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Perinatal exposure to environmental chemicals is proposed to reprogram development and alter disease susceptibility later in life. Supporting this, neonatal activation of the nuclear receptor constitutive androstane receptor (CAR) (Nr1i3) by TCPOBOP was previously reported to induce persistent expression of mouse hepatic Cyp2 genes into adulthood, and was attributed to long-term epigenetic memory of the early life exposure. Here, we confirm that the same high-dose neonatal TCPOBOP exposure studied previously (3 mg/kg, 15x ED50) does indeed induce prolonged (12 weeks) increases in hepatic Cyp2 expression; however, we show that the persistence of expression can be fully explained by the persistence of residual TCPOBOP in liver tissue. When the long-term presence of TCPOBOP in tissue was eliminated by decreasing the neonatal TCPOBOP dose 22-fold (0.67× ED50), strong neonatal increases in hepatic Cyp2 expression were still obtained but did not persist into adulthood. Furthermore, the neonatal ED50-range TCPOBOP exposure did not sensitize mice to a subsequent, low-dose TCPOBOP treatment. In contrast, neonatal treatment with phenobarbital, a short half-life (t 1/2 = 8 h) agonist of CAR and PXR (Nr1i2), induced high-level neonatal activation of Cyp2 genes and also altered their responsiveness to low-dose phenobarbital exposure at adulthood by either increasing (Cyp2b10) or decreasing (Cyp2c55) expression. Thus, neonatal xenobiotic exposure can reprogram hepatic Cyp2 genes and alter their responsiveness to exposures later in life. These findings highlight the need to carefully consider xenobiotic dose, half-life, and persistence in tissue when evaluating the long-term effects of early life environmental chemical exposures. [ABSTRACT FROM AUTHOR]

Background: While nuclear transcription and RNA processing and localization are well established for protein coding genes (PCGs), these processes are poorly understood for long non-coding (lnc)RNAs. Here, we characterize global patterns of transcript expression, maturation and localization for mouse liver RNA, including more than 15,000 lncRNAs. PolyA-selected liver RNA was isolated and sequenced from four subcellular fractions (chromatin, nucleoplasm, total nucleus, and cytoplasm), and from the chromatin-bound fraction without polyA selection. Results: Transcript processing, determined from normalized intronic to exonic sequence read density ratios, progressively increased for PCG transcripts in going from the chromatin-bound fraction to the nucleoplasm and then on to the cytoplasm. Transcript maturation was similar for lncRNAs in the chromatin fraction, but was significantly lower in the nucleoplasm and cytoplasm. LncRNA transcripts were 11-fold more likely to be significantly enriched in the nucleus than cytoplasm, and 100-fold more likely to be significantly chromatin-bound than nucleoplasmic. Sequencing chromatin-bound RNA greatly increased the sensitivity for detecting lowly expressed lncRNAs and enabled us to discover and localize hundreds of novel regulated liver lncRNAs, including lncRNAs showing sex-biased expression or responsiveness to TCPOBOP a xenobiotic agonist ligand of constitutive androstane receptor (Nr1i3). Conclusions: Integration of our findings with prior studies and lncRNA annotations identified candidate regulatory lncRNAs for a variety of hepatic functions based on gene co-localization within topologically associating domains or transcription divergent or antisense to PCGs associated with pathways linked to hepatic physiology and disease. [ABSTRACT FROM AUTHOR]

Growth hormone-activated STAT5b is an essential regulator of sex-differential gene expression in mouse liver; however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5b (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were downregulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were derepressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG methylation were seen in STAT5-deficient liver, where sex differences were abolished at 88% of; 1,500 sex-differentially methylated regions, largely due to increased DNA methylation upon STAT5 loss. STAT5-dependent CpG hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (histone H3 lysine 4 monomethylation [H3K4me1] and histone H3 lysine 27 acetylation [H3K27ac]), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. Thus, the sex-dependent binding of STAT5 to liver chromatin is closely linked to the sex-dependent demethylation of distal regulatory elements linked to STAT5-dependent genes important for liver sex bias. [ABSTRACT FROM AUTHOR]

Sex-specific transcription characterizes hundreds of genes in mouse liver, many implicated in sex-differential drug and lipid metabolism and disease susceptibility. While the regulation of liver sex differences by growth hormone-activated STAT5 is well established, little is known about autosomal genetic factors regulating the sex-specific liver transcriptome. Here we show, using genotyping and expression data from a large population of Diversity Outbred mice, that genetic factors work in tandem with growth hormone to control the individual variability of hundreds of sex-biased genes, including many long non-coding RNA genes. Significant associations between single nucleotide polymorphisms and sex-specific gene expression were identified as expression quantitative trait loci (eQTLs), many of which showed strong sex-dependent associations. Remarkably, autosomal genetic modifiers of sex-specific genes were found to account for more than 200 instances of gain or loss of sex-specificity across eight Diversity Outbred mouse founder strains. Sex-biased STAT5 binding sites and open chromatin regions with strain-specific variants were significantly enriched at eQTL regions regulating correspondingly sex-specific genes, supporting the proposed functional regulatory nature of the eQTL regions identified. Binding of the male-biased, growth hormone-regulated repressor BCL6 was most highly enriched at trans-eQTL regions controlling female-specific genes. Co-regulated gene clusters defined by overlapping eQTLs included sets of highly correlated genes from different chromosomes, further supporting trans-eQTL action. These findings elucidate how an unexpectedly large number of autosomal factors work in tandem with growth hormone signaling pathways to regulate the individual variability associated with sex differences in liver metabolism and disease. [ABSTRACT FROM AUTHOR]

Several thousand sex-differential distal enhancers have been identified in mouse liver; however, their links to sex-biased genes and the impact of any sex-differences in nuclear organization and chromatin interactions are unknown. To address these issues, we first characterized 1847 mouse liver genomic regions showing significant sex differential occupancy by cohesin and CTCF, two key 3D nuclear organizing factors. These sex-differential binding sites were primarily distal to sex-biased genes but rarely generated sex-differential TAD (topologically associating domain) or intra-TAD loop anchors, and were sometimes found in TADs without sex-biased genes. A substantial subset of sex-biased cohesin-non-CTCF binding sites, but not sex-biased cohesin-and-CTCF binding sites, overlapped sex-biased enhancers. Cohesin depletion reduced the expression of male-biased genes with distal, but not proximal, sex-biased enhancers by >10-fold, implicating cohesin in long-range enhancer interactions regulating sex-biased genes. Using circularized chromosome conformation capture-based sequencing (4C-seq), we showed that sex differences in distal sex-biased enhancer–promoter interactions are common. Intra-TAD loops with sex-independent cohesin-and-CTCF anchors conferred sex specificity to chromatin interactions indirectly, by insulating sex-biased enhancer–promoter contacts and by bringing sex-biased genes into closer proximity to sex-biased enhancers. Furthermore, sex-differential chromatin interactions involving sex-biased gene promoters, enhancers, and lncRNAs were associated with sex-biased binding of cohesin and/or CTCF. These studies elucidate how 3D genome organization impacts sex-biased gene expression in a non-reproductive tissue through both direct and indirect effects of cohesin and CTCF looping on distal enhancer interactions with sex-differentially expressed genes. [ABSTRACT FROM AUTHOR]

Xenobiotic exposure dysregulates hundreds of protein-coding genes in mammalian liver, impacting many physiological processes and inducing diverse toxicological responses. Little is known about xenobiotic effects on long noncoding RNAs (lncRNAs), many of which have important regulatory functions. Here, we present a computational framework to discover liver-expressed, xenobiotic-responsive lncRNAs (xeno-lncs) with strong functional, gene regulatory potential and elucidate the impact of xenobiotic exposure on their gene regulatory networks. We assembled the long noncoding transcriptome of xenobiotic-exposed rat liver using RNA-seq datasets from male rats treated with 27 individual chemicals, representing 7 mechanisms of action (MOAs). Ortholog analysis was combined with coexpression data and causal inference methods to infer lncRNA function and deduce gene regulatory networks, including causal effects of lncRNAs on protein-coding gene expression and biological pathways. We discovered > 1400 liver-expressed xeno-lncs, many with human and/or mouse orthologs. Xenobiotics representing different MOAs often regulated common xeno-lnc targets: 123 xeno-lncs were dysregulated by ≥ 10 chemicals, and 5 xeno-lncs responded to ≥ 20 of the 27 chemicals investigated; 81 other xeno-lncs served as MOA-selective markers of xenobiotic exposure. Xeno-lnc—protein-coding gene coexpression regulatory network analysis identified xeno-lncs closely associated with exposure-induced perturbations of hepatic fatty acid metabolism, cell division, or immune response pathways, and with apoptosis or cirrhosis. We also identified hub and bottleneck lncRNAs, which are expected to be key regulators of gene expression. This work elucidates extensive networks of xeno-lnc—protein-coding gene interactions and provides a framework for understanding the widespread transcriptome-altering actions of foreign chemicals in a key-responsive mammalian tissue. [ABSTRACT FROM AUTHOR]

