Hprt Locus Research Articles

Assessment of Biological Effect of Tritiated Water by Using Hypersensitive System

An exposure of human or animals to tritium radiation from nuclear fusion reactor is expected to be a low dose and with low dose-rate. We are focusing on the biological effects of tritiated water (HTO) given at low dose and/or with low dose rate. Hypersensitive assay systems for radiation biological experiments using cultured mammalian cells or transgenic mice have been established and their availability for assessment of biological effects of HTO were tested. A hypersensitive detection system for mutagenesis at Hprt locus was established by using hamster cells carrying a human X-chromosome. The cells present more than 50-fold sensitivity for Hprt-deficient mutation, and this allowed us to detect a significant elevation of mutation frequency by radiations at the dose of less than 0.2 Gy.Because the reverse dose rate effect has been reported for mutation induction by high LET radiation such as neutrons, we first tested whether the reverse dose rate effects could be seen for HTO (tritium beta-rays) or not. No significant change in mutation frequency by HTO was observed in the range of dose rates between 0.05 cGy/h and 2.0 cGy/h, whereas clear reverse dose rate effects was observed in the case of fission neutrons or carbon beam. The result suggests that the reverse dose rate effect does not apply in the case of mutation induction by HTO. In addition, mutation spectrum in the mutants induced by HTO was similar to those in spontaneously induced mutants, suggesting that exposure to a certain level of HTO could enhance the spontaneous mutagenesis.

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

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

Abstract 4195: Mechanisms involved in the HIF1 mediated acceleration of chemical carcinogenicity

Abstract Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens produced by the incomplete combustion of organic materials. The best studied PAH is the environmentally ubiquitous benzo[a]pyrene (B[a]P) whose mutagenic and carcinogenic potential has been demonstrated in both humans and animals. Recently we have demonstrated that activation of the transcription factor HIF1α amplifies the B[a]P induced mutagenic phenotype. More specifically, HIF1α appears to sequester a critical cofactor for carcinogen detoxification, HIF1β/ARNT. Reducing the availability of this cofactor for coupling with the aryl hydrocarbon receptor (AhR) results in decreased expression of enzymes responsible for detoxifying and removing the carcinogen. The primary mode of HIF1α activation is by low oxygen concentrations. Although not commonly observed in normal tissue, hypoxia can be induced by pathologies such as chronic obstructive pulmonary disease (COPD). Thus hypoxia may offer an attractive explanation for the observed increase in cancer incidence in patients with COPD. A secondary means to stabilize HIF1α is by oncogene activation or tumour suppressor loss. This mechanism is independent of oxygen levels and implies that HIF1α may play an important role in the progression of premalignant cells into a tumour. Although we observe a 3 fold increase in mutation frequency at the hprt locus in cells after B[a]P exposure under hypoxic conditions, the changes in DNA adduct formation are minimal. This suggests that other cellular response pathways are modified concurrently with HIF1α activation. We have identified three important cellular mechanisms that may be responsible for the increase in B[a]P mutagenicity upon HIF1α activation. These are changes to the metabolism of B[a]P; the transmembrane transport of B[a]P or its metabolites; or the capacity to repair the subsequent DNA damage. First, under conditions of low oxygen, the B[a]P-9,10-dihydrodiol metabolites decreased while the B[a]P-7,8-dihydrodiol and 3-OH-B[a]P metabolites are formed in higher quantities compared to normoxia. Furthermore, when HIF was upregulated either by hypoxia (0.2% O2) or CoCl2, the extracellular concentrations of the parent B[a]P compound were dramatically increased compared with cells treated under higher (20% and 5%) oxygen concentrations. And finally, using a modified comet assay to determine the DNA repair capacity, we observe that cells in which HIF1α is dysregulated (VHL mutants) have an approximately 10 fold decrease in the ability to repair B[a]P induced adducts. Taken together, these results show that all three of these processes may in part be responsible for the enhanced mutagenic phenotype under hypoxia. Insight into these pathways may provide further understanding of the intrinsic risks for mutagenic exposures and provide new targets and tools for therapies directed against cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4195. doi:10.1158/1538-7445.AM2011-4195

Vascular bed–specific regulation of the von Willebrand factor promoter in the heart and skeletal muscle

AbstractA region of the human von Willebrand factor (VWF) gene between −2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between −843 and −620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at −56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed–specific expression of VWF.

