Hypoxanthine-guanine Phosphoribosyltransferase Gene Research Articles

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N-ethyl-N-nitrosourea.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

BRD4 inhibition impairs DNA mismatch repair, induces mismatch repair mutation signatures and creates therapeutic vulnerability to immune checkpoint blockade in MMR-proficient tumors

BackgroundMismatch repair deficiency (dMMR) is a well-recognized biomarker for response to immune checkpoint blockade (ICB). Strategies to convert MMR-proficient (pMMR) to dMMR phenotype with the goal of sensitizing tumors to...

Establish an allele-specific real-time PCR for Leishmania species identification

BackgroundLeishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.MethodsA probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.ResultsThis new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.ConclusionsOur assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.Graphical

Association of Equiliberative Nucleoside Transporter (ENT) with liver fibrosis stage in Hepatitis-C.

To evaluate the pattern of equiliberative nucleoside transporters in viral hepatitis responding and non-responding patients, to correlate the liver fibrosis stage with the pattern among the non-responders, and to correlate the equiliberative nucleoside transporter status with housekeeping hypoxanthine-guanine phosphoribosyltransferase gene. The comparative cross-sectional study was conducted at the Molecular Biology and Genetics Department, Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, from March to August 2018, and comprised adult hepatitis C virus patients of either gender who completed six months of treatment. They were assessed for response to therapy in terms of the presence of the viral load in their serum by using real time polymerase chain reaction, and divided into responder group A and non-responder group B. The groups were compared and correlation between equiliberative nucleoside transporter expressions and liver fibrosis was evaluated. Data was analysed using SPSS 23. Of the 80 patients, 33(41.3%) were males and 47(58.8%) were females. The overall mean age was 37.46±10.61 years. In terms of response to treatment, there were 40(50%) in each of the two groups. Mean post-treatment duration was 15.38±30.09 weeks. Age was not significantly different with respect to gender (p>0.05), but the age pattern was significantly different between the two groups (p<0.001). Also, non-responders had significant post-treatment duration compared to the responders (p<0.001). Hypoxanthine-guanine phosphoribosyltransferase gene showed no significant difference between the groups (p=0.144). Equiliberative nucleoside transporter was significantly down-regulated in the non-responders (p<0.001) and showed correlation with the degree of liver fibrosis (p<0.034) compared to the responders. There was a significant association between equiliberative nucleoside transporters and liver fibrosis stage in hepatitis C virus non-responding patients.

Abstract 2075: DNA damage on HPRT as a biomarker of susceptibility to tobacco-induced cancer

Abstract Background: The prevention of cancer and the development of more effective strategies to detect cancer precursor lesions and early-stage cancers remain critical goals. It has been estimated that 50% - 60% of cancers could be prevented if known strategies were optimally used. Cancer risk is not uniform, but varies based on age, genetic susceptibility, exposures, existence of preneoplastic conditions across the population. The DNA is constantly damaged by exogenous and endogenous sources. DNA repair mechanisms maintain the integrity of the genome. DNA damage has emerged as a major culprit in tumorigenesis and progression. There are numerous strategies with inherent advantages and disadvantages that may be used for the evaluation of DNA damage and repair. We have developed a novel and highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify DNA damage in p53, the most frequently mutated gene in human cancer. Here, we have extended this assay to HPRT (hypoxanthine-guanine phosphoribosyltransferase), the gene most frequently analyzed in DNA damage studies. Aim: (1) To quantify the amount of DNA damage in HPRT in peripheral blood cells of never-smokers, smokers, and secondhand smokers. (2) To determine whether cancer patients have higher levels of DNA damage in HPRT than non-cancer patients. Methods: The assay, PADDA was used on a high-throughput setting to quantify DNA damage (q-PADDA) on the HPRT gene. DNA was extracted and damage was quantified by q-PADDA in peripheral blood cells collected from non-cancer patients [never-smokers (n=28), tobacco smokers (n=25), secondhand smokers (n=18)], and cancer patients (n=66). Pairwise comparisons in DNA damage among the diverse groups were performed using the Tukey's adjustment in a one-way ANOVA model. Linear regression models were also used to test difference in DNA damage between cancer and non-cancer groups. Results: Never-smokers, tobacco smokers, and secondhand smokers had similar age distribution. In non-cancer patients, the amount of DNA damage in the HPRT gene in smokers was significantly higher than in non-smokers (p=0.01). Secondhand smokers also showed higher levels of DNA damage than never-smokers but this difference did not reach significance. Among cancer patients (n=64), smokers (n=29) showed significantly higher levels of DNA damage (p=0.03) than non-smokers (n=16). Conclusion: Our preliminary data document PADDA's ability to quantify DNA damage on HPRT gene. Damage was significantly higher in smokers than never-smokers, both in cancer and non-cancer patients. Application of this assay to a larger population sample has a major potential to establish biomarkers of susceptibility to tobacco-induced cancer, which can guide preventive and diagnostic strategies. Funding: This work was supported by the NIH, NCI (1R33 CA202898-01, LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Mayilvanan Chinnaiyan, Daniel Brobst, Balaji Sadhasivam, Vengatesh Ganapathy, Sarah E. Johnston, Daniel Zhao, Greg A. Krempl, Lurdes Queimado. DNA damage on HPRT as a biomarker of susceptibility to tobacco-induced cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2075.

