ABSTRACT Exposure to toxic chemicals, especially chemotherapeutic drugs, may induce several DNA lesions, including DNA interstrand crosslinks. These crosslinks are considered toxic lesions to the dividing cells since they can induce mutations, chromosomal rearrangements, and cell death. Many DNA interstrand crosslinks lesions can be generated by platinum-based chemotherapeutic agents. Satraplatin is a novel orally administered platinum-based chemotherapeutic agent. In the present study, we investigated DNA interstrand crosslinks lesions induced by oxaliplatin and satraplatin in lymphocytes obtained from colorectal cancer patients and healthy volunteers. Satraplatin demonstrated an increase in interstrand crosslinks in a dose-dependent manner in the Comet assay (p < 0.001). In addition, satraplatin and oxaliplatin increased significantly the number of sister chromatid exchanges up to 8.5-fold and 5.1-fold (p < 0.001) respectively, when treated with 2 μM concentration in comparison to untreated colorectal cancer cells. Further, the γH2AX foci formation was investigated by an immunofluorescence assay with oxaliplatin and satraplatin. The γH2AX foci formation rate was increased by approximately 9-fold when lymphocytes were treated with 2 μM oxaliplatin. Satraplatin was found to significantly induce the number of γH2AX foci by 8.5-fold and 11-fold with both 0.2 μM and 2.0 μM, respectively, compared to the control volunteers that may indicate the repair system in cancer cells experiences a loss of ability to cope with the repair of DSBs. In conclusion, oxaliplatin and satraplatin effectively induced DNA interstrand crosslinks in lymphocytes obtained from colorectal cancer patients and healthy volunteers in vitro. Here, to the best of our knowledge we report for the first time evidence of DNA double strand breaks formation as a possible molecular mechanism of action for satraplatin.
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A. Alotaibi, A. Baumgartner, M. Najafzadeh, E. Cemeli and D. Anderson, "In Vitro Investigation of DNA Damage Induced by the DNA Cross-Linking Agents Oxaliplatin and Satraplatin in Lymphocytes of Colorectal Cancer Patients," Journal of Cancer Therapy, Vol. 3 No. 1, 2012, pp. 78-89. doi: 10.4236/jct.2012.31011.
 M. L. G. Dronkert and R. Kanaar, “Repair of DNA Interstrand Cross-Links,” Mutation Research/DNA Repair, Vol. 486, No. 4, 2001, pp. 217-247.
 D. M. Noll, T. M. Mason and P. S. Miller, “Formation and Repair of Interstrand Cross-Links in DNA,” Chemi- cal Reviews, Vol. 106, No. 2, 2005, pp. 277-301.
 C. F. O'Neill, B. Koberle, J. R. W. Masters and L. R. Kelland, “Gene-Specific Repair of Pt/DNA Lesions and Induction of Apoptosis by the Oral Platinum Drug JM216 in Three Human Ovarian Carcinoma Cell Lines Sensitive and Resistant to Cisplatin,” The British Journal of Cancer, Vol. 81, No. 8, 1999, pp. 1294-1303.
 L. Kelland, “The Resurgence of Platinum-Based Cancer Chemotherapy,” Nature Reviews Cancer, Vol. 7, No. 8, 2007, pp. 573-584. doi:10.1038/nrc2167
 C. Martin, T. Ellis, C. J. McGurk, T. C. Jenkins, J. A. Hartley, M. J. Waring and D. E. Thurston, “Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA: Footprinting and Enzyme Inhibition Studies,” Biochemistry, Vol. 44, No. 11, 2005, pp. 4135-4147.
