Phosphoribosyltransferase Gene Research Articles

A study on the relationship between TCTA tetranucleotide polymorphism of the HPRT gene and primary hyperuricemia

We examined polymorphism of the TCTA tetranucleotide sequence in the 3rd intron of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the Han population of Ningxia Province in China. We also looked for a possible relationship between STR polymorphism in the 3rd intron of the HPRT gene and primary hyperuricemia. We used Chelex-100 to extract DNA, then PCR, PAGE and silver staining for allele genotyping and DNA sequencing to obtain the distribution of the alleles. We found, for the first time, that there is high STR polymorphism in the 3rd intron of the HPRT gene. We detected 5 STR alleles in this intron in the Han population of Ningxia Province, with 15 genotypes in females; significant differences were observed in the distribution of alleles and genotypes between control and patient groups for both males and females. Alleles of the TCTA repeat in the 3rd intron of the HPRT gene were found to be associated with primary hyperuricemia; consequently, these alleles may be considered risk factors for primary hyperuricemia.

Characterization of amplicons that suppress the conditional lethal growth phenotype of a Leishmania donovani mutant lacking normal purine salvage mechanisms

A conditionally lethal mutant of Leishmania donovani that lacks both hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase exhibits a strikingly restricted growth phenotype, can only survive as the promastigote under pharmacological constraints, and is profoundly compromised in its ability to infect macrophages and mice. Interestingly, the conditionally lethal growth phenotype displayed by these mutant parasites can be suppressed in vitro by selection of strains that have markedly amplified the adenine phosphoribosyltransferase gene on extrachromosomal elements that are unique to these suppressor strains. Employing pulsed field gel electrophoresis, we have now determined that the amplicons in two of these suppressor lines are linear molecules by: (1) their pulse time-dependent mobility; (2) the failure of γ-irradiation to generate new discrete bands; (3) their susceptibility to λ exonuclease digestion; and (4) the presence of telomeric sequences. Pulsed field gel electrophoresis also shows these amplicons to be approximately 200–275 kb in size. However, quantitative polymerase chain reaction and Southern blot analyses demonstrated that the amplification units are ∼40 kb in length, implying that the formation of these amplicons involved additional chromosomal rearrangements or oligomerization.

Assessment of the cytotoxicity and genotoxicity of haloacetic acids using microplate-based cytotoxicity test and CHO/ HGPRT gene mutation assay

Haloacetic acids (HAAs) are the second most prevalent class of disinfection byproducts found in drinking water. The implications of HAAs presence in drinking water are a public health concern due to their potential mutagenic and carcinogenic effects. In the present study, we examined the cytotoxic and genotoxic effects of six common HAAs using a microplate-based cytotoxicity test and a hypoxanthine–guanine phosphoribosyltransferase ( HGPRT) gene mutation assay in Chinese hamster ovary K1 (CHO-K1) cells. We found that their chronic cytotoxicities (72 h exposure) to CHO-K1 cells varied, and we ranked their levels of toxicity in the following descending order: iodoacetic acid (IA) > bromoacetic acid (BA) > dibromoacetic acid (DBA) > chloroacetic acid (CA) > dichloroacetic acid (DCA) > trichloroacetic acid (TCA). The toxicity of IA is 1040-fold of that of TCA. All HAAs except TCA were shown to be mutagenic to CHO-K1 cells in the HGPRT gene mutation assay. The mutagenic potency was compared and ranked as follows: IA > DBA > BA > CA > DCA > TCA. There was a statistically significant correlation between cytotoxicity and mutagenicity of the HAAs in CHO-K1 cells. The microplate-based cytotoxicity assay and HGPRT gene mutation assay were suitable methods to monitor the cytotoxicity and genotoxicity of HAAs, particularly for comparing the toxic intensities quantitatively.

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Deltahgprt/Deltaxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084-16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Deltahgprt/Deltaxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2'-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Deltahgprt/Deltaxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Deltahgprt/Deltaxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Deltahgprt/Deltaxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Deltahgprt/Deltaxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Deltahgprt/Deltaxprt parasites displayed persistence and increased virulence in mice.

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6-thioguanine (6-TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6-TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb-A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb-B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity.

