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[Cancer Research 63, 3221-3227, June 15, 2003]
© 2003 American Association for Cancer Research

Experimental Therapeutics

Synchrotron Photoactivation of Cisplatin Elicits an Extra Number of DNA Breaks That Stimulate RAD51-mediated Repair Pathways1

Stéphanie Corde, Jacques Balosso2, Hélène Elleaume, Michel Renier, Aurélie Joubert, Marie-Claude Biston, Jean-François Adam, Anne-Marie Charvet, Thierry Brochard, Jean-François Le Bas, François Estève and Nicolas Foray

Equipe d’Accueil 2941 Rayonnement Synchrotron et Recherche Médicale, Unité IRM [S. C., J. B., H. E., A. J., M-C. B., J-F. A., A-M. C., J-F. L., F. E., N. F.], Département de Cancérologie et d’Hématologie [J. B.], and INSERM IFR 1 [H. E.], CHU A. Michallon, BP 217, F-38043 Grenoble cedex 09, France; European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble, France [M. R., T. B.]; and Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN19RQ, United Kingdom [N. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Combination of cis-platinum with ionizing radiation is one of the most promising anticancer treatments that appears to be more efficient than radiotherapy alone. Unlike conventional X-ray emitters, accelerators of high energy particles like synchrotrons display powerful and monochromatizable radiation that makes the induction of an Auger electron cascade in cis-platinum molecules [also called photoactivation of cis-platinum (PAT-Plat)] theoretically possible. Here, we examined the molecular consequences of one of the first attempts of synchrotron PAT-Plat, performed at the European Synchrotron Research Facility (Grenoble-France). PAT-Plat was found to result in an extra number of slowly repairable DNA double-strand breaks, inhibition of DNA-protein kinase activity, dramatic nuclear relocalization of RAD51, hyperphosphorylation of the BRCA1 protein, and activation of proto-oncogenic c-Abl tyrosine kinase.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is evidence that unrepaired or misrepaired DNA DSBs3 pose a threat to both genetic stability and cell survival (1 , 2) . In response to spontaneous or genotoxic stress-induced DSBs, mammalian cells activate two major signaling pathways to recover original DNA topology: (a) NHEJ, which depends on the protein kinase DNA-PK that is mainly activated after exposure to IR (1 , 3) ; (b) homologous recombination mediated by the RAD51 protein that functions after exposure to IR. Furthermore, it is noteworthy that RAD51 is involved in the early steps of repair of DNA adducts induced by certain alkylating agents like those used in chemotherapy (2 , 4) . Hence, concomitant chemoradiotherapy, one of the most effective anticancer treatment, raises the question of the interplay between these different DNA repair pathways.

Cisplatin, in its cDDP form, is a widely used therapeutic agent, notably for testicular and ovarian cancer chemotherapy (5 , 6) and a number of other solid tumors (7) . Cisplatin binds to DNA, particularly to linker regions via histone H1 protein, and forms inter- and intrastrand DNA adducts (8) . Cells treated with cDDP undergo repair of DNA adducts, cell cycle arrest, inhibition of replication, and mitotic or apoptotic cell death pathways (9, 10, 11, 12) . Cisplatin-DNA adducts were also recently shown to inhibit DNA-PK activity by preventing Ku, a subunit of the DNA-PK protein, to translocate on the duplex DNA, suggesting that treatment of tumors with platinated compounds leads to cellular sensitization by inhibiting NHEJ (13) . In parallel, cisplatin-DNA adducts promote the assembly of the breast and ovarian cancer-susceptibility BRCA1 and BRCA2 protein with RAD51 to participate in HR and repair of DNA adducts (12) . Consequently, cisplatin may be very efficient during anticancer treatments of syndromes associated with HR deficiency, or more generally to impaired RAD51-dependent repair pathways, by inducing irreparable DNA damage.

Another potential therapeutic advantage of cisplatin is that the disintegration of its metastable 193mPt form trigger Auger effect resulting in energy microdepositions sufficient to create DSBs in situ (Ref. 14, 15, 16, 17, 18, 19 and Refs. therein). The Auger effect consists in an electron cascade attributable to electronic rearrangements of unstable and/or excited atoms when electrons of deep shells (K, L, or M) are lacking. The Auger effect can be triggered by external photons; however, the beam fluence provided by conventional X-ray emitters is insufficient to induce such electron cascades (19, 20, 21, 22) . By contrast, accelerators of high energy particles like synchrotrons display powerful and easily monochromatizable radiation that makes the Auger effect in platinated cells theoretically possible, a phenomenon that may be designated as synchrotron PAT-Plat. A fundamental advantage of this technique, compared with the administration of isotopic Auger emitter, is the lower cytotoxicity obtained outside the tumor volumes (19 , 20) .

