT levels of DNA-PK, is competent for NHEJ, and does not display hypersensitivity to IR (35). Clonogenic colony-forming assays were done on these two cell lines and were consistent with the published values for IR sensitivity, with the MO59K and MO59J lines exhibiting LD50 values of 0.5 and 0.1 Gy, respectively (data not shown). The results presented in Fig. 2A reveal a nearly identical LD50 for cisplatin in each cell line (1.3 µmol/L). Median effect analysis was then done and the results are presented in Fig. 2B. The results obtained from the DNA-PK-positive MO59K cells (filled circles) revealed a curve that was similar to that observed for the A2780 cells, with an antagonistic interaction observed at the lower percentages of cells affected and clear synergism at higher percentages of cells affected. The results observed for the DNA-PK null MO59J cells (open circles) are significantly different, with the CI remaining very close to a value of 1 over the entire range of cells affected. No synergism was observed even at the highest fraction of cells affected. These data show that in DNA-PK null cells there was no antagonistic or synergistic interaction between cisplatin and IR; thus, the synergism between these two agents requires DNA-PK and a functional NHEJ pathway to repair the IR-induced DNA DSBs.



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  		FIGURE 1. Cisplatin and IR synergy in DNA-PK-positive A2780 ovarian carcinoma cells. A. Cells were treated with varying doses of IR and plated into colony-forming assays. Cell viability was determined by expressing the number of colonies as a percentage of the number of colonies present in mock-treated control plates and was plotted versus IR dose. B. Combined cisplatin and IR treatment was done as described in Materials and Methods. CIs were calculated from at least three separate experiments, and averages and SEs were plotted versus fraction of cells affected.

 


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  		FIGURE 2. Interaction of cisplatin and IR is dependent on DNA-PK status. A. Cells were treated with the indicated concentrations of cisplatin for 4 hours and were plated into colony-forming assays as described in Materials and Methods. Cell viability was determined by expressing the number of colonies as a percentage of the number of colonies present in mock-treated control plates and plotted versus cisplatin dose. •, MO59K cells; {circ}, MO59J cells. B. Combined cisplatin and IR treatment was done as described in Materials and Methods. CIs were calculated from at least three separate experiments, and averages and SEs were plotted versus fraction of cells affected.

 
MO59J cells that are devoid of DNA-PK also exhibit reduced expression of the ataxia telangiectasia mutated (ATM) protein (43). Although the importance of ATM in signaling the DNA damage response to IR is well established (44, 45), the levels of ATM do not correlate with radiosensitivity (43). If the synergism between IR and cisplatin involves DNA-PK and NHEJ, the inability to signal to this pathway may also be expected to reduce synergism. Therefore, we employed the ATBiva cell line, which is devoid of ATM, and analyzed the interaction between cisplatin and IR. The cisplatin toxicity curve revealed a LD50 value of 2.5 µmol/L for the ATBiva cells (Fig. 3A). The LD50 for IR in ATBiva cells was found to be 0.3 Gy, consistent with published data (data not shown). Median effect analysis revealed that the lack of ATM also resulted in a reduction in the ability of IR and cisplatin to synergize (Fig. 3B). Because ATM lies upstream of DNA-PK, the inability to signal to the NHEJ pathway and induce cell cycle arrest in response to IR suggests that activation of NHEJ is also required for the synergistic activity of IR and cisplatin. Although the CI was above unity at the lowest fraction of cells affected, this slight deflection above 1 suggests that there is only a slight degree of antagonism between cisplatin and IR in the absence of ATM. Importantly, the shape of the curve obtained with the ATM cells is similar to that obtained with the MO59K cells, whereas the MO59J cells display a dramatically different curve. This suggests that the reduction in ATM levels observed in MO59J cells cannot account for the difference in synergism observed at high total doses, which affect a larger fraction of cells, but may account in part for the decrease in antagonism observed at lower fractions of cells affected.



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  		FIGURE 3. Minimal interaction of cisplatin and IR in ATM cells. A. Cells were treated with cisplatin and plated into colony-forming assays as described in Materials and Methods. Cell viability was determined by expressing the number of colonies as a percentage of the number of colonies present in mock-treated control plates. B. Combined cisplatin and IR treatment was done as described in Materials and Methods. CIs were calculated from at least three separate experiments and averages and SEs were plotted versus fraction of cells affected.