Cyclophosphamide treatment on a medium-dose, intermittent chemotherapy (MEDIC) schedule activates both innate and adaptive immunity leading to major regression of implanted gliomas. Here, we show that this MEDIC treatment regimen induces tumor cell autonomous type-I interferon signaling, followed by release of soluble factors that activate interferon-stimulated genes in both tumor cells and tumor-infiltrating immune cells. In cultured GL261 and CT-2A glioma cells, activated cyclophosphamide stimulated production and release of type-I interferons, leading to robust activation of downstream gene targets. Antibody against the type-I interferon receptor IFNAR1 blocked the cyclophosphamide-stimulated induction of these genes in both cultured glioma cells and implanted gliomas. Furthermore, IFNAR1 antibody strongly inhibited the MEDIC cyclophosphamide-stimulated increases in tumor cell infiltration of macrophages, dendritic cells, B-cells, as well as natural killer cells and cytotoxic T-cells and their cytotoxic effectors. Finally, cyclophosphamide-treated dying glioma cells producing type-I interferons were an effective vaccine against drug-naïve glioma cells implanted in vivo. Thus, cyclophosphamide induces local, tumor cell-centric increases in type-I interferon signaling, which activates immunogenic cell death and is essential for the striking antitumor immune responses that MEDIC cyclophosphamide treatment elicits in these glioma models. [ABSTRACT FROM AUTHOR]

Constitutive androstane receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid, and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to later disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene), a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared with males. Early (1 day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipogenesis and xenobiotic metabolism and oxidative stress protection pathways; late (2 weeks) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for macrophage activation, immune response, and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to proinflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle induced carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Overall, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary gene responses in liver nonparenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP response genes include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease. [ABSTRACT FROM AUTHOR]

Conventional cytotoxic cancer chemotherapy is often immunosuppressive and associated with drug resistance and tumor regrowth after a short period of tumor shrinkage or growth stasis. However, certain cytotoxic cancer chemotherapeutic drugs, including doxorubicin, mitoxantrone, and cyclophosphamide, can kill tumor cells by an immunogenic cell death pathway, which activates robust innate and adaptive anti-tumor immune responses and has the potential to greatly increase the efficacy of chemotherapy. Here, we review studies on chemotherapeutic drug-induced immunogenic cell death, focusing on how the choice of a conventional cytotoxic agent and its dose and schedule impact anti-tumor immune responses. We propose a strategy for effective immunogenic chemotherapy that employs a modified metronomic schedule for drug delivery, which we term medium-dose intermittent chemotherapy (MEDIC). Striking responses have been seen in preclinical cancer models using MEDIC, where an immunogenic cancer chemotherapeutic agent is administered intermittently and at an intermediate dose, designed to impart strong and repeated cytotoxic damage to tumors, and on a schedule compatible with activation of a sustained anti-tumor immune response, thereby maximizing anti-cancer activity. We also discuss strategies for combination chemo-immunotherapy, and we outline approaches to identify new immunogenic chemotherapeutic agents for drug development. [ABSTRACT FROM AUTHOR]

The persistent environmental aryl hydrocarbon receptor agonist and hepatotoxin TCDD (2,3,7,8-tetrachlorodibenzo- p -dioxin) induces hepatic lipid accumulation (steatosis), inflammation (steatohepatitis) and fibrosis. Thousands of liver-expressed, nuclear-localized lncRNAs with regulatory potential have been identified; however, their roles in TCDD-induced hepatoxicity and liver disease are unknown. We analyzed single nucleus (sn)RNA-seq data from control and subchronic (4 wk) TCDD-exposed mouse liver to determine liver cell-type specificity, zonation and differential expression profiles for thousands of lncRNAs. TCDD dysregulated >4000 of these lncRNAs in one or more liver cell types, including 684 lncRNAs specifically dysregulated in liver non-parenchymal cells. Trajectory inference analysis revealed major disruption by TCDD of hepatocyte zonation, affecting >800 genes, including 121 lncRNAs, with strong enrichment for lipid metabolism genes. TCDD also dysregulated expression of >200 transcription factors, including 19 Nuclear Receptors, most notably in hepatocytes and Kupffer cells. TCDD-induced changes in cell–cell communication patterns included marked decreases in EGF signaling from hepatocytes to non-parenchymal cells and increases in extracellular matrix-receptor interactions central to liver fibrosis. Gene regulatory networks constructed from the snRNA-seq data identified TCDD-exposed liver network-essential lncRNA regulators linked to functions such as fatty acid metabolic process, peroxisome and xenobiotic metabolism. Networks were validated by the striking enrichments that predicted regulatory lncRNAs showed for specific biological pathways. These findings highlight the power of snRNA-seq to discover functional roles for many xenobiotic-responsive lncRNAs in both hepatocytes and liver non-parenchymal cells and to elucidate novel aspects of foreign chemical-induced hepatotoxicity and liver disease, including dysregulation of intercellular communication within the liver lobule. • Subchronic TCDD exposure dysregulates >4000 mouse liver-expressed lncRNAs • snRNA-seq-based gene regulatory networks identify TCDD-responsive regulatory lncRNAs • TCDD dysregulates >200 transcription factors in multiple liver cell types • TCDD disrupts intercellular communication within the liver lobule [ABSTRACT FROM AUTHOR]

The article discusses ales are more susceptible than (premenopausal) females to diet induced hepatic fat accumulation (steatosis) and progression to nonalcoholic fatty liver disease (NAFLD), liver fibrosis, and hepatocellular carcinoma, in both humans and rodents. Sex-specific differences in liver gene expression are primarily controlled by the sex-dependent temporal patterns of pituitary growth hormone (GH) secretion.

Cyclophosphamide administered on an intermittent metronomic schedule induces strong immune-dependent regression in several glioma models. Here we investigate whether this immunogenic chemotherapy can be potentiated by combination with the immune stimulatory TLR9 agonist CpG-1826. CpG-1826 treatment of GL261 gliomas implanted in immune competent mice induced tumor growth delay associated with increased tumor recruitment of macrophages and B cells. Anti-tumor responses varied between individuals, with CpG-1826 inducing robust tumor growth delay in ~50% of treated mice. Both high and low CpG-1826-responsive mice showed striking improvements when CpG-1826 was combined with cyclophosphamide treatment. Tumor-associated macrophages, B cells, dendritic cells, and cytotoxic T cells were increased, T regulatory cells were not induced, and long-term GL261 glioma regression with immune memory was achieved when CpG-1826 was combined with either single cyclophosphamide dosing (90 mg/kg) or metronomic cyclophosphamide treatment (two cycles at 45 mg/kg, spaced 12-days apart). B16F10 melanoma, a low immunogenic tumor model, also showed enhanced immune and anti-tumor responses to cyclophosphamide/CpG-1826 chemoimmunotherapy, but unlike GL261 tumors, did not regress. TLR9-based immunotherapy can thus be effectively combined with immunogenic cyclophosphamide treatment to enhance immune-based anti-tumor responses, even in poorly immunogenic cancer models. [ABSTRACT FROM AUTHOR]

