Small-Molecule Drugs Mimicking DNA Damage: A New Strategy for Sensitizing Tumors to Radiotherapy

Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. Experimental Design: A novel strategy for inhibiting DNA repair was tested. We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. Results: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1 and p53-binding protein 1 repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of “false” DNA damage signaling and repair inhibition. Conclusions: These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger “false” signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

[1]  M. Middleton,et al.  DNA repair pathways in drug resistance in melanoma , 2004, Anti-cancer drugs.

[2]  K. Manova,et al.  H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. , 2003, Developmental cell.

[3]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Richard C. Laughlin,et al.  Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention , 2005, Expert Reviews in Molecular Medicine.

[5]  Junjie Chen,et al.  Accumulation of Checkpoint Protein 53BP1 at DNA Breaks Involves Its Binding to Phosphorylated Histone H2AX* , 2003, Journal of Biological Chemistry.

[6]  Y. Miki,et al.  [PARP inhibitors for cancer therapy]. , 2011, Gan to kagaku ryoho. Cancer & chemotherapy.

[7]  Y. Pommier,et al.  Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes* , 2003, Journal of Biological Chemistry.

[8]  S. Elledge,et al.  53BP1, a Mediator of the DNA Damage Checkpoint , 2002, Science.

[9]  Ji-Hoon Lee,et al.  ATM Activation by DNA Double-Strand Breaks Through the Mre11-Rad50-Nbs1 Complex , 2005, Science.

[10]  D. Hallahan,et al.  DNA-dependent protein kinase is a molecular target for the development of noncytotoxic radiation-sensitizing drugs. , 2005, Cancer research.

[11]  W. Böcker,et al.  Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA‐PK in human cells exposed to ionizing radiation and treated with kinase inhibitors , 2005, Journal of cellular physiology.

[12]  J. Schiro,et al.  DNA-dependent protein kinase inhibitors as drug candidates for the treatment of cancer. , 2003, Molecular cancer therapeutics.

[13]  F. Lyko,et al.  DNA Methyltransferase Inhibitors for Cancer Therapy , 2007, Cancer journal.

[14]  Y. Shiloh.  ATM and related protein kinases: safeguarding genome integrity , 2003, Nature Reviews Cancer.

[15]  J. Sarkaria.  Identifying inhibitors of ATM and ATR kinase activities. , 2003, Methods in molecular medicine.

[16]  L. Milas,et al.  CpG Oligodeoxynucleotide Enhances Tumor Response to Radiation , 2004, Cancer Research.

[17]  P. Olive.  The comet assay. An overview of techniques. , 2002, Methods in molecular biology.

[18]  Michel C. Nussenzweig,et al.  Genomic Instability in Mice Lacking Histone H2AX , 2002, Science.

[19]  T. Paull,et al.  The Mre11/Rad50/Nbs1 Complex and Its Role as a DNA Double-Strand Break Sensor for ATM , 2005, Cell cycle.

[20]  V. Yamazaki,et al.  A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage , 2000, Current Biology.

[21]  Y. Adachi,et al.  Phosphorylation and Rapid Relocalization of 53BP1 to Nuclear Foci upon DNA Damage , 2001, Molecular and Cellular Biology.

[22]  U. Fischer,et al.  Glioblastoma multiforme: the role of DSB repair between genotype and phenotype , 2007, Oncogene.

[23]  H. Liber,et al.  Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation. , 2002, Cancer research.

[24]  E. Mazzon,et al.  Treatment with PARP-1 inhibitors, GPI 15427 or GPI 16539, ameliorates intestinal damage in rat models of colitis and shock. , 2005, European journal of pharmacology.

[25]  Thomas Helleday,et al.  DNA repair pathways as targets for cancer therapy , 2008, Nature Reviews Cancer.

[26]  P. Olive,et al.  Phosphorylation of histone H2AX as a measure of radiosensitivity. , 2004, International journal of radiation oncology, biology, physics.

[27]  J. Reichrath,et al.  UV damage and DNA repair in malignant melanoma and nonmelanoma skin cancer. , 2008, Advances in experimental medicine and biology.

[28]  Jesper Simonsen,et al.  An Overview of Techniques , 2009 .

[29]  E. Rogakou,et al.  Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo , 1999, The Journal of cell biology.

[30]  C. Deng,et al.  The Inhibition and Treatment of Breast Cancer with Poly (ADP-ribose) Polymerase (PARP-1) Inhibitors , 2006, International journal of biological sciences.

[31]  A. Krieg.  Antitumor applications of stimulating toll-like receptor 9 with CpG oligodeoxynucleotides , 2004, Current oncology reports.

[32]  H. Arai,et al.  Microarray analysis of temporal gene responses to ionizing radiation in two glioblastoma cell lines: up-regulation of DNA repair genes. , 2004, Journal of radiation research.

[33]  D. Boothman,et al.  Nonhomologous end joining is essential for cellular resistance to the novel antitumor agent, beta-lapachone. , 2007, Cancer research.

[34]  A. Pettitt,et al.  DNA-Dependent Protein Kinase Is a Therapeutic Target and an Indicator of Poor Prognosis in B-Cell Chronic Lymphocytic Leukemia , 2008, Clinical Cancer Research.

[35]  A. Kichler.  Gene transfer with modified polyethylenimines , 2004, The journal of gene medicine.

[36]  D. Fischer,et al.  Delivery of unmodified bioactive ribozymes by an RNA-stabilizing polyethylenimine (LMW-PEI) efficiently down-regulates gene expression , 2002, Gene Therapy.

[37]  A. Aigner,et al.  RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo , 2005, Gene Therapy.

[38]  A. Kauffmann,et al.  High expression of DNA repair pathways is associated with metastasis in melanoma patients , 2008, Oncogene.

[39]  P. Olive.  The Comet Assay , 2002 .

[40]  P. Jeggo,et al.  The life and death of DNA-PK , 2005, Oncogene.

[41]  F. Eckstein,et al.  Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides. , 1988, Nucleic acids research.

[42]  N. Curtin,et al.  Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441. , 2006, Cancer research.

[43]  O. Hammarsten,et al.  DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[44]  J. Behr,et al.  Intraperitoneal linear polyethylenimine (L-PEI)-mediated gene delivery to ovarian carcinoma nodes in mice , 2006, Cancer Gene Therapy.

[45]  P. Jeggo,et al.  ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation , 2004, Cancer Research.

[46]  Oscar Fernandez-Capetillo,et al.  Focusing on Foci: H2AX and the Recruitment of DNA-Damage Response Factors , 2003, Cell cycle.

[47]  N. Curtin,et al.  Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1. , 2003, Cancer research.

[48]  T. DeWeese,et al.  Enhanced radiation and chemotherapy-mediated cell killing of human cancer cells by small inhibitory RNA silencing of DNA repair factors. , 2003, Cancer research.

[49]  Michel Nussenzweig,et al.  H2AX: the histone guardian of the genome. , 2004, DNA repair.

[50]  T. Paull,et al.  Erratum: ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex (Science (April 22) (551)) , 2005 .

[51]  R. West,et al.  Productive and Nonproductive Complexes of Ku and DNA-Dependent Protein Kinase at DNA Termini , 1998, Molecular and Cellular Biology.