 
In Saccharomyces cerevisiae, it has been shown that cisplatin can radiosensitize cells containing a competent HR pathway but not Rad52 mutant cells defective in recombinational repair (46). Thus, we were interested in examining this effect in higher eukaryotes, where null mutants of Rad52 and Rad54 exist in chicken B lymphocyte DT40 cells (47, 48). Analysis of the vertebrate Rad orthologues reveal somewhat different effects in terms of recombination deficiencies in the various mutant lines, where Rad52 null cells were not hypersensitive to IR, methyl methanesulfonate, or cisplatin (47). Further analysis revealed that NHEJ, and specifically Ku70, plays a critical role in the repair of IR-induced DNA DSBs (49). Interestingly, these studies also indicated that Rad54 contributes to the repair of IR-induced DNA DSBs. To assess the potential role of HR in cisplatin radiosensitization, we first analyzed WT DT40 cells for a cisplatin-IR interaction. The results revealed that there was no synergistic activity, and in fact, considerable antagonism was displayed such that pretreatment with cisplatin resulted in resistance to IR (Fig. 4). As a control, we assessed the cisplatin-IR interaction in Rad52–/– cells, which despite displaying a recombination deficiency do not show an IR-sensitive phenotype (47). Interestingly, these cells also displayed an antagonistic cisplatin-IR interaction (Fig. 4). Similar results were obtained at varying doses of cisplatin (data not shown). These results are interesting in terms of the differential response to cisplatin and IR; however, the inability to detect a synergistic interaction in the control cells makes it impossible to determine the potential role of HR in this process.



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  		FIGURE 4. Cisplatin-IR interactions in DT40 cells. WT (circles) and Rad52 null DT40 (triangles) cells were mock treated (filled symbols) or cisplatin treated (open symbols) for 24 hours before IR treatment at the indicated doses. Cell viability was represented as a percent absorbance of mock IR-treated control cells with or without cisplatin treatment after the addition of CCK-8 dye. Points, averages from duplicate determinations from two independent treatments; bars, SDs.

 
Inhibition of DNA-PKcs by Terminal Cisplatin-DNA Lesions In vitro
The data obtained in our cell culture model for the cisplatin-IR interaction suggest that a functional NHEJ pathway and the ability to signal DSBs are required for the synergistic activity between these two agents. An in vitro approach was used to directly address how the NHEJ pathway responds to the presence of compound lesions, in this case cisplatin-DNA adducts, in proximity of a DNA DSB. To first assess the effect of the position of the cisplatin lesion with respect to the DNA termini on activities required for NHEJ, a series of in vitro analyses were done using either purified Ku or heterotrimeric DNA-PK and short, duplex DNA substrates. The DNA substrates were designed to contain a single cisplatin 1,2d (GpG) adduct at varying distances from the terminus where DNA-PK could bind. DNA-PK was blocked from binding to the opposite terminus by a streptavidin bound to the 5' biotin present on the undamaged, cDNA strand of the duplex (22, 23, 50). The results of Ku-binding experiments showed a 50% reduction in binding at subsaturating concentrations of Ku when the lesion was present 7 bp from the terminus, and only a minimal effect when the lesion was positioned 15 bp from the terminus (data not shown). The ability of the DNA to activate DNA-PKcs also revealed that the presence and position of the cisplatin lesion inhibited kinase activity (51). When the lesion was positioned 7 bp from the terminus, there was a 90% reduction in kinase specific activity, whereas a 50% reduction was observed when the lesion was placed 15 bp from the terminus. Therefore, we determined the effect of a cisplatin lesion positioned 7 bp from the terminus on NHEJ catalyzed repair.

Inhibition of NHEJ by Terminal Cisplatin-DNA Lesions In vitro
An in vitro assay was employed to test the ability of HeLa cell extracts to perform end joining of a DNA substrate that was either unmodified or contained a single intrastrand 1,2d (GpG) cisplatin adduct 7 bp distal to the dsDNA break. If the cisplatin lesion is processed by NHEJ machinery, one might expect to observe a slight reduction in the ability to join DNA termini with cisplatin lesions. If there is no processing of the lesions, a significant reduction in NHEJ would be expected, as DNA-PK catalytic activity is required for NHEJ (31).

