Oncogene (2000) 19, 5788 ± 5800 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Homologous recombination as a potential target for caeine radiosensitization in mammalian cells: reduced caeine radiosensitization in XRCC2 and XRCC3 mutants Nesrin A Asaad1, Zhao-Chong Zeng1, Jun Guan1, John Thacker2 and George Iliakis*,1 1Department of Radiation Oncology of Kimmel Cancer Center, Jeerson Medical College, Philadelphia, Pennsylvania, PA 19107, USA; 2Medical Research Council, Radiation and Genome Stability Unit, Harwell, Oxfordshire, OX11 ORD, UK The radiosensitizing eect of caeine has been associated Introduction with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also Caeine (1,3,7-trimethylxanthine) at submillimolar implicate inhibition of DNA repair. The role of DNA concentrations exerts a wide variety of physiological repair inhibition in caeine radiosensitization remains eects on dierent organisms from bacteria to man uncharacterized, and it is unknown which repair process, (Garattini, 1993; Timson, 1977). At higher concentra- or lesion, is aected. We show that a radiosensitive cell tions (0.5 ± 10 mM), it strongly enhances the cytotoxic line, mutant for the RAD51 homolog XRCC2 and eect of ionizing radiation and other physical or defective in homologous recombination repair (HRR), chemical agents that act by inducing damage in DNA displays signi®cantly diminished caeine radiosensitiza- (Kihlman, 1977; Murnane, 1995; Timson, 1977; tion that can be restored by expression of XRCC2. Waldren and Rasko, 1978). Radiosensitization is Despite the reduced radiosensitization, caeine eec- manifest at non-cytotoxic concentrations and therefore tively abrogates checkpoints in S and G2 phases in caeine is considered a model radiosensitizer. Eorts XRCC2 mutant cells indicating that checkpoint abroga- are underway to develop compounds with similar tion is not sucient for radiosensitization. Another mechanism of action but higher ecacy to permit radiosensitive line, mutant for XRCC3 and defective in application for the treatment of cancer. Understanding HRR, similarly shows reduced caeine radiosensitiza- of the mechanism of caeine action will greatly tion. On the other hand, a radiosensitive mutant (irs-20) facilitate these eorts. of DNA-PKcs with a defect in non-homologous end- The molecular mechanism of caeine radiosensitiza- joining (NHEJ) is radiosensitized by caeine to an tion remains unknown, but experiments carried out in extent comparable to wild-type cells. In addition, the last 30 years allow the development of working rejoining of radiation-induced DNA DSBs, that mainly models. A widely held view is that radiosensitization re¯ects NHEJ, remains unaected by caeine in derives from the ability of caeine to disrupt multiple XRCC2 and XRCC3 mutants, or their wild-type DNA damage-responsive cell cycle checkpoints. Indeed counterparts. These observations suggest that caeine caeine has been shown to reduce or eliminate p53 targets steps in HRR but not in NHEJ and that activation and G1-arrest (Kastan et al., 1991; Powell et abrogation of checkpoint response is not sucient to al., 1995; Valenzuela et al., 2000), S phase delay explain radiosensitization. Indeed, immortalized ®bro- (Griths et al., 1978; Murnane et al., 1980; Painter, blasts from AT patients show caeine radiosensitization 1980; Tolmach et al., 1977; Walters et al., 1974) and despite the checkpoint defects associated with ATM G2/M arrest (Jung and Streer, 1992; Kimler et al., mutation. We propose that caeine radiosensitization is 1982; Lau and Pardee, 1982; Lucke-Huhle, 1982; mediated by inhibition of stages in DNA DSB repair Rowley, 1992; Tomasovic and Dewey, 1978) in cells requiring HRR and that checkpoint disruption contri- exposed to ionizing radiation. Because checkpoints butes by allowing these DSBs to transit into irreparable provide extra time that can be used for repair states. Thus, checkpoints may contribute to genomic (Hartwell and Kastan, 1994; Hartwell and Weinert, stability by promoting error-free HRR. Oncogene 1989), abrogation by caeine is expected to enhance (2000) 19, 5788 ± 5800. the lethal and mutagenic eects of DNA damaging agents. In some cell systems this is also associated with Keywords: caeine; DNA; Double Strand Breaks an increase in apoptosis (Bernhard et al., 1996; (DSB); repair; homologous recombination; non-homo- Palayoor et al., 1995; Shinomiya et al., 1997; Yao et logous end joining al., 1996). Despite the obvious rationale for checkpoint abroga- tion as the mechanism for caeine radiosensitization, a signi®cant number of experiments cannot be fully interpreted without invoking a direct eect of caeine on DNA repair (Busse et al., 1977, 1978; Iliakis and Nusse, 1983a; Tolmach and