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Sulfur mustard [bis(2-chloroethyl)sulfide, SM] is a well-known DNA-damaging agent that has been used in chemical warfare since World War I, and is a weapon that could potentially be used in a terrorist attack on a civilian population. Dermal exposure to high concentrations of SM produces severe, long-lasting burns. Topical exposure to high concentrations of 2-(chloroethyl) ethyl sulfide (CEES), a monofunctional analog of SM, also produces severe skin lesions in mice. Utilizing a genetically engineered mouse strain, Big Blue, that allows measurement of mutation frequencies in mouse tissues, we now show that topical treatment with much lower concentrations of CEES induces significant dose- and time-dependent increases in mutation frequency in mouse skin; the mutagenic exposures produce minimal toxicity as determined by standard histopathology and immunohistochemical analysis for cytokeratin 6 and the DNA-damage induced phosphorylation of histone H2AX (γ-H2AX). We attempted to develop a therapeutic that would inhibit the CEES-induced increase in mutation frequency in the skin. We observe that multi-dose, topical treatment with 2,6-dithiopurine (DTP), a known chemical scavenger of CEES, beginning 1 hour post-exposure to CEES, completely abolishes the CEES-induced increase in mutation frequency. These findings suggest the possibility that DTP, previously shown to be non-toxic in mice, may be useful as a therapeutic agent in accidental or malicious human exposures to SM.

Microarray technologies have provided the ability to monitor the expression of whole genomes rapidly. However, concerns persist with regard to quantitation and reproducibility, and the detection limits for individual genes in particular arrays are generally unknown. This article describes a semiautomated PCR-based technology, Q-RAGE, which rapidly provides measurements of mRNA abundance with extremely high sensitivity using fluorescent detection of specific products separated by capillary electrophoresis. A linear relationship between template concentration and fluorescent signal can be demonstrated down to template concentrations in the low aM region, corresponding to approximately 0.04 zmol (24 molecules) per reaction. The technique is shown to be quantitative over five orders of magnitude of template concentration, and average mRNA abundances of approximately 0.01 molecule per cell can be detected. A single predefined set of 320 primers provides 90–95% coverage of all eukaryotic genomes. Analysis of a set of 19 p53-regulated genes in untreated cultures of normal human epithelial cells, derived from three different tissues, revealed a 600-fold range of apparent constitutive expression levels. For most of the genes assayed, good correlations were observed among the expression levels in normal mammary, bronchial, and epidermal epithelial cells.

Previous studies indicated that DNA adducts formed by a carcinogenic diol epoxide, 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. It was suggested that adduct-induced bends in the DNA were responsible for this behavior. The cell cycle–regulated transcription factor E2F is also known to bend DNA upon binding. When partially purified E2F was tested in a gel mobility-shift assay, binding to a target DNA containing two consensus E2F-binding sites was enhanced by prior modification of the DNA with BPDE. Recombinant human E2F1, E2F4, and DP1 fusion proteins were affinity purified from bacteria expressing these genes. A combination of either E2F1 or E2F4 with their dimerization partner, DP1, gave preparations that exhibited binding to the E2F site–containing DNA fragment. In both cases, the proteins exhibited much higher apparent affinity for BPDE-modified DNA than for unmodified DNA. In addition, BPDE-modified DNA was a better competitor for the binding than unmodified DNA. Heterologous DNA that contained no consensus E2F binding motifs also competed well for E2F binding when modified with BPDE. In contrast, a transcription factor that does not bend DNA appreciably (GAL4) did not show enhanced affinity for BPDE-modified DNA. These findings suggest that numerous transcription factors that bend DNA may bind with anomalously high affinity to sequences that contain carcinogen-DNA adducts. Mol. Carcinog. 20:216–223, 1997. © 1997 Wiley-Liss, Inc.