The DNA substrate was constructed from a 5-kbp plasmid that contained a single AvrII restriction site flanked by two SstI sites (Fig. 5A). A 30-mer linker molecule was designed to contain a single cisplatin adduct site as well as an AvrII/XbaI sticky end. The 34-base cDNA strand was 5'-labeled with [{gamma}-32P]ATP (Fig. 5B). The double-stranded 30-mers, cisplatin damaged or undamaged, were ligated to the plasmid at the AvrII site. This generated the linear NHEJ substrate containing a single cisplatin adduct 7 bp distal to the dsDNA ends and the 32P-radiolabeled 34 bases internal from each termini (Fig. 5C). SstI digestion of unligated molecules produce products of 111 and 129 bases termed the head and the tail, respectively (Fig. 5C). NHEJ of the substrate can be envisioned in a variety of orientations: head to head, head to tail, and tail to tail, each generating a different-sized product (222, 240, and 258 bp, respectively). This design presented several advantages, including the ability to ensure that every 32P-labeled DNA end contained one cisplatin adduct at a defined location and that the control substrate was identical, except for the absence of cisplatin damage.



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  		FIGURE 5. Cisplatin lesions on DNA termini abrogate NHEJ catalyzed repair of DNA DSBs. A. pGL-1 plasmid map showing a single AvrII site flanked by two SstI sites. B. SJC1.5C-Xba was 5' end-labeled with 32P and annealed to SJC1.5 that was damaged with cisplatin or undamaged. The position of the cisplatin site is indicated by the carat. C. The 30/34 duplex DNA, with or without the cisplatin adduct, was ligated to pGL-1 linearized with AvrII as described in Materials and Methods and the product is depicted. SstI digestion of the linear DNA substrate would yield products of 111 bp (Head) and 129 bp (Tail). D. NHEJ reactions (20 µL) contained 6 fmol DNA substrate and 0, 32, 80, or 160 µg HeLa cell-free extract. Reactions were done with either undamaged DNA (lanes 1-4) or cisplatin-damaged DNA (lanes 5-8). Following digestion of the products with SstI, ligation products were resolved on 10% native PAGE and visualized by autoradiography. Arrows, unligated head and tail products of 111 and 129 bp, respectively; bracket, ligation products of head to head, head to tail or tail to head, and tail to tail (222, 240, and 258, respectively).

 
NHEJ reactions were done with each substrate and the results are presented in Fig. 5D. On addition of the cell-free extract to reactions containing the undamaged DNA substrate, the expected ligation products were observed and showed an increase with increasing concentrations of extract (lanes 2-4). Dramatically, there was no detectable end joining of substrate containing the cisplatin lesions (lanes 6-8). The addition of wortmannin, an inhibitor of DNA-PK (52), completely blocked the end joining reaction of the non-cisplatin-treated DNA, demonstrating that joining is catalyzed by the DNA-PK-dependent NHEJ pathway (data not shown). To optimize the reaction, a variety of conditions were employed, including an increased substrate concentration. This increased the ligation of the undamaged DNA substrate 7-fold from that observed and still resulted in no end joining with the DNA substrate containing the cisplatin adduct (data not shown). Initial time course experiments were done and showed an increase in joining of the control substrate with increased time with no joining of cisplatin-damaged DNA being observed (data not shown). We also did mixing experiments in which the cisplatin-damaged and undamaged DNA substrates were combined. The results showed that the cisplatin-damaged DNA substrate did not inhibit joining of the undamaged DNA substrate (data not shown). Together, these data suggest that in a cell the presence of a cisplatin lesion at a DNA terminus will result in a persistent DNA DSB but may not significantly inhibit NHEJ catalyzed joining of DNA DSBs that do not have a cisplatin lesion in close proximity to the termini.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this report, we have investigated the mechanism by which cisplatin sensitizes cells to IR treatment. Cisplatin, in conjunction with IR, is under intense investigation in the treatment of a variety of cancers (53-56). Clinical trials of cervical cancer have shown the utility of this combined treatment, increasing survival and disease-free intervals, such that it is now part of the standard care for the treatment of cervical cancers (2, 3).

