Irreparable complex DNA double-strand breaks induce chromosome breakage in organotypic three-dimensional human lung epithelial cell culture

DNA damage and consequent mutations initiate the multistep carcinogenic process. Differentiated cells have a reduced capacity to repair DNA lesions, but the biological impact of unrepaired DNA lesions in differentiated lung epithelial cells is unclear. Here, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We showed, consistent with existing notions that the kinetics of loss of simple double-strand breaks (DSBs) were significantly reduced in organotypic 3D culture compared to kinetics of repair in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, a majority of complex DNA lesions were irreparable in organotypic 3D culture. Levels of expression of multiple DNA damage repair pathway genes were significantly reduced in the organotypic 3D culture compared with those in 2D culture providing molecular evidence for the defective DNA damage repair in organotypic culture. Further, when differentiated cells with unrepaired DNA lesions re-entered the cell cycle, they manifested a spectrum of gross-chromosomal aberrations in mitosis. Our data suggest that downregulation of multiple DNA repair pathway genes in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis.

[1]  David J. Chen,et al.  Mechanism of cluster DNA damage repair in response to high-atomic number and energy particles radiation. , 2011, Mutation research.

[2]  M. Gorospe,et al.  miR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. , 2011, Molecular cell.

[3]  J. Harper,et al.  Radiation induced DNA DSBs: Contribution from stalled replication forks? , 2010, DNA repair.

[4]  M. Lieber,et al.  The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. , 2010, Annual review of biochemistry.

[5]  Christopher M Waters,et al.  Epithelial repair mechanisms in the lung. , 2010, American journal of physiology. Lung cellular and molecular physiology.

[6]  E. Dogliotti,et al.  Mechanisms of dealing with DNA damage in terminally differentiated cells. , 2010, Mutation research.

[7]  R. Kanaar,et al.  Characteristics of DNA-binding proteins determine the biological sensitivity to high-linear energy transfer radiation , 2010, Nucleic acids research.

[8]  M. Lomax,et al.  Hierarchy of lesion processing governs the repair, double-strand break formation and mutability of three-lesion clustered DNA damage , 2009, Nucleic acids research.

[9]  C. Geard,et al.  Analysis of ionizing radiation‐induced DNA damage and repair in three‐dimensional human skin model system , 2009, Experimental dermatology.

[10]  L. Van Haute,et al.  Generation of lung epithelial-like tissue from human embryonic stem cells , 2009, Respiratory research.

[11]  M. Lomax,et al.  Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic , 2009, Nucleic acids research.

[12]  David J. Chen,et al.  Cellular responses to DNA double-strand breaks after low-dose γ-irradiation , 2009, Nucleic acids research.

[13]  J. Lieberman,et al.  miR-24–mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells , 2009, Nature Structural &Molecular Biology.

[14]  A. Georgakilas,et al.  Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells. , 2009, Mutation research.

[15]  Mina J Bissell,et al.  Unraveling the microenvironmental influences on the normal mammary gland and breast cancer. , 2008, Seminars in cancer biology.

[16]  L. Harrison,et al.  DNA repair of clustered lesions in mammalian cells: involvement of non-homologous end-joining , 2008, Nucleic acids research.

[17]  Yang Xie,et al.  Enhanced identification and biological validation of differential gene expression via Illumina whole-genome expression arrays through the use of the model-based background correction methodology , 2008, Nucleic acids research.

[18]  M. Hada,et al.  Formation of clustered DNA damage after high-LET irradiation: a review. , 2008, Journal of radiation research.

[19]  David J. Chen,et al.  Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts , 2008, Radiation research.

[20]  Frederick Grinnell,et al.  Fibroblast mechanics in 3D collagen matrices. , 2007, Advanced drug delivery reviews.

[21]  P. Degan,et al.  Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury , 2007, Proceedings of the National Academy of Sciences.

[22]  O. Kovalchuk,et al.  DNA double-strand breaks form in bystander cells after microbeam irradiation of three-dimensional human tissue models. , 2007, Cancer research.

[23]  T. Nouspikel.  DNA repair in differentiated cells: Some new answers to old questions , 2007, Neuroscience.

[24]  Genee Y. Lee,et al.  Three-dimensional culture models of normal and malignant breast epithelial cells , 2007, Nature Methods.