Previous studies indicated that DNA adducts formed by the carcinogenic diol epoxide 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) can increase the affinity of the transcription factor Sp1 for DNA sequences that are not normally specific binding sites. Whether adducts that form in the normal binding site, the GC box sequence, increase the affinity of Sp1 for the modified GC-box was not determined. Starting with a 23-nt sequence that contains two natural GC box sequences, site-specifically modified oligonucleotides were prepared with a single(+)-BPDE-deoxyguanosine adduct at one of three positions: the center of each GC-box or in between the two boxes. Four modified oligonucleotides were studied, two derived from cis addition of BPDE to the exocyclic amino group and two from trans addition. For three of these site-specifically modified oligonucleotides, there was a diminution in Sp1 affinity, whereas Sp1 binding to the fourth modified oligonucleotide was abolished. Furthermore, random modification of the oligonucleotide to a level of about 1 BPDE adduct per fragment slightly decreased the affinity for Sp1, and no evidence was found for a subpopulation of molecules with high affinity. These findings suggest that BPDE modification of the GC box does not lead to an increased affinity for Sp1. This is consistent with a model in which a BPDE-induced bend in the DNA mimics the conformation of the normal GC box:Sp1 complex, leading to high-affinity binding of Sp1 to non-Gc box sites.

Female transgenic mice that constitutively overexpress the transcription factor ATF3 in the basal epithelium of the mammary gland develop mammary carcinomas with high frequency, but only if allowed to mate and raise pups early in life. This transgenic mouse model system reproduces some features of human breast cancer in that about 20% of human breast tumor specimens exhibit overexpression of ATF3 in the tumor cells. The ATF3-induced mouse tumors are phenotypically similar to mammary tumors induced by overexpression of activating Wnt/β-catenin pathway genes. We now show that the Wnt/β-catenin pathway is indeed activated in ATF3-induced tumors. β-catenin is transcriptionally up-regulated in the tumors, and high levels of nuclear β-catenin are seen in tumor cells. A reporter gene for Wnt/β-catenin pathway activity, TOPGAL, is up-regulated in the tumors and several downstream targets of Wnt signaling, including Ccnd1, Jun, Axin2 and Dkk4, are also expressed at higher levels in ATF3-induced tumors compared to mammary glands of transgenic females. Several positive-acting ligands for this pathway, including Wnt3, Wnt3a, Wnt7b, and Wnt5a, are significantly overexpressed in tumor tissue, and mRNA for Wnt3 is about 5-fold more abundant in transgenic mammary tissue than in non-transgenic mammary tissue. Two known transcriptional targets of ATF3, Snai1 and Snai2, are also overexpressed in the tumors, and Snail and Slug proteins are found to be located primarily in the nuclei of tumor cells. In vitro knockdown of Atf3 expression results in significant decreases in expression of Wnt7b, Tcf7, Snai2 and Jun, suggesting that these genes may be direct transcriptional targets of ATF3 protein. By chromatin immunoprecipitation analysis, both ATF3 and JUN proteins appear to bind to a particular subclass of AP-1 sites upstream of the transcriptional start sites of each of these genes.

Sulfur mustard (bis-(2-chloroethyl)sulfide) has been used in chemical warfare since World War I and is well known as an acutely toxic vesicant. It has been implicated as a carcinogen after chronic low-level exposure and is known to form interstrand cross-links in DNA. Sulfur and nitrogen mustards are currently of interest as potential chemical threat agents for terrorists because of ease of synthesis. Sulfur mustard and monofunctional analogues (half-mustards, 2-[chloroethyl] alkyl sulfides) react as electrophiles, damaging cellular macromolecules, and thus are potentially subject to scavenging by nucleophilic agents. We have determined rate constants for the reaction of four purine derivatives that contain nucleophilic thiol moieties with several sulfur-half-mustards. Three of these compounds, 2,6-dithiopurine, 2,6-dithiouric acid, and 9-methyl-6-mercaptopurine, exhibit facile reaction with the electrophilic mustard compounds. At near neutral pH, these thiopurines are much better nucleophilic scavengers of mustard electrophiles than other low molecular weight thiols such as N-acetyl cysteine and glutathione. Progress curves calculated by numerical integration techniques indicate that equimolar concentrations of thiopurine provide significant reductions in the overall exposure to the episulfonium ions, which are the major reactive, electrophiles produced when sulfur mustards are dissolved in aqueous solution.

