Hypoxanthine-guanine Phosphoribosyl Transferase Locus Research Articles

Molecular Configuration of Human Genome Neighboring Megabase-Sized Large Deletions Induced by X-Ray Irradiation.

The genomic landscape neighboring large deletions including the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on human X chromosome in 6-thioguanine-resistant mutants originating from immortalized human fibroblast cells exposed to X rays was characterized by real-time quantitative PCR (qPCR)-based analyses. Among the 13 mutant clones with large deletions extending over several Mb, including the HPRT locus, revealed by 10 conventional sequence-tagged site (STS) markers, three clones bearing the largest deletions were selected for further qPCR analysis using another 21 STS markers and 15 newly designed PCR primer pairs. The results indicated that the major deletions were in very specific regions between the 130-Mb and 140-Mb positions containing the HPRT locus on the X chromosome and, contrary to our initial expectations, additional minor deletions were distributed in a patchwork pattern. These findings strongly indicate that the complex deletion patterns in the affected chromosome are related to the radiation track structure with spatially heterogeneous energy deposition and the specific structure of the chromatin-nuclear membrane complex. The uncovered complex deletion patterns are in agreement with the idea of complex chromatin damage, which is frequently associated with carcinogenesis.

Lethal and mutagenic bystander effects in human fibroblast cell cultures subjected to low-energy-carbon ions

Purpose: We studied lethal and mutagenic bystander effects in normal human fibroblasts irradiated with low-energy-carbon ions.Materials and methods: After cells reached confluence, cells were irradiated with initial energies of 6 MeV/n carbon ions. The residual energy and LET value were 4.6 MeV/n and 309 keV/µm. The doses used for survival and mutational studies were 0.082 and 0.16 Gy. Irradiation was carried out using 4 different irradiation conditions and plating conditions: (1) The entire cell area on the Mylar film was irradiated (We abbreviate as ‘all irradiation’); (2) Irradiated and unirradiated cells were pooled in a 1:1 ratio and plated as a single culture until the plating for lethal and mutagenic experiments (We abbreviate as ‘mixed population’); (3) Only half of the area on the Mylar film were irradiated using an ion-beam stopper (We abbreviate as ‘half irradiation’); and (4) Only half of the area of the cells were irradiated, and a specific inhibitor of gap junctions was added to the culture (We abbreviate as ‘half irradiation with inhibitor’). Cell samples were analyzed for lethal and mutagenic bystander effects, including a PCR evaluation of the mutation spectrum.Results: The surviving fraction of all irradiation was the same as the half irradiation case. The surviving fractions of both mixed population and the half irradiation with inhibitor were the same level and higher than those of all irradiation and half irradiation. The mutation frequencies at the HPRT (the hypoxanthine-guanine phosphoribosyl transferase) locus of all irradiation and half irradiation were at the same level and were higher than those of mixed population and half irradiation with inhibitor, respectively.Conclusion: There is evidence that the bystander effects for both lethality and mutagenicity occurred in the unirradiated half of the cells, in which only half of the cells were irradiated with the carbon ions. These results suggest that the bystander cellular effects via gap-junction-mediated cell-cell communication are induced by high-LET-carbon ions.

A polymorphism in intron I of the human angiotensinogen gene (hAGT) affects binding by HNF3 and hAGT expression and increases blood pressure in mice

Angiotensinogen (AGT) is the precursor of one of the most potent vasoconstrictors, peptide angiotensin II. Genome-wide association studies have shown that two A/G polymorphisms (rs2493134 and rs2004776), located at +507 and +1164 in intron I of the human AGT (hAGT) gene, are associated with hypertension. Polymorphisms of the AGT gene result in two main haplotypes. Hap-I contains the variants -217A, -6A, +507G, and +1164A and is pro-hypertensive, whereas Hap-II contains the variants -217G, -6G, +507A, and +1164G and does not affect blood pressure. The nucleotide sequence of intron I of the hAGT gene containing the +1164A variant has a stronger homology with the hepatocyte nuclear factor 3 (HNF3)-binding site than +1164G. Here we found that an oligonucleotide containing +1164A binds HNF3β more strongly than +1164G and that Hap-I-containing reporter gene constructs have increased basal and HNF3- and glucocorticoid-induced promoter activity in transiently transfected liver and kidney cells. Using a knock-in approach at the hypoxanthine-guanine phosphoribosyltransferase locus, we generated a transgenic mouse model containing the human renin (hREN) gene and either Hap-I or Hap-II. We show that transgenic animals containing Hap-I have increased blood pressure compared with those containing Hap-II. Moreover, the transcription factors glucocorticoid receptor, CCAAT enhancer-binding protein β, and HNF3β bound more strongly to chromatin obtained from the liver of transgenic animals containing Hap-I than to liver chromatin from Hap-II-containing animals. These findings suggest that, unlike Hap-II variants, Hap-I variants of the hAGT gene have increased transcription rates, resulting in elevated blood pressure.

Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

Restoring Ureagenesis in Hepatocytes by CRISPR/Cas9-mediated Genomic Addition to Arginase-deficient Induced Pluripotent Stem Cells.

Urea cycle disorders are incurable enzymopathies that affect nitrogen metabolism and typically lead to hyperammonemia. Arginase deficiency results from a mutation in Arg1, the enzyme regulating the final step of ureagenesis and typically results in developmental disabilities, seizures, spastic diplegia, and sometimes death. Current medical treatments for urea cycle disorders are only marginally effective, and for proximal disorders, liver transplantation is effective but limited by graft availability. Advances in human induced pluripotent stem cell research has allowed for the genetic modification of stem cells for potential cellular replacement therapies. In this study, we demonstrate a universally-applicable CRISPR/Cas9-based strategy utilizing exon 1 of the hypoxanthine-guanine phosphoribosyltransferase locus to genetically modify and restore arginase activity, and thus ureagenesis, in genetically distinct patient-specific human induced pluripotent stem cells and hepatocyte-like derivatives. Successful strategies restoring gene function in patient-specific human induced pluripotent stem cells may advance applications of genetically modified cell therapy to treat urea cycle and other inborn errors of metabolism.

Data in support of the mutagenic potential of the isoflavone irilone in cultured V79 cells.

The isoflavone irilone is found in human plasma after ingestion of red clover-based dietary supplements, but information allowing safety assessment is rare. Here, data in support of the mutagenic potential of irilone in cultured V79 cells [1] are presented. These data include (i) a quantitative assessment of irilone in the culture medium during the cell culture experiments, (ii) changes in the mutation spectrum in cDNA of the hypoxanthine-guanine phosphoribosyltransferase locus of irilone-treated V79 cells, (iii) occurrence of karyorrhexis and apoptosis as well as (iv) number of micronucleated cells containing whole chromosomes or chromosomal fragments. Also exemplary micrographs, used for the fluorescence microscopic assessment of (iii) and (iv) are presented.

Low-radiation environment affects the development of protection mechanisms in V79 cells.

Very little is known about the influence of environmental radiation on living matter. In principle, important information can be acquired by analysing possible differences between parallel biological systems, one in a reference-radiation environment (RRE) and the other in a low-radiation environment (LRE). We took advantage of the unique opportunity represented by the cell culture facilities at the Gran Sasso National Laboratories of the Istituto Nazionale di Fisica Nucleare, where environment dose rate reduction factors in the underground (LRE), with respect to the external laboratory (RRE), are as follows: 10(3) for neutrons, 10(7) for directly ionizing cosmic rays and 10 for total γ-rays. Chinese hamster V79 cells were cultured for 10months in both RRE and LRE. At the end of this period, all the cultures were kept in RRE for another 6months. Changes in the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPX) and spontaneous mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus were investigated. The results obtained suggest that environmental radiation might act as a trigger of defence mechanisms in V79 cells, specifically those in reference conditions, showing a higher degree of defence against endogenous damage as compared to cells grown in a very low-radiation environment. Our findings corroborate the hypothesis that environmental radiation contributes to the development of defence mechanisms in today living organisms/systems.

Genetic changes in progeny of bystander human fibroblasts after microbeam irradiation with X-rays, protons or carbon ions: The relevance to cancer risk