The growth hormone (GH)-activated transcription factor signal transducer and activator of transcription 5b (STAT5b) is a key regulator of sexually dimorphic gene expression in the liver. Suppression of hepatic STAT5b signaling is associated with lipid metabolic dysfunction leading to steatosis and liver cancer. In the companion publication, a STAT5b biomarker gene set was identified and used in a rank-based test to predict both increases and decreases in liver STAT5b activation status/function with high (≥ 97%) accuracy. Here, this computational approach was used to identify chemicals and hormones that activate (masculinize) or suppress (feminize) STAT5b function in a large, annotated mouse liver and primary hepatocyte gene expression compendium. Exposure to dihydrotestosterone and thyroid hormone caused liver masculinization, whereas glucocorticoids, fibroblast growth factor 15, and angiotensin II caused liver feminization. In mouse models of diabetes and obesity, liver feminization was consistently observed and was at least partially reversed by leptin or resveratrol exposure. Chemical-induced feminization of male mouse liver gene expression profiles was a relatively frequent phenomenon: of 156 gene expression biosets from chemically-treated male mice, 29% showed feminization of liver STAT5b function, while <1% showed masculinization. Most (93%) of the biosets that exhibited feminization of male liver were also associated with activation of one or more xenobiotic-responsive receptors, most commonly constitutive activated receptor (CAR) or peroxisome proliferator-activated receptor alpha (PPARα). Feminization was consistently associated with increased expression of peroxisome proliferator-activated receptor gamma (Pparg) but not other lipogenic transcription factors linked to steatosis. GH-activated STAT5b signaling in mouse liver is thus commonly altered by diverse chemicals, and provides a linkage between chemical exposure and dysregulated gene expression associated with adverse effects on the liver. [ABSTRACT FROM AUTHOR]

Exposure to Bisphenol A (BPA) during the critical window of uterine development has been proposed to program the uterus for increased disease susceptibility based on well-documented effects of the potent xenoestrogen diethylstilbestrol. To investigate this proposal, we reviewed 37 studies of prenatal and/or perinatal BPA exposure in animal models and evaluated evidence for: molecular signatures of early BPA exposure; the development of adverse uterine health effects; and epigenetic changes linked to long-term dysregulation of uterine gene expression and health effects. We found substantial evidence for adult uterine effects of early BPA exposure. In contrast, experimental support for epigenetic actions of early BPA exposure is very limited, and largely consists of effects on Hoxa gene DNA methylation. Critical knowledge gaps were identified, including the need to fully characterize short-term and long-term uterine gene responses, interactions with estrogens and other endogenous hormones, and any long-lasting epigenetic signatures that impact adult disease. [ABSTRACT FROM AUTHOR]

Background: Cyclophosphamide treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms whereby metronomic cyclophosphamide induces innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the immune responses linked to tumor regression. Methods: Untreated and metronomic cyclophosphamide-treated human U251 glioblastoma xenografts were analyzed on human microarrays at two treatment time points to identify responsive tumor cell-specific factors and their upstream regulators. Mouse microarray analysis across two glioma models (human U251, rat 9L) was used to identify host factors and gene networks that contribute to the observed immune and tumor regression responses. Results: Metronomic cyclophosphamide increased expression of tumor cell-derived DNA damage, cell stress, and cell death genes, which may facilitate innate immune activation. Increased expression of many host (mouse) immune networks was also seen in both tumor models, including complement components, toll-like receptors, interferons, and cytolysis pathways. Key upstream regulators activated by metronomic cyclophosphamide include members of the interferon, toll-like receptor, inflammatory response, and PPAR signaling pathways, whose activation may contribute to anti-tumor immunity. Many upstream regulators inhibited by metronomic cyclophosphamide, including hypoxia-inducible factors and MAP kinases, have glioma-promoting activity; their inhibition may contribute to the therapeutic effectiveness of the six-day repeating metronomic cyclophosphamide schedule. Conclusions: Large numbers of responsive cytokines, chemokines and immune regulatory genes linked to innate immune cell recruitment and tumor regression were identified, as were several immunosuppressive factors that may contribute to the observed escape of some tumors from metronomic CPA-induced, immune-based regression. These factors may include useful biomarkers that facilitate discovery of clinically effective immunogenic metronomic drugs and treatment schedules, and the selection of patients most likely to be responsive to immunogenic drug scheduling. [ABSTRACT FROM AUTHOR]

Background: Cyclophosphamide treatment on a six-day repeating metronomic schedule induces a dramatic, innate immune cell-dependent regression of implanted gliomas. However, little is known about the underlying mechanisms whereby metronomic cyclophosphamide induces innate immune cell mobilization and recruitment, or about the role of DNA damage and cell stress response pathways in eliciting the immune responses linked to tumor regression. Methods: Untreated and metronomic cyclophosphamide-treated human U251 glioblastoma xenografts were analyzed on human microarrays at two treatment time points to identify responsive tumor cell-specific factors and their upstream regulators. Mouse microarray analysis across two glioma models (human U251, rat 9L) was used to identify host factors and gene networks that contribute to the observed immune and tumor regression responses. Results: Metronomic cyclophosphamide increased expression of tumor cell-derived DNA damage, cell stress, and cell death genes, which may facilitate innate immune activation. Increased expression of many host (mouse) immune networks was also seen in both tumor models, including complement components, toll-like receptors, interferons, and cytolysis pathways. Key upstream regulators activated by metronomic cyclophosphamide include members of the interferon, toll-like receptor, inflammatory response, and PPAR signaling pathways, whose activation may contribute to anti-tumor immunity. Many upstream regulators inhibited by metronomic cyclophosphamide, including hypoxia-inducible factors and MAP kinases, have glioma-promoting activity; their inhibition may contribute to the therapeutic effectiveness of the six-day repeating metronomic cyclophosphamide schedule. Conclusions: Large numbers of responsive cytokines, chemokines and immune regulatory genes linked to innate immune cell recruitment and tumor regression were identified, as were several immunosuppressive factors that may contribute to the observed escape of some tumors from metronomic CPA-induced, immune-based regression. These factors may include useful biomarkers that facilitate discovery of clinically effective immunogenic metronomic drugs and treatment schedules, and the selection of patients most likely to be responsive to immunogenic drug scheduling. [ABSTRACT FROM AUTHOR]

The administration of chemotherapy at reduced doses given at regular, frequent time intervals, termed ‘metronomic’ chemotherapy, presents an alternative to standard maximal tolerated dose (MTD) chemotherapy. The primary target of metronomic chemotherapy was originally identified as endothelial cells supporting the tumor vasculature, and not the tumor cells themselves, consistent with the emerging concept of cancer as a systemic disease involving both tumor cells and their microenvironment. While anti-angiogenesis is an important mechanism of action of metronomic chemotherapy, other mechanisms, including activation of anti-tumor immunity and a decrease in acquired therapeutic resistance, have also been identified. Here we present evidence supporting a mechanistic explanation for the improved activity of cancer chemotherapy when administered on a metronomic, rather than an MTD schedule and discuss the implications of these findings for further translation into the clinic. [ABSTRACT FROM AUTHOR]

Metronomic cyclophosphamide (CPA) treatment activates robust innate anti-tumor immunity and induces major regression of large, implanted brain tumor xenografts when administered on an intermittent, every 6-day schedule, but not on a daily low-dose or a maximum-tolerated dose CPA schedule. Here, we used an implanted GL261 glioma model to compare five intermittent metronomic CPA schedules to elucidate the kinetics and schedule dependence of innate immune cell recruitment and tumor regression. Tumor-recruited natural killer cells induced by two every 6-day treatment cycles were significantly ablated 1 day after a third CPA treatment, but largely recovered several days later. Natural killer and other tumor-infiltrating innate immune cells peaked 12 days after the last CPA treatment on the every 6-day schedule, suggesting that drug-free intervals longer than 6 days may show increased efficacy. Metronomic CPA treatments spaced 9 or 12 days apart, or on an alternating 6 and 9 day schedule, induced extensive tumor regression, similar to the 6-day schedule; however, the tumor-infiltrating natural killer cell responses were not sustained, leading to rapid resumption of tumor growth after day 24, despite ongoing metronomic CPA treatment. Increasing the CPA dose prolonged the period of tumor regression on the every 9-day schedule, but natural killer cell activation was markedly decreased. Thus, while several intermittent metronomic CPA treatment schedules can activate innate immune cell recruitment leading to major tumor regression, sustained immune and anti-tumor responses are only achieved on the 6-day schedule. However, even with this schedule, some tumors eventually relapse, indicating a need for further improvements in this immunogenic metronomic therapy. [ABSTRACT FROM AUTHOR]

