Developing new radiotherapy techniques using linac based gamma radiation sources

A major challenge in cancer radiotherapy is to deliver a lethal dose of radiation to the target volume while minimizing damage to the surrounding normal tissue. We have proposed a model on how treatment efficacy might be improved by interfering with biological responses to DNA damage using exogenous electric fields as a strategy to drastically reduce radiation doses in cancer therapy. This approach is demonstrated at this Laboratory through case studies with prokaryotes (bacteria) and eukaryotes (yeast) cells, in which cellkilling rates induced by both gamma radiation and exogenous electric fields were measured. It was found that when cells exposed to gamma radiation are immediately submitted to a weak electric field, cell death increases more than an order of magnitude compared to the effect of radiation alone. This finding suggests, although does not prove, that DNA damage sites are reached and recognized by means of long-range electric DNA-protein interaction, and that exogenous electric fields could destructively interfere with this process. As a consequence, DNA repair is avoided leading to massive cell death. Here we are proposing the use this new technique for the design and construction of novel radiotherapy facilities associated with linac generated gamma beams under controlled conditions of dose and beam intensity.

O6-Methylguanine Repair by O6-Alkylguanine-DNA Alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O6-methylguanine (O6mG) in DNA that is known to cause Mutation and cancer. On the basis of Calculations performed using density functional theory involving the active site of AGT, a mechanism for catalytic demethylation of O6mG to guanine has been proposed. In this mechanism, roles of six amino acids, i.e., Cys145, His 146, Glu172, Tyr114, Lys165, and Ser159 in catalytic demethylation of O6mG are involved. This mechanism has three steps as follows. At the first step, Cys145 in the Cys145-water-His146-Glu172 tetrad is converted to cysteine thiolate anion while at the second step, abstraction of the Tyr114 proton by the N3 site of O6mG occurs in a barrierless manner. In the third step, abstraction of Lys165 proton by deprotonated Tyr114 and transfer of the methyl group of O6mG to the thiolate group of Cys145 anion Occur simultaneously. As AGT is a major target in cancer therapy, identification of the roles of the different amino acids in demethylation of O6mG is expected to be useful in designing efficient AGT inhibitors.

Mutagenicity associated with O6-methylguanine-DNA damage and mechanism of nucleotide flipping by AGT during repair

Methylated guanine damage at O6 position (i.e. O6MG) is dangerous due to its mutagenic and carcinogenic character that often gives rise to G:C-A:T mutation. However, the reason for this mutagenicity is not known precisely and has been a matter of controversy. Further, although it is known that O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6MG paired with cytosine in DNA, the complete mechanism of target recognition and repair is not known completely. All these aspects of DNA damage and repair have been addressed here by employing high level density functional theory in gas phase and aqueous medium. It is found that the actual cause of O6MG mediated mutation may arise due to the fact that DNA polymerases incorporate thymine opposite to O6MG, misreading the resulting O6MG:T complex as an A:T base pair due to their analogous binding energies and structural alignments. It is further revealed that AGT mediated nucleotide flipping occurs in two successive steps. The intercalation of the finger residue Arg 128 into the DNA double helix and its interaction with the O6MG: C base pair followed by rotation of the O6MG nucleotide are found to be crucial for the damage recognition and nucleotide flipping.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knock-down studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

DNA mismatch repair and the DNA damage response to ionizing radiation:making sense of apparently conflicting data

The DNA mismatch repair (MMR) pathway detects and repairs DNA replication errors. While DNA MMR-proficiency is known to play a key role in the sensitivity to a number of DNA damaging agents, its role in the cytotoxicity of ionizing radiation (IR) is less well characterized. Available literature to date is conflicting regarding the influence of MMR status on radiosensitivity, and this has arisen as a subject of controversy in the field. The aim of this paper is to provide the first comprehensive overview of the experimental data linking MMR proteins and the DNA damage response to IR. A PubMed search was conducted using the key words "DNA mismatch repair" and "ionizing radiation". Relevant articles and their references were reviewed for their association between DNA MMR and IR. Recent data suggest that radiation dose and the type of DNA damage induced may dictate the involvement of the MMR system in the cellular response to IR. In particular, the literature supports a role for the MMR system in DNA damage recognition, cell cycle arrest, DNA repair and apoptosis. In this review we discuss our current understanding of the impact of MMR status on the cellular response to radiation in mammalian cells gained from past and present studies and attempt to provide an explanation for how MMR may determine the response to radiation.