Identification of an intergenic region that is not essential for replication of goatpox virus

A recombinant goatpox virus was constructed in which an enhanced green fluorescent protein gene was inserted under the control of the 11K late promoter, a guanine phosphoribosyltransferase gene was inserted under the control of the 7.5K early/late promoter, and exogenous genes were inserted into an intergenic region between loci gp_24 and gp_24.5 of the recombinant genome. Analysis of protein expression showed that LT cells infected with only the recombinant virus produced specific fluorescence. A comparative growth assay demonstrated the stability of the recombinant virus at the replication level. These results suggest that the intergenic region is not essential for replication of goatpox virus.

Real-time PCR detection of Enterococcus faecalis associated with amyloid arthropathy

To develop a RT-PCR method for detection of the multilocus sequence type 82 of Enterococcus faecalis associated with amyloid arthropathy (AA) in layers. Bacteria were selected from lesions including AA in layers. The primers were designed based on the phosphate ATP binding cassette transporter (pstS) and xanthine phosphoribosyltransferase (xpt) genes and first tested against three isolates with known base pairs at the specific sites. Subsequently, 12 isolates were selected from our collection by one researcher, and RT-PCR was performed blinded. The sequence type (ST) was then confirmed by multilocus sequence analysis. Two single-nucleotide polymorphisms in the pstS and xpt genes allowed an unambiguous identification of ST82. As an alternative to DNA extraction, a boiling method for release of DNA from cells was used. The real-time PCR targeting ST82 enables rapid screening of Ent. faecalis cultured from suspect cases with results available after a few hours, much faster than multilocus sequence typing and pulse field gel electrophoresis. The new method allows a rapid screening of isolates with results available after only few hours. This RT-PCR method could be a useful tool for molecular epidemiological studies on the spread of arthropathic and amyloidogenic Ent. faecalis within and between birds more efficiently.

Targeted Cytosine Deaminase-Uracil Phosphoribosyl Transferase Suicide Gene Therapy Induces Small Cell Lung Cancer–Specific Cytotoxicity and Tumor Growth Delay

Small cell lung cancer (SCLC) is a highly malignant cancer for which there is no curable treatment. Novel therapies are therefore in great demand. In the present study we investigated the therapeutic effect of transcriptionally targeted suicide gene therapy for SCLC based on the yeast cytosine deaminase (YCD) gene alone or fused with the yeast uracil phosphoribosyl transferase (YUPRT) gene followed by administration of 5-fluorocytosine (5-FC) prodrug. The YCD gene or the YCD-YUPRT gene was placed under regulation of the SCLC-specific promoter insulinoma-associated 1 (INSM1). Therapeutic effect was evaluated in vitro in SCLC cell lines and in vivo in SCLC xenografted nude mice using the nonviral nanoparticle DOTAP/cholesterol for transgene delivery. INSM1-YCD/5-FC and INSM1-YCD-YUPRT/5-FC therapy induced high cytotoxicity in a range of SCLC cell lines. The highest therapeutic effect was obtained from the YCD-YUPRT fusion gene strategy. No cytotoxicity was induced after treatment of cell lines of other origin than SCLC. In addition the INSM1-YCD-YUPRT/5-FC therapy was superior to an established suicide gene system consisting of the herpes simplex virus thymidine kinase (HSVTK) gene and the prodrug ganciclovir. The superior effect was in part due to massive bystander cytotoxicity of YCD-YUPRT-produced toxins. Finally, INSM1-YCD-YUPRT/5-FC therapy induced significant tumor growth delay in SCLC xenografts compared with control-treated xenografts. The current study is the first to test cytosine deaminase-based suicide gene therapy for SCLC and the first to show an antitumor effect from the delivery of suicide gene therapeutics for SCLC in vivo.

Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specific modification of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modification strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the efficiency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fluorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.