Although successful clinically for a subset of cancer types, only recently has the molecular mechanism of the radiosensitization activity of cisplatin been studied. This is at least in part a result of more recent advances in our understanding of the many pathways involved in signaling the cellular response to DNA damage and our detailed understanding of the mechanisms involved in repairing various types of DNA damage. An analysis of DSB formation revealed that the number of IR-induced DNA DSBs was largely independent of cisplatin pretreatment (57). It was convincingly shown that the repair of IR-induced DNA DSBs was altered by pretreatment with cisplatin, with a clear dose effect being observed (5). The authors showed that low concentrations of cisplatin resulted in stimulation of DSB repair, whereas inhibition was only observed at higher doses of cisplatin (5). These results are consistent with the median effect analysis results in this study where antagonism is observed at low combined doses and synergy is observed at high doses in cells with a functional NHEJ pathway (Figs. 1 and 2). The results we have obtained with DNA-PK-deficient cells suggest a role for NHEJ in cisplatin-dependent radiosensitization. These results are supported by previous data, which showed that there was no dependence of cisplatin on the repair of ssDNA breaks (5), consistent with the role of NHEJ in processing DNA DSBs. A role for NHEJ in cisplatin radiosensitization has been observed in Ku null cells, which were inefficiently sensitized to IR by cisplatin; however, the investigators could not rule out a limitation of their assay in detecting differences in viability as a result of the extreme sensitivity of the Ku null cells to IR (4). The use of median effect analysis allowed us to circumvent this limitation and using DNA-PK null cells show the influence of NHEJ on the interaction of cisplatin and IR over the entire range of cells affected. The observation that ATM cells also are defective in cisplatin-dependent radiosensitization further supports the NHEJ pathway in the potentiation of this effect.

DNA damage signaling pathways have been studied in a variety of systems and involve an array of sensor, mediator, and effector proteins (58, 59). The phosphatidylinositol 3-kinase–like kinase family members ATM and ATR are thought to be the major transducers of DNA damage capable of phosphorylating a series of downstream target proteins, including the chk2 and chk1 kinases, in response to DNA damage. ATM is primarily responsible for signaling DNA DSBs, whereas ATR signals stalled replication forks and UV-induced DNA damage. The combination of IR and cisplatin treatment is likely to result in the activation of both pathways, as cisplatin has been shown to increase ATR activity (60), and our data suggest that the final effect will depend on the total dose. The differential interactions seen between cisplatin and IR as a function of doses, antagonism at low doses and synergy at high doses (Fig. 2B), could be the result of cross-talk between the two signaling pathways. At low doses of cisplatin, activation of the ATR pathway resulting in downstream activation of chk1, phosphorylation of p53, and inhibition of cdc25A phosphatase activity could contribute to the subsequent sensing of IR-induced DNA DSBs via ATM. ATM activation and chk2 phosphorylation would also result in p53 activation and cdc25A inhibition. This overlap could be envisioned to result in antagonism, as the sensor and effector pathways leading to establishing cell cycle checkpoints would result in increased survival of the cells treated with both agents compared with the individual treatments. In contrast, at higher combined doses, there are a greater number of cisplatin-DNA lesions when the cells are exposed to IR treatment. Assuming a completely random distribution of IR-induced DNA DSBs following cisplatin treatment, we calculated that ~0.01% of the treated cells would have a cisplatin lesion within 7 bp of a DNA terminus generated from the DSB at the levels of cisplatin and doses of IR used in this study. However, previously published data suggest that the assumption of random IR-induced DNA DSBs of cisplatin-damaged DNA may be incorrect. It was noted that cisplatin treatment did not affect the total number of DNA DSBs, although the DNA extractability was altered (57). This indicates that there was some, yet to be described, change in the DNA or its association with nuclear proteins. We propose that the position of an IR-induced DNA DSBs is altered such that the breaks occur within the vicinity of a preexisting cisplatin lesion. This hypothesis is supported by in vitro studies using the radiomimetic agent bleomycin, which showed that the positions of the bleomycin-induced DNA DSBs were altered when the DNA was pretreated with cisplatin (61). This supposition is also supported circumstantially in experiments that show that the order of treatment is critical as cisplatin must be given before IR treatment from maximum synergy (40). If the position of IR-induced DNA DSBs are also modified dependent on cisplatin, then the substrates for NHEJ catalyzed repair of the DSBs can be influenced by the cisplatin lesion. Therefore, at higher cisplatin-DNA adduct levels and higher doses of IR, the likelihood that a DNA DSB occurs in the vicinity of a cisplatin lesion significantly increases. Our in vitro results show that this lesion represents an absolute block to NHEJ, which in vivo will represent a persistent DNA DSB. Considering that a single persistent DNA DSB may be sufficient to induce cell death (62), the presence of a cisplatin-DNA lesion near a terminus would be expected to be lethal to the cell. The in vitro data of kinase activation confirm the importance of cisplatin adduct position with respect to the DNA terminus (23). Clearly, the effect of adduct position on NHEJ catalyzed is an important issue to be addressed.