Involvement of APOBEC3B in mutation induction by irradiation.

To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

Differential Expression of IGF1, IGFBP5, MSTN, and MYH1 Across Different Age Classes in American Quarter Horses

The objective of this study was to determine the influence of age on expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein (IGFBP5), myostatin (MSTN), and myosin (MYH1) genes which are related to growth and muscle development in the American Quarter Horse. Thus, horses (n = 10) from weanling, yearling, 2-, 3-, and 10-year-old age classes were sampled and gene expression was assessed by RT-qPCR. ΔCT was calculated using the hypoxanthine-guanine phosphoribosyltransferase gene as an internal normalizer. The generalized linear model was used to determine differentially expressed genes, by pairwise comparison between ages. Among technical replicates, the coefficient of variation ranged from 1.0 to 5.2% and was lower than the variation observed between biological replicates (2.1–12.9%). IGF1 demonstrated significantly lower expression in the 3-year-old age class than in weanlings and yearlings, but the 10-year-old age class displayed a significantly higher level than 2- and 3-year-old age classes. Expression of IGFBP5 was highest in weanlings compared with all other age classes. Expression of MSTN was significantly higher in weanlings than in other age classes, whereas 10-year-old horses had an intermediate level of expression, but significantly different from yearlings, 2- and 3-year-old fillies. Finally, expression of MYH1 was lower in 2- and 10-year-old horses than in weanlings and yearlings, whereas 3-year-old fillies demonstrated an intermediate level of expression. Differential expression patterns observed in this preliminary study provide insight into the physiological changes occurring throughout the life span of horses. These patterns could also help explain the variation in performance and endurance between individuals at different developmental stages.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Hypoxanthine-Guanine Phosphoribosyltransferase Is Dispensable for Mycobacterium smegmatis Viability.

Purine metabolism plays a ubiquitous role in the physiology of Mycobacterium tuberculosis and other mycobacteria. The purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is essential for M. tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is unclear. Membrane-permeable prodrugs of specifically designed HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds. Here, we investigated the purine salvage pathway in the model organism Mycobacterium smegmatis Using genomic deletion analysis, we confirmed that HGPRT is the only guanine and hypoxanthine salvage enzyme in M. smegmatis but is not required for in vitro growth of this mycobacterium or survival under long-term stationary-phase conditions. We also found that prodrugs of M. tuberculosis HGPRT inhibitors displayed an unexpected antimicrobial activity against M. smegmatis that is independent of HGPRT. Our data point to a different mode of mechanism of action for these inhibitors than was originally proposed.IMPORTANCE Purine bases, released by the hydrolytic and phosphorolytic degradation of nucleic acids and nucleotides, can be salvaged and recycled. The hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the formation of guanosine-5'-monophosphate from guanine and inosine-5'-monophosphate from hypoxanthine, represents a potential target for specific inhibitor development. Deletion of the HGPRT gene (Δhgprt) in the model organism Mycobacterium smegmatis confirmed that this enzyme is not essential for M. smegmatis growth. Prodrugs of acyclic nucleoside phosphonates (ANPs), originally designed against HGPRT from Mycobacterium tuberculosis, displayed anti-M. smegmatis activities comparable to those obtained for M. tuberculosis but also inhibited the ΔhgprtM. smegmatis strain. These results confirmed that ANPs act in M. smegmatis by a mechanism independent of HGPRT.