BCR-ABL Gene Expression Is Required for Its Mutations in a Novel KCL-22 Cell Culture Model for Acquired Resistance of Chronic Myelogenous Leukemia

Acquired resistance through genetic mutations is a common phenomenon in several cancer therapies using molecularly targeted drugs, best exemplified by the BCR-ABL inhibitor imatinib in treating chronic myelogenous leukemia (CML). Overcoming acquired resistance is a daunting therapeutic challenge, and little is known about how these mutations evolve. To facilitate understanding the resistance mechanisms, we developed a novel culture model for CML acquired resistance in which the CML cell line KCL-22, following initial response to imatinib, develops resistant T315I BCR-ABL mutation. We demonstrate that the emergence of BCR-ABL mutations do not require pre-existing BCR-ABL mutations derived from the original patient as the subclones of KCL-22 cells can form various BCR-ABL mutations upon imatinib treatment. BCR-ABL mutation rates vary from cell clone to clone and passages, in contrast to the relatively stable mutation rate of the hypoxanthine-guanine phosphoribosyltransferase gene. Strikingly, development of BCR-ABL mutations depends on its gene expression because BCR-ABL knockdown completely blocks KCL-22 cell relapse on imatinib and acquisition of mutations. We further show that the endogenous BCR-ABL locus has significantly higher mutagenesis potential than the transduced randomly integrated BCR-ABL cDNA. Our study suggests important roles of BCR-ABL gene expression and its native chromosomal locus for acquisition of BCR-ABL mutations and provides a new tool for further studying resistance mechanisms.

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated.

Intermediate frequency magnetic fields generated by an induction heating (IH) cooktop do not affect genotoxicities and expression of heat shock proteins

The aim of this study is to evaluate the effects of intermediate frequency (IF) fields generated by induction heating (IH) cooktops from the perspective of cellular genotoxicity and stress responses. We evaluated the effects of exposure to 23 kHz magnetic fields at 6.05 mT(rms) for 2 h on cellular genotoxicity and stress responses in vitro. The maximum output power in most IH cooktops is at this frequency. The magnetic flux density is approximately 1000 times higher than the reference level in the International Commission on Non-ionising Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cell growth, comet assay, micronucleus formation and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation. Heat shock protein (Hsp) 27, 70, 105 and phosphorylated Hsp27 were evaluated as indicators of the stress responses. We did not detect any effects of the IF magnetic fields on cell growth, comet assay, micronucleus formation, HPRT gene mutation, expression of phosphorylated Hsp27, or nuclear translocation of Hsp27, 70 or 105. Our results indicate that exposure to an IF magnetic field at 6.05 mT(rms) for 2 h does not cause detectable cellular genotoxicity, and does not induce detectable cellular stress.

Heterochromatinization as a Potential Mechanism of Nickel-Induced Carcinogenesis

Epigenetics refers to heritable patterns of gene expression that do not depend on alterations of the genomic DNA sequence. Nickel compounds have demonstrated carcinogenicity without any associated mutagenesis, suggesting that its mechanism of carcinogenesis is epigenetic in nature. One such potential mechanism is the heterochromatinization of chromatin within a region of the genome containing a gene sequence, inhibiting any further molecular interactions with that underlying gene sequence and effectively inactivating that gene. We report here the observation, by atomic force microscopy and circular dichroism spectropolarimetry, that nickel ion (Ni(2+)) condenses chromatin to a greater extent than the natural divalent cation of the cell, magnesium ion (Mg(2+)). In addition, we use a model experimental system that incorporates a transgene, the bacterial xanthine guanine phosphoribosyl transferase gene (gpt), differentially near, and far from, a heterochromatic region of the genome, in two cell lines, the Chinese hamster V79-derived G12 and G10 cells, respectively, to demonstrate by a DNase I protection assay that nickel treatement protects the gpt gene sequence from DNase I exonuclease digestion in the G12 cells, but not in the G10 cells. We conclude that condensation of chromatin by nickel is a potential mechanism of nickel-mediated gene regulation.