The inability of the NHEJ pathway to process a cisplatin lesion in the vicinity of a DNA DSB has numerous interesting ramifications, the first of which is that these lesions are irreparable and will persist until the cell dies. This model is supported by the results of the median effect analysis revealing the lack of interaction between cisplatin and IR in DNA-PK null cells. The finding that there was no synergism in the DNA-PK null cells indicates that a functional NHEJ pathway is required for cisplatin-dependent radiosensitization. What remains unknown is the role other DSB repair pathways potentially play in repairing compound lesions. If other pathways are capable of repairing a compound cisplatin-DNA-DSB lesion, for example, HR, these would reduce the degree of synergy. Interestingly, recent evidence has shown a competition between NHEJ and HR in the repair of DNA DSBs (63). It was shown using a specific inhibitor of DNA-PK, IC86621, that inactivation of DNA-PKcs results in decreased DSB-induced repair via HR. The authors concluded that an inactive DNA-PKcs positioned at a DNA terminus could inhibit both NHEJ and HR as a result of the lack of autophosphorylation and dissociation of the subunit from the DNA end. The fact that a terminal cisplatin lesion results in inhibition of DNA-PKcs catalyzed kinase and autophosphorylation activity (64, 65) indicates that both NHEJ and HR catalyzed repair of DNA DSBs containing cisplatin lesions would be inhibited and therefore contribute to the radiosensitization activity of cisplatin.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture and Treatment
Mammalian cells were grown in DMEM supplemented with L-glutamine (Atlanta Biologicals, Atlanta, GA), penicillin/streptomycin, and 10% fetal bovine serum (Atlanta Biologicals). Incubations were done at 37°C in a 5% CO2 humidified atmosphere. Cisplatin (Sigma, St. Louis, MO) was added to complete medium at the indicated concentrations for 4 hours at 37°C. Following incubation, the medium containing cisplatin was removed and replaced with fresh complete medium lacking cisplatin or PBS in the case of IR treatments. IR treatments were done on ice using a HP Faxitron series X-ray generator (Hewlett-Packard, McMinnville, OR) to deliver the indicated doses. The X-rays were filtered through a 0.07 mm aluminum filter resulting in a dose rate of 0.15 Gy/min. Dosimetry was done using a Radcal dosimeter (Monrovia, CA). Before IR treatment, medium was removed and replaced with PBS. Cell viability was assessed by a clonogenic colony-forming assay. Briefly, cells were removed from the plates with trypsin, suspended in complete medium, counted, and plated at two densities in triplicate. Alternatively, cells were plated in triplicate, treated, and then placed directly into the incubator. Similar results were obtained with each procedure. Plates were incubated at 37°C for 7 to 14 days, stained with 1% methylene blue in 70% ethanol, and washed with water and colonies containing at least 50 cells in size were counted using an Acolyte cell colony counter (Synbiosis, Cambridge, United Kingdom). Averages and SEs were then plotted versus dose (IR or cisplatin), and LD50 values for each treatment were determined for each cell line.

Chicken B lymphocyte DT40 cells, WT and HR mutant, were kindly provided by R. Fishel (Ohio State University, Columbus, OH). Cells were grown in RPMI 1640 supplemented with L-glutamine, penicillin/streptomycin, 50 µmol/L ß-mercaptoethanol, 10% fetal bovine serum, and 5% chicken serum (Life Technologies, Grand Island, NY) at 37°C. Cisplatin (20 µmol/L) was added to complete medium for 24 hours at 37°C followed by washing with PBS and resuspension in complete medium lacking cisplatin. IR treatments were done on ice as noted above. Cells were incubated for 24 hours after IR treatment and then transferred to 96 well plates, where cell viability was measured with CCK-8 (Dojindo Molecular Technologies, Gaithersburg, MD) according to standard protocol. The data were represented as the percent absorbance of mock IR-treated controls with or without cisplatin treatment.

