Hypoxanthine Phosphoribosyltransferase Locus Research Articles

Targeted Knock-in of Transgenes into the CHO Cell Genome Using CRISPR-mediated Integration Systems

Biopharmaceutical proteins are usually produced by culturing recombinant Chinese hamster ovary (CHO) cells. High producer cell lines are screened from transfected cells with random integration of target genes. Since transgene expression is susceptible to the surrounding environment of the integrated genomic locus, producer cell lines should be selected from a large number of recombinant cells with heterogeneous transgene insertion. In contrast, targeted integration into a characterized genomic locus allows for predictable transgene expression and less clonal variability, and thus stable production of target proteins can be expected. Genome editing technology based on programmable nucleases has recently emerged as a versatile tool for precise editing of target locus in the cell genome. Here, we demonstrated targeted knock-in of transgenes into the hypoxanthine phosphoribosyltransferase (hprt) locus of CHO cells using CRISPR/Cas9 and CRISPR-mediated precise integration into target chromosome (PITCh) systems. We also generated knock-in CHO cells based on the homology-independent targeted integration (HITI) system. We evaluated the knock-in efficiency of transgenes into the hprt locus using these systems.

Determination of Mutational Spectra Induced by Environmental Toxicants in Complex Human Cell Populations.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and have potent mutagenic and carcinogenic activities. Studies of mutations induced by these compounds in human cells can help acquire an understanding of their mutagenic pathways. In this chapter, independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with benzo(a)pyrene (BaP) or dibenzo(a,l)pyrene (DBP), and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine resistance (6TGR). The kinds and positions of the mutations occurring in the third exon of the HPRT gene were analyzed in the mixed HPRTR mutant cell populations using a combination of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Mutant bands were excised from the gel, amplified using PCR, and sequenced to determine the kinds and positions, or spectrum of mutations.

ATR Kinase Activity Limits Mutagenesis and Promotes the Clonogenic Survival of Quiescent Human Keratinocytes Exposed to UVB Radiation

The ATR protein kinase has well-described roles in maintaining genomic integrity during the DNA synthesis phase of the cell cycle. However, ATR function in cells that are not actively replicating DNA remains largely unexplored. Using HaCaT and telomerase-immortalized human keratinocytes maintained in a confluent, nonreplicating state in vitro, ATR was found to be robustly activated in response to UVB radiation in a manner dependent on the nucleotide excision repair factor and DNA translocase XPB. Inhibition of ATR kinase activity under these conditions negatively impacted acute cell survival and cytotoxicity and severely inhibited the ability of UVB-irradiated HaCaT keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiated UVB mutagenesis at the hypoxanthine phosphoribosyltransferase locus. Though ATR inhibition did not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA, elevated levels of PCNA mono-ubiquitination and chromatin-associated PCNA and RPA indicate that excision gap-filling synthesis was altered in the absence of ATR signaling. These results indicate that the ATR kinase plays important roles in preventing mutagenesis and in promoting the proliferative potential of quiescent keratinocytes exposed to UVB radiation.

Role of Human N-Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen-Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay.

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc.

ATR kinase inhibition sensitizes quiescent human cells to the lethal effects of cisplatin but increases mutagenesis

The ATR protein kinase is known to protect cells from DNA damage induced during the replicative phase of the cell cycle. Small molecule ATR kinase inhibitors have therefore been developed to improve the effectiveness of DNA damage-based chemotherapy regimens aimed at killing rapidly proliferating tumor cells. However, whether ATR functions in a similar manner in non-replicating cells has not been examined and is important considering the fact that most cells in the body, including cancer stem cells in solid tumors, normally reside in either a quiescent or differentiated non-replicating state. Using cultured human cell lines maintained in a quiescent or slowly growing state in vitro, ATR was found to be activated following treatment with the common anti-cancer drug cisplatin in a manner dependent on the nucleotide excision repair (NER) system. Moreover, treatment with the ATR kinase inhibitors VE-821 and AZD6738 enhanced quiescent cell killing and apoptotic signaling induced by cisplatin. However, ATR kinase inhibition in quiescent cells treated with a low concentration of cisplatin also elevated the level of mutagenesis at the hypoxanthine phosphoribosyltransferase locus and resulted in increased levels of PCNA mono-ubiquitination. These results suggest that the excision gaps generated by NER may require a greater utilization of potentially mutagenic translesion synthesis polymerases in the absence of ATR kinase function. Thus, though ATR kinase inhibitors can aid in the killing of cisplatin-treated quiescent cells, such treatments may also result in a greater reliance on alternative mutagenic DNA polymerases to complete the repair of cisplatin-DNA adducts.