Hence, the synchrotron PAT-Plat may theoretically enhance the biological efficiency of the drug by producing additional DNA damage in treated cells. Here, we examined the molecular consequences of a treatment with cDDP combined with synchrotron irradiation. cDDP-treated SQ20B carcinoma cell lines were irradiated at energy just above (A energy) or below (B energy) the platin-K-edge peak (78.4 keV) that is delivered by the ESRF (Grenoble, France). Synchrotron PAT-Plat was found to result in an extra number of slowly repairable DSBs. Moreover, synchrotron PAT-Plat inhibits DNA-PK activity but stimulates the RAD51-mediated homologous recombination repair pathways. A model in which synchrotron PAT-Plat implies the RAD51, c-Abl, and BRCA1 proteins is discussed. Altogether, these data represent the molecular basis of a promising anticancer treatment combining synchrotron radiation and chemotherapy with platinated compounds.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The SQ20B (BRCA1-positive) cell line is derived from a human head and neck squamous carcinoma (23) and the HCC1937 (BRCA1-mutated) cell line from a human ductal carcinoma bearing a BRCA1 5382insC mutation in one allele and a deletion of the second allele (24) . Cells were cultured in DMEM supplemented with antibiotics and FCS. All of the experiments have been performed with cells on exponential phase of growth.

Synchrotron Irradiation and Treatment with cDDP.
Treatment with cisplatin was performed by incubating cells in DMEM supplemented with 3 or 30 µM cDDP (Cysplatyl; Rhône-Poulenc Rhorer, Bellon, France) for 6 h. Cell culture flasks were then incubated on ice for at least 30 min, immediately prior to irradiation. At that time, cells incubated with 30 µM cDDP contained 3 x 108 atoms of platinum per cell as measured by mass spectrometry as described previously (19) . Irradiations were carried out at ESRF’s medical beamline (ID17) by using the set-up for coronary angiography, normally devoted to humans (25) . The two-thirds filling mode of the ESRF storage ring provided the highest intensity achievable (storage ring current ~200 m A with a lifetime of more than 50 h). The resulting synchrotron radiation was filtered and transformed by a silicium fixed-exit monochromator to obtain photons energies tuned just 400 eV above or below the 78.39-keV platinum K-absorption edge, as detailed previously (26) . The most homogeneous part of the beam was 4 cm wide and 1.5 mm high. Because of this small size, cells were continuously scanned through the beam, with a vertical, up-and-down translating device. The motor speed was 12.5 mm/s. With this particular configuration, the mean dose rate was around 1 cGy per scan. Doses were continuously monitored during irradiation with nitrogen-filled ion chambers. For DNA strand breaks assay, cells in plateau phase were irradiated at 4°C, in sets of three 12.5-cm2 flasks in the vertical position. The refrigerating system was obtained by liquid nitrogen vapor diffusion on samples and was monitored by a thermocouple. This setup was also used for the nuclear extracts study, but cells were irradiated in suspension in small, permanently rotating cryotubes. For immunofluorescence, small boxes containing coverslips covered with low-density growing cells were filled with cell culture medium and were also scanned in the beam but at room temperature.

DSB and SSB Assays.
Cells were prepared for a DSB assay as described elsewhere (27) . Briefly, cells were labeled with 370 Bq/ml [14C]thymidine (NEN DuPont de Nemours; 248 MBq/mmol). Before irradiation, culture flasks were precooled for at least 30 min and kept at 4°C throughout the irradiation period. Incubation was then applied at 37°C for the indicated repair times. Cells were trypsinized on ice, and agarose plugs of cells containing 2 x 106 cells/ml were prepared. The cells in the plugs were lysed in 2% L-laurylsarcosine (1 mg/ml)-0.5 M EDTA (pH 8) at 50°C for 38 h. Migration of DNA fragments was performed with pulsed field gel electrophoresis (CHEF DRIII; Bio-Rad, Hercules, CA) in megabase-size region after a 4-day migration program as detailed previously (27) . The FAR into the lane was calculated from: FAR = cpmlane/(cpmlane + cpmwell). For assessing SSBs, the same procedures were applied, with the notable exceptions of the pH of the lysis solution (pH 12) and of the migration program (24 h, 1.5V/cm).

Nuclear Extracts.
Nuclear extracts were prepared using a standard protocol described previously (28) . Briefly, cells were collected and were rinsed by centrifugation in cold PBS. The pellet was suspended in hypotonic buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride] supplemented with 50 mM NaF, 2 mM sodium orthovanadate, and protease inhibitors (Calbiochem, Darmstadt, Germany) for 10 min at 4°C. After centrifugation at 4,000 rpm, nuclei were then resuspended in hypertonic buffer [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) supplemented with 0.6% Triton X-100, 50 mM NaF, 2 mM sodium orthovanadate, and protease inhibitors. Nuclear extracts were collected after stirring for 15 min at 4°C and centrifugation at 10,000 rpm (28) . Treatment to {lambda}-phosphatase was performed by following the manufacturer’s instructions (Calbiochem).