Purpose: Radiation-induced bystander effects have important implications in radiotherapy. Their persistence in normal cells may contribute to risk of health hazards, including cancer. This study investigates the role of radiation quality and gap junction intercellular communication (GJIC) in the propagation of harmful effects in progeny of bystander cells.Materials and methods: Confluent human skin fibroblasts were exposed to microbeam radiations with different linear energy transfer (LET) at mean absorbed doses of 0.4 Gy by which 0.036–0.4% of the cells were directly targeted by radiation. Following 20 population doublings, the cells were harvested and assayed for micronucleus formation, gene mutation and protein oxidation.Results: Our results showed that expression of stressful effects in the progeny of bystander cells is dependent on LET. The progeny of bystander cells exposed to X-rays (LET ∼6 keV/μm) or protons (LET ∼11 keV/μm) showed persistent oxidative stress, which correlated with increased micronucleus formation and mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus. Such effects were not observed after irradiation by carbon ions (LET ∼103 keV/μm). Interestingly, progeny of bystander cells from cultures exposed to protons or carbon ions under conditions where GJIC was inhibited harbored reduced oxidative and genetic damage. This mitigating effect was not detected when the cultures were exposed to X-rays.Conclusions: These findings suggest that cellular exposure to proton and heavy charged particle with LET properties similar to those used here can reduce the risk of lesions associated with cancer. The ability of cells to communicate via gap junctions at the time of irradiation appears to impact residual damage in progeny of bystander cells.

A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

Abstract 5454: Genotoxic consequences of exposure to mixtures of food-derived chemical carcinogens

Abstract It has been shown that mutagenic chemicals can form naturally in foods during cooking. Examples include Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon generated by incomplete combustion of organic substances such as lipids, leading to contamination of numerous foodstuffs (Lijinski and Shubik, Science 1964, 145, 53), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine that is formed during the cooking of meat (Murray, et al. J Chromatog 1993, 616, 211), and glycidamide (GA) is the ultimate genotoxic metabolite of acrylamide (AC), which forms when foods, such as potatoes and cereals, are cooked at temperatures exceeding 100°C, in a reaction between the amino acid asparagine and reducing sugars that are naturally present in these foods (Tornqvist, Adv Exp Biol Med 2005, 561, 1). These chemicals have individually been shown to be genotoxic but the biological consequences of exposure to mixtures of these chemicals have not been systematically examined. The aim of the current study was to examine the biological response of MCL-5 cells (a metabolically competent human lymphoblastoid cell line) to mixtures of these genotoxic chemicals at concentrations that are relevant to human exposure (mM to sub-nM). Cells were exposed to the chemicals individually or in mixtures for 24h and mutagenicity was assessed through resistance to trifluorothymidine at the thymidine kinase (TK) locus and 6-thioguanine at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mixture exposures gave modified dose-response curves from the individual chemicals, with increased mutation frequency (MF) at some low concentration combinations that were not mutagenic individually, and decreased MF at some combinations of higher concentrations that did produce a mutation response individually. Responses also differed between the TK and HPRT loci, with a higher MF observed for high concentration combinations at HPRT compared to TK. Ethoxyresorufin-O-deethylase (EROD) activity was measured and was found to correspond to the MF at the TK locus suggesting the involvement of CYP1A family in the mutation response. These data suggest that mixtures of genotoxic chemicals produce different mutation responses to the individual chemicals. The increase in MF at some low concentration combinations where the individual chemicals are not genotoxic is of significance when considering the carcinogenic potential of food. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5454. doi:1538-7445.AM2012-5454

Susceptibility of Human Lymphocytes to 2-Nitrofluorene-Induced Gene Mutation is Strongly Dependent on Metabolic Genotypes

Genetic polymorphisms in drug metabolism contribute largely to adverse drug reactions in susceptible individuals due to excess production of reactive metabolites and active oxygen species. We conducted an in vitro study that clearly demonstrates that functional impact of common polymorphisms in the metabolic genes, such as N-acetyltransferase 2 (NAT2), can be easily reflected in mutant induction in the gene coding for hypoxanthineguanine phosphoribosyl transferase (HPRT) in T-lymphocytes isolated from human peripheral blood. NAT2 is involved in the metabolic activation of 2-nitrofluorene (2-NF) to the known aromatic amide carcinogen N-acetyl- 2-aminofluorene. Subsequent deactivation through glutathione conjugation involves glutathione S-transferase M1 (GSTM1). We obtained a clear dose-related increase in the HPRT mutant frequency after treating mitogen-stimulated lymphocytes isolated from a normal blood donor with 2-NF (up to 5 fold at 400 μg/mL, 24h exposure), while no HPRT mutant induction was observed using cells from another blood donor. The susceptible cells turned out to have the NAT2 rapid and GSTM1 null genotype combination (capable of activation, with insufficient deactivation), while the resistant cells had the NAT2 slow and GSTM1 positive combination. Although the contribution of GSTM1 genotype is less clear, our finding suggests that functional polymorphisms in key metabolic genes do affect induction of gene mutations at the HPRT locus and at least the NAT2 genotype plays a critical role in determining the susceptibility of human cells to genotoxicity of 2-NF. Further, human peripheral T-lymphocytes and the in vitro HPRT gene mutation assay can be utilized to study the functional impact of common genetic polymorphisms in drug metabolism and to identify risk genotypes susceptible for drug toxicity and somatic mutations.

