Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines
2012; Elsevier BV; Volume: 40; Issue: 10 Linguagem: Inglês
10.1016/j.exphem.2012.06.001
ISSN1873-2399
AutoresBenigno C. Valdez, Yago Nieto, David Murray, Yang Li, Guiyun Wang, Richard E. Champlin, Börje S. Andersson,
Tópico(s)Acute Myeloid Leukemia Research
ResumoHematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ∼IC10 drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (∼65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated–CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2′-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2′-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre–hematopoietic stem cell transplantation conditioning for lymphoma. Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M), and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ∼IC10 drug concentrations (57 μM B, 1 μM M and 0.02 μM G), which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (0.6 μM) to this combination resulted in a marked (∼65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated–CHK2 pathway, phosphorylation of KRAB-associated protein-1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2′-deoxycytidine to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2′-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre–hematopoietic stem cell transplantation conditioning for lymphoma. Lymphomas are heterogeneous hematologic neoplasms of the lymphoid system [1Murawski N. Pfreundschuh M. New drugs for aggressive B-cell and T-cell lymphomas.Lancet Oncol. 2010; 11: 1074-1085Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar]. The intricacy of their pathogenesis and molecular defects requires complex treatment modalities, but in spite of recent improvements, a significant proportion of patients still suffer from progressive disease. In this situation, hematopoietic stem cell transplantation (HSCT) is becoming an accepted treatment option [2Klyuchnikov E. Bacher U. Kröger N. et al.The role of allogeneic stem cell transplantation in relapsed/refractory Hodgkin's lymphoma patients.Adv Hematol. 2011; 2011: 974658Crossref PubMed Scopus (4) Google Scholar]. The success of HSCT requires an effective preparative regimen. DNA alkylating agents and nucleoside analogs are commonly used in these regimens [3Salit R.B. Bishop M.R. Pavletic S.Z. 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Newer anticancer agents.Med Clin North Am. 1964; 48: 501-527PubMed Google Scholar]. It is commonly assumed that their cytotoxic effects are related to the induction of DNA interstrand cross links, which block replication forks and cause DNA strand breaks when cells attempt to repair the DNA lesions [11Hanada K. Budzowska M. Modesti M. et al.The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks.EMBO J. 2006; 25: 4921-4932Crossref PubMed Scopus (233) Google Scholar]. Nucleoside analogs, on the other hand, inhibit DNA synthesis when incorporated into an elongating nascent DNA strand [12Plunkett W. Gandhi V. Purine and pyrimidine nucleoside analogs.Cancer Chemother Biol Response Modif. 2001; 19: 21-45PubMed Google Scholar]. Their ability to inhibit ribonucleotide reductase depletes the cellular deoxynucleotide pools and results in a preferential incorporation of the nucleoside analogs into DNA strands, leading to autopotentiation of the drugs [13Parker W.B. Shaddix S.C. Chang C.H. et al.Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5′-triphosphate.Cancer Res. 1991; 51: 2386-2394PubMed Google Scholar]. The differences in their mechanisms of cytotoxicity underscore the potential relevance of combining DNA alkylators and nucleoside analogs. In addition, repair of DNA alkylator-mediated damage is expected to be compromised due to decreased deoxynucleotide pools caused by nucleoside analogs, as shown by Yamauchi et al. [14Yamauchi T. Nowak B.J. Keating M.J. Plunkett W. DNA repair initiated in chronic lymphocytic leukemia lymphocytes by 4-hydroperoxycyclophosphamide is inhibited by fludarabine and clofarabine.Clin Cancer Res. 2001; 11: 3580-3589Google Scholar]. The efficacy of DNA alkylator and nucleoside analog combinations is further demonstrated by recent preclinical and clinical studies on the synergistic effects of clofarabine (Clo), fludarabine (Flu), and busulfan (B) combinations in myeloid leukemia [15Valdez B.C. Li Y. Murray D. Champlin R.E. Andersson B.S. The synergistic cytotoxicity of clofarabine, fludarabine and busulfan in AML cells involves ATM pathway activation and chromatin remodeling.Biochem Pharmacol. 2011; 81: 222-232Crossref PubMed Scopus (36) Google Scholar, 16Andersson B.S. Valdez B.C. de Lima M. et al.Clofarabine ± fludarabine with once daily i.v. busulfan as pretransplant conditioning therapy for advanced myeloid leukemia and MDS.Biol Blood Marrow Transplant. 2011; 17: 893-900Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. Based on these data, we proposed a model whereby Clo and Flu cause chromatin relaxation and facilitate access of busulfan to genomic DNA for alkylation [17Valdez B.C. Andersson B.S. Interstrand crosslink inducing agents in pretransplant conditioning therapy for hematologic malignancies.Environ Mol Mutagen. 2010; 51: 659-668PubMed Google Scholar, 18Valdez B.C. Murray D. Nieto Y. Li Y. Wang G. Champlin R.E. Andersson B.S. Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and SAHA in lymphoma cell lines.Leuk Lymphoma. 2012; 53: 973-981Crossref PubMed Scopus (19) Google Scholar]. We recently extended this approach to lymphoma cell lines, also including gemcitabine (G) in the combinations, and observed similar efficacies of [Clo+Flu+B], [Clo+G+B] and [Flu+G+B] in some (but not all) cell types [18Valdez B.C. Murray D. Nieto Y. Li Y. Wang G. Champlin R.E. Andersson B.S. Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and SAHA in lymphoma cell lines.Leuk Lymphoma. 2012; 53: 973-981Crossref PubMed Scopus (19) Google Scholar]. We used combinations of two nucleoside analogs and a DNA alkylator in these studies. Interestingly, J45.01 cells did not respond well to these combinations. Based on the observed resistance of J45.01 cells to two nucleoside analogs and a DNA alkylator, we postulated that an enhanced cytotoxicity would be obtained if we combined one nucleoside analog and two DNA alkylators. In this study, we therefore studied the effects of combinations of busulfan (B), melphalan (M) and gemcitabine (G) (i.e., B±M±G) on the J45.01 T-cell lymphoma cell line. Because of the possibility that combining two alkylators, especially at high drug concentrations, might cause excessive normal tissue cytotoxicity, it was important that anticancer effects be achievable using reduced doses of the individual drugs. We report in the present study that low drug concentrations of BMG in combination with a chemosensitizer, namely the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA), do indeed provide very effective killing of lymphoma cells. These findings suggest a role for epigenetic changes in sensitizing lymphoma cells to BMG combinations. Similar responses were observed in myeloid leukemia cell lines, suggesting a generality of the observed effect and providing a basis for a clinical trial on using this four-drug combination as part of pretransplantation therapy for lymphoma patients undergoing HSCT. Lymphoma cell lines J45.01 and Daudi were obtained from the American Type Culture Collection (Manassas, VA, USA). KBM7/B5/Bu2506 is a busulfan-resistant chronic myelogenous leukemia (CML) cell line developed in our laboratory [19Valdez B.C. Murray D. Ramdas L. et al.Altered gene expression in busulfan-resistant human myeloid leukemia.Leuk Res. 2008; 32: 1684-1697Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. All cells were cultured in RPMI-1640 (Mediatech, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Inc., Lawrenceville, GA, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin (Mediatech) at 37°C in a humidified atmosphere of 5% CO2. Busulfan, melphalan, and 5-aza-2′-deoxycytidine (DAC; Sigma-Aldrich, St Louis, MO, USA) were dissolved in dimethyl sulfoxide; gemcitabine (Eli Lilly, Indianapolis, IN, USA), and SAHA (Cayman Chemical Co., Ann Arbor, MI, USA) were dissolved in phosphate-buffered saline (PBS) and ethanol, respectively. The final concentrations of dimethyl sulfoxide and ethanol in all experiments did not exceed 0.08% by volume. Cell suspensions were aliquoted (100 μL of 2 × 105 cells/mL) into 96-well plates in the presence of drug(s) or solvent alone and continuously incubated at 37°C for 96 hours. The cells were analyzed for cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [20Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Methods. 1983; 65: 55-63Crossref PubMed Scopus (44685) Google Scholar]. The advantage of using a 96-hour treatment in this assay is that the cells proliferate for several cell cycle times without being disturbed before staining with MTT. With the relatively short half-lives of the drugs in cell culture medium at 37°C (e.g., busulfan ∼16 hours [21Hassan Z. Hassan M. Hellström-Lindberg E. The pharmacodynamic effect of busulfan in the P39 myeloid cell line in vitro.Leukemia. 2001; 15: 1240-1247Crossref PubMed Scopus (30) Google Scholar]; melphalan ∼60 min [22Bosanquet A.G. Bird M.C. Degradation of melphalan in vitro: rationale for the use of continuous exposure in chemosensitivity assays.Cancer Chemother Pharmacol. 1988; 21: 211-215Crossref PubMed Scopus (14) Google Scholar]), we assumed there would be negligible residual drug concentrations after a 48-hour exposure. Graphical analyses including calculations of IC20 values (the concentration of drug required for 20% growth inhibition) were done using Prism 5 (GraphPad Software, San Diego, CA, USA). Cells in logarithmic growth phase (5 × 105 cells/mL) were continuously incubated with the indicated concentrations of the drug(s) at 37°C for 48 hours. Cells were centrifuged, resuspended in 70% ethanol in PBS, and fixed at −20°C overnight. Fixed cells were pelleted at 3000g at room temperature, washed with PBS, and treated with 0.25 mL 500 U/mL RNAse A in PBS containing 1.12% sodium citrate at 37°C for 30 min. After addition of 0.25 mL propidium iodide (50 μg/mL) solution, the cells were kept in subdued light for at least 1 hour. The DNA content of at least 10,000 cells was analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and the proportion of cells in the different phases of the cell cycle was determined using CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA). Histograms were analyzed for the proportion of cells with sub-G1 DNA content using ModFit LT (Verity Software House, Topsham, ME, USA). Cell death by apoptosis after a 48-hour drug exposure was determined by flow cytometric measurements of phosphatidylserine externalization [23Martin S.J. Reutelingsperger C.P. McGahon A.J. et al.Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.J Exp Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2525) Google Scholar] with the Annexin-V-FLUOS staining kit (Roche Diagnostics, Indianapolis, IN, USA) using the FACSCalibur instrument. The extent of cleavage of poly(ADP-ribose) polymerase (PARP) −1 and caspase 3, determined by Western blot, was also used as an indicator of apoptosis. J45.01 cells were exposed to the indicated concentrations of drugs for 48 hours, washed with PBS, and analyzed for the enzymatic activities of caspases 3 and 9 using colorimetric assay kits from Millipore (Billerica, MA, USA). Cells were incubated with the drug(s) at 37°C for 48 hours, centrifuged, washed with PBS, and lysed with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Western blot analysis was done by separating protein extracts on polyacrylamide sodium dodecyl sulfate gels and blotting onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Immunoblot analyses by chemiluminescence