Median Effect Analysis
Median effect analysis was employed to quantify the interaction of cisplatin and IR. For combined treatments, the ratio of IR dose to cisplatin dose was kept constant based on the LD50, and cells were treated with increasing total doses. A plot of the log of the total dose versus log of the reciprocal of the fraction of cells affected minus 1 yielded linear plots. The slope and y-intercept from these plots were used to calculate the CI as described previously (36).

In vitro NHEJ Assay
Preparation of DNA substrates for NHEJ assays. The NHEJ substrate consisted of linearized pGL-1 (5,032 bp) ligated with 32P-labeled 30-mer linker molecules containing a single intrastrand 1,2d (GpG) cisplatin adduct. Oligonucleotide SJC1.5 was platinated as described previously (66) and oligonucleotide SJC1.5C-Xba was 5' labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and unincorporated nucleotide was removed using spin column chromatography. 32P-labeled SJC1.5C-Xba was annealed to platinated SJC1.5C (1:2 molar ratio) in buffer containing 10 mmol/L Tris (pH 7.9), 50 mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L DTT followed by HaeIII digestion (8 units; New England Biolabs, Beverly, MA). Products were separated on 10% native gels and annealed. HaeIII-resistant DNA was isolated, eluted in buffer containing 0.3 mol/L sodium acetate, 1 mmol/L EDTA, and 0.1% SDS, ethanol precipitated, and stored in buffer containing 10 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, and 1 mmol/L EDTA. A control unplatinated DNA was made in the same manner but was not digested with HaeIII. The plasmid pGL-1 was linearized with AvrII (New England Biolabs) in buffer containing 10 mmol/L Tris (pH 7.9), 50 mmol/L NaCl, 10 mmol/L MgCl2, and 1 mmol/L DTT. Linker DNA with or without the cisplatin adduct was mixed with plasmid DNA in a 2:1 molar ratio, ethanol precipitated, and resuspended in buffer containing 10 mmol/L Tris (pH 7.9), 50 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L DTT, 1 mmol/L ATP, 100 µg/mL bovine serum albumin, T4 DNA ligase (300 units), AvrII (3 units), and Xba (7.5 units; New England Biolabs). Ligation reactions proceeded for 16 hours at 18°C. Ligated product was separated from unligated linker by agarose gel electrophoresis (0.8%) followed by purification using gel extraction spin columns (Qiagen, Valencia, CA). The NHEJ substrate was eluted and stored at –20°C in 10 mmol/L Tris (pH 8.5). NHEJ reactions (20 µL) were done in buffer containing 50 mmol/L HEPES (pH 8.0), 40 mmol/L potassium acetate, 1 mmol/L magnesium acetate, 1 mmol/L DTT, 100 µg/mL bovine serum albumin, 2 mmol/L ATP, and 5 µmol/L IP6. Cell-free extracts were prepared from exponentially growing HeLa cells as described previously (67). Extracts were incubated for 10 minutes at 37°C before the addition of 32P-labeled NHEJ DNA substrate (6 fmol). Reactions proceeded for 2 hours at 37°C and were terminated by the addition of 500 µL buffer containing 0.3 mol/L sodium acetate, 1 mmol/L EDTA, 0.1% SDS, and proteinase K (10 µg). These reactions were incubated at 45°C for 30 minutes after which the 32P-labeled DNA was extracted with 500 µL phenol/chloroform/isoamyl alcohol (50:49:1) and ethanol precipitated. The DNA products were digested with SstI (Invitrogen, Carlsbad, CA) for 30 minutes at 37°C and were analyzed by electrophoresis on 10% native polyacrylamide gels. Following electrophoresis for 2 hours at 160 V, gels were dried and quantified by PhosphorImager analysis.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank the other members of the laboratory for their helpful discussions and editing of the article.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: NIH grant R01-CA82741 (J.J. Turchi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: H.J. Boeckman and K.S. Trego contributed equally to this work.

Received 2/20/04; revised 2/18/05; accepted 3/21/05.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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