Abstract: Cancer stem-like cells were isolated from several human tumor cell lines by limiting dilution assays and holoclone morphology, followed by assessment of self-renewal capacity, tumor growth, vascularity, and blood perfusion. H460 holoclone-derived tumors grew slower than parental H460 tumors, but displayed significantly increased microvessel density and tumor blood perfusion. Microarray analysis identified 177 differentially regulated genes in the holoclone-derived tumors, of which 47 were associated with angiogenesis. The dysregulated genes include several small leucine-rich proteoglycans that may modulate angiogenesis and serve as novel therapeutic targets for inhibiting cancer stem cell-driven angiogenesis. [Copyright &y& Elsevier]

Chromatin state maps were developed to elucidate sex differences in chromatin structure and their impact on sex-differential chromatin accessibility and sex-biased gene expression in mouse liver. Genes in active, inactive, and poised chromatin states exhibited differential responsiveness to ligand-activated nuclear receptors and distinct enrichments for functional gene categories. Sex-biased genes were clustered by chromatin environments and mapped to DNase-hypersensitive sites (DHS) classified by sex bias in chromatin accessibility and enhancer modifications. Results were integrated with genome-wide binding data for five transcription factors implicated in growth hormone-regulated, sex-biased liver gene expression, leading to the following findings. (i) Sex-biased DHS, but not sex-biased genes, are frequently characterized by sex-differential chromatin states, indicating distal regulation. (ii) Trimethylation of histone H3 at K27 (H3K27me3) is a major sex-biased repressive mark at highly female-biased but not at highly male-biased genes. (iii) FOXA factors are associated with sex-dependent chromatin opening at male-biased but not female-biased regulatory sites. (iv) Sex-biased STAT5 binding is enriched at sex-biased DHS marked as active enhancers and preferentially targets sex-biased genes with sex-differences in local chromatin marks. (v) The male-biased repressor BCL6 preferentially targets female biased genes and regulatory sites in a sex-independent chromatin state. (vi) CUX2, a female-specific repressor of male-biased genes, also activates strongly female-biased genes, in association with loss of H3K27me3 marks. Chromatin states are thus a major determinant of sex-biased chromatin accessibility and gene expression, with FOXA pioneer factors proposed to confer sex-dependent chromatin opening and STAT5, but not BCL6, regulating sexbiased genes by binding to sites in a sex-biased chromatin state. [ABSTRACT FROM AUTHOR]

Abstract: The anti-tumor activity, metronomic chemotherapy sensitization potential and metastatic effects of the endogenous angiogenesis inhibitors thrombospondin-1 and PEDF were investigated in KM12 colon adenocarcinoma xenografts. Thrombospondin-1 and PEDF decreased KM12 tumor microvessel density, increased macrophage infiltration, and improved responsiveness to metronomic cyclophosphamide (CPA) treatment, but did not activate the anti-tumor innate immunity that metronomic CPA induces in other tumor models. Moreover, thrombospondin-1, but not PEDF, significantly increased KM12 metastasis to the lung, while PEDF augmented the anti-metastatic activity of metronomic CPA. Thus, while thrombospondin-1 and PEDF both increase the KM12 tumor responsiveness to metronomic CPA, they have disparate effects on tumor metastasis. [Copyright &y& Elsevier]

In metronomic chemotherapy, frequent drug administration at lower than maximally tolerated doses can improve activity while reducing the dose-limiting toxicity of conventional dosing schedules. Although the antitumor activity produced by metronomic chemotherapy is attributed widely to antiangiogenesis, the significance of this mechanism remains somewhat unclear. In this study, we show that a 6-day repeating metronomic schedule of cyclophosphamide administration activates a potent antitumor immune response associated with brain tumor recruitment of natural killer (NK) cells, macrophages, and dendritic cells that leads to marked tumor regression. Tumor regression was blocked in nonobese diabetic/severe combined immunodeficient (NOD/SCID-γ) mice, which are deficient or dysfunctional in all these immune cell types. Furthermore, regression was blunted by NK cell depletion in immunocompetent syngeneic mice or in perforin-deficient mice, which are compromised for NK, NKT, and T-cell cytolytic functions. Unexpectedly, we found that VEGF receptor inhibitors blocked both innate immune cell recruitment and the associated tumor regression response. Cyclophosphamide administered at a maximum tolerated dose activated a transient, weak innate immune response, arguing that persistent drug-induced cytotoxic damage or associated cytokine and chemokine responses are required for effective innate immunity-based tumor regression. Together, our results reveal an innate immunity-based mechanism of tumor regression that can be activated by a traditional cytotoxic chemotherapy administered on a metronomic schedule. These findings suggest the need to carefully evaluate the clinical effects of combination chemotherapies that incorporate antiangiogenesis drugs targeting VEGF receptor. [ABSTRACT FROM AUTHOR]

Background: Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression. Methods: Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3. Results: We demonstrate that, under standard culture conditions, ∼10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, FAM65B, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, MFI2 and LEF1, that are consistently down regulated. This molecular profile, FAM65Bhigh/MFI2low/LEF1low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin a2b1. PC3 tumors seeded with holoclones showed dramatic down regulation of FAM65B and dramatic up regulation of MFI2 and LEF1, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis. Conclusions: These findings support the proposal that PC3 tumors are sustained by a small number of tumorinitiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern FAM65Bhigh/MFI2low/LEF1low. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3. [ABSTRACT FROM AUTHOR]

Poloxamer 407 (P-407) is a copolymer surfactant that induces a dose-controlled dyslipidemia in both mice and rats. Human macrophages cultured with P-407 exhibit a concentration-dependent reduction in cholesterol efflux to apolipoprotein A1 (apoA1) linked to downregulation of the ATP-binding cassette transporter A1 (ABCA1). Activators of peroxisome proliferator-activated receptor gamma (PPARγ), as well as PPARα, increase expression of liver X receptor alpha (LXRα) in macrophages and promote the expression of ABCA1, which, in turn, mediates cholesterol efflux to apoA1. The present study investigated whether P-407 interferes with this signaling pathway. A transactivation assay was used to evaluate whether P-407 can either activate or inhibit the transcriptional activity of PPARγ. Because thiazolidinedione drugs (PPARγ agonists) improve glycemic control in type 2 diabetes by reducing blood glucose concentrations, P-407 was also evaluated for its potential to alter plasma insulin and blood glucose concentrations in wild-type (C57BL/6) and PPARγ-deficient mice. Additionally, because thiazolidinediones attenuate release of free fatty acids (FFAs) from adipocytes and, consequently, decrease circulating plasma levels of FFAs, plasma concentrations of circulating FFAs were also determined in P-407-treated mice. P-407 was unable to modulate PPARγ activity in cell-based transactivation assays. Furthermore, P-407 did not perturb plasma insulin and blood glucose concentrations after administration to mice. However, by an as yet unidentified mechanism, P-407 caused a significant increase in the serum concentration of FFAs in mice beginning 3 h after administration and lasting more than 24 h postdosing. It is concluded that P-407 does not interfere with the functional activity of PPARγ after administration to mice. Le poloxamère 407 (P-407) est un surfactant copolymère qui induit une dyslipidémie fonction de la dose chez les souris et les rats. Des macrophages humains cultivés en présence de P-407 présentent une réduction concentration-dépendante de l’efflux de cholestérol vers l’apolipoprotéine A1 (apoA1) associée à une diminution du transporteur ABC transporteur ABC (ATP binding cassette) A1. Les activateurs du récepteur gamma activé de la prolifération des peroxysomes (PPARγ), ainsi que du PPARα, augmentent l’expression du récepteur alpha X (LRXα) dans les macrophages et favorisent l’expression du transporteur ABCA1, qui, lui, véhicule l’efflux de cholestérol vers l’apoA1. La présente étude examine si P-407 interfère avec cette voie de signalisation. Un test de transactivation a été utilisé pour évaluer si P-407 peut activer ou inhiber l’activité transcriptionnelle de PPARγ. Comme les thiazolidinediones (agonistes de PPARγ) améliorent la régulation glycémique du diabète de type 2 en diminuant les concentrations de glucose sanguin, la capacité de P-407 de modifier les concentrations d’insuline plasmatique et de glucose sanguin après son administration à des souris de type sauvage et à des souris déficientes en PPARγ a aussi été évaluée. De plus, les triazolinediones atténuent la libération des acides gras libres (AGL) des adipocytes et, par conséquent, les taux d’AGL plasmatiques circulants. Les concentrations d’AGL plasmatiques circulants ont donc aussi été déterminées chez des souris traitées au P-407. Le P-407 n’a pu moduler l’activité de PPARγ dans les tests de transactivation cellulaire. Il n’a pas non plus perturbé les concentrations d’insuline plasmatique et de glucose sanguin après son administration aux souris. Toutefois, il a provoqué une augmentation significative de la concentration d’AGL sérique chez les souris, qui a débuté 3 h après l’administration et a duré >24 h, et ce, par un mécanisme encore inconnu. On conclut que P-407 n’interfère pas avec l’activité fonctionnelle de PPARγ après son administration aux souris. [ABSTRACT FROM AUTHOR]