Cre-Mediated Transgene Integration in Chinese Hamster Ovary Cells Using Minicircle DNA Vectors.

Bacterial backbone sequences of conventional plasmid vectors have been reported to exhibit negative effects on transgene expression in mammalian cells, such as cytotoxicity and gene silencing. Minicircle DNA vectors can be employed to overcome these issues and to improve the transfection efficiency because of their smaller size. In this study, transgenes are integrated into the hypoxanthine phosphoribosyltransferase (hprt) locus of Chinese hamster ovary (CHO) cells by the Cre-loxP system using minicircle DNA vectors as transgene donors. The targeted transgene integration efficiency is improved 2-3-fold (≈1.4%) using minicircle DNA vectors compared with conventional plasmid vectors. Moreover, clones with expected structures after transgene integration are obtained with a high frequency. When a transgene together with bacterial sequences derived from a plasmid vector is integrated into the hprt locus, the cell growth rate and antibody titer decrease. These results indicate that minicircle DNA vectors are more suitable than conventional plasmid vectors for transgene delivery in recombinant protein production using CHO cells.

X-Ray Induced Mutation Frequency at the &amp;lt;i&amp;gt;Hypoxanthine Phosphoribosyltransferase&amp;lt;/i&amp;gt; Locus in Clinically Relevant Radioresistant Cells

To elucidate the molecular mechanisms underlying cellular radioresistance, clinically relevant radioresistant cell lines were established via long-term exposure to X-rays with stepwise dose escalation. Established cells continue to proliferate despite exposure to 2 Gy X-rays/day for more than 30 days, a standard protocol in cancer radiotherapy. DNA repair fidelity in radioresistant and the parental cells by evaluating the mutation frequency at the hypoxanthine phosphoribosyltransferase (HPRT) locus after exposure to X-rays was determined. Mutation spectrum at the HPRT locus was examined by multiplex polymerase chain reaction. Rejoining kinetics of X-ray-induced DNA double strand breaks (dsbs) was evaluated by the detection of phosphorylated histone H2AX (γH2AX) after X-irradiation. The fold increase in the HPRT mutation frequency due to acute radiation was similar between radioresistant and the parental cell lines. However, fractionated radiation (FR) consisting of 2 Gy X-rays/day increased the mutation frequency at the HPRT locus in parental but not in radioresistant cells. Analysis of the FR-induced mutations at the HPRT locus revealed a high frequency of deletion mutations (>70%) in parental but not in radioresistant cells. As assessed by γH2AX immunostaining, DNA dsbs induced by acute exposure to 10 Gy of X-rays were repaired to the control level within 7 days in radioresistant but not in the parental cells. Moreover, 2 Gy × 5 FR increased the number of γH2AX-positive cells in parental cultures but not in radioresistant cultures. DNA dsbs induced by 2 Gy/day FR are repaired with fidelity in radioresistant but not in parental cells.