Predictive and prognostic markers for the outcome of chemotherapy in advanced colorectal cancer, a retrospective analysis of the phase III randomised CAIRO study

We have tested several biomarkers [dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), thymidylate synthase (TS) and excision cross-complementing gene (ERCC1)] for their prognostic and predictive value in relation to the outcome of chemotherapy in tumour tissues of 556 advanced colorectal cancer (ACC) patients who were randomised between sequential treatment and combination treatment in the CApecitabine, IRinotecan, Oxaliplatin (CAIRO) study. DPD expression showed a statistically significant predictive value for combination treatment with capecitabine plus irinotecan with low versus high values resulting in an improved median progression-free survival (PFS) and median overall survival (OS) of 8.9 (95% confidence interval (CI) 8.3–9.9) versus 7.2 months (95% CI 6.5–8.1, p = 0.006), and 21.5 months (95% CI 17.9–26.5) versus 16.9 months (95% CI 13.0–19.1, p = 0.04), respectively. In the overall patient population a high OPRT expression in stromal cells was a favourable prognostic parameter for OS, with 21.5 months (95% CI 17.9–27.3) versus 17.2 months (95% CI 15.1–18.6, p = 0.036), respectively. A similar effect was observed for PFS. In a multivariate analysis that included known prognostic factors these results remained significant and also showed that a high OPRT expression in tumour cells was an unfavourable prognostic parameter for PFS and OS. In conclusion, in this largest study on capecitabine with or without irinotecan to date we found a predictive value of DPD expression. Our results on the prognostic value of OPRT expression warrant further studies on the role of stromal cells in the outcome of treatments. The divergent results of ours and previous studies underscore the complexity of these biomarkers and currently prevent the routine use of these markers in daily clinical practice.

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.

Bystander effect from cytosine deaminase and uracil phosphoribosyl transferase genes in vitro: A partial contribution of gap junctions

Among gene therapy strategies elaborated to kill cancer cells, one uses the CodA gene, coding for cytosine deaminase (CD) that converts 5-fluorocytosine (5-FC) into toxic 5-fluorouracil (5-FU). To enhance 5-FC metabolic activation, we prepared a vector carrying CodA and upp (uracil phosphoribosyl transferase) genes which rendered HeLa cells sensitive to 5-FC and enhanced a bystander effect not mediated by gap junctions. However, 1% CD +–UPP + cells were able to kill 40% of the cell population if the cells were communicating. This suggests that, at very low percentages of CD +–UPP + cells, CodA and upp induce a bystander effect through gap junction-dependent mechanisms.

Fertility Alternation of Thermo-Photo-Sensitive Genic Male Sterile (TGMS) Wheat Line C412S and its Association with Adenine Phosphoribosyltransferase Gene Expression

C412S, a novel thermo-photo-sensitive genic male sterile (TGMS) wheat ( Triticum aestivum L.) line, is derived from a cross between TGMS line C49S and the conventional line C412. The objectives of this study were to examine the fertility alternation of C412S planted at different sowing dates and to investigate the possible function of APRT1 and APRT2 in the fertility alternation of thermo-photo-sensitive wheat. Under the 10 treatments of seeding date from 26 October to 10 December in 2005–2007 growing seasons, C412S showed fertility variations from complete sterility to high sterility, semisterility, low sterility, and normal fertility, which were achieved by adjusting the environmental temperature and photoperiod conditions during the development of male sex cells. Smear and squash microscopic technique was employed to follow the developmental progress of pollen mother cell (PMC) meiosis and microsporogenesis. Fertility-sensitive period of C412S was from PMC formation stage to mature pollen stage, especially from PMC formation to PMC meiosis stage. C412S pollen abortion, which belongs to spherical abortion type, reached peak level at later mononucleate stage of microsporogenesis. Moreover, the full length of adenine phosphoribosyltransferase gene ( APRT) was cloned from C412S and its control line C412. Sequences analysis showed that there were a few base differences in APRT1 between C412S and C412. However, the deducted amino acid sequences of C412S and C412 are identical. According to the results of semiquantitative reverse transcription PCR (RT-PCR), the transcriptional level of APRT genes in the young spikes of the sterile line C412S was lower than that of the fertile line C412 from PMC formation to mononucleate microspore stage. This indicates that the transcriptional level of APRT in young spikes may associate with fertility alternation in TGMS wheat.