Immunoprecipitation and Immunoblotting.
Immunoprecipitation and immunoblotting procedures were described previously (28) . Briefly, immunoprecipitation was performed by stirring 300 µg of nuclear extracts, 0.5–1 µg of specific monoclonal antibody, and protein A-Sepharose beads (Pharmacia, Buckinghamshire, United Kingdom) in NET-N buffer [50 mM Tris (pH 8), 1 mM EDTA, 120 mM NaCl, and 0.5% Nonidet NP40) supplemented with phosphatase and protease inhibitors (see above) for 4 h at 4°C. After washes in NET-N, immunoprecipitates were collected and were kept in loading buffer before separation. Preimmune rabbit (DAKO, Glostrup, Denmark) and mouse IgG (Jackson Immunoresearch, West Grove, PA) were used as controls.

For immunoblotting, 100 µg of cell extracts were boiled in loading buffer and were separated by SDS-PAGE (28) . The protein transfer to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) was performed with a semidry transfer system (Bio-Rad) and blocked in 5% milk TBS-T solution[100 mM Tris (pH 8), 150 mM NaCl, and 1% Tween 20; Ref. 28 ). Primary antibodies were applied for 6 h in 2% milk-TBS-T solution. Anti-BRCA1, anti-c-Abl, and anti-RAD51 antibodies were from Oncogene Research (Darmstadt, Germany). After washing for 1 h in TBS-T, antirabbit or antimouse secondary antibodies were applied in 2% milk-TBS-T solution, and the membrane was rewashed against TBS-T. Blotting was revealed using an ECL kit (Amersham, Buckinghamshire, United Kingdom).

DNA-PK Kinase Activity Assay.
Three hundred µg of nuclear extracts were subjected to immunoprecipitation with anti-DNA-PK antibody (Oncogene Research) for 2 h at 4°C. Immunocomplexes were then washed in DNA-PK kinase buffer [10 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, and 5 µM ATP], supplemented with 50 mM NaF, 2 mM sodium orthovanadate, 50 mM ß-glycerophosphate and protease inhibitors, and were incubated for 30 min at 30°C in the same buffer with 2 µCi [{alpha}-32P]ATP and 5 µg of GST-p53 (residues 1–40) as DNA-PK substrate peptide. Protein A-Sepharose beads that were bound to substrates were collected. DNA-PK kinase activity was quantified by liquid scintillation counting. In all of these experiments, an assay was routinely validated by using phosphospecific anti-p53Ser15 antibody.

C-Abl Kinase Activity Assay.
The c-Abl kinase assay has been described previously (28) . Briefly, 100 µg of lysates were subjected to immunoprecipitation with anti-c-Abl antibody (Ab-3; Oncogene Research) for 2 h at 4°C. Kinase assays were performed by incubating immunoprecipitates with 2 µCi [{alpha}-32P]ATP and 20 µM substrate peptide EAIYAAPFAKKK (New England Biolabs, Beverly, MA) in c-Abl kinase buffer [50 mM Tris (pH 7.5), 10 mM MnCl2, and 1 mM DTT) for 20 min at 30°C. Protein A-Sepharose beads that were bound to 32P-labeled substrates were collected and 32P activity was quantified by liquid scintillation counting. In all of these experiments, equal amounts of c-Abl immunoprecipitates were routinely controlled (28) .