344. Targeting piggyBac Transposition Into a User-Defined Chromosomal Locus in Human Cells

The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into the genomes of human cells. We have previously demonstrated biased PB integration into an artificial target site engineered into human cells (Kettlun et al., Molecular Therapy, 2011). We undertook the current investigation to see if we could engineer PB to target integration into a user-defined chromosomal locus in human cells. We chose to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on chromosome X. HPRT-deficient cells can be selected by growth in 6-thioguanine (6TG), so PB-mediated targeted knockout of HPRT can be used to isolate cells with targeted integration events. We used HT-1080 human fibrosarcoma cells which harbor one × chromosome for our studies. We engineered a series of 4 zinc finger proteins (ZFP) and 4 transcription activator-like (TAL) proteins fused to the PB transposase. The transposon used contained a splice-acceptor sequence followed by a beta-galactosidase-neomycin resistance fusion gene (b-Geo) which allowed measurement of gene transfer activity (measuring neomycin resistant colonies after transposition) as well as HPRT knockout efficiency (due to the splice-acceptor sequence). All HPRT-targeted ZFP-PB and TAL-PB proteins exhibited transposition activity. Chromatin immunoprecipitation assays revealed varied targeting-binding of the different PB chimeras to the HPRT locus. One ZFP-PB and one TAL-PB chimera demonstrated higher HPRT gene targeting via reproducible production of a higher number of 6TG resistant cells. Deep sequencing analysis revealed integration efficiency of 0.42% and 1% for the ZFP-PB and TAL-PB chimeric transposases in 6TG-resistant HT-1080 cells. Our study provides new insights into engineering PB to target locations in the human genome which will permit further refinements to improve targeting efficiency. Additionally, selection of cells with targeted integration events should allow isolation of “targeted-only” cells for consideration of therapeutic purposes.ss

Evaluation of the cytotoxic and mutagenic potential of three ginkgolic acids.

Ginkgolic acids (GAs) are alkylphenols which can be found in the fruits and leaves of Ginkgo biloba L. (Ginkgoaceae) used in herbal teas, drugs and food supplements. Standardized leaf extracts of G. biloba are widely used in the therapy of cognitive decline including Alzheimer's diseases. However, GAs are known to have cytotoxic and allergenic potential and are suspected to possess genotoxic properties. Therefore, we examined in this study the cytotoxicity and mutagenicity of three major GAs with different alkyl or alkenyl groups (13:0, 15:1, 17:1). Cytotoxicity was assessed in male Chinese hamster lung fibroblasts (V79 cells) using the resazurin reduction assay. The substances showed concentration dependent cytotoxic effects after 24h of incubation at concentrations of 50μM and higher. Mutagenicity was determined by using the Ames fluctuation assay in different Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) with and without exogenous metabolic activation (S9 mix). Furthermore, we analyzed the mutagenic potency of the three major GAs in V79 cells by performing the hypoxanthine phosphoribosyl transferase (HPRT) assay which detects gene mutations at the HPRT locus. None of the mutagenic assays showed any increase in mutagenicity above background. Therefore, these data provide evidence that the GAs tested have some cytotoxic potency but are not mutagenic. Thus, our findings contribute to the risk assessment of preparations containing plant extracts from G. biloba.