 E. Reed, “ERCC1 Measurements in Clinical Oncology,” The New England Journal of Medicine, Vol. 355, No. 10, 2006, pp. 1054-1055. doi:10.1056/NEJMe068162
 E. Donzelli, M. Carfi, M. Miloso, A. Strada, S. Galbiati, M. Bayssas, G. Griffon-Etienne, G. Cavaletti, M. G. Petruccioli and G. Tredici, “Neurotoxicity of Platinum Compounds: Comparison of the Effects of Cisplatin and Oxaliplatin on the Human Neuroblastoma Cell Line SH-SY5Y,” Journal of Neuro-Oncology, Vol. 67, No. 1-2, 2007, pp. 65-73. doi:10.1023/B:NEON.0000021787.70029.ce
 A. Stachurska, M. Dudkowska, A. Czopek, M. Manteuffel-Cymborowska and B. Grzelakowska-Sztabert, “Cisplatin Up-Regulates the in Vivo Biosynthesis and Degradation of Renal Polyamines and c-Myc Expression,” Biochimica et Biophysica Acta (BBA)—Molecular Basis of Disease, Vol. 1689, No. 3, 2004, pp. 259-266.
 M. Raschle, P. Knipsheer, M. Enoiu, T. Angelov, J. C. Sun, J. D. Griffith, T. E. Ellenberger, O. D. Scharer and J. C. Walter, “Mechanism of Replication-Coupled DNA Interstrand Crosslink Repair,” Cell, Vol. 134, No. 6, 2008, pp. 969-980. doi:10.1016/j.cell.2008.08.030
 X. Wang, C. A. Peterson, H. Y. Zheng, R. S. Nairn, R. J. Legerski and L. Li, “Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair,” Molecular and Cellular Biology, Vol. 21, No. 3, 2001, pp. 713-720. doi:10.1128/MCB.21.3.713-720.2001
 D. M. Kweekel, H. Gelderblom and H. J. Guchelaar, “Pharmacology of Oxaliplatin and the Use of Pharmacogenomics to Individualize Therapy,” Cancer Treatment Reviews, Vol. 31, No. 2, 2005, pp. 90-105.
 L. R. Kelland, G. Abel, M. J. McKeage, M. Jones, P. M. Goddard, M. Valenti, B. A. Murrer and K. R. Harrap, “Preclinical Antitumor Evaluation of Bis-acetato-amminedichloro-cyclohexylamine Platinum(IV)—An Orally Active Platinum Drug,” Cancer Research, Vol. 53, No. 11, 1993, pp. 2581-2586.
 H. Choy, C. Park and M. Yao, “Current Status and Future Prospects for Satraplatin, an Oral Platinum Analogue,” Clinical Cancer Research, Vol. 14, No. 6, 2008, pp. 1633-1638. doi:10.1158/1078-0432.CCR-07-2176
 L. Martelli, F. Di Mario, E. Ragazzi, P. Apostoli, R. Leone, P. Perego and G. Fumagalli, “Different Accumulation of Cisplatin, Oxaliplatin and JM216 in Sensitive and Cisplatin-Resistant Human Cervical Tumour Cells,” Biochemical Pharmacology, Vol. 72, No.6 , 2006, pp. 693-700.
 K. Wosikowski, L. Lamphere, G. Unteregger, V. Jung, F. Kaplan, J. P. Xu, B. Rattel and M. Caligiuri, “Preclinical Antitumor Activity of the Oral Platinum Analog Satraplatin,” Cancer Chemotherapy and Pharmacology, Vol. 60, No. 4, 2007, pp. 589-600.
 K. Wosikowski, F. Obermayr and B. Rattel, “Efficacy of Satraplatin, an Oral Platinum Analogue in Human Breast Cancer Models: Synergistic Activity with Capecitabine,” Breast Cancer Research and Treatment, Vol. 100, 2006, pp. S74-S74.
 P. R. Twentyman, K. A. Wright, P. Mistry, L. R. Kelland and B. A. Murrer, “sensitivity to Novel Platinum Compounds of Panels of Human Lung-Cancer Cell-Lines with Acquired and Inherent Resistance to Cisplatin,” Cancer Research, Vol. 52, 1992, pp. 5674-5680.
 NIC Registry, “Cancer Incidence and Mortality,” 2010.
 M. Kalimutho, A. Minutolo, S. Grelli, A. Formosa, G. Sancesario, A. Valentini, G. Federici and S. Bernardini, “Satraplatin (JM-216) Mediates G2/M Cell Cycle Arrest and Potentiates Apoptosis via Multiple Death Pathways in Colorectal Cancer Cells thus Overcoming Platinum Chemo-Resistance,” Cancer Chemotherapy and Pharmacology, Vol. 67, No. 6, 2011, pp. 1299-1312.