were done using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Bedford, MA, USA). All Western blots were performed at least twice, and representative data are shown. All antibodies, their sources, and other relevant information are listed in Table 1.Table 1List of primary antibodies, their sources, and dilutionsAntigenSource/Catalog no.Clone type∗pAb = polyclonal antibody; used anti-rabbit IgG (or anti-goat as indicated) for secondary antibody from Bio-Rad Lab; mAb = monoclonal antibody; used anti-mouse IgG for secondary antibody from Bio-Rad Lab.Dilution†Fold dilution in PBS with 0.05% Tween 20.AcH3K9Active Motif/39138pAb3500AIB1/SR3Active Motif/39798pAb2500ATMSanta Cruz Biotech/23921mAb750ATRCell Signaling/2790pAb2500Caspase 3Cell Signaling/9661pAb1500CHK1Cell Signaling/2345pAb2000CHK2Cell Signaling/2662pAb2500Cyclin ECell Signaling/4129mAb3000DNMT1Santa Cruz Biotech/10222pAb700DNMT3ACell Signaling/3598mAb3000DNMT3BSanta Cruz Biotech/10236pAb700ELP3Active Motif/39950pAb3000HDAC4Cell Signaling/5392mAb3000HDAC5Cell Signaling/2083pAb2500KAP1Bethyl Laboratories A300-774ApAb3000p53Santa Cruz Biotech/126mAb1000Poly(ADP-ribose)Alexis BiochemicalsmAb3000PARP1Santa Cruz Biotech/8007mAb1000P-ATM (Ser1981)Rockland/200-301-4000mAb2000P-ATR (Ser428)Cell Signaling/2853pAb2500P-CHK1 (Ser317)Cell Signaling/2344pAb2500P-CHK2 (Ser19)Cell Signaling/2666pAb2500P-KAP1 (Ser824)Bethyl Laboratories A300-767ApAb1500P-p53 (Ser15)Cell Signaling/9284pAb2000P-SMC1 (Ser957)Novus Biolog/NB100-205pAb2000P-SMC1 (Ser966)Abcam/1276pAb3000SMC1Cell Signaling/4802pAb2500SRC1Cell Signaling/2191mAb2500XIAPCell Signaling/2045mAb2000β-ACTINSigma/A5316mAb10000γ-H2AXUpstate Biotech/05-636mAb30003MeH3K27Active Motif/39155pAb3500∗ pAb = polyclonal antibody; used anti-rabbit IgG (or anti-goat as indicated) for secondary antibody from Bio-Rad Lab; mAb = monoclonal antibody; used anti-mouse IgG for secondary antibody from Bio-Rad Lab.† Fold dilution in PBS with 0.05% Tween 20. Open table in a new tab Immunofluorescence staining with anti–5-methylcytidine antibody and flow cytometry were used to determine the level of DNA methylation according to a protocol described previously [24Habib M. Fares F. Bourgeois C.A. et al.DNA global hypomethylation in EBV-transformed interphase nuclei.Exp Cell Res. 1999; 249: 46-53Crossref PubMed Scopus (61) Google Scholar]. Briefly, J45.01 cells exposed to drugs for 48 hours were washed with PBS + 0.1% Tween 20 + 1% bovine serum albumin (PBST-BSA) then fixed with 0.25% paraformaldehyde (Polysciences, Inc., Warrington, PA, USA) in PBS at 37°C for 10 min. The cell membrane was permeabilized by adding nine volumes of 88% methanol in PBS and maintaining at −20°C for 0.5 to 1.0 hour. Cells were washed twice with PBST-BSA and treated with 2 N HCl at 37°C for 30 min, followed by 0.1 M sodium borate (pH 8.5) for 5 min. Samples were treated with 10 μg/mL RNase A in PBST-BSA at 37°C for 30 min and washed twice with PBST-BSA. Cells were resuspended in a blocking solution (10% fetal bovine serum in PBST-BSA) and incubated at 37°C for 20 min, followed by incubation with 1 μg/mL anti–5-methylcytidine antibody (AbD Serotec, Raleigh, NC, USA) at 37°C for 45 min. Samples were successively washed three times with PBST-BSA, probed with 1:4000 diluted anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen Corp., Carlsbad, CA, USA), and washed two times with PBST-BSA. A third wash was done by tumbling the samples at 4°C overnight. Cells were stained with 50 μg/mL propidium iodide in PBST-BSA for 30 min before flow cytometric analysis using the FACSCalibur instrument. Results are presented as the mean of three to four independent experiments and statistical analysis was done using a Student's paired t test with a two-tailed distribution. We recently reported greater resistance of J45.01, a T-cell lymphoma cell line, to the combinations of busulfan and two nucleoside analogs when compared with Daudi and U937, which are B-cell lymphoblastic and histiocytic lymphoma cell lines, respectively [18Valdez B.C. Murray D. Nieto Y. Li Y. Wang G. Champlin R.E. Andersson B.S. Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and SAHA in lymphoma cell lines.Leuk Lymphoma. 2012; 53: 973-981Crossref PubMed Scopus (19) Google Scholar]. In the present study, J45.01 showed greater sensitivity to the combinations of two DNA alkylating agents and a nucleoside analog than it did to the combinations of busulfan and two nucleoside analogs [18Valdez B.C. Murray D. Nieto Y. Li Y. Wang G. Champlin R.E. Andersson B.S. Synergistic cytotoxicity of the DNA alkylating agent busulfan, nucleoside analogs and SAHA in lymphoma cell lines.Leuk Lymphoma. 2012; 53: 973-981Crossref PubMed Scopus (19) Google Scholar]. Exposure of J45.01 cells individually to 57 μM busulfan (B), 1 μM melphalan (M), or 0.02 μM gemcitabine (G) for 96 hours did not show significant effects on cell proliferation or on the proportion of cells in sub-G1 phase (Fig. 1A). However, exposure of this cell line to the [B+M+G] combination resulted in 52% inhibition of proliferation and 39% of cells with sub-G1 DNA content, suggesting strong synergism (Fig. 1A). These effects were negated when the drug concentrations were reduced by approximately half; thus, exposure of J45.01 cells to the combination of 20 μM B, 0.7 μM M, and 0.01 μM G had negligible effects on cell proliferation (BMG in Fig. 1B). Addition of 0.6 μM SAHA (S) to this triple-drug combination dramatically inhibited cell proliferation by 65% (BMGS in Fig. 1B), even though 0.6 μM SAHA alone had no effect on the proliferation of J45.01 cells. These results suggest that addition of SAHA sensitizes J45.01 cells to the cytotoxic effects of low concentrations of BMG. To determine whether the proliferation of other cell lines is similarly inhibited by the BMG±S combinations, we exposed Daudi (B-cell lymphoma), KBM7/B5/Bu2506 (busulfan-resistant CML), and OCI-AML3 (acute myelogenous leukemia) cells to the same drugs at concentrations close to their IC20 values (Fig. 2A). The cytotoxicity of BMG±S in these cell lines is shown by the cleavage of PARP1 and phosphorylation of histone 2AX (Fig. 2B), indicative of activation of apoptosis and the DNA damage response, respectively; this will be discussed here later. SAHA alone did not affect the proliferation of these cells and BMG decreased their relative proliferation to 40% to 72% of control (Fig. 2C). When SAHA was combined with BMG in these cell lines, cell proliferation further decreased to 8% to 32% of control, again suggesting that SAHA sensitizes these cells to the three-drug combination (Fig. 2C). Overall, these results suggest that the HDAC inhibitor SAHA sensitizes both lymphoid and myeloid cancer cell lines to low concentrations of the BMG combination. DNA alkylators and nucleoside analogs have been shown to cause DNA strand breaks and other types of damage that activate the DNA damage response (DDR) when cells attempt to process such DNA lesions [25Drabløs F. Feyzi E. Aas P.A. et al.Alkylation damage in DNA and RNA-repair mechanisms and medical significance.DNA Repair (Amst). 2004; 3: 1389-1407Crossref PubMed Scopus (474) Google Scholar, 26Ewald B. Sampath D. Plunkett W. Nucleoside analogs: molecular mechanisms signaling cell death.Oncogene. 2008; 27: 6522-6537Crossref PubMed Scopus (169) Google Scholar]. Among the early steps associated with the cellular DDR are activation of the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases. As shown in Figure 3, exposure of J45.01 cells to 20 μM B, 0.7 μM M, 0.01 μM G, or 0.6 μM SAHA, either alone or in two-drug combinations, slightly increased the phosphorylation of ATM, a serine-threonine protein kinase that phosphorylates itself (at Ser1981) as well as other substrates. Triple-drug combinations, such as MGS and BGS, further increased the level of phosphorylated ATM; however, the highest levels of phosphorylated ATM were observed in cells exposed to BMG and BMGS (Fig. 3). The efficiency of BMGS in activating the DDR is further indicated by the phosphorylations of histone 2AX (H2AX) and CHK2, both of which are known substrates for the activated ATM kinase [26Ewald B. Sampath D. Plunkett W. Nucleoside analogs: molecular mechanisms signaling cell death.Oncogene. 