Pig-a mutations in bone marrow erythroblasts of rats treated with 7,12-dimethyl-benz[a]anthracene

Flow cytometry-based phenotypic detection of red blood cells (RBCs) deficient in surface markers anchored by glycosylphosphatidylinositol (GPI) is an efficient tool for monitoring somatic mutation in mammalian species. Biochemical considerations suggest that GPI-anchored marker-deficient RBCs found in peripheral blood are due to mutations in the endogenous X-linked phosphatidylinositolglycan, class A gene (Pig-a gene). Yet the linkage between the detected mutant phenotype and the actual mutation in the Pig-a gene is difficult to establish directly in mammalian RBCs that are naturally free of genomic DNA and may have only traces of heavily degraded mRNA. We have traced the origin of the marker-deficient RBC phenotype in the precursors of peripheral RBCs, bone marrow erythroid cells (BMEs, also known as erythroblasts), in rats treated by gavage with 75 mg/kg of the potent mutagen, 7,12-dimethyl-benz[a]anthracene (DMBA). The frequencies of marker-deficient BMEs were significantly increased in DMBA-treated rats. We identified Pig-a mutations in sorted mutant phenotype BMEs. The spectrum of DMBA-induced Pig-a mutations in erythroid lineage cells was identical to the spectra of mutations previously determined for the Pig-a and for another X-linked reporter gene, hypoxanthine-guanine phosphoribosyltransferase gene, in cells of lymphoid lineage, spleen T-lymphocytes. Our observations lend additional support to the hypothesis that GPI-anchored marker-deficient RBCs are true Pig-a mutants.

GENOTOXICITY OF N-HYDROXY AND AMINOPHENOL METABOLITES OF 2,6- AND 3,5-DIMETHYLANILINE AT THE HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE LOCUS IN TK6 CELLS

Objective: The objective of this study as to characterize the genotoxicity of reactive metabolites of 2,6-dimethylaniline (2,6-DMA) and 3,5-DMA in the hypoxanthineguanine phosphoribosyltransferase (HPRT) gene of human lymphoblastoid TK6 cells.Methods: Cultures were exposed to N-hydroxylamine and aminophenol metabolites of 2,6- and 3,5-DMA for 1 h in serum-free medium. Cell survival 24 h after exposure was determined by trypan blue exclusion. Cells were then subcultured for 7–10 days to allow to phenotypic expression of HPRT mutants. After the expression period, cells were plated in the presence of 2 μg/ml 6-thioguanine for the selection of HPRT mutants. Plating efficiency was determined and mutant fraction calculated. Electron paramagnetic resonance (EPR) was also used to determine whether 3,5- dimethylaminophenol (DMAP) produced reactive oxygen species (ROS).Results: All of the metabolites tested were cytotoxic to these cells but exhibited a considerable variation in potency. The aminophenol metabolites of 2,6- and 3,5-DMA were considerably more toxic than the corresponding N-hydroxylamines. Furthermore, each metabolite of 3,5-DMA was more toxic than its 2,6-DMA counterpart; N-OH-3,5-DMA and 3,5-DMAP were clearly mutagenic at a level of 50 μM. EPR studies showed intracellular oxidative stress induced under 3,5-DMAP treatment.Conclusions: Our findings suggest that genotoxic responses of 2,6- and 3,5-DMA are mediated through the generation of ROS by hydroxylamine and/ or aminophenol metabolites.

Prioritization of Deleterious Variations in the Human Hypoxanthine-Guanine Phosphoribosyltransferase Gene

Prioritization of Deleterious Variations in the Human Hypoxanthine-Guanine Phosphoribosyltransferase Gene

Mutagenicity of silver nanoparticles in CHO cells dependent on particle surface functionalization and metabolic activation

