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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Montreal Centre for Experimental Therapeutics in Cancer-Lady Davis Institute for Medical Research, Sir Mortimer B Davis-Jewish General Hospital, McGill University, Montreal, Quebec, Canada (L.A., M.L., A.-C.G., M.D., R.A., L.P.); and Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, Montreal, Quebec, Canada (B.J.-C.)
Received December 8, 2006; accepted March 6, 2007.
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Abstract |
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H2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G2/M phase arrest, and NU7026 increases this CLB-induced G2/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as H2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.
).
CLL is an excellent malignancy for in vitro studies, because there is easy access to a homogenous population of malignant B lymphocytes, and there is a good correlation between in vitro cytotoxicity of CLB [as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] and in vivo response in CLL patients (Panasci et al., 2001). CLB cytotoxicity is mediated by the introduction of DNA interstrand cross-links (ICLs) into the DNA of treated cells. Interstrand cross-linking agents may induce double-strand breaks (DSBs) as an intermediate step during ICL repair. DSBs are repaired by nonhomologous DNA end-joining (NHEJ) and/or by homologous recombinational repair (HR) pathways (De Silva et al., 2000; McHugh et al., 2001). It has been proposed that enzyme-mediated repair of DSBs is a major mechanism of resistance to both ionizing radiation (IR) and drugs that cause DSBs as intermediates in repair processes (Jackson, 2002). Histone H2AX is phosphorylated at serine 139 (H2AX) following the introduction of DSBs (Rogakou et al., 1998; Riballo et al., 2004). H2AX forms foci at the DSB sites, facilitates the recruitment and retention of damage-responsive proteins, and influences the efficiency of DSB repair (Celeste et al., 2003; Downs et al., 2004; Fernandez-Capetillo et al., 2004; Thiriet and Hayes, 2005). We and others have demonstrated that both NHEJ and HR are involved in the repair of chlorambucil-induced DNA damage in CLL lymphocytes (Christodoulopoulos et al., 1999; Bello et al., 2002; Noll et al., 2006). Important components in these repair pathways are the phosphatidylinositol 3-kinase (PI3-K)-related protein kinase family of enzymes. These DNA damage-activated serine/threonine protein kinases include DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated kinase (ATM), and ataxia telangiectasia Rad3-related kinase (Durocher and Jackson, 2001). We have reported that there is a strong linear correlation between the sensitivity of CLL primary lymphocytes to chlorambucil and DNA-PK activity (Christodoulopoulos et al., 1998; Muller et al., 1998). Moreover, we have demonstrated that inhibition of DNA-PK, a major component of NHEJ, with the nonspecific PI3-K inhibitor wortmannin sensitizes CLL lymphocytes to chlorambucil (Christodoulopoulos et al., 1998). NU7026, a specific DNA-PK inhibitor, radiosensitizes both proliferating and quiescent mouse embryonic fibroblast cells to IR and inhibits DSB repair. Use of this inhibitor in cell lines proficient or deficient for DNA-PK suggests that NU7026 mediates its effect on IR-induced cytotoxicity and DSB repair, specifically, via inhibition of DNA-PK (Veuger et al., 2003). Furthermore, NU7026 potentiates etoposide-induced cytotoxicity in two leukemia derivative cell lines, the acute myeloid leukemia cell line ML1 and the chronic myeloid leukemia cell line K562 (Willmore et al., 2004).