Sulfur mustard (bis-(2-chloroethyl)sulfide) is a well-known chemical warfare agent that induces debilitating cutaneous toxicity in exposed individuals. It is also known to be carcinogenic and mutagenic because of its ability to damage DNA via electrophilic attack. We previously showed that a nucleophilic scavenger, 2,6-dithiopurine (DTP), reacts chemically with several electrophilic carcinogens, blocking DNA damage in vitro and in vivo and abolishing tumor formation in a two-stage mouse skin carcinogenesis model. To assess the potential of DTP as an antagonist of sulfur mustard, we have utilized monofunctional chemical analogues of sulfur mustard, 2-chloroethyl ethyl sulfide (CEES) and 2-chloroethyl methyl sulfide (CEMS), to induce toxicity and mutagenesis in a cell line, NCTC2544, derived from a human skin tumor. We show that DTP blocks cytotoxicity in CEMS- and CEES-treated cells when present at approximately equimolar concentration. A related thiopurine, 9-methyl-6-mercaptopurine, is similarly effective. Correlated with this, we find that DTP is transported into these cells and that adducts between DTP and CEES are found intracellularly. Using a shuttle vector-based mutagenesis system, which allows enumeration of mutations induced in the skin cells by a blue/white colony screen, we find that DTP completely abolishes the mutagenesis induced by CEMS and CEES in human cells.

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its ability to react with electrophiles. Acrolein, an electrophilic metabolite of cyclophosphamide (CP) involved in the toxicities of this anticancer drug, can be scavenged by DTP. The present study examined the effect of DTP treatment on CP-mediated bladder and lung toxicity in male ICR mice. Mice fed a diet containing 4% DTP that were treated intraperitoneally (i.p.) with 350 mg/kg CP showed no significant bladder damage (measured as bladder blood content at 48 h) with respect to the group fed a control diet. DTP (50 and 100 mg/kg), given i.p. 0.5 and 7 h after the initial injection of CP, also prevented the bladder damage when compared with the group receiving CP alone. Surprisingly, although neither parenteral CP nor DTP alone caused any mortality at these doses, the combined treatment resulted in 67% mortality within 3 days. At 24 h after CP+DTP, blood urea nitrogen was elevated 6-fold and urine volumes decreased by 70%. Histopathological analyses revealed a diffuse myocardial degeneration and necrosis, severe granular degeneration in the liver, abundant cellularity and infiltrates in interalveolar spaces in the lung and swollen nephron epithelial cells with some necrosis. All mice survived treatment when the dose of CP was lowered to 250 and 25–75 mg/kg DTP was given i.p. 0.5 and 7 h after CP. These DTP regimens reduced the degree of CP-induced lung toxicity, measured by [3H]thymidine incorporation into lung DNA 7 days after CP, in a dose-dependent manner. DTP (75 mg/kg) also reduced CP-induced lung fibrosis estimated by lung hydroxyproline content 28 days after CP. Analyses of urine from mice given CP+DTP revealed large amounts of the metabolic product dithiouric acid, smaller amounts of the parent DTP and several smaller peaks. The major unique metabolite peak was collected and analyzed by mass spectrometry, but did not correspond to either acrolein-DTP or acrolein-dithiouric acid. Thus, either very small amounts of an acrolein adduct are generated, the adduct is broken down to an unidentified product, or the ability of DTP to prevent CP-induced lung and bladder damage is related to some other mechanism. The possibility that mercapturic acid metabolites of acrolein released the parent electrophile in the urine was not supported by the finding that probenecid did not prevent CP-induced bladder toxicity.