The potential of engineered nanomaterials to induce genotoxic effects is an important aspect of hazard identification. In this study, cytotoxicity and mutagenicity as a function of metabolic activation of three silver nanoparticle (AgNP) preparations differing in surface coating were determined in Chinese hamster ovary (CHO) subclone K1 cells. Three silver nanoparticle preparations (x 90,0 <30 nm) stabilized with polyoxyethylene glycerol trioleate and polyoxyethylene sorbitan monolaurate (AgPure™), citrate (Citrate-Ag), and polyvinylpyrrolidone (PVP-Ag) were used for the experiments. The cytotoxic effect of AgNPs was assessed with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) test using different concentrations of nanoparticles, while the mutagenicity was evaluated using the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. The cytotoxicity of all three AgNPs was lower in a cell culture medium containing 10% fetal calf serum (FCS) than in medium without FCS. The HPRT test without metabolic activation system S9 revealed that compared to the other AgNP formulations, citrate-coated Ag showed a lower genotoxic effect. However, addition of S9 increased the mutation frequency of all AgNPs and especially influenced the genotoxicity of Citrate-Ag. The results showed that exogenous metabolic activation of nanosilver is crucial even if interactions of the metabolic activation system, nanosilver, and cells are not really understood up to now.

Generation of Hprt-disrupted rat through mouse←rat ES chimeras.

We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.

Evaluation of Sister Chromatid Exchanges and Hypoxanthine guanine phosphoribosyl transferase Gene Mutation Assay in Peripheral Lymphocytes of Workers Exposed to Radiation at Al-Tuwaitha Site

The present study aims at using the biological techniques in a genotoxicity assessment of low doses of radiation in samples of workers in Al-Tuwaitha site due to decommissioning to radioactive contamination as a result of work. This study included 50 male blood samples, aged 35 - 63 years as well as 35 blood samples from non-smokers and non-alcoholic as control group which included 25 male and 10 female aged 25 -57 years. The endpoints used were sister chromatid exchange (SCE) and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation assay. The sister chromatid exchange in the human lymphocyte for radiation worker was significantly (P&lt;0.05) higher than in the control group. While the results of mutation for HPRT were non-significant when compared with the control group. In conclusion, the results indicated the possibility of using the changes in sister chromatid exchange as useful biomarkers for the detection of human exposure to ionizing radiation. In conclusion, the increase frequencies of sister chromatid exchange in radiation workers indicate the cumulative effect of low-level chronic exposure to ionizing radiation.

In vitro evaluation of genotoxic effects under magnetic resonant coupling wireless power transfer.

Wireless power transfer (WPT) technology using the resonant coupling phenomenon has been widely studied, but there are very few studies concerning the possible relationship between WPT exposure and human health. In this study, we investigated whether exposure to magnetic resonant coupling WPT has genotoxic effects on WI38VA13 subcloned 2RA human fibroblast cells. WPT exposure was performed using a helical coil-based exposure system designed to transfer power with 85.4% efficiency at a 12.5-MHz resonant frequency. The magnetic field at the positions of the cell culture dishes is approximately twice the reference level for occupational exposure as stated in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. The specific absorption rate at the positions of the cell culture dishes matches the respective reference levels stated in the ICNIRP guidelines. For assessment of genotoxicity, we studied cell growth, cell cycle distribution, DNA strand breaks using the comet assay, micronucleus formation, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation, and did not detect any significant effects between the WPT-exposed cells and control cells. Our results suggest that WPT exposure under the conditions of the ICNIRP guidelines does not cause detectable cellular genotoxicity.

Epigenetic regulation in amyloid precursor protein and the Lesch-Nyhan syndrome

Lesch-Nyhan syndrome (LNS) is a neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine–guanine phosphoribosyltransferase (HPRT) is defective. A major unsolved question is how the loss of HPRT enzyme function affects the brain to cause the neurobehavioural syndrome in LNS and its attenuated variants (LNVs). To address this issue, a search for a link between LNS and the amyloid precursor protein (APP) is developed. Here, I identified, for the first time in fibroblasts from normal subjects as well as from LNS and LNV patients: (a) several APP-mRNA isoforms encoding divers APP protein isoforms ranging from 120 to 770 amino acids (with or without mutations and/or deletions) accounted for epigenetic mechanisms in the regulation of alternative APP pre-mRNA splicing and (b) five novel independent polymorphisms in the APP promoter: −956A>G, −1023T>C, −1161A>G, −2224G>A, −2335C>T relative to the transcription start site. A role for epistasis between mutated HPRT and APP genes affecting the regulation of alternative APP pre-mRNA splicing in LNS is suggested. An accurate quantification of various APP isoforms in brain tissues for detection of initial pathological changes or pathology development is needed. My findings may provide new directions not only for investigating the role of APP in neuropathology associated with HPRT-deficiency in LNS but also for the research in neurodevelopmental and neurodegenerative disorders by which various APP isoforms involved in the pathogenesis of the diseases such as Alzheimer’s disease.