The effect of the nonspecific irreversible inhibitor wortmannin on DNA-PK activity in cell incubations can be determined using the classical DNA-PK pull-down assay (Christodoulopoulos et al., 1998). However, the effect of NU7026, a specific reversible DNA-PK inhibitor, has not been directly examined in cell incubations, because NU7026-induced inhibition of DNA-PK in cell incubations is lost in the preparation of the nuclear extracts (Deriano et al., 2005). Following activation, DNA-PK autophosphorylates the threonine 2609 site (T2609) of DNA-PK catalytic subunit. It has been demonstrated that T2609 phosphorylation is required for DSB repair by the NHEJ pathway (Chan et al., 2002). Using an antibody specific to this phosphorylated site, activated DNA-PK can be visualized as nuclear foci or analyzed by flow cytometry. Using these techniques, we determined the inhibitory effect of NU7026 on chlorambucil-activated DNA-PK vis-à-vis chlorambucil cytotoxicity in CLL malignant lymphocytes. Our results demonstrate that NU7026 sensitizes CLL lymphocytes to chlorambucil and that this sensitization correlates with inhibition of DNA-PK phosphorylation, increased accumulation of H2AX, and prolongation of G2/M arrest.
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). Cells were maintained as a suspension culture in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) in a 5%CO2 humidified atmosphere. Patients. Nineteen patients with a diagnosis of B-CLL followed at the Jewish General Hospital of Montreal were enrolled in the study after informed consent. Patients were either untreated (n = 14) or treated with CLB (n = 5) for various times.
Cytotoxicity Assay. Lymphocytes were isolated from the peripheral blood using Ficoll-Hypaque (GE Healthcare, Piscataway, NJ) as described previously (Christodoulopoulos et al., 1998, 1999; Bello et al., 2002). The T-lymphocyte contamination in the isolated B-lymphocytes population was 6.32 ± 3.84 (expressed as a mean percentage ± S.D. and determined by flow cytometry analysis). The CLL lymphocytes (1.5 x 106 cells/ml) and the B-CLL cell line I83 (1.5 x 105 cells/ml) were plated in RPMI 1640 medium supplemented with 10% FBS, and samples were incubated in the presence of various concentrations (0–100 µM) of NU7026 alone (Sigma-Aldrich, St. Louis, MO), chlorambucil alone (Sigma-Aldrich), or in combination as indicated. Control samples were incubated with the greatest volume of DMSO. The MTT assay was performed 72 h after treatment as described previously (Christodoulopoulos et al., 1998). Synergy was determined by the formula a/A + b/B = I, where a is the CLB IC50 (concentration resulting in 50% of control) in combination with NU7026 at concentration b, A is the CLB IC50 without NU7026, and B is the NU7026 IC50 in the absence of CLB. If the IC50 of NU7026 was greater than 100 µM, 100 µM was used as the IC50 of NU7026. According to the formula, when I < 1, the interaction is synergistic; when I = 1, the interaction is additive; and when I > 1, there is an antagonistic interaction (Christodoulopoulos et al., 1998).
Flow Cytometry Analysis. I83 cells were plated in RPMI 1640 medium with 10% FBS (1.5 x 105 cells/ml) and treated with vehicle (DMSO), 5 µM CLB, CLB IC50, 10 µM NU7026, or the combination of both drugs for 0, 6, 24, and 48 h. Cell cycle distribution, apoptosis, DNA-PK phosphorylation, and H2AX determination were determined as described below, and they are expressed as a percentage of cells in each phase of the cycle. DNA content was analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) equipped with CellQuest software (BD Biosciences).
Cell Cycle Analysis. Cell cycle progression was analyzed as described previously (Loignon et al., 1997; Aloyz et al., 2004). In brief, the cells were harvested at the indicated times, fixed with 70% ethanol, washed with PBS, stained overnight at 4°C in PBS containing 50 µg/ml propidium iodide (Molecular Probes, Eugene, OR) and 0.02 mg/ml DNase-free RNase A (Hoffman-La Roche, Nutley, NJ), and then analyzed as described above.