 J. T. Reardon, A. Vaisman, S. G. Chaney and A. Sancar, “Efficient Nucleotide Excision Repair of Cisplatin, Oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamineplatinum (IV) (JM216) Platinum Intrastrand DNA Diadducts,” Cancer Research, Vol. 59, No. 16, 1999, pp. 3968-3971.
 D. Fink, S. Nebel, S. Aebi, H. Zheng, B. Cenni, A. Nehme, R. D. Christen and S. B. Howell, “The Role of DNA Mismatch Repair in Platinum Drug Resistance,” Cancer Research, Vol. 56, No. 21, 1996, pp. 4881-4886.
 A. Vaisman, S. E. Lim, S. M. Patrick, W. C. Copeland, D. C. Hinkle, J. J. Turchi and S. G. Chaney, “Effect of DNA Polymerases and High Mobility Group Protein 1 on the Carrier Ligand Specificity for Translesion Synthesis Past Platinum-DNA Adducts,” Biochemistry, Vol. 38, No. 34, 1999, pp. 11026-11039. doi:10.1021/bi9909187
 S. P. Jackson and J. Bartek, “The DNA-Damage response in Human Biology and Disease,” Nature, Vol. 461, No. 7267, 2009, pp. 1071-1078. doi:10.1038/nature08467
 K. K. Khanna and S. P. Jackson, “DNA Double-Strand Breaks: Signaling, Repair and the Cancer Connection,” Nature Genetics, Vol. 27, No. 3, 2001, pp. 247-254.
 N. Kongruttanachok, C. Phuangphairoj, A. Thongnak, W. Ponyeam, P. Rattanatanyong, W. Pornthanakasem and A. Mutirangura, “Research Replication Independent DNA Double-Strand Break Retention May Prevent Genomic Instability,” Molecular Cancer, Vol. 9, No. 1, 2010, pp.
 E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova and W. M. Bonner, “DNA Double-Stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139,” Journal of Biological Chemistry, Vol. 273, No. 10, 1998, pp. 5858-5868. doi:10.1074/jbc.273.10.5858
 Y. Shiloh, “ATM and Related Protein Kinases: Safeuarding Genome Integrity,” Nature Reviews Cancer, Vol. 3, No. 3, 2003, pp. 155-168. doi:10.1038/nrc1011
 I. H. Ismail and M. J. Hendzel, “The Gamma-H2A.X: Is It Just a Surrogate Marker of Double-Strand Breaks or Much More?” Environmental and Molecular Mutagenesis, Vol. 49, No. 1, 2008, pp. 73-82. doi:10.1002/em.20358
 R. S. Vasireddy, M. M. Tang, L.-J. Mah, G. T. Georgiadis, A. El-Osta and T. C. Karagiannis,” Evaluation of the Spatial Distribution of GammaH2AX Following Ionizing Radiation,” Journal of Visualized Experiments, No. 47, 2010, pp. 4-7.
 I. H. Ismail, T. I. Wadhra and O. Hammarsten, “An Optimized Method for Detecting Gamma-H2AX in Blood Cells reveals a Significant Interindividual Variation in the Gamma-H2AX Response among Humans,” Nucleic Acids Research, Vol. 35, No. 2, 2007, p. e36.
 M. Stucki, J. A. Clapperton, D. Mohammad, M. B. Yaffe, S. J. Smerdon and S. P. Jackson, “MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks,” Cell, Vol. 123, No. 7, 2005, pp. 1213-1226.
 T. M. Marti, E. Hefner, L. Feeney, V. Natale and J. E. Cleaver, “H2AX phosphorylation within the G(1) Phase after UV Irradiation Depends on Nucleotide Excision Repair and Not DNA Double-Strand Breaks,” Proceedings of the National Academy of Sciences of USA, Vol. 103, No. 26, 2000, pp. 9891-9896.