Abstract P5-05-15: Transgenic Mice Expressing Either p200, p110 or p75 CUX1 Isoform Develop Mammary Tumors of Various Histological Types

Abstract Short isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. The full-length CUX1 protein, p200 CUX1, is proteolytically processed into p110 CUX1 by a nuclear isoform of cathepsin L. Another isoform, p75 CUX1, was found to be aberrantly expressed in breast tumors. The full-length and short CUX1 isoforms exhibit distinct biochemical and cellular activities. They bind DNA differently and regulate transcription in different ways. Over-expression of p110 or p75 CUX1 accelerates the start of DNA replication, promotes genetic instability and stimulates cell motility. In contrast, the full-length p200 CUX1 isoform appears to be inactive in these cell-based assays. To assess and compare the ability of the full-length and short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the hprt locus to generate transgenic mice expressing p200, p110 or 75 CUX1 under the control of the mouse mammary tumor virus (MMTV) long terminal repeat. We report that mammary tumors of various histopathologies developed in all transgenic lines after a long latency period. Mammary tumors were classified as solid carcinomas with or without papillary differentiation, adenosquamous carcinomas, adenomyoepithelioma or tubular/acinar adenoma and many displayed pathophysiological features similar to that of human basal-like breast cancers. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, while higher expression of various Wnt genes and activation of the β-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, shRNA-mediated knockdown and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/β-catenin autocrine loop. To our surprise, transgenic mice expressing p200 CUX1 developed mammary tumors with a penetrance equivalent to that of p75 and p110 CUX1 transgenic mice. Preliminary results showed that the expression and activity of p110 CUX1 was elevated in tumors from p200 CUX1 transgenics. Accordingly, cathepsin L was over-expressed in 7 out of 8 tumors from p200 CUX1 transgenic mice, but in only 1 out 11 tumors from p110 CUX1 mice. Moreover, in a p200 CUX1 tumor an activating point mutation has been identified at codon 61 of the k-ras gene. Activation of the ras pathway has previously been shown to lead to over-expression of cathepsin L. These results suggest that molecular events that lead to cathepsin L overexpression cooperate in tumorigenicity by stimulating the proteolytic processing of p200 CUX1 into the active p110 CUX1 isoform. Overall, these results confirm the oncogenic potential of CUX1 in the mammary gland and reveal some of the cooperating events and molecular pathways involved in mammary tumorigenesis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-15.

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.

1-Methoxy-3-indolylmethyl glucosinolate; a potent genotoxicant in bacterial and mammalian cells: Mechanisms of bioactivation

1-Methoxy-3-indolylmethyl (1-MIM) glucosinolate, contained in many Brassica vegetables, is strongly mutagenic in Salmonella typhimurium TA100 when activated by myrosinase. Here, we describe the synthesis and evaluation of two breakdown products, 1-MIM nitrile and 1-MIM alcohol. 1-MIM nitrile was not mutagenic and 1-MIM alcohol showed low direct mutagenicity in TA100, indicating that other breakdown products mediated the mutagenicity of 1-MIM glucosinoate/myrosinase in this strain. However, 1-MIM alcohol was strongly mutagenic to a TA100-derived strain expressing human sulphotransferase SULT1A1. Likewise, 1-MIM glucosinolate (with myrosinase) showed 10 times higher mutagenic activity in TA100-SULT1A1 than in strain TA100. Identical adducts, N 2-(1-MIM)-dG and N 6-(1-MIM)-dA, were detected in both strains, but the levels were higher in TA100-hSULT1A1. A similar influence of SULT1A1 was observed in recombinant V79-hSULT1A1 cells compared to parental SULT-deficient Chinese hamster V79 cells. 1-MIM glucosinolate (with myrosinase) as well as 1-MIM alcohol induced sister chromatid exchange in both cell lines, but with clearly higher efficiency in V79-hSULT1A1 cells. Gene mutation assays were conducted at the HPRT locus with 1-MIM alcohol in V79-hSULT1A1 cells, and with 1-MIM glucosinolate/myrosinase in V79 parental cells. In both cases, the result was clearly positive. Thus, 1-MIM glucosinolate is mutagenic in bacterial and mammalian cells via at least two different metabolites.