Abstract 5457: Cytarabine leaves a mutational fingerprint in relapsing leukemia

Abstract Cytarabine (1-β-D-arabinofuranosylcytosine; Ara-C) forms the backbone of chemotherapeutic regimes for acute myeloid leukemia (AML) and has a high success rate in inducing complete remission. However, the majority of AML patients will relapse within 2 years, often with an aggressive, chemoresistant form of the disease. Mechanisms associated with the development of relapsed disease remain to be fully elucidated, although recent whole-genome deep sequencing data derived from matched primary AML-relapse pairs demonstrated that relapse is associated with gain of novel mutations not seen at initial diagnosis [1] . Furthermore, the nature of relapse-specific mutations suggests these to be shaped, at least in part, by the chemotherapy used to induce and maintain remission. In order to investigate this further we have assessed the mutagenicity of Ara-C. We show here that the frequency of induction of mutations at thymidine kinase (TK) and hypoxanthine phosphoribosyltransferase (HPRT) loci in vitro is increased following exposure to Ara-C, confirming this agent to be mutagenic in human cells. In addition, molecular characterization of Ara-C-induced mutations at the HPRT locus revealed that this commonly used nucleoside analogue induces a mutational fingerprint in vitro that can also be identified in vivo. Specifically, our data demonstrate that Ara-C is able to induce both deletion and base substitution mutations and has a strong propensity to induce mutations at 5′TGA3′/5′TCA3′ sequences in the genome, and particularly at the central G:C position consistent with its function as a cytosine analogue (p&amp;lt;0.001). Subsequent analysis of 4039 somatic AML mutations (3587 at disease presentation and 452 at relapse) derived from genome-wide deep sequencing data revealed that the frequency of putative Ara-C-associated G:C mutations in 5′TGA3′/5′TCA3′ sequences was significantly higher in AML relapse following Ara-C therapy compared to chemotherapy-naïve leukemia at initial presentation (OR 2.2, 95% CI 1.4-3.6; p=0.003). Given that most cancer patients die from progressive disease, understanding the etiology of somatic mutations responsible for driving the evolution of relapse is a substantial challenge in the management of malignant disease. Although long suspected to exist, our study is the first to identify a chemotherapy-induced mutational fingerprint in a relapsing cancer and demonstrates that chemotherapy plays a role in driving evolution of the somatic cancer genome. The approaches we present could be used to discern the contribution of other treatments to evolution of relapsed disease, informing both personalized medicine and drug development strategies. [1] Ding L et al. Nature 2012; 481(7382):506-10 Citation Format: Sarah E. Fordham, Michael Cole, Julie A. Irving, James M. Allan. Cytarabine leaves a mutational fingerprint in relapsing leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2014-5457

Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat.

Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease

The thiopurines azathioprine and 6-mercaptopurine (6-MP) are effective immune modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the hypoxanthine phosphoribosyltransferase (HPRT) locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). ANOVA and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment, and total therapeutic dose of azathioprine and 6-MP. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared with 63 subjects not treated with thiopurines. This increase was related to both total dose (P < 0.001) and duration of treatment (P < 0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (P < 0.001), particularly G:C to A:T transitions (P < 0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation, and T-cell receptor beta CDR3 region unique gene sequence also showed a significant thiopurine-dependent increase in mutant cell clonal proliferation (P < 0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Integrin-mediated axoglial interactions initiate myelination in the central nervous system

All but the smallest-diameter axons in the central nervous system are myelinated, but the signals that initiate myelination are unknown. Our prior work has shown that integrin signaling forms part of the cell–cell interactions that ensure only those oligodendrocytes contacting axons survive. Here, therefore, we have asked whether integrins regulate the interactions that lead to myelination. Using homologous recombination to insert a single-copy transgene into the hypoxanthine phosphoribosyl transferase (hprt) locus, we find that mice expressing a dominant-negative β1 integrin in myelinating oligodendrocytes require a larger axon diameter to initiate timely myelination. Mice with a conditional deletion of focal adhesion kinase (a signaling molecule activated by integrins) exhibit a similar phenotype. Conversely, transgenic mice expressing dominant-negative β3 integrin in oligodendrocytes display no myelination abnormalities. We conclude that β1 integrin plays a key role in the axoglial interactions that sense axon size and initiate myelination, such that loss of integrin signaling leads to a delay in myelination of small-diameter axons.

The impact of NAT2 acetylator genotype on mutagenesis and DNA adducts from 2-amino-9H-pyrido[2,3-b]indole.

2-amino-9H-pyrido[2,3-b]indole (AalphaC) is a carcinogenic heterocyclic aromatic amine (HAA) that is produced in high quantities in tobacco smoke and that also forms in charred meats. The bioactivation of AalphaC occurs by cytochrome P450-mediated (P450 1A2) N-oxidation of the exocyclic amine group, to form 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AalphaC). The HONH-AalphaC metabolite can then undergo further activation by phase II enzymes to form the penultimate ester species, which bind to DNA. Some epidemiological studies suggest a role for NAT2 genetic polymorphisms in human susceptibilities to various cancers from tobacco smoke and from consumption of well-done meats, where the exposures to AalphaC can be substantial. In this investigation, we have measured the genotoxicity of AalphaC in nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human P450 1A2 and either the NAT2*4 (rapid, wild-type acetylator) or the NAT2*5B (the most common slow acetylator) allele, to determine the role of NAT2 phenotype in the biological effects of AalphaC. Mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus were induced in a dose-dependent manner by AalphaC and were found to be highest in cells transfected with P450 1A2 and NAT2*4, followed by cells transfected with P450 1A2 and NAT2*5B. The level of formation of the deoxyguanosine (dG) adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AalphaC) paralleled the mutagenic potency in these cell lines. However, AalphaC did not form DNA adducts or induce mutations in untransfected CHO cells or in cells only expressing P450 1A2. These findings clearly demonstrate that NAT2 genetic polymorphism plays a major role in the genotoxic potency of AalphaC.