 L. Henderson, A. Wolfreys, J. Fedyk, G. Bourner and S. Windebank, “The Ability of the Comet Assay to Discriminate between Genotoxins and Cytotoxins,” Mutagenesis, Vol. 13, No. 1, 1998, pp. 89-94.
 R. R. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu and Y. F. Sasaki, “Single Cell Gel/Comet Assay: Guidelines for in Vitro and in Vivo Genetic Toxicology Testing,” Environmental and Molecular Mutagenesis, Vol. 35, No. 3, 2000, pp. 206-221.
 P. L. Olive, P. J. Johnston, J. P. Banath and R. E. Durand, “The Comet Assay: A New Method to Examine Heterogeneity Associated with Solid Tumors,” Nature Medicine, Vol. 4, No. 1, 1998, pp. 103-105.
 M. Volpato, J. Seargent, P. M. Loadman and R. M. Phillips, “Formation of DNA Interstrand Cross-Links as a Marker of Mitomycin C Bioreductive Activation and Chemo Sensitivity,” European Journal of Cancer, Vol. 41, No. 9, 2005, pp. 1331-1338.
 A. Baumgartner, T. E. Schmid, E. Cemeli and D. Anderson, “Parallel Evaluation of Doxorubicin-Induced Genetic Damage in Human Lymphocytes and Sperm Using the Comet Assay and Spectral Karyotyping,” Mutagenesis, Vol. 19, No. 4, 2004, pp. 313-318.
 D. Meynard, V. Le Morvan, J. Bonnet and J. Robert, “Functional Analysis of the Gene Expression Profiles of Colorectal Cancer Cell Lines in Relation to Oxaliplatin and Cisplatin Cytotoxicity,” Oncology Reports, Vol. 17, No. 5, 2007, pp. 1213-1221.
 M. Fenech, “Cytokinesis-Block Micronucleus Cytome Assay,” Nature Protocols, Vol. 2, 2007, pp. 1084-1104.
 S. Wolff and P. Perry, “Differential Giemsa Staining of Sister Chromatids and Study of Sister Chromatid Exchanges without Autoradiography,” Chromosoma, Vol. 48, No. 4, 1974, pp. 341-353. doi:0.1007/BF00290991
 P. H. Clingen, J. Y. H. Wu, J. Miller, N. Mistry, F. Chin, P. Wynne, K. M. Prise and J. A. Hartley, “Histone H2AX Phosphorylation as a Molecular Pharmacological Marker for DNA Interstrand Crosslink Cancer Chemotherapy,” Biochemical Pharmacology, Vol. 76, No. 1, 2008, pp. 19-27. doi:10.1016/j.bcp.2008.03.025
 T. Nikolova, M. Ensminger, M. Lobrich and B. Kaina, “Homologous Recombination Protects Mammalian Cells from Replication-Associated DNA Double-Strand Breaks Arising in Response to Methyl Methanesulfonate,” DNA Repair, Vol. 9, No. 10, 2010, pp. 1050-1063.
 F. I. Raynaud, P. Mistry, A. Donaghue, G. K. Poon, L. R. Kelland, C. F. J. Barnard, B. A. Murrer and K. R. Harrap, “Biotransformation of the Platinum Drug JM216 Following Oral Administration to Cancer Patients,” Cancer Chemotherapy and Pharmacology, Vol. 38, No. 2, 1996, pp. 155-162. doi:10.1007/s002800050464
 K. J. Mellish, C. F. J. Barnard, B. A. Murrer and L. R. Kelland, “DNA-Binding Properties of Novel cis- and trans Platinum-Based Anticancer Agents in 2 Human Ovarian-Carcinoma Cell-Lines,” International Journal of Cancer, Vol. 62, No. 6, 1995, pp. 717-723.
 G. M. Almeida, T. L. Duarte, W. P. Steward and G. D. D. Jones, “Detection of Oxaliplatin-Induced DNACrosslinks in Vitro and in Cancer Patients Using the Alkaline Comet Assay,” DNA Repair, Vol. 5, No. 2, 2006, pp. 219-225.