Methoxyacetic acid (MAA), the active biological oxidation product of the industrial solvent ethylene glycol monomethyl ether (EGME), causes acute toxicity in several species including humans. MAA primarily affects tissues with rapidly dividing cells and high rates of energy metabolism, including testes, thymus and the fetus. Testicular toxicity, one of the most prominent consequences of EGME, and MAA, exposure, results from apoptosis of primary spermatocytes and is associated with changes in the expression of various genes and signalling pathways. This review of EGME metabolism and its organ-specific toxicities emphasizes genes and signalling pathways that are modulated by EGME exposure and their relevance to the molecular mechanisms underlying EGME and MAA toxicity. Of particular importance are the genes that code for oxidative stress response factors, protein kinases, and nuclear hormone receptors. Nuclear receptors and protein kinases regulate multiple cellular processes and are critical for signalling events required for spermatogenesis. De-regulation of their activity by EGME or MAA leads to inappropriate signalling in testicular cells. Oxidative stress in spermatocytes exposed to MAA triggers mitochondrial release of cytochrome C, activation of caspases and ultimately apoptosis. Detailed investigation of the molecular responses to MAA exposure may help elucidate the overall impact and extent of toxicity seen following EGME exposure. Finally, given the effects of EGME on multiple genes and signalling pathways in the testis, mixture studies combining EGME, or MAA, with other testicular toxicants may help identify toxicities that are aggravated by EGME exposure. [ABSTRACT FROM AUTHOR]

Computational solvent mapping moves small organic molecules as probes around a protein surface, finds favorable binding positions, clusters the conformations, and ranks the clusters on the basis of their average free energy. Prior mapping studies of enzymes, crystallized in either substrate-free or substrate-bound form, have shown that the largest number of solvent probe clusters invariably overlaps in the active site. We have applied this method to five cytochromes P450. As expected, the mapping of two bacterial P450s, P450 cam (CYP101) and P450 BM-3 (CYP102), identified the substrate-binding sites in both ligand-bound and ligand-free P450 structures. However, the mapping finds the active site only in the ligand-bound structures of the three mammalian P450s, 2C5, 2C9, and 2B4. Thus, despite the large cavities seen in the unbound structures of these enzymes, the features required for binding small molecules are formed only in the process of substrate binding. The ability of adjusting their binding sites to substrates that differ in size, shape, and polarity is likely to be responsible for the broad substrate specificity of these mammalian P450s. Similar behavior was seen at “hot spots” of protein—protein interfaces that can also bind small molecules in grooves created by induced fit. In addition, the binding of S-warfarin to P450 2C9 creates a high-affinity site for a second ligand, which may help to explain the prevalence of drug—drug interactions involving this and other mammalian P450s. [ABSTRACT FROM AUTHOR]

Abstract: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that stimulates transcription directed by xenobiotic response elements upstream of target genes. Recently, AhR ligands were reported to induce formation of an AhR–estrogen receptor (ER) complex, which can bind to estrogen response elements (EREs) and stimulate transcription of ER target genes. Presently, we investigate the effect of the AhR ligands 3-methylcholanthrene (3MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3′,4,4′,5-pentachlorobiphenyl (BZ126) on ERE-regulated luciferase reporter activity and endogenous ER target gene expression. In MCF-7 human breast cancer cells, 3MC induced transcription of ER reporter genes containing native promoter sequences of the ER-responsive genes complement 3 and pS2 and heterologous promoters regulated by isolated EREs. Dose–response studies revealed that the concentration of 3MC required to half-maximally activate transcription (EC50) was >100-fold higher for an ER reporter (27–57 μM) than for an AhR reporter (86–250 nM) in both MCF-7 cells and in human endometrial cancer Ishikawa cells. 3MC also stimulated expression of the endogenous ER target genes amphiregulin, cathepsin D and progesterone receptor, albeit to a much lower extent than was achieved following stimulation with 17β-estradiol. In Ishikawa cells, 3MC, but not BZ126 or TCDD, stimulated ERα-dependent reporter activity but did not induce expression of endogenous ER target genes. Finally, studies carried out in the AhR-positive rat hepatoma cell line 5L and the AhR-deficient variant BP8 demonstrated that ER reporter activity could be induced by 3MC in a manner that was independent of AhR and thus distinct from the AhR-ER ‘hijacking’ mechanism described recently. 3MC may thus elicit estrogenic activity by multiple mechanisms. [Copyright &y& Elsevier]

Abstract: Cancer chemotherapeutic prodrugs, such as the oxazaphosphorines cyclophosphamide and ifosfamide, are metabolized by liver cytochrome P450 enzymes to yield therapeutically active, cytotoxic metabolites. The effective use of these prodrugs is limited by host toxicity associated with the systemic distribution of cytotoxic metabolites formed in the liver. This problem can, in part, be circumvented by implementation of cytochrome P450 gene-directed enzyme prodrug therapy (P450 GDEPT), a prodrug activation strategy for cancer treatment that augments tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating cytochrome P450 enzyme. P450 GDEPT has been exemplified in preclinical rodent and human tumor models, where chemosensitivity to a P450 prodrug can be greatly increased by introduction of a prodrug-activating P450 gene. Further enhancement of the efficacy of P450-based gene therapy can be achieved: by co-expression of P450 with the flavoenzyme NADPH-P450 reductase, which provides electrons required for P450 metabolic activity; by metronomic (anti-angiogenic) scheduling of the prodrug; by localized delivery of the prodrug to the tumor; and by combination with anti-apoptotic factors, which slow the death of the P450 ‘factory’ cells and thereby enhance the bystander cytotoxic response. P450 GDEPT has several important features that make it a clinically attractive strategy for cancer treatment. These include: the substantial bystander cytotoxicity of P450 prodrugs such as cyclophosphamide and ifosfamide; the ability to use human P450 genes and thereby avoid an immune response to the therapeutic gene; the use of well-established conventional chemotherapeutic prodrugs, as well as bioreductive drugs activated by P450/P450 reductase in a hypoxic tumor environment; and the potential to decrease systemic exposure to active drug metabolites by selective inhibition of hepatic P450 activity. Recent advances in this area of research are reviewed, and two proof-of-concept clinical trials that highlight the utility of this strategy are discussed. [Copyright &y& Elsevier]

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are members of the nuclear receptor superfamily. These transcription factors are predominantly expressed in the liver, where they are activated by structurally diverse compounds, including many drugs and endogenous substances. CAR and PXR regulate the expression of a broad range of genes, which contribute to transcellular transport, bioactivation, and detoxification of numerous xenochemicals and endogenous substances. This article discusses the importance of these receptors for pharmacology and toxicology, emphasizing the role of individual drugs and natural products as agonists, indirect activators, inverse agonists, and antagonists of CAR and PXR. [ABSTRACT FROM AUTHOR]

The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH naïve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH-GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STATSb continued for ∼30–40 min, however, indicating that GHR signaling and deactivation of the GH·GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH·GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteascme in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling. [ABSTRACT FROM AUTHOR]

There is growing concern about the human-health impact of environmental chemicals that have the potential to disrupt normal endocrine function. Endocrine disrupting chemicals (EDCs) include structurally diverse organochlorine pesticides, polychlorinated biphenyls (PCBs), plasticizers, fungicides, herbicides and pharmaceutical compounds, and can have a profound impact on development, and on reproductive, neurological and immune system functions. While many studies have focused on the role of androgen receptor, estrogen receptor and aryl hydrocarbon receptor in mediating the effects of EDCs, other nuclear receptors that regulate steroid hormone action and metabolism may also serve as targets of EDC action. This review focuses on two classes of EDCs, PCBs and phthalate monoesters, both of which have been shown to interact with pregnane X receptor (PXR), a member of the nuclear receptor superfamily that regulates a large number of target genes, many of which have important roles in steroid metabolism and transport. Recent findings on the ability of PCBs and phthalate monoesters to activate PXR are discussed and the potential role of PXR and other intracellular receptor proteins in mediating toxicities associated with EDC exposure is considered. Finally, we discuss several gaps in our knowledge regarding the actions of EDCs and the difficulties associated with the evaluation of risks associated with exposure to these endocrine active environmental chemicals. [ABSTRACT FROM AUTHOR]