Immunofluorescence.
Cells were fixed in 3% paraformaldehyde-2% sucrose PBS for 10 min at room temperature and were permeabilized in 20 mM HEPES (pH 7.4), 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100 for 5 min at 4°C. Thereafter, coverslips were washed in PBS before immunostaining. Primary antibody incubations were performed for 20 min at 37°C at 1:100 in PBS supplemented with 2% bovine serum fraction V albumin, which was followed by PBS washing. Secondary antibody incubations were also performed for 20 min at 37°C at 1:100 in PBS supplemented with 2% bovine serum fraction V albumin and followed by PBS washing. After PBS washing, nuclei were counterstained by DAPI (0.000025% in PBS) for 10 min at 4°C. Coverslips were mounted in Vectashield and examined with Zeiss microscope.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Synchrotron PAT-Plat Results in an Extra Number of Slowly Repairable DSBs.
The combination of treatment with cisplatin and synchrotron irradiation at energy higher than the platin K-absorption edge may theoretically lead to an Auger electron cascade, known to induce DSBs in situ (15, 16, 17, 18, 19) . To examine whether the synchrotron PAT-Plat is associated with an extra number of DSBs, the human head and neck squamous carcinoma SQ20B cells were subjected to different concentrations of cDDP (3 and 30 µM) and exposed to synchrotron radiation tuned 400 eV above (A energy) or below (B energy) the 78.39 keV platin K-absorption edge. A 4-day electrophoretic migration program allowing the detection of DNA fragments size up to 10 Mbp was used (27) . As described previously, the dose-response curves for DSB induction, expressed as FAR, show, generally, a threshold for FAR lower than 15% and are linearly dose dependent up to 60%. However, these values may change according the migration program and the DNA condensation (Ref. 27 and Refs. therein). With the notable exception of 16-Gy data, no significant difference was detected between A- and B-energy-induced DSBs yields in untreated or 3-µM-cDDP-treated cells. By contrast, after treatment with 30 µM cDDP, the amount of A-energy-induced DSBs was found to be significantly higher (by three times) than those assessed with B energy, suggesting that synchrotron PAT-Plat produces an extra number of DSBs (Fig. 1A)Citation . Under our conditions, the 20% excess of FAR observed between the two energies that combined to 30 µM cDDP corresponds to the effect of an extra dose of 8 Gy delivered with 30 µM cDDP [i.e., an excess of about 300 DSBs per cell, with 40 DSBs induced per Gy per cell (27 , 29) ].



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  		Fig. 1. DSBs induced by synchrotron photoactivation of cDDP. A, induction of DSBs in cells treated with cDDP (circles, control; triangles, 3 µM; squares, 30 µM) and exposed to synchrotron radiation (open symbols, energy A; closed symbols, energy B). FAR is plotted as a function of dose. Each data point, the mean ± SD of triplicate experiments. B, repair of DSBs in cells treated with 30 µM cDDP and exposed to synchrotron radiation (open symbols, energy A; closed symbols, energy B). Percentage of FAR remaining is plotted as a function of repair time. Each data point, the mean ± SD of triplicate experiments.

 
The repairability of the DSBs induced by the synchrotron PAT-Plat was then examined. Incubations at 37°C (1 and 4 h) were applied after each treatment. The resulting FAR, expressed as percentage of initial FAR value, was plotted against repair times (Fig. 1B)Citation . DSB repair kinetics exhibited a higher amount of unrepaired DSBs in cDDP-treated cells 4 h after A-energy than after B-energy synchrotron irradiation, raising the possibility that DNA fragments induced in excess by PAT-Plat are slowly repairable if they are repairable (Fig. 1B)Citation .

Synchrotron PAT-Plat Is Associated with an Extra Number of SSBs.
Both induction and repair rates of SSB induced by each treatment were investigated by pulsed-field gel electrophoresis by using an alkaline lysis buffer (29) . Like DSB data, no significant difference between A- and B-energy-induced SSB yields was detected in untreated cells and in cells treated with 3 µM cDDP. After treatment with 30 µM cDDP, the amount of A-energy-induced SSB was found to be about four times higher than those assessed with B energy, suggesting that synchrotron PAT-Plat creates an extra number of SSBs (Fig. 2A)Citation . Interestingly, the excess of FAR observed after 16 Gy irradiation corresponds to the effect of an extra dose of 10 Gy for SSBs [i.e., an excess of about 11,000 SSBs/cell with 1,100 SSBs induced per Gy/cell (29) ]. Because the same experimental conditions induce an equivalent of 8 Gy with regard to DSBs, the number of PAT-Plat-induced SSBs is, therefore, higher than the number expected with the complete transformation of PAT-Plat-induced DSBs into SSBs, suggesting that the synchrotron PAT-Plat is associated with production of both additional DSBs and additional SSBs.



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  		Fig. 2. SSBs induced by synchrotron photoactivation of cDDP. A, induction of SSBs in cells treated with cDDP (circles, control; triangles, 3 µM; squares, 30 µM) and exposed to synchrotron radiation (open symbols, energy A; closed symbols, energy B). FAR is plotted as a function of dose. Each data point, the mean ± SD of duplicate experiments. B, repair of SSBs in cells treated with 30 µM cDDP and exposed to synchrotron radiation (open symbols, energy A; closed symbols, energy B). Percentage of FAR remaining is plotted as a function of repair time. Each data point, the mean ± SD of duplicate experiments.

 
In human cells, the majority of radiation-induced SSB are generally repaired in 10–15 min (e.g., see Ref. 30 ). Unfortunately, our experimental conditions did not allow us to prepare plugs for early times postirradiation. Nevertheless, the SSB repair rate assessed for similar repair times as used in DSB repair assays showed no significant difference between different treatments (Fig. 2B)Citation .