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-beta superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-beta pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.

Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex, a specific inducer of N3-methyladenine

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased ( p = 0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50 μМ Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939 ± 0.182) was approximately 10-fold higher than in CHO-9 (0.129 ± 0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25 μM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.

Report From the Jerusalem Workshop on Lynch Syndrome-Hereditary Nonpolyposis Colorectal Cancer

A Workshop was held in Jerusalem, Israel, on October 26 and 27, 2009 to discuss the management of Lynch syndrome-hereditary nonpolyposis colorectal cancer (CRC), with the primary goal to develop consensus for the optimal management of this disease. A second goal was to identify areas of research with the potential to advance the clinical management of Lynch syndrome. The perspectives and recommendations from the workshop are meant to be a platform for discussion and deliberation. The Workshop was organized by Moshe Shike (Memorial Sloan Kettering Cancer Center, New York) and sponsored by The Colon Cancer Foundation. More details of each presentation are available in an on-line supplement.

Conditional expression of tissue-specific antigen regulates CD8 T cell tolerance induction (101.3)

Abstract We have developed a carefully-designed transgenic model to learn how expression of an autoantigen influences the degree and nature of tolerance. The surrogate antigen, directed to thyroid by a tissue-specific promoter, consists of a cell-surface glycoprotein (human interleukin-2 receptor α chain, Tac) to which well-defined B cell, CD8+ T cell, and CD4+ T cell epitopes have been appended in order to assess all three lymphocyte subset responses. A LoxP-flanked stop cassette was inserted between the tissue-specific promoter and the transgene open reading frame (ORF) to allow for conditional expression. To avoid positional and copy number effects in traditional transgenesis, the transgene was targeted to HPRT locus with a single-copy insertion. Here we show that embryonic but not adult expression of Tac transgene results in a profound epitope-specific CD8 T cell tolerance. However, prolonged expression of postnatal antigen is able to facilitate tolerance induction. Moreover, the ability to induce Tac-specific immune tolerance correlates with the loss of susceptibility to rejection of Tac-expressing tumor cells. These findings provide direct evidence of dynamic tolerance induction to normal or tumor antigens.

Small scale genetic alterations contribute to increased mutability at the X-linked Hprt locus in vivo in Blm hypomorphic mice

BLM, the gene mutated in Bloom syndrome (BS), encodes an ATP-dependent RecQ DNA helicase that is involved in the resolution of Holliday junctions, in the suppression of crossovers and in the management of damaged replication forks. Cells from BS patients have a characteristically high level of sister chromatid exchanges (SCEs), and increased chromosomal aberrations. Fibroblasts and lymphocytes of BS patients also exhibit increased mutation frequency at the X-linked reporter gene HPRT, suggesting that BLM also plays a role in preventing small scale genomic rearrangements. However, the nature of such small scale alterations has not been well characterized. Here we report the characterization of Hprt mutations in vivo in Blm hypomorphic mice, Blm tm1Ches / Blm tm3Brd . We found that the frequency of Hprt mutants was increased about 6-fold in the Blm tm1Ches / Blm tm3Brd mice when compared to Blm tm3Brd heterozygous mice or wildtype mice. Molecular characterization of Hprt gene in the mutant clones indicates that many of the mutations were caused by deletions that range from several base pairs to several thousand base pairs. While deletions in BLM-proficient somatic cells are often shown to be mediated by direct repeats, all three deletion junctions in Hprt of Blm tm1Ches / Blm tm3Brd mice were flanked by inverted repeats, suggesting that secondary structures formed during DNA replication, when resolved improperly, may lead to deletions. In addition, single base pair substitution and insertion/deletion were also detected in the mutant clones. Taken together, our results indicated that BLM function is important in preventing small scale genetic alterations. Thus, both large scale and small scale genetic alterations are elevated when BLM is reduced, which may contribute to loss of function of tumor suppressor genes and subsequent tumorigenesis.