Cytochrome P450 2A2 (CYP2A2) is an adult male-specific rat liver steroid hydroxylase whose sex-dependent expression is regulated at the transcriptional level by sexually dimorphic pituitary growth hormone (GH) secretory patterns. In contrast to CYP2C11 and other male-specific, plasma GH pulse-inducible liver genes, CYP2A2 is highly expressed in hypophysectomized rat liver, despite the absence of GH stimulation. CYP2A2 promoter fragments 0.9-6.2 kb long exhibited unusually high basal promoter activity when transfected into the liver cell line HepG2. A further ∼2.5-fold increase in activity was obtained by cotransfection of hepatocyte nuclear factor (HNF) 3γ or HNF4α. CYP2A2 promoter activity was inhibited ∼85% by transfection of HNF3β or HNF6, both of which are more highly expressed in female than male liver and can strongly trans-activate the female-specific CYP2C12 promoter. The male GH pulse-activated transcription factor STAT5b had no effect on CYP2A2 promoter activity, either alone or in combination with HNF3γ and HNF4α, consistent with the GH pulse-independence of CYP2A2 expression. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4α toward two other male-specific liver target genes, Cyp2d9 and CYP8B1. Furthermore, STAT5b in combination with the HNF4α coactivator peroxisome proliferator-activated receptor γ coactivator-1α strongly enhanced the transcriptional activity of HNF4α toward CYP8B1 but not toward CYP2A2. These findings support the hypothesis that sex-dependent HNFs contribute to the sexually dimorphic expression of CYP2A2 and other liver CYPs and highlight the ability of STAT5b to act in concert with HNF4α to regulate select male-specific liver CYP genes. [ABSTRACT FROM AUTHOR]

The transcription factors peroxisome proliferator-activated receptor (PPAR) and signal transducer and activator of transcription 5 (STAT5) activate genes involved in fatty acid metabolism (PPARα) and adipogenesis (PPARγ) and mediate hormonal responses important for body growth, liver gene expression, and mammary gland development (STAT5a and STAT5b). These seemingly disparate pathways are subject to mutually inhibitory crosstalk, with growth hormone (GH)-activated STAT5 able to inhibit PPAR-regulated gene transcription by approximately 80%, and conversely, ligand-activated PPAR able to inhibit STAT5-regulated transcription to a similar degree. Given the co-expression of PPAR and STAT5 in multiple tissues, we investigated whether one of the factors dominates the inhibitory crosstalk. A PPAR-responsive Renilla luciferase reporter was constructed and used to monitor PPAR transcriptional activity in COS-1 cells co-transfected with a STAT5 firefly luciferase reporter. In cells co-stimulated with GH and a PPAR agonist, STAT5b inhibited expression of the PPAR-regulated Renilla luciferase reporter, whereas PPARα and PPARγ inhibited transcription of the STAT5b-regulated firefly luciferase reporter. The extent of the inhibitory crosstalk was dependent on the relative levels of expression of each transcription factor and on the relative concentrations of GH and PPAR agonist. Dose-response studies revealed that STAT5b was inhibited at an approximately 7-fold lower concentration of the PPARγ ligand troglitazone than was required for activation of PPARγ, indicating that only a portion of cellular PPARγ is needed for STAT5b inhibition. Similarly, mono-(2-ethylhexyl)phthalate (MEHP), a reproductive toxicant and primary metabolite of the environmental chemical di-(2-ethylhexyl)phthalate (DEHP), inhibited STAT5b transcriptional activity with an EC50 value of 1.1 μM, corresponding to an approximately 10-fold lower concentration than required for activation of PPARγ-dependent transcription. We conclude that the cross-inhibition between PPAR and STAT5 proceeds in a simultaneous, bidirectional manner. Exposure to phthalates and other environmental chemical activators of PPARs may thus lead to alteration of hormone-induced, STAT5-regulated gene expression in tissues such as liver, fat and breast, where both transcription factors are expressed. Conversely, STAT5-activating hormones and cytokines may modulate the responsiveness of PPARs to their foreign chemical ligands. [Copyright &y& Elsevier]

Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC50 values of 7–8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals. [Copyright &y& Elsevier]

The liver is a sexually dimorphic organ in many species, including humans. In rodent models, dramatic sex differences characterize the expression of numerous plasma proteins, receptors and other signaling molecules, and enzymes of steroid and foreign compound metabolism, including members of the cytochrome P450 (CYP) superfamily. The sexual dimorphism of liver gene expression is dictated by the temporal pattern of plasma growth hormone (GH) stimulation, which is intermittent and highly pulsatile in males and more frequent in females. Many liver-specific genes, including CYP genes, are regulated by the coordinated action of multiple hepatic nuclear factors (HNFs) through a complex transcriptional hierarchy. These HNFs are proposed to collaborate with the GH pulse-activated latent cytoplasmic transcription factor STAT5b to regulate the sex-dependent expression of liver CYPs. This hypothesis is supported by the finding that certain HNFs are regulated by GH and exhibit a differential responsiveness to the sex-specific pattern of GH secretion. In particular, recent studies of an HNF4αdeficient mouse model demonstrate an essential role for this nuclear receptor in regulating several liver-enriched transcription factors and sexually dimorphic CYPs in liver in vivo. Further studies on the mechanisms by which HNF4α and other liver factors respond to GH may expand our understanding of the mechanisms by which GH, via the coordinated action of HNFs and STAT5b, regulate sexually dimorphic liver gene expression. [ABSTRACT FROM AUTHOR]

Dimethyl sulfoxide (DMSO) is reported to induce hepatocyte redifferentiation. The impact of DMSO on liver transcription factors, cytochromes P450 (CYPs), and nuclear receptors regulating CYP expression was assayed in primary rat hepatocytes by QPCR. CYP 2B1, 3A1, and 4A1 mRNAs were reduced to 10–30% of initial liver levels without DMSO and restored at or above liver levels by DMSO treatment. In contrast, CYP1A1 mRNA increased ∼5-fold during the course of culture, independent of DMSO. DMSO enhanced expression of the nuclear receptors CAR, PXR, and PPARα 2- to 5-fold, which may contribute to the increase in basal CYP expression. Without DMSO, liver transcription factors were decreased (HNF4, C/EBPα), largely unchanged (HNF1α, HNF3α, and C/EBPβ) or elevated (HNF3β, HNF6) compared to intact liver. DMSO largely restored hepatic levels of HNF4 and C/EBPα, partially suppressed the elevated levels of HNF6, increased HNF1α ∼2-fold, and had little effect on HNF3α, HNF3β, and C/EBPβ. Overall, DMSO helped maintain normal hepatic transcription factor patterns and basal CYP and nuclear receptor profiles, suggesting that hepatocytes cultured with DMSO may be useful for CYP metabolic studies under conditions where the endogenous liver phenotype is preserved. [Copyright &y& Elsevier]

Provides information on sexual dimorphism of hepatic gene expression. Functions of the regulator of sex limitation (Rsl) locus; Details of the cloning of Rsl1 and Rsl2; Depression of gene silencing by male growth hormone pulsation.