Synchrotron PAT-Plat Results in the Inhibition of DNA-PK Activity.
Radiation-induced DSBs were previously shown to recruit the heterodimeric Ku protein on DNA ends and to stimulate the activation of DNA-PK protein kinase, triggered by the association of Ku heterodimer and the DNA-PKcs (3) . By contrast, cisplatin-DNA adducts decrease the ability of Ku to translocate away from the DNA ends, resulting, therefore, in the inhibition of the DNA-PK activity (13) . Here, the DNA-PK activity was examined in PAT-Plat conditions. DNA-PKcs immunoprecipitates from treated and untreated cells were subjected to the standard DNA-PK activity assay using 32P-labeled specific substrate. Single synchrotron irradiation followed by incubation at 37°C results in activation of the DNA-PK protein kinase, in agreement with previous reports related to radiation-induced DNA-PK activity (e.g., Ref. 31 ). This activation occurs to a similar extent whatever the energy emitted (A or B; Fig. 3Citation ). By contrast, cDDP treatment inhibits DNA-PK activity (~2-fold decrease) whether combined with synchrotron irradiation or not and whatever the radiation energy, suggesting that 30 µM cDDP may prevent the repair of DSBs by NHEJ (Fig. 3)Citation .



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  		Fig. 3. DNA-PK activity in cells treated with cDDP and exposed to synchrotron irradiation. The DNA-PK activity was assessed from DNA-PKcs immunoprecipitates from cells subjected to 30 µM cDDP treatment and exposed to 16 Gy of A- or B-energy synchrotron radiation. Repair time (4 h) has been applied as indicated. Results are expressed in counts per minutes (cpm) of [{alpha}-32P]ATP activity. Each data point, the mean ± SE of duplicate experiments.

 
Synchrotron PAT-Plat Results in a Dramatic Relocalization of RAD51 Protein.
The RAD51 protein was shown to be a single-strand DNA binding protein that participates with the early steps of the HR and the DNA adducts repair pathways (32) . After separated IR and cDDP treatments, the RAD51 protein relocalizes in cell nuclei to discrete foci, which were hypothesized to represent repair factories or "repairosomes" (33) . Besides, a number of HR-deficient cells do not form RAD51 foci after genotoxic treatments, suggesting that RAD51 relocalization may serve as an end point for HR activity and the RAD51-DNA binding property (e.g., Ref. 34 ). Hence, to examine whether synchrotron PAT-Plat stimulates RAD51-mediated HR process, we subjected cells to immunofluorescence with an anti-RAD51 antibody (Fig. 4)Citation . By considering the number of foci per cell as an end point, A-energy irradiation combined with cDDP treatment (i.e., PAT-plat) results in a larger relocalization of RAD51 foci (~50 foci/cell) than when induced after B-energy irradiation combined with cDDP treatment (~26 foci/cell; Fig. 4Citation ). These data, therefore, suggest that the DNA damage induced in excess by synchrotron PAT-Plat and recognized by RAD51 may stimulate RAD51-mediated repair pathways, which is like the HR process.



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  		Fig. 4. RAD51 nuclear relocalization in cells treated with cDDP and exposed to synchrotron irradiation. Representative examples of the percentage of cells with RAD51 foci scored in at least 300 cells (A), RAD51 nuclear foci observed after 30 µM cDDP treatment followed by 10 Gy of A- or B-energy synchrotron radiation and 1 h for repair. Each data point, the mean ± SE of duplicate experiments. B, examples of RAD51 foci in cells treated with 30 µM cDDP and irradiated with A or B energy (10 Gy). Nuclei were counterstained with DAPI.

 
Synchrotron PAT-Plat Results in a Hyperphosphorylation of BRCA1 Protein.
It has previously been demonstrated that the breast cancer-susceptibility BRCA1 protein is involved in RAD51-mediated HR and that BRCA1-negative cells are HR repair-deficient (35) . Furthermore, the BRCA1 protein is phosphorylated after each of the IR and cDDP treatments separately, suggesting that combining the treatments may result in an additive effect by increasing the subset of phosphorylated BRCA1 forms (12 , 33) . BRCA1 phosphorylation has been examined after exposure to synchrotron radiation and/or cDDP treatment. BRCA1-immunoblots revealed a slower migrated form of the BRCA1 protein after separated exposures to synchrotron radiation (no significant difference between A- and B-energy; data not shown) and to cDDP (30 µM; Fig. 5ACitation ). Furthermore, nuclear extracts from cells treated with both synchrotron radiation and cDDP elicit hyperphosphorylation. However, BRCA1 hyperphosphorylation is more pronounced after synchrotron PAT-Plat (A-energy irradiation + cDDP), in agreement with the findings that BRCA1 protein participates in the response to both cisplatin and radiation throughout RAD51-dependent homologous recombination repair pathways (Fig. 5ACitation ; Refs. 11 and 12 ). It is noteworthy that a constitutive phosphorylated form of BRCA1 is observed (likely because of the proportion of the S-phase cells) as indicated by untreated cell extracts. This form disappears after treatment with phosphatase (Fig. 5A)Citation .