Phosphorylated DNA-PK and H2AX Flow Cytometry Analysis. After fixation with 1% paraformaldehyde at 4°C for 15 min, cells were washed with PBS and then permeabilized in 70% ice-cold ethanol for 20 min in ice. After washing in PBS, the cells were incubated 30 min at room temperature in dilution buffer (PBS, 1% bovine serum albumin, and 0.1% Triton X-100), and then they were incubated overnight at 4°C with a mouse anti-phosphorylated DNA-PK (T2609) antibody (Cedarlane, Hornby, ON, Canada) 1:200 in dilution buffer or a rabbit anti-H2AX (Ser139) antibody (Upstate Biotechnology, Lake Placid, NY) (1:500) in dilution buffer. The cells were washed in PBS, and then they were incubated for 90 min with a goat anti-mouse or chicken anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes) diluted 1:500 in dilution buffer, washed with PBS, stained overnight at 4°C with 7-aminoactinomycin D (BD Biosciences PharMingen, San Diego, CA), and then analyzed by flow cytometry as described previously. I83 cells and two untreated CLL lymphocyte samples were analyzed.
Apoptosis Analysis. Apoptosis was analyzed using the anti-single-stranded DNA/Apostain antibody (Bender MedSystems, Vienna, Austria) following the manufacturer's instructions. In brief, the cells were harvested at 24 and 48 h, fixed with methanol, washed with PBS, and incubated for 15 min in dilution buffer (PBS and 1% milk) containing the F7-26 Apostain antibody. The cells were washed with PBS and incubated with a goat anti-mouse IgM Alexa Fluor 647 secondary antibody (Molecular Probes) (1:200). Before analysis by flow cytometry, the DNA was stained using YO-PRO-1 (Molecular Probes).
Phosphorylated DNA-PK Foci Determination. I83 cells were treated with vehicle (DMSO), CLB IC50, 10 µM NU7026, or the combination of both drugs for 48 h. Aliquots (50 µl) of the cell suspension were centrifuged onto clean glass slides at 1500 rpm for 10 min in a cytospin (Hettich, Ramsey, MN). After cytocentrifugation, the slides were fixed in methanol/acetone [1:1 (v/v)] for 10 min at –20°C, rinsed thrice in PBS, and then incubated for 30 min in blocking solution (PBS, 0.5% BSA, and 0.5% Triton X-100) followed by an overnight incubation at 4°C with an anti-phosphorylated DNA-PK (T2609) antibody (1:200). The slides were washed thrice in PBS, incubated for 1 h with goat anti-mouse Alexa Fluor 488 secondary antibody (1:500), and then washed in PBS. Slides were mounted using Sigma mounting medium and photographed by microscopy using a Leica DM LB 2 microscope equipped with a Leica DFC480 camera (Leica Microsystems, Inc., Deerfield, IL).
Statistical Analysis. Differences between mean values were assessed by two-tailed t test. Results are expressed as a mean ± S.D.
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Results |
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The MTT assay was used to determine the cytotoxicity of CLB alone, NU7026 alone, or the combination in lymphocytes from CLL patients. Our results, summarized in Table 2, show that CLB IC50 alone ranged from 7.14 to 61.17 µM. NU7026 was not toxic in 50% of the CLL patients at the highest concentration (100 µM) used. In the remaining 50% of the patients' lymphocytes tested, NU7026 IC50 alone ranged from 17.35 to 67.48 µM. When used at 5 or 10 µM, NU7026 sensitized CLL lymphocytes to CLB in all the patients but one patient (U8). In most of the samples, NU7026 sensitization to CLB was synergistic. In patients previously treated clinically with CLB, synergy with CLB was observed in 80 and 100% of samples incubated with 5 and 10 µM NU7026, respectively. At a concentration as low as 1 µM, NU7026 induced sensitization in most samples (except for patient T4). In the CLL patients tested in this study, the sensitization effect of NU7026 on chlorambucil-induced cytotoxicity does not correlate with constitutive levels of DNA-PK catalytic subunit, Ku70, and Ku80 (data not shown).