Targeted transgenesis at the HPRT locus: an efficient strategy to achieve tightly controlled in vivo conditional expression with the tet system

The tet-inducible system has been widely used to achieve conditional gene expression in genetically modified mice. To alleviate the frequent difficulties associated with recovery of relevant transgenic founders, we tested whether a controlled strategy of transgenesis would support reliable cell-specific, doxycycline (Dox)-controlled transgene expression in vivo. Taking advantage of the potent hypoxanthine-aminopterin-thymidine selection strategy and an embryonic stem (ES) cell line supporting efficient germ-line transmission, we used hypoxanthine phosphoribosyltransferase (HPRT) targeting to insert a single copy tet-inducible construct designed to allow both glucocorticoid receptor (GR) and beta-galactosidase (beta-Gal) expression. Conditional, Dox-dependent GR and beta-Gal expression was evidenced in targeted ES cells. Breeding ES-derived single copy transgenic mice with mice bearing appropriate tet transactivators resulted in beta-Gal expression both qualitatively and quantitatively similar to that observed in mice with random integration of the same construct. Interestingly, GR expression in mice was dependent on transgene orientation in the HPRT locus while embryonic stem cell expression was not. Thus, a conditional construct inserted in single copy and in predetermined orientation at the HPRT locus demonstrated a Dox-dependent gene expression phenotype in adult mice suggesting that controlled insertion of tet-inducible constructs at the HPRT locus can provide an efficient alternative strategy to reproducibly generate animal models with tetracycline-induced transgene expression.

A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

The human angiotensinogen (hAGT) gene contains an A/G polymorphism at -217, and frequency of -217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant -217A almost always occurs with -532T, -793A, and -1074T, whereas variant -217G almost always occurs with -532C, -793G, and -1074G. Since allele -6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, -6A:-217A (AA) and -6A:-217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by approximately 40%, and plasma angiotensin II level is increased by approximately 50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure.

Generation of mice with conditionally activated transforming growth factor beta signaling through the TβRI/ALK5 receptor

We generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a latent constitutively active TGFbeta type I receptor (TbetaRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken beta-actin) promoter, is repressed by a floxed transcriptional "Stop" (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the "Stop" is excised to allow TbetaRI(CA) transgene expression. We showed that restricted expression of TbetaRI(CA) in T lymphocytes efficiently activates TGFbeta signaling and rescues the T-cell autoimmune disorders of TGFbetaRII conditional knockouts. Unexpectedly, our study reveals that TGFbeta signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFbeta effects on T-cell biology, LSL-TbetaRI(CA) mouse constitutes an attractive tool to address the effect of TGFbeta signaling upregulation in any cell type expressing the cre-recombinase.