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  		Fig. 5. Phosphorylation of the BRCA1 protein and c-Abl tyrosine kinase activity in cells treated with cDDP and exposed to synchrotron irradiation. A, BRCA1-immunobloting of SQ20B nuclear extracts from cells treated or not with 30 µM cDDP and/or exposed to synchrotron radiation (16 Gy delivered by A or B energy). No significant difference in the BRCA1 phosphorylation pattern was detected after radiation treatment with A or B energy (data not shown). Untreated extracts have been subjected to phosphatase (PPase) treatment and used as control. B, nuclear extracts from HCC1937 (BRCA1-negative) and SQ20B (BRCA1-positive) cells were subjected to anti-BRCA1 and anti-c-Abl antibodies. Here, the c-Abl protein level serves as loading control. C, the c-Abl activity was assessed from c-Abl immunoprecipitates from SQ20B and HCC1937 cells subjected to 30 µM cDDP treatment and exposed to 16 Gy of A- or B-energy synchrotron radiation followed by 4 h for repair.

 
Synchrotron PAT-Plat Results in the BRCA1-dependent Activation of c-Abl Kinase Activity.
Recently, we have reported that the BRCA1 phosphorylation leads to the disruption of the complex formed by BRCA1 and the proto-oncogenic tyrosine kinase c-Abl (28) . The release of the BRCA1–c-Abl complex was shown to enhance the tyrosine kinase activity of c-Abl. Besides, the BRCA1-negative cells show constitutively high c-Abl tyrosine kinase activity (28) . Activated c-Abl is involved in the injury response to DNA damage, notably after IR and cisplatin treatments, by mediating the c-jun NH2-terminal kinase (JNK)-dependent apoptosis pathways (36) . We have, therefore, examined whether synchrotron PAT-Plat influences the c-Abl tyrosine kinase activity in BRCA1-negative (HCC1937) and BRCA1-positive (SQ20B) cells during synchrotron PAT-Plat. The HCC1937 cells showed very low amounts of BRCA1 protein compared with SQ20B cells, whereas the levels of c-Abl were similar in both cell lines (Fig. 5B)Citation . The constitutive c-Abl tyrosine kinase activity was found to be clearly lower in SQ20B than in HCC1937 cells (data not shown). SQ20B cells exhibit a higher c-Abl activity after PAT-Plat than after concomitant cDDP and B-energy radiation treatment. This difference is absent in HCC1937 cells, suggesting that PAT-Plat overstimulation of the c-Abl tyrosine kinase is BRCA1-dependent (Fig. 5C)Citation .

Altogether, our data provide evidence that synchrotron PAT-Plat is associated with (a) the production of an extra number of SSBs and of slowly repaired DSBs; (b) the inhibition of the NHEJ pathway; (c) the stimulation of the RAD51-dependent HR repair pathways associated with dramatic relocalization of RAD51 foci, hyperphosphorylation of the BRCA1 protein, and activation of c-Abl tyrosine kinase only in a BRCA1-dependent manner.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The ESRF Synchrotron: A Real Opportunity to Test Feasibility of PAT-Plat.
A combination of radiotherapy with halogenated chemotherapeutic agents was shown to significantly enhance the cellular response and to improve the prognosis of a number of malignancies more than radiation and chemotherapy treatments alone (e.g., Ref. 7 ). In addition to additive or supra-additive effects produced by concomitant chemoradiotherapy, there is potential interest in using certain halogenated drugs because their excited forms are generally capable of producing an Auger electron cascade after appropriate photoexcitation and, consequently, to enhance the therapeutic index by inducing additional DNA damage. Besides, a cytotoxic effect has previously been reported after treatment with 193mPt, bromo- and iododeoxyuridine combined with external X-rays beams (19, 20, 21 , 37) . However, the use of polychromatic sources of radiation was insufficient to provide maximal Auger-effect radiation, and the choice of both powerful and monochromatic light for triggering the Auger effect was, therefore, crucial. Because electron-binding energy increases according to its subshell level and the atom mass, only the K shell of sufficiently heavy atoms has the energy level high enough to represent a realistic preclinical model for external excitation irradiation. ESRF is presently the only synchrotron to produce sufficient fluence at high energy to make K-edge photoactivation possible with heavy atoms. It represented, therefore, an opportunity to examine the feasibility of PAT-Plat. Here are presented the first molecular data of a synchrotron PAT-Plat developed at ESRF. These data suggest that the synchrotron PAT-Plat consists in a supra-additive effect with an overproduction of DNA breaks, notably DSBs, that are preferentially repaired by RAD51-related repair pathways.