The sensitivity of tumors to cyclophosphamide (CPA) and other anticancer prodrugs can be substantially enhanced by transduction of tumors with a prodrug-activating mammalian cytochrome P450 (CYP) enzyme in combination with the flavoenzyme P450 reductase. This gene therapy strategy provides for intratumoral prodrug activation, but is also associated with a high level of hepatic prodrug activation, which reduces the extent of intratumoral prodrug activation and contributes to systemic drug toxicity. To address this issue, five P450 inhibitors were tested for their ability to block liver CYP2C-catalyzed CPA activation selectively, i.e., without inhibiting the corresponding intratumoral activation of CPA catalyzed by a transduced CYP2B enzyme. In vitro studies revealed that the P450 inhibitors 1-aminobenzotriazole and DDEP were preferentially inhibitory toward CYP2C-dependent liver microsomal CPA activation, whereas the P450 inhibitor SKF-525A inhibited CYP2C- and CYP2B-dependent CPA activation without P450 form selectivity. By contrast, the P450 inhibitors chloramphenicol and metyrapone preferentially inhibited CYP2Bdependent CPA activation. Rat pharmacokinetic studies confirmed the inhibitory action of these compounds in vivo, with up to a 4-fold decrease in C[sub max] and a 7-fold increase in apparent half-life of the activated CPA metabolite, 4-hydroxy-CPA, seen in the case of 1-aminobenzotriazole. Although the rate of hepatic CPA activation could thus be decreased substantially by P450 inhibitor treatment, the net extent of hepatic CPA activation was only modestly decreased, as judged by plasma area-under-the-curve values for 4-hydroxy-CPA. Moreover, P450 inhibitor treatment did not decrease CPA's host toxicity and did not enhance the tumor growth delay response to CPA in rats bearing CYP2B1-transduced gliosarcomas. These findings are discussed in the context of P450-based gene therapy strategies and ongoing efforts to enhance anticancer drug activity by... [ABSTRACT FROM AUTHOR]

Cytochrome P450-based cancer gene therapy is a novel prodrug activation strategy for cancer treatment that has substantial potential for improving the safety and efficacy of cancer chemotherapeutics. The primary goal of this strategy is to selectively increase tumor cell exposure to cytotoxic drug metabolites generated locally by a prodrug-activating P450 enzyme. This strategy has been exemplified for the alkylating agents cyclophosphamide and ifosfamide, which are bioactivated by select P450 enzymes whose expression is generally high in liver and deficient in tumor cells. Transduction of tumors with a prodrug-activating P450 gene, followed by prodrug treatment, greatly increases intratumoral formation of activated drug metabolites. This leads to more efficient killing of the transduced tumor cells without a significant increase in host toxicity. P450 gene therapy is accompanied by substantial bystander cytotoxicity which greatly enhances the therapeutic effect by extending it to nearby tumor cells not transduced with the therapeutic P450 gene. Although endogenous P450 reductase is not expected to be a limiting factor in prodrug activation in tumor cells that express moderate levels of an exogenous P450 gene, P450 reductase transduction has recently been found to substantially enhance intratumoral prodrug activation and its associated therapeutic effects. Using this gene combination, an overall 50- to 100-fold increase in tumor cell kill in vivo over that provided by hepatic drug activation alone has been observed. Striking improvements in therapeutic effects can thus be achieved using an established anticancer drug in an intratumoral prodrug activation strategy based on the combination of a cytochrome P450 gene with the gene encoding NADPH-P450 reductase. This strategy is readily extendable to several other widely used P450-activated cancer chemotherapeutic prodrugs, as well as to prodrugs that undergo P450 reductase-dependent bioreductive activation and which... [ABSTRACT FROM AUTHOR]

Spatial Frequency Domain Imaging (SFDI) can provide longitudinal, label-free, and widefield hemodynamic and scattering measurements of murine tumors in vivo. Our previous work has shown that the reduced scattering coefficient (μ′s) at 800 nm, as well as the wavelength dependence of scattering, both have prognostic value in tracking apoptosis and proliferation during treatment with anti-cancer therapies. However, there is limited work in validating these optical biomarkers in clinically relevant tumor models that manifest specific treatment resistance mechanisms that mimic the clinical setting. It was recently demonstrated that metronomic dosing of cyclophosphamide induces a strong anti-tumor immune response and tumor volume reduction in the E0771 murine breast cancer model. This immune activation mechanism can be blocked with an IFNAR-1 antibody, leading to treatment resistance. Here we present a longitudinal study utilizing SFDI to monitor this paired responsive-resistant model for up to 30 days of drug treatment. Mice receiving the immune modulatory metronomic cyclophosphamide schedule had a significant increase in tumor optical scattering compared to mice receiving cyclophosphamide in combination with the IFNAR-1 antibody (9% increase vs 10% decrease on day 5 of treatment, p < 0.001). The magnitude of these differences increased throughout the duration of treatment. Additionally, scattering changes on day 4 of treatment could discriminate responsive versus resistant tumors with an accuracy of 78%, while tumor volume had an accuracy of only 52%. These results validate optical scattering as a promising prognostic biomarker that can discriminate between treatment responsive and resistant tumor models. [ABSTRACT FROM AUTHOR]

Sex differences in gene expression are widespread in the liver, where many autosomal factors act in tandem with growth hormone signaling to regulate individual variability of sex differences in liver metabolism and disease. Here, we compare hepatic transcriptomic and epigenetic profiles of mouse strains C57BL/6J and CAST/EiJ, representing two subspecies separated by 0.5–1 million years of evolution, to elucidate the actions of genetic factors regulating liver sex differences. We identify 144 protein coding genes and 78 lncRNAs showing strain-conserved sex bias; many have gene ontologies relevant to liver function, are more highly liver-specific and show greater sex bias, and are more proximally regulated than genes whose sex bias is strain-dependent. The strain-conserved genes include key growth hormone-dependent transcriptional regulators of liver sex bias; however, three other transcription factors, Trim24, Tox, and Zfp809, lose their sex-biased expression in CAST/EiJ mouse liver. To elucidate the observed strain specificities in expression, we characterized the strain-dependence of sex-biased chromatin opening and enhancer marks at cis regulatory elements (CREs) within expression quantitative trait loci (eQTL) regulating liver sex-biased genes. Strikingly, 208 of 286 eQTLs with strain-specific, sex-differential effects on expression were associated with a complete gain, loss, or reversal of the sex differences in expression between strains. Moreover, 166 of the 286 eQTLs were linked to the strain-dependent gain or loss of localized sex-biased CREs. Remarkably, a subset of these CREs apparently lacked strain-specific genetic variants yet showed coordinated, strain-dependent sex-biased epigenetic regulation. Thus, we directly link hundreds of strain-specific genetic variants to the high variability in CRE activity and expression of sex-biased genes and uncover underlying genetically-determined epigenetic states controlling liver sex bias in genetically diverse mouse populations. Author summary: Male-female differences in liver gene expression confer sex differences in diverse biological processes relevant to human health and disease but are difficult to model in inbred mice given their identical genetic backgrounds. Outbred mice provide some variability, but cross-strain studies of sex bias in rodents have not been well studied. Here we elucidate the actions of genetic factors regulating liver sex differences in two Diversity Outbred mouse founder mouse strains, C57BL/6 and CAST/EiJ. We find that many of the strain differences in sex-biased gene expression can be linked to the gain or loss of a cis regulatory element associated with one or more strain-specific sequence variants. Strikingly, in many cases, the associated cis regulatory element lacked strain-specific variants, yet was subject to coordinated, strain-dependent epigenetic regulation. Thus, harnessing the power of naturally occurring genetic diversity of Diversity Outbred mice, we integrated biological data at the genetic, epigenetic, and transcriptomic levels across evolutionary divergent mouse strains to discover hundreds of localized genomic regions that control phenotypic sex differences in the liver. These findings may serve as a model for studies of human genetic variation and the effect of population-wide variation on sex differences in health and disease. [ABSTRACT FROM AUTHOR]

Cancer chemotherapy using cytotoxic drugs can induce immunogenic tumor cell death; however, dosing regimens and schedules that enable single-agent chemotherapy to induce adaptive immune-dependent ablation of large, established tumors with activation of long-term immune memory have not been identified. Here, we investigate this issue in a syngeneic, implanted GL261 glioma model in immune-competent mice given cyclophosphamide on a 6-day repeating metronomic schedule. Two cycles of metronomic cyclophosphamide treatment induced sustained upregulation of tumor-associated CD8+cytotoxic T lymphocyte (CTL) cells, natural killer (NK) cells, macrophages, and other immune cells. Expression of CTL- and NK–cell-shared effectors peaked on Day 6, and then declined by Day 9 after the second cyclophosphamide injection and correlated inversely with the expression of the regulatory T cell (Treg) marker Foxp3. Sustained tumor regression leading to tumor ablation was achieved after several cyclophosphamide treatment cycles. Tumor ablation required CD8+T cells, as shown by immunodepletion studies, and was associated with immunity to re-challenge with GL261 glioma cells, but not B16-F10 melanoma or Lewis lung carcinoma cells. Rejection of GL261 tumor re-challenge was associated with elevated CTLs in blood and increased CTL infiltration in tumors, consistent with the induction of long-term, specific CD8+T-cell anti-GL261 tumor memory. Co-depletion of CD8+T cells and NK cells did not inhibit tumor regression beyond CD8+T-cell depletion alone, suggesting that the metronomic cyclophosphamide-activated NK cells function via CD8a+T cells. Taken together, these findings provide proof-of-concept that single-agent chemotherapy delivered on an optimized metronomic schedule can eradicate large, established tumors and induce long-term immune memory. [ABSTRACT FROM PUBLISHER]

