The goal of this project is the development of therapeutic radiopharmaceuticals based on targeting the decay of Auger electron emitting radioisotopes to specific sequences in DNA (genes) using triplex forming oligonucleotides as delivery vehicles. The principal innovation in our approach is that it is the specific DNA sequence of a gene within the genome of a cell that becomes the target of radiotherapy, not the total DNA of that cell. Gene-specific radiotherapy optimally utilizes the sub-nanometer effect range of Auger emitters to allow targeting of most of the radiodamage to a selected gene sequence while producing minimal damage to the rest of the genome and other cell components. This approach requires a carrier molecule that exhibits enough specificity for a selected DNA sequence to deliver the radionuclide to that specific sequence and not to other sites in the genome. As our initial carrier molecule we selected short synthetic oligonucleotides that are able to form a sequence-specific triple helix with the target sequence, so-called triplex-forming oligonucleotides (TFO). This year we focused on the improvement of intracellular delivery of TFO via conjugation with nuclear localization signal (NLS) peptide. As an important step in the progression of gene-specific radiotherapy we have demonstrated the ability of 125I-TFO-NLS conjugates to produce double strand breaks in a specific site in the human multidrug resistance (mdr1) gene within live cultured cells. We also studied the distribution of DNA strand breaks produced by decay of 125I and the repair of these breaks by protein extracts from mammalian cells. We found that the repair of the radiodecay-produced breaks was orders of magnitude less effective than that of the breaks produced by restriction enzymes and was always associated with deletions at the target site. The above findings prove the principle of gene-specific radiotherapy. To further improve the efficiency of our approach we are currently developing of a new class of delivery molecules based on peptide nucleic acids (PNA). In addition, we are developing a new mutation-based cell culture system for fast evaluation of Auger emitter carrying molecules. We have also completed development and characterization of a proposed in vitro DSB repair assay employing DNA substrates bearing authentic DSB damage. The assay has been evaluated for optimal biochemical conditions, and tested with a variety of cellular extraction techniques and human DSB repair enzyme preparation methods. Nonhomologous end joining (NHEJ), the primary human DSB repair pathway, has been shown to be responsible for DSB repair observed in our assay, and the assay has been used to demonstrate tumor progression dependent changes in NHEJ activity with human breast cell lines. These results suggest a potential role for this assay in individualization of cancer therapies by directly testing the DSB repair capacity of patient tumors. We have also employed our in vitro DSB repair assay to establish that the structure of the DSB produced by different DNA damaging agents (enzymatic, chemical, low-LET radiation, and 125-I) directly affects the ability of human enzymes to repair breaks. These findings are significant because the biological effects of radiation are thought to be a direct effect of the chemical structure of the DSBs produced by radiation, in conjunction with the inherent DSB repair capacity of the cells in which the breaks occur. Consequently, detailed knowledge of the chemical structure of a radiation induced DSB would not only permit analysis of the biochemical mechanisms involved in its repair, but may permit application of such structural information to the direct manipulation of the cellular mechanism (DSB repair) responsible for resistance to many antineoplastic agents. Thus we have begun a study to map, and define the complete spectrum and distribution of DNA lesions associated with 125-II-TFO-induced DSBs. Initial work from this study indicates 125-I-TFO-induced DSBs to be associated with base damage and other DNA lesions proximal to the DSB ends. Using our in vitro DSB repair assay, we have shown such structures to be strong inhibitors of human NHEJ repair. Completion of the 125-I DSB structural model will open many new avenues of investigation, including DSB structural effects on NHEJ, intracellular signaling cascades, apoptosis, and cellular sensitivity to DNA damaging agents. They may also allow molecular analysis of repair processing at highly complex DSB structures. Such studies are not currently possible due to a lack of knowledge concerning the actual structure of a complex radiation-induced DSB, and what aspects of its structure are biologically important.

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Pastwa, Elzbieta; Somiari, Richard I; Malinowski, Mariusz et al. (2009) In vitro non-homologous DNA end joining assays--the 20th anniversary. Int J Biochem Cell Biol 41:1254-60
Datta, Kamal; Weinfeld, Michael; Neumann, Ronald D et al. (2007) Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures. Radiat Res 167:152-66
Sokolov, Mykyta V; Neumann, Ronald D; Panyutin, Igor G (2007) Effects of DNA-targeted ionizing radiation produced by 5-[125I]iodo-2'-deoxyuridine on global gene expression in primary human cells. BMC Genomics 8:192
Feinendegen, Ludwig E; Pollycove, Myron; Neumann, Ronald D (2007) Whole-body responses to low-level radiation exposure: new concepts in mammalian radiobiology. Exp Hematol 35:37-46
Medvedeva, Natalia G; Panyutin, Irina V; Panyutin, Igor G et al. (2007) Phosphorylation of histone H2AX in radiation-induced micronuclei. Radiat Res 168:493-8
Sokolov, M V; Smirnova, N A; Camerini-Otero, R D et al. (2006) Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide. Gene 382:47-56
Feinendegen, L E; Neumann, R D (2006) The issue of risk in complex adaptive systems: the case of low-dose radiation induced cancer. Hum Exp Toxicol 25:11-7
Datta, Kamal; Neumann, Ronald D; Winters, Thomas A (2006) An in vitro nonhomologous end-joining assay using linear duplex oligonucleotides. Anal Biochem 358:155-7
Sokolov, M; Panyutin, I G; Neumann, R (2006) Genome-wide gene expression changes in normal human fibroblasts in response to low-LET gamma-radiation and high-LET-like 125IUdR exposures. Radiat Prot Dosimetry 122:195-201
Panyutin, Irina V; Sedelnikova, Olga A; Bonner, William M et al. (2005) DNA damage produced by 125I-triplex-forming oligonucleotides as a measure of their successful delivery into cell nuclei. Ann N Y Acad Sci 1058:140-50

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