Production of an Extra Number of DNA Strand Breaks by Synchrotron PAT-Plat.
The direct consequence of an Auger electron cascade is the production of an extra number of DSBs and SSBs in situ. Besides, the Auger electrons were longer used for the calibration of techniques assessing the actual number of radiation-induced DSBs, notably by using 125I, with which each ß disintegration induces one DSB (Refs. 15, 16, 17, 18, 19 and Refs. therein). Because tens and hundreds electron volts were shown to produce SSBs and DSBs (38) , respectively, the electrons emitted by photoactivated platin are largely sufficient to induce an extra set of both DSBs and SSBs. In our conditions, the PAT-plat consists in the production of ~300 DSBs and 11,000 SSBs in excess per cell, corresponding to an extra dose of radiation of 8–12 Gy (27 , 29) . Because the PAT-Plat-induced DNA strand breaks are likely to be induced in the close vicinity of cisplatin molecules, PAT-Plat may result in the production of LMDSs, composed of DNA adducts, SSBs, and DSBs (39) . Hence, because RAD51 foci have been associated with both DSBs and DNA adducts repair sites, separately or together (12) , the dramatic accumulation of RAD51 foci may be a direct consequence of the formation of LMDSs by PAT-Plat.

The Synchrotron PAT-Plat: An Interplay between Two Major DSB Repair Pathways.
NHEJ is the predominant DSB repair pathway in human cells and NHEJ-deficient cells generally exhibit a characteristic shape of DSB repair kinetics with a large amount of unrepaired DNA damage (1 , 2 , 40) . The plateau of unrepaired DSBs observed after synchrotron PAT-Plat suggests, therefore, an impairment of the NHEJ pathway. This assumption is consistent with the inhibition of DNA-PK kinase observed in the presence of cisplatin (Fig. 3)Citation and with recent in vitro data (13) . However, DSB repair kinetics after treatment with cisplatin combined with B-energy synchrotron radiation do not show large amounts of unrepaired DSBs, maybe because of the high radioresistance and/or capacity of SQ20B tumor cells to repair DSBs (41) . Lastly, the extra number of DSBs induced by PAT-Plat cannot be attributed to apoptosis because the p53-mutated SQ20B cells do not undergo apoptosis and because the maximal repair time used in this study (4 h) is insufficient for assessing a significant apoptotic signal (41) .

The presence of cisplatin-DNA adducts inhibits the translocation of Ku protein and prevents the association of DNA-PKcs to form and activate the DNA-PK complex (13) . Under our conditions, if the penetration of cisplatin into cells was complete, 30 µM cisplatin would create a maximum of 60 DNA adducts/Mbp [16 Gy induces 600 DSBs (i.e., an average of 0.1 DSB/Mbp)], raising the possibility that cisplatin may inhibit the DNA-PK activity directly on the LMDSs but also throughout other pathways like binding to Ku protein itself (13) . The interpretation that PAT-Plat-induced DSBs are repaired by pathways different from NHEJ, e.g., like HR, is supported by both massive RAD51 relocalization and stimulation of c-Abl kinase activity, both sensors of the HR process (32 , 42) . Moreover, synchrotron PAT-Plat dose-dependently induces hyperphosphorylation of BRCA1 (e.g., Ref. 33 ), and our findings are, therefore, consistent with the extra dose produced specifically by PAT-Plat. Lastly, the BRCA1 hyperphosphorylation consists in the disruption of the BRCA1–c-Abl complex was shown to exacerbate the c-Abl activity (28) . Altogether, our findings suggest a model in which PAT-Plat creates LMDS notably composed by SSB and severe DSBs induced after Auger electron emission. All of the DNA strand breaks are produced in the close vicinity of cisplatin molecules that inhibit their repair by the NHEJ pathway by blocking Ku protein on the DNA. However, similarly to DNA adducts, the PAT-Plat-induced DNA strand breaks may result in stimulating HR; RAD51 is, therefore, recruited on damage site; BRCA1 is hyperphosphorylated; and c-Abl kinase is activated (Fig. 6)Citation .



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  		Fig. 6. Model for synchrotron PAT-Plat. Combination of synchrotron radiation and cisplatin treatment generally results in the induction of DSBs and DNA adducts that stimulate predominantly DNA-PK and RAD51-dependent repair pathways, respectively. The presence of DNA adducts leads to the inhibition of NHEJ by blocking the translocation of Ku protein on the DNA. PAT-Plat results in an additional number of SSBs and severe DSBs that are likely to be formed in LMDSs, very close to the cis-platinum molecules. the RAD51 protein is dramatically relocalized to the damage sites as discrete foci; BRCA1 is hyperphosphorylated; and c-Abl kinase, released from BRCA1, is overactivated. P, phosphorylated.