Busse, 1980). Support for DNA repair inhibition as a contributor to caeine *Correspondence: G Iliakis, Thomas Jeerson University, radiosensitization also comes from studies demonstrat- Department of Radiation Oncology, Thompson Bldg. Rm. B-1, ing a lack of correlation between abrogation of cell Philadelphia, PA 19107, USA Received 11 August 2000; revised 18 September 2000; accepted 22 cycle checkpoints and the level of caeine radio- September 2000 sensitization (Harvey and Savage, 1994; Musk, 1991; Caffeine and homologous recombination NA Asaad et al 5789 Musk and Steel, 1990; Ribeiro et al., 1999; Walters et To examine this possibility, we tested caeine al., 1974). In addition, caeine is less eective in cells radiosensitization in irs1 cells, and extended our with functional p53 although it inhibits the induction experiments to include irs1SF, a radiosensitive cell line of this protein and abrogates the G1 checkpoint (Fan (Fuller and Painter, 1988) with a defect in the RAD51 et al., 1995; Kastan et al., 1991; Powell et al., 1995; homolog XRCC3 (Liu et al., 1998), and an associated Russell et al., 1995; Taylor et al., 1993; Valenzuela et defect in DNA DSB repair by HRR (Pierce et al., al., 2000). 1999). We reasoned that if caeine acts as a radio- It is likely therefore that caeine radiosensitization is sensitizer by targeting steps in HRR, it should be less the combined result of checkpoint abrogation and eective in cells with genetic defects in this pathway. In DNA repair inhibition, as is strongly suggested by order to examine whether HRR is speci®cally targeted extensive studies on cell radiosensitization throughout by caeine, the results with these mutants are the cell cycle (Beetham and Tolmach, 1982; Busse et compared to those of irs20, a radiosensitive mutant al., 1978). Based on its chemical properties, caeine is with defects in NHEJ (Stackhouse and Bedford, 1993a) expected to have multiple molecular targets and a as a result of mutation in DNA-PKcs (Priestley et al., particular anity for protein kinases (Jung and 1998). Experiments on caeine radiosensitization to Streer, 1992). Checkpoint abrogation and DNA killing are complemented with an evaluation of the repair inhibition may therefore be the result of eect of caeine on checkpoint abrogation in HRR inactivation of distinct, though possibly interacting, de®cient mutants, and with studies on caeine radio- protein kinases. While targets aecting DNA repair, if sensitization in AT cells. The results of these present, remain to be identi®ed, recent data point to experiments are used to develop a model for the ATM and ATR as kinases directly inhibited by caeine mechanism of caeine radiosensitization that invokes and partly provide a mechanistic explanation for the DNA DSB repair inhibition and checkpoint abrogation observed checkpoint disruption (Blasina et al., 1999; and suggests possible interrelationships between the Hall-Jackson et al., 1999; Sarkaria et al., 1999; Zhou et two processes. al., 2000). For a complete understanding of the mechanism of caeine radiosensitization, it is impor- tant to con®rm and elucidate the basis of its inhibitory Results eects on DNA repair, identify the lesions and the repair processes aected and examine potential inter- Caffeine radiosensitization is reduced in cells defective in actions between DNA repair processes and checkpoint homologous recombination control. The recent molecular characterization of genetic To examine whether caeine sensitizes cells to ionizing defects in radiosensitive mutants of cells from verte- radiation by inhibiting HRR, we carried out experi- brates (Jeggo, 1998; Smith and Jackson, 1999; Thacker, ments using irs1 cells. Because caeine radiosensitiza- 1999; Thompson and Schild, 1999) allows a re- tion critically depends on drug concentration and interpretation of old caeine radiosensitization data treatment duration (Busse et al., 1997, 1978; Tolmach and the formulation of hypotheses regarding potential and Busse, 1980), we carried out preliminary experi- molecular targets for the repair eects of caeine. ments to standardize these parameters. The results of Experiments with synchronized rodent or human cells these experiments (not shown) demonstrate that show a ¯attening of the cell cycle-dependent variation caeine at 2 ± 4 mM and 10 ± 12 h postirradiation in radiosensitivy to killing after treatment with caeine. incubation time gives nearly maximal radiosensitization This is interpreted as inhibition of a repair process, with acceptable toxicity in both the parental V79 and repair of potentially lethal damage (Iliakis, 1988), the irs1 mutant. On the basis of these results further which occurs with a variable ecacy throughout the experiments were carried