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NU7026 Sensitizes the CLL Cell Line I83 to CLB. Based on the MTT assay with CLL lymphocytes from patients, we evaluated the effect of NU7026 in CLB cytotoxicity in the I83 cell line. Our results show that NU7026 synergistically sensitizes I83 cells to CLB 3.5-fold (Table 3). Although NU7026 alone does not result in significant cytotoxicity at the highest concentration used (100 µM), treatment of these cells with chlorambucil alone requires 11.69 µM to produce an IC50. However, both drugs together result in synergy with an IC50 of 3.35 µM for CLB in the presence of 10 µM NU7026 (I = 0.29; p < 0.002) (Table 3). Similar results were obtained using a cell count assay (Table 4).
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NU7026 Decreases Immunofluorescence-Detected CLB-Induced DNA-PK Phosphorylation. Immunofluorescence experiments showed that CLB induced phosphorylated DNA-PK (T2609) nuclear foci 48 h after treatment (Fig. 1b). In contrast, DNA-PK T2609 nuclear foci were not detected either in vehicle (control) (Fig. 1a) or NU7026 alone-treated cells. Using the same technique, we found that CLB-induced DNA-PK T2609 nuclear foci were inhibited by NU7026 (Fig. 1c). To quantify the effect of NU7026 in CLB-induced DNA-PK phosphorylation, we performed flow cytometry using the same antibody as described below.
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H2AX staining. NU7026 alone has no effect on H2AX phosphorylation. CLB alone or in combination with NU7026 induced the accumulation of H2AX over time, peaking at 24 h in S phase and 48 h in G2/M. The CLB-induced H2AX was enhanced in cells treated with CLB in combination with NU7026 (Fig. 5).
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Discussion |
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; Bramson et al., 1995a,b; Muller et al., 1998; Christodoulopoulos et al., 1999). Specifically, DNA-PK activity is low in the chlorambucil-sensitive B lymphocytes of untreated CLL patients (Muller et al., 1998). The role of DNA-PK inhibitors in cancer therapy is expanding. Inhibition of DNA-PK sensitizes tumors to radiotherapy (Allen et al., 2003; Kashishian et al., 2003; Shinohara et al., 2005) and leukemia cells to chemotherapy. Inhibition of DNA-PK sensitizes cells to DNA topoisomerase II poisons in acute myeloid leukemia and chronic myeloid leukemia derivative cell lines (Willmore et al., 2004), and ionizing radiation in both mouse embryonic fibroblast (Veuger et al., 2003) and B-CLL primary lymphocytes (Deriano et al., 2005) by inhibition of DSB repair in DNA. We investigated the effect of NU7026 on chlorambucil-induced cytotoxicity in the I83 cell line and malignant B lymphocytes from CLL patients. In the B-CLL derivative cell line and the majority of samples from CLL patients, NU7026 synergistically sensitizes cells to CLB. To determine whether this synergistic effect was due to DNA-PK inhibition, we assessed indirectly the steady-state activity of DNA-PK by assessing DNA-PK phosphorylation at threonine 2609 in I83 cells and CLL lymphocytes using immunofluorescence and flow cytometry. We find that in I83 cells and lymphocytes from two CLL patients, NU7026 inhibited CLB-induced DNA-PK phosphorylation (T2609) to the same extent (50–40%). Importantly, equal lethal doses of CLB were used in the aforementioned experiments (i.e., the respective CLB IC50). However, the cell cycle profiles associated with changes in the steady state of phosphorylated DNA-PK (T2609) differ between primary lymphocytes and I83 cells. These differences in cell cycle progression after the treatment are explained by the fact that I83 is an immortalized proliferative cell line; therefore, the mitotic index of I83 cells was higher than primary CLL-lymphocytes, which are largely nonproliferative. Taken together, our results suggest that NU7026 sensitization to CLB in both I83 cells and primary CLL lymphocytes is mediated by DNA-PK activity inhibition.