Assessment of the genotoxicity of the rat carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat liver epithelial cells in vitro

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a disinfection by-product in chlorinated drinking water, is a multisite carcinogen in rats. One main target organ is the liver. The mechanism of the tumorigenicity was evaluated by testing the genotoxicity of MX in rat liver epithelial cell line cells. In the studies, the single cell gel/Comet assay and the hypoxanthine phosphoribosyl transferase locus assay to 6-thioguanine resistance were used. MX induced a dose-related genotoxic response in the comet assay. The lowest effective concentration was 120 microM when the exposure was in medium plus supplements and 3.75 microM when the exposure was in phosphate-buffered salt solution. MX also increased the frequency of TG(r) mutants, when the cells were treated in phosphate-buffered salt solution, at a concentration range of 2.3-9.2 microM. The present results show for the first time that MX causes DNA damage and gene mutations in rat liver epithelial cells, the target cells of MX's tumorigenicity in rats. We have earlier shown that MX also inhibits gap junctional intercellular communication in the same cells. The genotoxic effects were induced starting at about 60 times higher concentration, in identical exposure conditions, compared with the lowest concentration of MX causing the tumor promoter effect.

Observation of radiation-specific damage in cells exposed to depleted uranium: hprt gene mutation frequency

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. Recent animal studies have also shown that DU is leukemogenic and genotoxic. DU possesses both a radiological (alpha particle) and chemical (metal) component. Since DU has a low specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. The potential contribution of radiation to DU-induced biological effects is unknown, and the involvement of radiation in DU-induced biological effects could have significant implications for current risk estimates for internalized DU exposure. The purpose of the current study was to measure the induction of mutagenic damage in V79 cells and to determine if radiation plays a role in the induction of that damage. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. There was a dose-dependent increase in mutagenic response following DU exposure ( 10 – 50 μ m ) ; the average increase in mutagenicity above background ranged from 2.54 ± 1.19 to 8.75 ± 1.8 ( P < 0.05 ) . Using the same concentration ( 25 μ M ) of two uranyl nitrate compounds that have different uranium isotopic concentrations and, therefore, different specific activities, we examined the effect on hprt mutant frequency in vitro. V79 cells were exposed to either U 238 -uranyl nitrate, specific activity 0.33 μ Ci / g , or DU-uranyl nitrate, specific activity 0.44 μ Ci / g , delivered at a concentration of 25 μ M for 24 h. Results showed, that at equal uranium concentration, a 1.33-fold increase in specific activity resulted in a 1.27 ± 0.11 -fold ( P < 0.05 ) increase in hprt mutant frequency. Taken together these data support earlier results showing that radiation can play a role in DU-induced biological effects in vitro.

2‐amino‐1‐methyl‐6‐phenylimidazo [4,5‐b] pyridine‐induced DNA adducts and genotoxicity in chinese hamster ovary (CHO) cells expressing human CYP1A2 and rapid or slow acetylator N‐acetyltransferase 2

Heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) are present in diet and cigarette smoke. Bioactivation in humans includes N-hydroxylation catalyzed by cytochrome P4501A2 possibly followed by O-acetylation catalyzed by N-acetyltransferase 2 (NAT2). Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. CYP1A2 and NAT2 catalytic activities were undetectable in untransfected CHO cell lines. CYP1A2 catalytic activity levels did not differ significantly (P > 0.05) among the CYP1A2-transfected cell lines. Cells transfected with NAT2*4 had significantly higher levels of N-acetyltransferase (P = 0.0001) and N-hydroxy-PhIP O-acetyltransferase (P = 0.0170) catalytic activity than cells transfected with NAT2*5B. PhIP caused dose-dependent decreases in cell survival and significant (P < 0.001) increases in mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in all the CYP1A2-transfected cell lines. Transfection with NAT2*4 or NAT2*5B did not further increase hprt mutagenesis. PhIP-induced hprt mutant cDNAs were sequenced, and 80% of the mutations were single base substitutions at G:C base pairs. dG-C8-PhIP DNA adduct levels were dose-dependent in the order: untransfected < transfected with CYP1A2 < transfected with CYP1A2 and NAT2*5B < transfected with CYP1A2 and NAT2*4. Following incubation with 1.2 microM PhIP, DNA adduct levels were significantly (P < 0.05) higher in CHO cells transfected with CYP1A2/NAT2*4 versus CYP1A2/NAT2*5B. These results strongly support an activation role for CYP1A2 in PhIP-induced mutagenesis and DNA damage and suggest a modest effect of human NAT2 and its genetic polymorphism on PhIP DNA adduct levels.