 R. C. Todd and S. J. Lippard, “Inhibition of Transcription by Platinum Antitumor Compounds,” Metallomics, Vol. 1, No. 4, 2009, pp. 280-291. doi:10.1039/b907567d
 V. Brabec, “DNA Modifications by Antitumor Platinum and Ruthenium Compounds: Their Recognition and Repair,” In: Progress in Nucleic Acid Research and Molecular Biology, Academic Press, Cambridge, 2002, 1-68.
 S. S. Hah, R. A. Sumbad, R. W. de Vere White, K. W. Turteltaub and P. T. Henderson, “Characterization of Oxaliplatin-DNA Adduct Formation in DNA and Differentiation of Cancer Cell Drug Sensitivity at Microdose Concentrations,” Chemical Research in Toxicology, Vol. 20, No. 12, 2007, pp. 1745-1751. doi:10.1021/tx700376a
 G. Boysen, B. F. Pachkowski, J. Nakamura and J. A. Swenberg, “The Formation and Biological Significance of N7-Guanine Adducts,” Genetic Toxicology and Environmental Mutagenesis, Vol. 678, No. 2, 2009, pp. 76-94.
 F. Faust, F. Kassie, S. Knasmüller, R. H. Boedecker, M. Mann and V. Mersch-Sundermann, “The Use of the Alkaline Comet Assay with Lymphocytes in Human Biomonitoring Studies,” Mutation Research/Reviews in Mutation Research, Vol. 566, No. 3, 2004, pp. 209-229.
 D. J. McKenna, S. R. McKeown and V. J. McKelvey-Martin, “Potential Use of the Comet Assay in the Clinical Management of Cancer,” Mutagenesis, Vol. 23, No. 3, 2008, pp. 183-190. doi:10.1093/mutage/gem054
 J. F. Hartwig and S. J. Lippard, “DNA-Binding Properties of [Pt(NH3)(C6H11NH2)Cl2], a Metabolite of an Orally Active Platinum Anticancer Drug,” Journal of American Chemical Society, Vol. 114, No. 14, 1992, pp. 5646-5654.
 E. D. Scheeff, J. M. Briggs and S. B. Howell, “Molecular Modeling of the Intrastrand Guanine-Guanine DNA Adducts Produced by Cisplatin and Oxaliplatin,” Molecular Pharmacology, Vol. 56, No. 3, 1999, pp. 633-643.
 S. D. Webley, R. J. Francis, R. B. Pedley, S. K. Sharma, R. H. J. Begent, J. A. Hartley and D. Hochhauser, “Measurement of the Critical DNA Lesions Produced by Antibody-Directed Enzyme Prodrug Therapy (ADEPT) in Vitro, in Vivo and in Clinical Material,” British Journal of Cancer, Vol. 84, No. 12, 2001, pp. 1671-1676.
 E. Cemeli, E. Mirkova, G. Chiuchiarelli, E. Alexandrova and D. Anderson, “Investigation on the Mechanisms of Genotoxicity of Butadiene, Styrene and Their Combination in Human Lymphocytes Using the Comet Assay,” Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 664, No. 1-2, 2009, pp. 69-76.
 G. Chipitsyna, D. Slonina, K. Siddiqui, F. Peruzzi, T. Skorski, K. Reiss, B. E. Sawaya, K. Khalili and S. Amini, “HIV-1 Tat Increases Cell Survival in Response to Cisplatin by Stimulating Rad51 Gene Expression,” Oncogene, Vol. 23, No. 15, 2004, pp. 2664-2671.
 A. Vaisman and S. G. Chaney, “The Efficiency and Fidelity of Translesion Synthesis Past Cisplatin and Oxaliplatin GpG Adducts by Human DNA Polymerase Beta,” The Journal of Biological Chemistry, Vol. 275, No. 17, 2000, pp. 13017-13025.
 S. G. Chaney, S. L. Campbell, E. Bassett and Y. B. Wu, “Recognition and Processing of Cisplatin- and Oxaliplatin-DNA Adducts,” Critical Reviews in Oncology/Hematology, Vol. 53, No. 1, 2005, pp. 3-11.