It has been demonstrated that NHEJ repair occurs in all phases of the cell cycle (Poot et al., 1991). In agreement with this, we show that CLB induces DNA-PK phosphorylation in all phases of the cell cycle. DNA-PK activity increases in G2/M concomitant with the induction of G2/M arrest after CLB treatment. Furthermore, the combination of CLB and NU7026 results in a more pronounced G2/M arrest. The concentration of NU7026 used does not affect cell cycle progression of the I83 cells in the absence of CLB (data not shown), as has been reported previously (Willmore et al., 2004). A similar G2/M arrest has been previously shown when NU7026 is used in combination with etoposide (Willmore et al., 2004) or cryptoleptine (Zhu and Gooderham, 2006).
Another member of the PI3-K family, ATM, together with DNA-PK is known to be a key regulator of the cellular response to DSBs (Shiloh, 2003). Consistently, we find that after CLB treatment ATM is phosphorylated in I83 cells (see Supplemental Data). Importantly, NU7026 did not affect CLB-induced ATM phosphorylation, suggesting that inhibition of ATM could potentially sensitize malignant B lymphocytes to CLB (see Supplemental Data). Noteworthy, it has been shown that specific DNA-PK or ATM inhibitors sensitize breast carcinoma cells to IR-induced death. Nevertheless, when used together the inhibitors did not display synergistic or additive effect (Cowell et al., 2005).
ICL repair may proceed through a DSB intermediate, and because inhibition of DNA-PK phosphorylation has been demonstrated to inhibit repair of DSBs by NHEJ (Noll et al., 2006), we speculate that the prolongation of G2/M arrest by NU7026 is probably due to an accumulation of DNA damage. Previous studies show that chemosensitivity correlates with accumulation of H2AX phosphorylation after DSB-inducing drugs (Banáth and Olive, 2003). Consistent with these hypotheses, we found that CLB induces H2AX phosphorylation and that, moreover, CLB in combination with NU7026 enhances H2AX phosphorylation, suggesting that NU7026 inhibits the repair of CLB-induced DNA damage, resulting in the accumulation of DSBs. We also showed that expression of phosphorylated histone H2AX inversely correlates with DNA-PK phosphorylation. Rothkamm et al. (2003) suggest a model whereby NHEJ and HR preferentially repair radiation-induced DSBs in different phases of the cell cycle. In this model, NHEJ predominates in G1/early S, whereas both NHEJ and HR contribute to DSB repair during late S/G2. Based on this model, we can hypothesize that NHEJ offers a reduced contribution to the repair of CLB-induced damage in the presence of NU7026, therefore slowing the repair process, which in turn contributes to a prolonged G2/M arrest. We have shown that inhibition of HR pathway also results in an increase of CLB cytotoxicity in CLL lymphocytes (Aloyz et al., 2004). Compensation, cross-talk, or both between the NHEJ and HR pathways have been suggested previously (Allen et al., 2003). To assess this point, we calculate the density of Rad51 nuclear foci induced by CLB in the absence or presence of NU7026 as described previously (Aloyz et al., 2004). Our results suggest that inhibition of DNA-PK does not affect CLB-induced Rad51 foci (Supplemental Data).
In summary, we have demonstrated that NU7026 synergistically increases CLB cytotoxicity in CLL lymphocytes associated with decreased activation of DNA-PK accompanied by increased DSBs, and, in I83 cell line, G2/M arrest. Specific inhibition of DNA-PK may be useful as an adjunct to CLB therapy in CLL patients.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CLL, chronic lymphocytic leukemia; CLB, chlorambucil; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ICL, interstrand cross-link; DSB, double-strand break; NHEJ, nonhomologous DNA end-joining; HR, homologous recombinational repair; NU7026, 2-(morpholin-4-yl)-benzo[h]chomen-4-one; IR, ionizing radiation; H2AX, phosphorylated histone H2AX; PI3-K, phosphatidylinositol 3-kinase; DNA-PK, DNA-dependent protein kinase; ATM, ataxia telangiectasia-mutated kinase; I, synergy value; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Lawrence Panasci, Montreal Centre for Experimental Therapeutics in Cancer-Lady Davis Institute for Medical Research, Sir Mortimer B Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada. E-mail: lpanasci{at}hotmail.com
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