Functional studies on DNA double-strand break repair proteins (MmRad51, Ku80) in mice

RecA in Escherichia coli and it's homologue, ScRad51 in Saccharomyces cerevisiae, play important roles in recombinational repair. ScRad51 homologues have been discovered in a wide range of organisms including Schizosaccharomyces pombe, lily, chicken, mouse and human. To date there is no direct evidence to describe that mouse Rad51(MmRad51) is involved in DNA double-strand break repair. In order to elucidate the role of MmRad51 in vivo, it was mutated by the embryonic stem (ES) cell/gene targeting technology in mice. The mutant embryos arrested in development shortly after implantation. There was a decrease in cell proliferation followed by programmed cell death, and trophectoderm-derived cells were sensitive to $\gamma$-radiation. Severe chromosome loss was observed in most mitotically dividing cells. The mutant embryos lived longer and developed further in a p53 mutant background; however, double-mutant embryonic fibroblasts failed to proliferate in tissue culture, reflecting the embryos limited life span. Based on these data, MmRad51 repairs DNA damage induced by $\gamma$-radiation, is needed to maintain euplody, and plays an important role in proliferating cells.^ Ku is a heterodimer of 70 and 80 kDs subunit, which binds to DNA ends and other altered DNA structures such as hairpins, nicks, and gaps. In addition, Ku is required for DNA-PK activity through a direct association. Although the biochemical properties of Ku and DNA-PKcs have been characterized in cells, their physiological functions are not clear. In order to understand the function of Ku in vivo, we generated mice homozygous for a mutation of the Ku80 gene. Ku80-deficient mice, like scid mice, showed severe immunodeficiency due to a impairment of V(D)J recombination. Mutant mice were semiviable and runted, cells derived from mutant embryos displayed hypersensitivity to $\gamma$-radiation, a decreased growth rate, a slow entry into S phase, altered colony size distributions, and a short life span. Based on these results, mutant cells and mice appeared to prematurely age. ^

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

Valor prognóstico de polimorfismos nos genes de reparo do DNA XRCC3 E RAD51 em pacientes com carcinoma epidermóide oral e de orofaringe

Faults in the genes responsible for repairs to the DNA can influence the onset of cancer or affect the response to treatment. This research evaluated the frequency of three single nucleotide polymorphisms (SNPs) in two repair genes DNA RAD51 172g> T (rs1801321), RAD51 135G> C (rs1801320) and XRCC3 T241M (rs861539) in individuals without cancer (n = 130) and patients with oral squamous cell carcinoma (OSC) and carcinoma oropharyngeal squamous (ORSC) (n = 126) and investigated possible relationships of these findings with clinical and pathological data and clinical outcomes: tumor response to radiotherapy and chemotherapy, disease-free survival, and overall survival. It was found that the allele and genotype frequencies were in equilibrium Hard-Weinberg equilibrium. The presence of at least one polymorphic allele in XRCC3 (rs861539) gene is associated with histological grade (WHO) higher (p = 0.007). We observed a higher recurrence rate trend (p = 0.08) and more advanced stage (p = 0.08) in the group that had at least one polymorphic allele of RAD51 gene (rs1801321). The presence of the analyzed SNPs not proved to be a risk factor for the development of CEO or CEOR; however, when combined with smoking or drinking, increased the risk of developing cancer from three to one hundred and fifty times. The tumor response to radiotherapy and chemotherapy was similar in patients with and without SNPs. No polymorphism showed statistical significance in relation to recurrence-free survival or overall survival. We conclude that the presence of at least one polymorphic allele of the SNPs rs861539 in XRCC3 gene, rs1801320 and rs1801321 in the RAD51 gene increase the risk of development of OSC and ORSC, when associated with the habit of drinking or smoking. Polymorphisms studied in XRCC3 and RAD51 genes are not associated with response to radiation therapy, relapse-free survival or overall survival.