The Wnt/β-catenin signaling pathway is up-regulated in mammary tumors that arise with 70% incidence in parous female BK5.ATF3 transgenic mice. Nuclear accumulation of β-catenin is seen in both basal and supra-basal tumor cells in this model, whereas nuclear expression of the transcription factor ATF3 is only seen in the basal cell layer of the tumors. If ATF3 acted as a positive regulator of one or more Wnt ligands in the basal cells, one might expect paracrine upregulation of the Wnt/β-catenin pathway in neighboring supra-basal cells. To test this, we measured mRNA expression levels for 9 Wnt genes in BK5.ATF3 induced mammary tumors and normal mammary glands. The genes for five known positive acting Wnt ligands, Wnt3, Wnt3a, Wnt5a, Wnt7b, and Wnt10b, were significantly upregulated in tumors as compared to transgenic mammary glands. One of these, Wnt3, was significantly down-regulated in non-transgenic glands. In a mouse mammary tumor cell line, EMT6, in which the endogenous Atf3 gene is overexpressed, the only Wnt gene for which expression could be detected by qPCR was Wnt7b. ChIP assays demonstrated binding of both Atf3 and Jun proteins to two putative AP-1 sites upstream of the Wnt7b transcription start site in these cells. ATF3 and JUN are known in other systems to form heterodimers that exhibit positive transactivation through AP-1 sites, and we have identified a physical interaction between ATF3 and JUN in BK5.ATF3-induced tumors. 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 2022. doi:10.1158/1538-7445.AM2011-2022

We previously showed that in a transgenic model (BK5.ATF3) in which the transcription factor ATF3 is overexpressed in cytokeratin 5 (CK5)-expressing cells, parous female mice develop mammary carcinomas with about 70% incidence between 6 months and one year of age. In these mice, ATF3 is overexpressed in cell nuclei in the myoepithelial cell layer (basal layer) of normal mammary glands, and in the basal cell layers of the tumors, in addition to expression in epidermis and other CK5-expressing epithelia. Non-parous, female transgenic mice exhibited a high incidence of squamous metaplastic lesions in the mammary glands between 14 and 32 weeks of age. These lesions showed dysregulation of cytokeratin expression, but no other molecular defects in non-parous mammary glands have been described. Several genes in the EGFR-MAPK pathway and in the Wnt/β-catenin pathway are misregulated in the mammary tumors that arise in parous transgenic mice. When expression of these genes was compared between normal mammary glands from transgenic and non-transgenic mice, the only genes that showed significant differences were Lef1 and Tcf7, two members of the family of transcription factors that dimerize with β-catenin to mediate Wnt-induced transcription. These genes were overexpressed by 2.5-3-fold in the transgenic mammary glands. A mouse mammary tumor cell line, EMT6, was found to have high levels of expression of both Atf3 and Tcf7, but not Lef1. Expression of Atf3 was knocked down by 97% in EMT6 cells 48 hrs post-treatment with Atf3-specific siRNA. This resulted in about a 70% decrease in expression of Tcf7, and a similar decrease in expression of cJun, a known target of Wnt/β-catenin signaling. This is consistent with the possibility that Tcf7 is a target gene for Atf3, and may be involved in the early stages of tumorigenesis in this model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1241.

The responses of a line of normal human mammary epithelial cells, HME87, to treatment with the ultimate carcinogen benzo[a]pyrene diol epoxide (BPDE) were analyzed using a directed gene expression analysis technique, RAGE. Under conditions where cell number was decreased by 50% 24 or 48 h post-treatment, flow cytometry demonstrated no establishment of a G(1)/S arrest nor induction of apoptosis; cells continued to enter S phase from G(1) for at least 24 h but were blocked at G(2)/M. Using the RAGE technique, changes in gene expression were assayed for over 1000 genes, and multiple time-point data were collected for approximately 90 genes. In accord with the cell cycle studies, expression of the p21-WAF1 gene, the major mediator of p53-dependent G(1)/S arrest, did not increase until 24 h post-treatment. The expression of other target genes for transactivation by p53 was increased at early time points, including GADD45, an effector of the G(2)/M checkpoint, and WIP1. Analyses of proteins in treated cells indicated that p53 was phosphorylated at Ser15 but not at Ser20 within 30 min of treatment, and this correlated with an increase in the total amount of p53 protein. Significant expression changes were noted in a number of transcription factor genes, including ATF3 and E2A, genes that have not been previously connected to a response to DNA damage involving bulky chemical adducts. In addition, expression of the XPC gene was induced by BPDE treatment; the XPC product is thought to be involved in recognition of DNA damage by the nucleotide excision repair system.