2008; 27: 6522-6537Crossref PubMed Scopus (169) Google Scholar, 27Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.J Biol Chem. 1998; 273: 5858-5868Crossref PubMed Scopus (4015) Google Scholar]. P-CHK2 mediates the cell cycle checkpoint functions of the ATM pathway [28Kastan M.B. Lim D.S. The many substrates and functions of ATM.Nat Rev Mol Cell Biol. 2000; 1: 179-186Crossref PubMed Scopus (648) Google Scholar], whereas phosphorylated H2AX (γ-H2AX) is believed to hold broken chromosomal DNA ends in close proximity and recruit DNA damage response proteins [29Bassing C.H. Alt F.W. H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity.Cell Cycle. 2004; 3: 149-153Crossref PubMed Scopus (2) Google Scholar]. We also looked at KRAB-associated protein-1 (KAP1), which is phosphorylated by ATM after certain types of DNA damage and alters chromatin structure to provide access for DNA repair machinery [30Ziv Y. Bielopolski D. Galanty Y. et al.Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway.Nat Cell Biol. 2006; 8: 870-876Crossref PubMed Scopus (540) Google Scholar]. The highest level of KAP1 phosphorylation was seen in cells exposed to BMG and BMGS (Fig. 3), which is broadly consistent with the pattern of activation of ATM and of other ATM substrates. Other known substrates for the ATM kinase include SMC1 and p53, which are both phosphorylated in response to the various drug combinations used in the present study (Fig. 3). We also examined the effects of each drug combination on activation of the ATR-CHK1 pathway in J45.01 cells. Figure 3 shows that phosphorylation of ATR at Ser428 occurred in the presence of each individual drug, although this did not significantly increase for any drug combination. Unlike the autophosphorylation of ATM at Ser1981 (which is a widely used marker for ATM activation), none of the known phosphorylations of ATR have been established as reliable markers of ATR activation [31Cimprich K.A. Cortez D. ATR: an essential regulator of genome integrity.Nat Rev Mol Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1274) Google Scholar]. We therefore monitored downstream events such as the phosphorylation of CHK1 at Ser317, which appears to be a reasonable indicator of ATR activity [31Cimprich K.A. Cortez D. ATR: an essential regulator of genome integrity.Nat Rev Mol Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1274) Google Scholar, 32Zhao H, Piwnica-Worms H. ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. 2001;21:4129–4139.Google Scholar]. As shown in Figure 3, phosphorylation of CHK1 at Ser317 parallels the phosphorylation of ATR; thus, the ATR-CHK1 pathway in J45.01 cells is responsive to the individual drugs, but is not further activated by combinations of the drugs. In general, cells of hematological origin undergo apoptosis in response to an overwhelming DDR. The observed activation of the ATM-CHK2 and ATR-CHK1 pathways by these drugs (Fig. 3) is strongly suggestive of such a substantial DDR that may, among other responses, trigger the apoptotic pathway. Indeed, flow cytometric analysis of J45.01 cultures exposed to BMGS showed almost 70% of the cells with sub-G1 DNA content and ∼56% cells positive for Annexin-V staining, both hallmarks of apoptosis pathway activation (Fig. 4A). These results parallel the elevated cleavage of PARP1 and caspase 3 in cells exposed to the four-drug combination compared with any other drug combination (Fig. 4B). The BMGS-mediated activation of the apoptotic pathway is further suggested by the observed decrease in the level of XIAP1,
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