 
The Synchrotron PAT-Plat: A Promising Anticancer Treatment?
Here, we have presented the first molecular basis of a potential anticancer therapy based on PAT-Plat by external irradiation. Are the radiation doses and cDDP concentrations used in this study clinically relevant? Testicular and ovarian tumors are generally treated with doses from 25 to 45 Gy delivered as a fractionated course (equivalent to doses from 10 to 16 Gy delivered in one fraction), i.e., in the range of tens Gy delivered in this study.4 With regard to cisplatin, 1 µM cDDP delivered in vitro is known to produce biological effects similar to routine treatments (i.v. route, regular doses of 75–100 mg/m2 cDDP), corresponding to about 3 x 106 cDDP molecules/cell (19) . Consequently, a dose of 30 µM cDDP, is higher than the usual i.v. concentration, 100-fold higher than those measured by Ortega et al. (43) , but interestingly of the same magnitude as 7 x 108 iodine molecules/cell introduced by Laster et al. (22) to obtain a significant effect with irradiation above the K-shell edge of iodine. Although it is difficult to imagine platinum doses 30 times higher than 100 mg/m2 in humans, it is noteworthy that exposure to cDDP after an i.p. route was shown to be three times higher than the cumulated i.v. dose of free platinum (44) . Hence, radiation and cDDP doses are reasonably close to those used in routine treatments. The proportionality of biochemical effects with radiation dose and drug concentration has been increasingly documented, notably for the amount of induced DNA damage, but care must be taken in comparing clinical, biological, and biochemical end points (27 , 29) . Nevertheless, it is possible that in the case of a cell like SQ20B in which the RAD51-dependent recombination pathways are overstimulated, no significant PAT-Plat-induced sensitization occurs (19) . Therefore, the genotypic status of tumor appears to be crucial for therapeutic index enhancement. The known involvement of BRCA1 and BRCA2 genes in some cancers like breast and ovarian cancers, generally associated with HR deficiency and impaired DNA-binding properties of RAD51, suggests an elective application of PAT-Plat to these neoplasms with a rationale for selecting patients. More generally, because large volumes could be treatable, any other solid tumors with impaired RAD51-dependent repair pathway might be submitted to PAT-plat.

However, some limits of feasibility have to be underlined. Despite its significant molecular effects, cisplatin is known to elicit a poor capacity to enter into cells at high concentrations and to induce cytotoxic effects; additional experiments are, therefore, required to test other platinum compounds also (45 , 46) . Finally, the use of giant synchrotron accelerators such as the ESRF allows in vivo applications to test feasibility of PAT treatment as for coronary imaging (47) . However, a routine use of synchrotron light for human treatment will necessitate the development of new X-ray monochromatic sources devoted to medical use. The next decade should be productive in developing such technology.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. V. Favaudon (Institut Curie Recherche, INSERM, U350, France), to Dr. M. Décorps (INSERM, U438, Grenoble, France), and to Drs B. Kysela and M. Chovanec (University of Sussex, Brighton, United Kingdom). We thank Richard Coleman for technical help.


   FOOTNOTES
 
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.

1 Supported by the Région Rhône-Alpes and the Fondation pour la Recherche Médicale (to S. C.), Electricité de France (Comité de Radioprotection) (to J. B.), the Ligue Nationale Contre le Cancer (Comité de l’Isère) (to J. B.) and the Association pour la Recherche contre le Cancer (to J. B. and N. F.). All experiments were made possible thanks to the ESRF ID17 beam shift allocation and the help of beamline staff. Back

2 To whom requests for reprints should be addressed, at Equipe d’Accueil n°2941 "Rayonnement Synchrotron et Recherche Médicale," Département de Cancérologie et d’Hématologie, CHU A. Michallon, BP 217, F-38043 Grenoble, France. Phone: 33-4-76-76-54-35; Fax: 33-4-76-76-56-29; E-mail: JBalosso{at}chu-grenoble.fr./ Back

3 The abbreviations used are: DSB, double-strand break; NHEJ, nonhomologous end joining; HR, homologous recombination; PK, protein kinase; IR, ionizing radiation; cDDP, cis-diamminedichloroplatinum(II); PAT-Plat, photoactivation of cisplatin; ESRF, European Synchrotron Radiation Facility; SSB, single-strand break; FAR, fraction of activity released; TBS, Tris-buffered saline; DNA-PKcs, DNA-PK catalytic subunit; GST, glutathion S-transferase; DAPI, 4,6-diamidino-2-phenylindole; LMDS, local multiple-damage site. Back

4 J. Balosso, personal communication. Back

Received 9/17/02. Accepted 4/16/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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