 A. Vaisman, M. W. Warren and S. G. Chaney, “The Effect of DNA Structure on the Catalytic Efficiency and Fidelity of Human DNA polymerase Beta on Templates with Platinum-DNA Adducts,” The Journal of Biological Chemistry, Vol. 276, 2001, pp. 18999-19005.
 E. Bassett, A. Vaisman, K. A. Tropea, C. M. McCall, C. Masutani, F. Hanaoka and S. G. Chaney, “Frameshifts and Deletions during in Vitro Translesion Synthesis Past Pt-DNA Adducts by DNA Polymerases β and η,” DNA Repair, Vol. 1, No. 12, 2002, pp. 1003-1016.
 J. M. Havener, S. A. N. McElhinny, E. Bassett, M. Gauger, D. A. Ramsden and S. G. Chaney, “Translesion Synthesis Past Platinum DNA Adducts by Human DNA Polymerase μ,” Biochemistry, Vol. 42, No. 6, 2003, pp. 1777-1788. doi:10.1021/bi0270079
 J. P. Banath and P. L. Olive, “Expression of Phosphorylated Histone H2AX as a Surrogate of Cell Killing by Drugs That Create DNA Double-Strand Breaks,” Cancer Research, Vol. 63, No. 15, 2003, pp. 4347-4350.
 R. Sánchez-Olea, M. R. Calera and A. Degterev, “Molecular Pathways Involved in Cell Death after Chemically Induced DNA Damage,” In: A. Luch, Ed., Molecular, Clinical and Environmental Toxicology, Birkh?user, Basel, 2009, pp. 209-230.
 W. G. McGregor, “DNA Repair, DNA Replication, and UV Mutagenesis,” Journal of Investigative Dermatology Symposium Proceedings, Vol. 4, 1999, pp. 1-5.
 B. Kaina, “The Interrelationship between SCE Induction, Cell-Survival, Mutagenesis, Aberration Formation and DNA Synthesis Inhibition in V79 Cells Treated with N-methyl-N-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine,” Mutation Research Letters, Vol. 142, No. 1-2, 1985, pp. 49-54. doi:10.1016/S0165-7992(85)80012-9
 I. M. Ward and J. Chen, “Histone H2AX Is Phosphorylated in an ATR-Dependent Manner in Response to Replicational Stress,” The Journal of Biological Chemistry, Vol. 276, No. 51, 2001, pp. 47759-47762.
 A. J. Osborn, S. J. Elledge and L. Zou, “Checking on the Fork: the DNA-Replication Stress-Response Pathway,” Trends in Cell Biology, Vol. 12, No. 11, 2002, pp. 509-516. doi:10.1016/S0962-8924(02)02380-2
 B. Pascucci, M. T. Russo, M. Crescenzi, M. Bignami and E. Dogliotti, “The Accumulation of MMS-Induced Single Strand Breaks in G1 Phase is Recombinogenic in DNA Polymerase β Defective Mammalian Cells,” Nucleic Acids Research, Vol. 33, No. 1, 2005, pp. 280-288.
 O. Staszewski, T. Nikolova and B. Kaina, “Kinetics of γH2AX focus Formation upon Treatment of Cells with UV Light and Alkylating Agents,” Environmental and Molecular Mutagenesis, Vol. 49, No. 9, 2008, pp. 734-740.
 W. M. Bonner, C. E. Redon, J. S. Dickey, A. J. Nakamura, O. A. Sedelnikova, S. Solier and Y. Pommier, “γH2AX and Cancer,” Nature Reviews Cancer, Vol. 8, No. 12, 2008, pp. 957-967. doi:10.1038/nrc2523
 A. Sak, S. Grehl, P. Erichsen, M. Engelhard, A. Granna?, S. Levegrun, C. Pottgen, M. Groneberg and M. Stuschke, “Gamma-H2AX Foci Formation in Peripheral Blood Lymphocytes of Tumor Patients after Local Radiotherapy to Different Sites of the Body: Dependence on the Dose-Distribution, Irradiated Site and Time from Start of Treatment,” International Journal of Radiation Biology, Vol. 83, No. 10, 2007, pp. 639-652.