Activation of the nuclear receptor and transcription factor CAR (Nr1i3) by its specific agonist ligand TCPOBOP (1, 4-bis[2- (3, 5-dichloropyridyloxy)]benzene) dysregulates hundreds of genes in mouse liver and is linked to male-biased hepatocarcinogenesis. To elucidate the genomic organization of CAR-induced gene responses, we investigated the distribution of TCPOBOP-responsive RefSeq coding and long noncoding RNA (lncRNA) genes across the megabase-scale topologically associating domains (TADs) that segment the genome, and which provide a structural framework that functionally constrains enhancer-promoter interactions. We show that a subset of TCPOBOP-responsive genes cluster within TADs, and that TCPOBOP-induced genes and TCPOBOP-repressed genes are often found in different TADs. Further, using DNase-seq and DNase hypersensitivity site (DHS) analysis, we identified several thousand genomic regions (ADHS) where short-term exposure to TCPOBOP induces localized changes (increases or decreases) in mouse liver chromatin accessibility, many of which cluster in TADs together with TCPOBOP-responsive genes. Sites of chromatin opening were highly enriched nearby genes induced by TCPOBOP and chromatin closing was highly enriched nearby genes repressed by TCPOBOP, consistent with TCPOBOP-responsive ADHS serving as enhancers and promoters that positively regulate CAR-responsive genes. Gene expression changes lagged behind chromatin opening or closing for a subset of TCPOBOP-responsive ADHS. ADHS that were specifically responsive to TCPOBOP in male liver were significantly enriched for genomic regions with a basal male bias in chromatin accessibility; however, the malebiased response of hepatocellular carcinoma-related genes to TCPOBOP was not associated with a correspondingly male-biased ADHS response. These studies elucidate the genome-wide organization of CAR-responsive genes and of the thousands of associated genomic sites where TCPOBOP exposure induces both rapid and persistent changes in chromatin accessibility. [ABSTRACT FROM AUTHOR]

Sex-dependent pituitary growth hormone (GH) secretory profiles—pulsatile in males and persistent in females—regulate the sex-biased, STAT5-dependent expression of hundreds of genes in mouse liver, imparting sex differences in hepatic drug/lipid metabolism and disease risk. Here, we examine transcriptional and epigenetic changes induced by continuous GH infusion (cGH) in male mice, which rapidly feminizes the temporal profile of liver STAT5 activity. cGH repressed 86% of male-biased genes and induced 68% of female-biased genes within 4 days; however, several highly female-specific genes showed weak or no feminization, even after 14 days of cGH treatment. Female-biased genes already in an active chromatin state in male liver generally showed early cGH responses; genes in an inactive chromatin state often responded late. Early cGH-responsive genes included those encoding two GH/STAT5-regulated transcriptional repressors: male-biased BCL6, which was repressed, and female-specific CUX2, which was induced. Male-biased genes activated by STAT5 and/or repressed by CUX2 were enriched for early cGH repression. Female-biased BCL6 targets were enriched for early cGH derepression. Changes in sex-specific chromatin accessibility and histone modifications accompanied these cGHinduced sex-biased gene expression changes. Thus, the temporal, sex-biased gene responses to persistent GH stimulation are dictated by GH/STAT5-regulated transcription factors arranged in a hierarchical network and by the dynamics of changes in sex-biased epigenetic states. [ABSTRACT FROM AUTHOR]

Mutations in histone acetyltransferases (HATs) are among the most common mutations in diffuse large B-cell lymphoma (DLBCL). We previously showed that two human DLBCL cell lines, RC-K8 and SUDHL2, express C-terminally truncated, HAT domain-deficient p300 proteins (p300ΔC) that are required for optimal cell proliferation. Microarray analysis of mRNA expression in RC-K8 cells following p300ΔC knockdown shows upregulation of NF-κB and p53 gene expression programs and downregulation of a MYC gene expression program. Experiments indicate that these gene expression changes are due to inhibitory effects of p300ΔC on NF-κB activity and on p53 protein levels and stimulatory effects on MYC protein levels, suggesting that p300ΔC mutants enhance the proliferation of DLBCL cells by adjusting the transcriptional output of cell-specific oncoproteins. We propose that p300/CBP gene truncation represents a new class of oncogenic mutation that optimizes the activity of context-specific oncogenic transcription factors. We propose ‘oncogenic modifier’ to describe such mutations. [ABSTRACT FROM PUBLISHER]

Background: Cyclophosphamide (CPA) can activate immunogenic tumor cell death, which induces immune-based tumor ablation and long-term anti-tumor immunity in a syngeneic C57BL/6 (B6) mouse GL261 glioma model when CPA is given on a 6-day repeating metronomic schedule (CPA/6d). In contrast, we find that two other syngeneic B6 mouse tumors, LLC lung carcinoma and B16F10 melanoma, do not exhibit these drug-induced immune responses despite their intrinsic sensitivity to CPA cytotoxicity.Methods: To elucidate underlying mechanisms, we investigated gene expression and molecular pathway changes associated with the disparate immune responsiveness of these tumors to CPA/6d treatment.Results: Global transcriptome analysis indicated substantial elevation of basal GL261 immune infiltration and strong CPA/6d activation of GL261 immune stimulatory pathways and their upstream regulators, but without preferential depletion of negative immune regulators compared to LLC and B16F10 tumors. In LLC tumors, where CPA/6d treatment is shown to be anti-angiogenic, CPA/6d suppressed VEGFA target genes and down regulated cell adhesion and leukocyte transendothelial migration genes. In GL261 tumors implanted in adaptive immune-deficient scid mice, where CPA/6d-induced GL261 regression is incomplete and late tumor growth rebound can occur, T cell receptor signaling and certain cytokine-cytokine receptor responses seen in B6 mice were deficient. Extending the CPA treatment interval from 6 to 9 days (CPA/9d) - which results in a strong but transient natural killer cell response followed by early tumor growth rebound - induced fewer cytokines and increased expression of drug metabolism genes.Conclusions: These findings elucidate molecular response pathways activated by intermittent metronomic CPA treatment and identify deficiencies that characterize immune-unresponsive tumor models and drug schedules. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 5b (STAT5b) is a growth hormone (GH)-activated transcription factor and a master regulator of sexually dimorphic gene expression in the liver. Disruption of the GH hypothalamo-pituitary-liver axis controlling STAT5b activation can lead to metabolic dysregulation, steatosis, and liver cancer. Computational approaches were developed to identify factors that disrupt STAT5b function in a mouse liver gene expression compendium. A biomarker comprised of 144 STAT5b-dependent genes was derived using comparisons between wild-type male and wild-type female mice and between STAT5b-null and wild-type mice. Correlations between the STAT5b biomarker gene set and a test set comprised of expression datasets (biosets) with known effects on STAT5b function were evaluated using a rank-based test (the Running Fisher algorithm). Using a similarity p-value ≤ 10−4, the test achieved a balanced accuracy of 99% and 97% for detection of STAT5b activation or STAT5b suppression, respectively. The STAT5b biomarker gene set was then used to identify factors that activate (masculinize) or suppress (feminize) STAT5b function in an annotated mouse liver and primary hepatocyte gene expression compendium of ~1,850 datasets. Disruption of GH-regulated STAT5b is a common phenomenon in liver in vivo, with 5% and 29% of the male datasets, and 11% and 13% of the female datasets, associated with masculinization or feminization, respectively. As expected, liver STAT5b activation/masculinization occurred at puberty and suppression/feminization occurred during aging and in mutant mice with defects in GH signaling. A total of 70 genes were identified that have effects on STAT5b activation in genetic models in which the gene was inactivated or overexpressed. Other factors that affected liver STAT5b function were shown to include fasting, caloric restriction and infections. Together, these findings identify diverse factors that perturb the hypothalamo-pituitary-liver GH axis and disrupt GH-dependent STAT5b activation in mouse liver. [ABSTRACT FROM AUTHOR]