[25]  J. Emerman,et al.  Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes , 1977, In Vitro.

[26]  Dudley T Goodhead,et al.  Energy deposition stochastics and track structure: what about the target? , 2006, Radiation protection dosimetry.

[27]  J. Minna,et al.  A three-dimensional model of differentiation of immortalized human bronchial epithelial cells. , 2006, Differentiation; research in biological diversity.

[28]  F. A. Cucinotta,et al.  Immunofluorescence Detection of Clustered γ-H2AX Foci Induced by HZE-Particle Radiation , 2005, Radiation research.

[29]  Peter O'Neill,et al.  Molecular dynamics simulation of clustered DNA damage sites containing 8‐oxoguanine and abasic site , 2005, J. Comput. Chem..

[30]  David J. Chen,et al.  Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks* , 2005, Journal of Biological Chemistry.

[31]  F A Cucinotta,et al.  Immunofluorescence detection of clustered gamma-H2AX foci induced by HZE-particle radiation. , 2005, Radiation research.

[32]  J. Pollack,et al.  Immortalization of Human Bronchial Epithelial Cells in the Absence of Viral Oncoproteins , 2004, Cancer Research.

[33]  M. Lomax,et al.  Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts. , 2004, Biochemistry.

[34]  Peter O'Neill,et al.  Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation. , 2004, Nucleic acids research.

[35]  M. Scholz,et al.  The Increased Biological Effectiveness of Heavy Charged Particles: From Radiobiology to Treatment Planning , 2003, Technology in cancer research & treatment.

[36]  Kai Rothkamm,et al.  Pathways of DNA Double-Strand Break Repair during the Mammalian Cell Cycle , 2003, Molecular and Cellular Biology.

[37]  C. D. de Lara,et al.  Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19), hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites. , 2002, Nucleic acids research.

[38]  T. Sofuni,et al.  Application of mFISH for the analysis of chemically-induced chromosomal aberrations: a model for the formation of triradial chromosomes. , 2002, Mutation research.

[39]  G. Dianov,et al.  Repair of Clustered DNA Lesions , 2002, The Journal of Biological Chemistry.

[40]  Peter O'Neill,et al.  Efficiency of incision of an AP site within clustered DNA damage by the major human AP endonuclease. , 2002, Biochemistry.

[41]  G. Dianov,et al.  Repair of Clustered DNA Lesions SEQUENCE-SPECIFIC INHIBITION OF LONG-PATCH BASE EXCISION REPAIR BY 8-OXOGUANINE* , 2002 .

[42]  Junjie Chen,et al.  Threonine 68 of Chk2 Is Phosphorylated at Sites of DNA Strand Breaks* , 2001, The Journal of Biological Chemistry.

[43]  P O'Neill,et al.  Computational Approach for Determining the Spectrum of DNA Damage Induced by Ionizing Radiation , 2001, Radiation research.

[44]  S. Wallace,et al.  Base excision repair processing of radiation-induced clustered DNA lesions. , 2001, Radiation protection dosimetry.

[45]  T. Halazonetis,et al.  P53 Binding Protein 1 (53bp1) Is an Early Participant in the Cellular Response to DNA Double-Strand Breaks , 2000, The Journal of cell biology.

[46]  M. Löbrich,et al.  Formation and repair of DNA double-strand breaks in gamma-irradiated K562 cells undergoing erythroid differentiation. , 2000, Mutation research.

[47]  T Hyslop,et al.  DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. , 2000, Cancer research.

[48]  B M Sutherland,et al.  Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[49]  S. Wallace,et al.  In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites. , 1999, Journal of molecular biology.

[50]  S. Wallace,et al.  Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII. , 1998, Nucleic acids research.

[51]  D T Goodhead,et al.  Initial events in the cellular effects of ionizing radiations: clustered damage in DNA. , 1994, International journal of radiation biology.

[52]  D J Brenner,et al.  Constraints on energy deposition and target size of multiply damaged sites associated with DNA double-strand breaks. , 1992, International journal of radiation biology.

[53]  J. Ward,et al.  DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability. , 1988, Progress in nucleic acid research and molecular biology.

[54]  H Stein,et al.  Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. , 1984, Journal of immunology.