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO2) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO2 NPs. The purpose of this study is to evaluate the genotoxicity of TiO2 NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO2 NPs (75±15nm) through intragastric administration at 0, 10, 50 and 200mg/kg body weight every day for 30 days. The γ-H2AX assay showed TiO2 NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO2 NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO2 NPs at the dose of 0, 5, 10, 20, 50 and 100μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24h and 48h exposure. Significant DNA damage was only observed at the concentration of 100μg/mL after 24h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100μg/mL after 2h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO2 NPs, and TiO2 NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO2 NPs induced genotoxic effects.

Abstract A08: Analysis of MRGBP and PPIL1 genes in colon cancer

Abstract Colorectal cancer is one of the most common causes of cancer death throughout the world. Colorectal carcinogenesis is a multi-step process and in addition to genetic changes significant changes in DNA methylation and epigenetic events such as histone modifications can also lead to deregulation of gene expression. The histone proteins play a dynamic role in the chromatin structure and transcription. Aberrant protein acetylation, particularly on histones, has been related to cancer while abnormal expression of histone deacetlytransferases (HDACs) are observed in a broad range of cancer types. Therefore, HDACs have emerged as promising targets in cancer therapeutics. The MRG-binding protein (MRGBP / C20orf20) is a component of the NuA4 histone acetyltransferase (HAT) complex and is involved in the transcriptional activation of specific genes. The MRGBP gene is frequently upregulated in tumors and is thought to play a role in cancer development. On the other hand removal of introns from pre-mRNA is carried out by a large macromolecular spliceosome complex. PPIL1 (Peptidyl-prolylisomerase-like 1) is a component of the human spliceosome and plays a primary role in pre-mRNA splicing for catalyzing isomerization of the peptide bonds. Overexpression of the PPIL1 protein in cancer suggest a potential role of PPIL1 in the in the development and promotion of colon cancer. In the present study we analyzed MRGBP and PPIL1 gene expression levels in matched tumor and normal tissue samples from patients with colorectal cancer. Tissue samples were collected from 51 patients with colorectal cancer who underwent surgery for tumor resection. The normal tissue specimens used in the study were histologically confirmed to be free of cancer. The HPRT (hypoxanthine-Guanine phosphoribosyltransferase) gene was used as reference. The target and reference genes were amplified in the same multiplex PCR using the LightCycler 480 System. 8-9 nucleotide long UPL probes were labeled with fluorescein (FAM) and TAMRA at the 5′ and 3′ ends, respectively. Expression levels were determined using the Basic Relative Quantification analysis software and the results were evaluated by the Mann-Whitney U test. Although the median MRGBP expression levels in the tumor tissue were higher than normal tissue (1,72 vs.1,41) the difference was not significant. The median expression levels of PPIL1 in the tumors and normal tissue samples (1,97 vs 1,86) were also similar. Our results indicate that neither MRGBP nor PPIL1 gene expression levels are upregulated in colon cancer cells indicating that these are not directly associated with colorectal cancer. Citation Format: Ebru Akisik, Nejat Dalay, Sumer Yamaner, Dursun Bugra. Analysis of MRGBP and PPIL1 genes in colon cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A08.

Research progress in hypoxanthine-guanine phosphoribosyltrans-ferase

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a cytoplasmic enzyme which is widely distributed in the body. It not only involves in the purine salvage pathway, but also relates to the metabolism of purine analogues drugs. It is a critical transferase regulating the pharmacological effects and toxicity of purine analogues drugs. The mutations of the gene for HPRT, which influence its activity, may cause metabolic diseases with different clinical symptoms, and influence the metabolism of purine analogues. The HPRT gene, also a housekeeping gene, can serve diagnostic markers for many disorders. This paper reviews the recent progresses on HPRT researches in promoting the individual treatment of analogues drugs and the development of new drugs and improving the diagnosis and therapy of inherited metabolic disease caused by HPRT mutations.