High Orotate Phosphoribosyltransferase Gene Expression Predicts Complete Response to Chemoradiotherapy in Patients with Squamous Cell Carcinoma of the Esophagus

Objective: Chemoradiotherapy (CRT) is a possible alternative to surgery for esophageal cancer. As complete response (CR) to CRT is essential for a good prognosis, potential biomarkers predictive of CR were explored. Methods: Endoscopic tumor biopsies were obtained from 41 patients with stage II–III esophageal squamous cell carcinoma before 5-fluorouracil/cisplatin-based definitive CRT. cDNA was derived from RNA isolated from microdissected tumor cells. mRNA expression levels of 10 genes involved in CRT or tumor biology were measured using quantitative real-time RT-PCR. Results: Expression levels of orotate phosphoribosyltransferase (OPRT) and dihydrofolate reductase mRNA were significantly higher in the CR group compared with the non-CR group (p = 0.0206 and 0.0191, respectively). Matrix metalloproteinase 9 mRNA expression was significantly lower in the CR group (p = 0.0436). CR rates were significantly higher in patients with node-negative disease and high expression levels of OPRT and dihydrofolate reductase genes (p = 0.0448, 0.0104 and 0.0104, respectively). No significant difference in CR rates was observed for other variables. Multivariate analysis revealed that high OPRT gene expression was an independent predictive factor of CR (p = 0.0192). It was also significantly associated with good prognosis (p = 0.0450). Conclusion: High OPRT gene expression may be a predictive factor of CR to 5-fluorouracil/cisplatin-based CRT in esophageal cancer.

Oncolytic Gene Therapy Combined with Double Suicide Genes for Human Bile Duct Cancer in Nude Mouse Models

The prognosis of bile duct cancer is quite poor because of the low resection rate and the tolerance of the cancer to chemotherapy and radiotherapy. We investigated the feasibility of an oncolytic adenovector with two suicide genes for the treatment of bile duct cancer. We developed a new conditionally replicating adenovirus (AxE1CAUT) with the uracil phosphoribosyltransferase (UPRT) gene and the herpes simplex virus thymidine kinase (HSV-tk) gene, and compared its antitumor effects with a replication defective adenovector (AxCAUT) that has both the UPRT and HSV-tk genes. We evaluated the effects of these adenoviruses with 5-fluorouracil (5-FU) and/or ganciclovir (GCV) on human cholangiocarcinoma cells (HuCCT1, with mutant p53) in vitro and in vivo. The drug sensitivity of HuCCT1 cells to 5-FU and/or GCV was increased with an increase in the multiplicity of infection (MOI). The antitumor effect increased when 5-FU and GCV were given at the same time. Subcutaneous tumors of nude mice directly injected with AxCAUT showed a higher response to 5-FU/GCV than 5-FU or GCV alone, but there was no difference between AxCAUT and AxE1CAUT. However, AxE1CAUT with 5-FU/GCV produced a decrease in tumor weight and better survival than AxCAUT in a peritoneal dissemination model infected by intraperitoneal administration of the adenovectors. Oncolytic double suicide gene therapy is effective against human cholangiocarcinoma cells in nude mouse models.

Intermediate frequency magnetic fields generated by an induction heating (IH) cooktop do not affect genotoxicities and expression of heat shock proteins

Purpose: The aim of this study is to evaluate the effects of intermediate frequency (IF) fields generated by induction heating (IH) cooktops from the perspective of cellular genotoxicity and stress responses.Materials and methods: We evaluated the effects of exposure to 23 kHz magnetic fields at 6.05 mTrms for 2 h on cellular genotoxicity and stress responses in vitro. The maximum output power in most IH cooktops is at this frequency. The magnetic flux density is approximately 1000 times higher than the reference level in the International Commission on Non-ionising Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cell growth, comet assay, micronucleus formation and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation. Heat shock protein (Hsp) 27, 70, 105 and phosphorylated Hsp27 were evaluated as indicators of the stress responses.Results: We did not detect any effects of the IF magnetic fields on cell growth, comet assay, micronucleus formation, HPRT gene mutation, expression of phosphorylated Hsp27, or nuclear translocation of Hsp27, 70 or 105.Conclusions: Our results indicate that exposure to an IF magnetic field at 6.05 mTrms for 2 h does not cause detectable cellular genotoxicity, and does not induce detectable cellular stress.