Targeted insertion of two Mthfr promoters in mice reveals temporal- and tissue-specific regulation

Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C→T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues.

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01 Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1–4 Gy) of high-LET IR (carbon-ion: 20 and 40 keV/μm, neon-ion: 150 keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine–guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.

Abstract 5413: Is a non-conjugated triple-helix forming oligonucleotide targeting the genomic HPRT locus capable of sequence specific mutagenesis

Abstract Oligonucleotide strands have been shown to be capable of binding to duplex DNA in a sequence specific manner to form a triple-helix or triplex structure. Such phenomenons have been shown to induce mutation, as demonstrated using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). These studies have engendered concern from the European Medicines Agency (EMEA) in regard to biotechnology derived pharmaceuticals such that antisense oligonucleotides may also form triplex structures in a sequence specific manner at genomic DNA with potentially mutagenic effects. In this study, we have examined the mutagenic potential of a non-conjugated triplex forming oligonucleotide (TFO27) targeting the genomic hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The mutagenicity of TFO27 was assessed using the human lymphoblastoid TK6 cell line through 6-thioguanine resistance (6TGr). Electrophoretic mobility shift assays were used to demonstrate triplex formation by TFO27 at the target motif at sub-nanomolar concentrations. As expected, a control oligonucleotide, SCR27, failed to form a triplex at the target motif. Subsequently, a range of transfection facilitators were evaluated for optimal delivery of a fluorescently labelled oligonucleotide probe. In preliminary experiments treating 3×106 cells, TFO27 appeared to produce a dose dependent increase in 6TGr mutants. Furthermore, TFO27 failed to induce mutation at the non-targeted thymidine kinase (TK) locus suggesting locus specificity for its mode of action. Moreover, SCR27 failed to induce mutation at both loci. However, TFO27 failed to induce mutation at the HPRT locus in experiments treating 10×106 cells. Increasing the oligonucleotide concentration had no effect on the number of 6TGr recovered. These contradictory findings are difficult to explain, but emphasise the importance of using appropriate target cell numbers in mutation assays. Overall, our data would suggest that triplex formation could occur in cells but chromatin structure, intranuclear pH, ion concentrations and nucleases are all potential barriers that probably influence the dynamics of triplex formation and stability. In view of these factors, we suggest that studies of the mutagenic potential of triplex formation are better performed at genomic loci rather than extra-chromosomal test systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5413. doi:10.1158/1538-7445.AM2011-5413

Abstract 4630: Cruciferous vegetable glucosinolate hydrolysis products protect against Benzo[a]pyrene-induced genotoxicity

Abstract Recent epidemiology studies have indicated that cruciferous vegetable intake is inversely correlated with lung cancer risk among smokers (Tang et al., BMC Cancer 2010, 10, 162). Benzo[a]pyrene (BaP) is a prominent contaminant of cigarette smoke, a model genotoxic carcinogen and powerful mutagen. Glucosinolates found in cruciferous vegetables are known to be hydrolysed to compounds that have been shown to be chemopreventive in both animal and cell-based systems, while, paradoxically, evidence is gathering that high concentrations of some of these hydrolysis products are genotoxic. The aim of the current study was to investigate whether pre-treatment of MCL-5 cells (a metabolically competent human lymphoblastoid cell line) with glucosinolate hydrolysis products conferred protection against the cytotoxicity and genotoxicity induced by a subsequent exposure to BaP. In the case of AITC (allyl isothiocyanate), a model glucosinolate hydrolysis product, toxicity and mutagenicity to MCL-5 cells was established for a range of concentrations. Mutagenicity was assessed through resistance to trifluorothymidine at the thymidine kinase (TK) locus and 6-thioguanine at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. The chemical treatments revealed a consistent trend for reduced cytotoxicity in cells pre-treated with AITC prior to BaP compared to those treated with BaP alone. Mutation frequency at the TK locus was consistently lower in the cells pre-treated with AITC compared to those treated with BaP alone. High concentrations of AITC induced a prolonged, dose-dependent cytotoxicity with cells only returning to normal growth rate after 7 days. These data suggest that pre-treatment of MCL-5 cells with glucosinolate hydrolysis products such as AITC affords protection against the genotoxic insult induced by mutagens such as BaP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4630. doi:10.1158/1538-7445.AM2011-4630