Efficient integration of transgenes into a defined locus in human embryonic stem cells

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.

Differential roles for ETS, CREB, and EGR binding sites in mediating VEGF receptor 1 expression in vivo

AbstractVascular endothelial growth factor receptor 1 (VEGFR1) is a marker for endothelial-specific gene expression. We previously reported that the human VEGFR1 promoter (between −748 and +284) contains information for expression in the intact endothelium of transgenic mice. The objective of this study was to dissect the cis-regulatory elements underlying VEGFR1 promoter activity in vitro and in vivo. In primary endothelial cells, binding sites for E74-like factor 1 (ELF-1; between −49 and −52), cyclic adenosine monophosphate response element binding (CREB; between −74 and −81), and early growth response factor 1/3 (EGR-1/3; between −16 to −25) were shown to play a positive role in gene transcription, whereas a putative E26 transformation-specificsequence (ETS) motif between −36 and −39 had a net negative effect on promoter activity. When targeted to the Hprt locus of mice, mutations of the ELF-1 binding site and the CRE element reduced promoter activity in the embryonic vasculature and resulted in a virtual loss of expression in adult endothelium. Postnatally, the EGR binding site mutant displayed significantly reduced promoter activity in a subset of vascular beds. In contrast, mutation of the −39 ETS site resulted in increased LacZ staining in multiple vascular beds. Together, these results provide new insights into the transcriptional regulatory mechanisms of VEGFR1.

Anomalous genotoxic responses induced in mouse lymphoma L5178Y cells by potassium bromate

Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.

Mouse Mammary Tumor Virus p75 and p110 CUX1 Transgenic Mice Develop Mammary Tumors of Various Histologic Types

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the beta-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA-mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/beta-catenin autocrine loop.

High frequency of genomic deletions induced by Me-lex, a sequence selective N3-adenine methylating agent, at the Hprt locus in Chinese hamster ovary cells

We have investigated the mutagenicity induced at the Hprt locus in Chinese hamster ovary (CHO) cells treated with increasing concentrations of Me-lex, a minor groove selective methylating agent that efficiently generates more than 90–95% of 3-MeA DNA adducts. Me-lex treatment was cytotoxic but weakly mutagenic, resulting in up to 7-fold induction above background in the Hprt mutation frequency. The molecular nature of 43 Hprt mutations induced by Me-lex was determined by sequence analysis of the Hprt cDNA and genomic analysis of the gene locus. Base pair substitutions represented about 25% of Me-lex induced mutations. The mutation spectrum revealed a high percentage of genomic deletions (51%) comprising single/multiple exon(s) and even the loss of the complete locus. When the distribution of mutations among different classes was considered, the difference between the spontaneous and Me-lex induced CHO spectra was statistically significant ( p < 0.012), indicating that the sites where mutations occurred were Me-lex specific. Based upon these results we hypothesize that a large proportion of mutations may result from the processing of 3-MeA, the main adduct induced by Me-lex, within A/T rich sequences in non-coding regions of the Hprt gene. The processing of these lesions by DNA polymerases could result in recombination and genomic deletions, thus representing a severe threat for genome integrity.

The Down syndrome critical region gene 1 short variant promoters direct vascular bed–specific gene expression during inflammation in mice

Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4-7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A-sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s-deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed-specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.

Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP

Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at −1607 bp, which creates the sequence 5′-GGAA-3′(2G allele), and which is an ETS binding site. Compared to the 1G allele (5′-GAA-3′), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity.Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with −4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ß-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ß-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.