Cellular Response to Cytarabine Is Modulated by the DNA Mismatch Repair Pathway: Implications for Treatment of Acute Myeloid Leukemia

AbstractAbstract 1819The DNA mismatch repair (MMR) pathway is responsible for repair of spontaneous errors arising during DNA replication, thus maintaining the integrity of the genome. DNA MMR is frequently dysregulated in some forms of leukemia. We and others have shown that microsatellite instability, the hallmark of dysfunctional DNA MMR, is present in up to 90% of therapy-related myeloid leukemia, 50% of relapsed myeloid leukemia, but is rarely seen in de novo leukemia. Paradoxically, functional MMR mediates the cytotoxicity of certain chemotherapeutic agents, particularly methylating agents and the nucleoside analogue 6-thioguanine (6-TG), and dysregulation of the MMR pathway confers tolerance to these agents. In the present study, using cell lines harboring defects in MMR components, we show that MMR status also modulates response to the nucleoside analogue cytarabine (Ara-C) and to other therapeutic nucleoside analogues commonly used in the treatment of leukemia. We initially determined gene and protein expression levels of the major MMR components (MSH2, MSH3, MSH6, MLH1 and PMS2) in two psuedo-isogenic cell line pairs, and investigated the ability of cell extracts to bind to defined mismatches in electrophoretic mobility shift assays. In cytotoxicity assays, the cell lines HL-60R (which demonstrates 200-fold overexpression of MSH3) and MT-1 (which lacks functional MSH6 due to bi-allelic gene mutation) were tolerant to the cytotoxic effects of a methylating agent, methylnitrosourea (MNU), and 6-TG, relative to their respective parental cell lines. We also generated a panel of fully isogenic MMR-defective cell lines in which either MSH2, MSH3 or MSH6 protein was reduced to almost negligible levels using short hairpin RNA-mediated gene knockdown. Knockdown of either MSH2 or MSH6 conferred tolerance to the killing effects of MNU and 6-TG by virtue of loss of MutSα activity (a heterodimer of MSH2 and MSH6 responsible for recognition of base:base mispairs), whereas knockdown of MSH3 did not affect cellular response to these agents. Consistent with a role for MMR in affecting cellular response to other nucleoside analogues used to treat leukemia, the cell lines also displayed differential toxicity to the killing effects of Ara-C, clofarabine, cladribine and fludarabine compared to their MMR-proficient parental counterparts, however the exact response was dependent on the specific nature of the MMR defect. Cell lines with a reduction in MSH2 protein demonstrated hypersensitivity to cytotoxicity induced by these nucleoside analogues. Conversely, MSH3 knockdown conferred resistance to the cytotoxic effects of these agents. These data suggest that DNA MMR can affect response to nucleoside analogues via multiple mechanisms, and may also involve interaction of DNA MMR components with other DNA repair pathways. One possibility is that these agents induce base lesions in DNA recognized by DNA MMR components. Consistent with this model, we have shown that Ara-C induces DNA polymerase slippage in vitro, generating a substrate potentially recognized by MutSβ (a heterodimer of MSH2 and MSH3 responsible for recognition of small insertions and extrahelical loops). Furthermore, we have also shown that Ara-C is mutagenic at the thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase loci in the TK6 cell line. Taken together, these data suggest that cellular MMR status affects response to nucleoside analogues and furthermore, the specific nature of the defect is important in determining the exact response. These findings have implications for the use of nucleoside analogues in the treatment of cancers where MMR dysfunction has been identified to occur with high frequency, such as therapy-related and relapsed acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02–0.1 Gy X-rays, followed 6 h later by challenging 1 Gy heavy-ion radiation (carbon-ion: 20 and 40 keV/μm, neon-ion: 150 keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

Abstract 6: A triple-helix forming oligonucleotide targeting a genomic locus is capable of sequence specific mutagenesis in human lymphoblastoid TK6 cells

Abstract It has been reported that DNA triplex formation induces mutagenesis as determined using plasmid-based reporter constructs (Wang et al 1996, Science, 271, p802). Triplex mediated mutagenesis has been shown to include point mutations, deletions, small insertions and homologous recombination. To the best of our knowledge, no study has successfully examined the mutagenic potential of a non-conjugated triplex-forming oligonucleotide (TFO) targeting a genomic sequence. In this study, we have designed a TFO that targets the hemizygous hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus, in the human lymphoblastoid TK6 cell line, and assessed mutagenicity through resistance to 6-thioguanine. Our TFO, TFO27, has been designed to form a triplex with a purine tract in exon 3 of the HPRT gene. Triplex formation at the target motif was shown to occur at nanomolar concentrations, confirmed by Electrophoretic Mobility Shift Assays. A scrambled oligonucleotide, SCR27, failed to form a triplex at the target motif. A range of transfection reagents were evaluated for facilitated delivery of TFO27, and resulted in variable cellular toxicity. Transfection of high concentrations of oligonucleotide, with acceptable levels of cytotoxicity, resulted in HPRT mutation with TFO27 but not SCR27. Similar experiments failed to result in mutation at the non-targeted thymidine kinase (TK) locus, suggesting locus specificity for the mode of action of TFO27. The target specificity and sequence context of these mutagenic events is being determined to establish the mechanism of mutation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 6.

Dihydropyridines decrease X-ray-induced DNA base damage in mammalian cells

Compounds with the structural motif of 1,4-dihydropyridine display a broad spectrum of biological activities, often defined as bioprotective. Among them are L-type calcium channel blockers, however, also derivatives which do not block calcium channels exert various effects at the cellular and organismal levels. We examined the effect of sodium 3,5-bis-ethoxycarbonyl-2,6-dimethyl-1,4-dihydropyridine-4-carboxylate (denoted here as DHP and previously also as AV-153) on X-ray-induced DNA damage and mutation frequency at the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in Chinese hamster ovary CHO-K1 cells. Using formamido-pyrimidine glycosylase (FPG) comet assay, we found that 1-h DHP (10 nM) treatment before X-irradiation considerably reduced the initial level of FPG-recognized DNA base damage, which was consistent with decreased 8-oxo-7,8-dihydro-2′-deoxyguanosine content and mutation frequency lowered by about 40%. No effect on single strand break rejoining or on cell survival was observed. Similar base damage-protective effect was observed for two calcium channel blockers: nifedipine (structurally similar to DHP) or verapamil (structurally unrelated). So far, the specificity of the DHP-caused reduction in DNA damage – practically limited to base damage – has no satisfactory explanation.

Bystander effect-induced mutagenicity in HPRT locus of CHO cells following BNCT neutron irradiation: Characteristics of point mutations by sequence analysis

To investigate bystander mutagenic effects induced by alpha particles during boron neutron capture therapy (BNCT), we mixed cells that were electroporated with borocaptate sodium (BSH), which led to the accumulation of 10B inside the cells, with cells that did not contain the boron compound. BSH-containing cells were irradiated with α particles produced by the 10B(n,α) 7Li reaction, whereas cells without boron were only affected by the 1H(n,γ) 2H and 14N(n,ρ) 14C reactions. The frequency of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was examined in Chinese hamster ovary (CHO) cells irradiated with neutrons (Kyoto University Research Reactor: 5 MW). Neutron irradiation of 1:1 mixtures of cells with and without BSH resulted in a survival fraction of 0.1, and the cells that did not contain BSH made up 99.4% of the surviving cell population. Using multiplex polymerase chain reactions (PCRs), molecular structural analysis indicated that most of the mutations induced by the bystander effect were point mutations and that the frequencies of total and partial deletions induced by the bystander effect were lower than those resulting from the α particles produced by the 10B(n,α) 7Li reaction or the neutron beam from the 1H(n,γ) 2H and 14N(n,ρ) 14C reactions. The types of point mutations induced by the BNCT bystander effect were analyzed by cloning and sequencing methods. These mutations were comprised of 65.5% base substitutions, 27.5% deletions, and 7.0% insertions. Sequence analysis of base substitutions showed that transversions and transitions occurred in 64.7% and 35.3% of cases, respectively. G:C→T:A transversion induced by 8-oxo-guanine in DNA occurred in 5.9% of base substitution mutants in the BNCT bystander group. The characteristic mutations seen in this group, induced by BNCT α particles, differed from those typical of gamma ray radiation.