Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression.

One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.

Imaging of metastatic breast cancer: distribution and radiological assessment at presentation.

This prospective study was performed to document the distribution of sites of disease in breast cancer patients with newly diagnosed metastatic disease, and to identify those with assessable or measurable disease by International Union Against Cancer (UICC) criteria. Data were collected on a consecutive series of 100 patients presenting with metastatic breast cancer. Imaging findings recorded included whether patients had assessable or measurable disease and which potentially assessable sites were rendered unassessable by radiotherapy. Radiologically diagnosable complications were recorded. Skeletal metastases comprised the majority, with 67 patients having skeletal involvement, although of these only 33 (49%) had assessable disease and 24 (36%) measurable disease. Sixteen (24%) patients had radiographically occult metastases. Liver ultrasound examination showed metastatic disease in 32 patients, of whom 28 (88%) had measurable lesions and 12% diffuse disease. Chest radiographs demonstrated metastatic disease in 42 patients, with assessable disease in 39 (93%) and measurable disease in 18 (43%). In total, 80 patients had radiologically assessable disease, with five rendered unassessable by the administration of radiotherapy to the only assessable site. Therefore, of the 100 patients, 75% (95% confidence interval (CI) 65-83) had radiologically assessable disease, with 55% (95% 45-65 CI) having measurable lesions by UICC criteria.

Accessibility and activity of the promoter for a dioxin-inducible ecto-ATPase gene.

We have analyzed the core promoter for a dioxin-inducible ecto-ATPase gene in mouse hepatoma cells. The transcriptional initiation site maps to a region that contains neither a TATA sequence nor a consensus initiator sequence nor a downstream promoter element. The core promoter has constitutive activity that does not require either the aromatic hydrocarbon receptor or its heterodimerization partner Arnt. Two GC-rich regions contribute approximately equally to the constitutive activity. Proteins constitutively occupy the GC-rich regions in chromatin. The promoter assumes a non-nucleosomal configuration in its native chromosomal setting in both uninduced and dioxin-induced cells. Our findings imply that the GC-rich regions together with their cognate binding proteins carry out core promoter functions for the ecto-ATPase gene. The promoter is constitutively accessible in situ, and chromatin structure is not a limiting factor for dioxin-inducible ecto-ATPase transcription in intact cells.

Dioxin-inducible transactivation in a chromosomal setting. Analysis of the acidic domain of the Ah receptor.

We analyzed the transactivation function of the acidic segment of the Ah receptor (amino acids 515-583) by reconstituting AhR-defective mouse hepatoma cells with mutants. Our data reveal that both hydrophobic and acidic residues are important for transactivation and that these residues are clustered in two regions of the acidic segment of AhR. Both regions are crucial for function, because disruption of either one substantially impairs transactivation of the chromosomal CYP1A1 target gene. Neither region contains an amino acid motif that resembles those reported for other acidic activation domains. Furthermore, proline substitutions in both regions do not impair transactivation in vivo, a finding that implies that alpha-helix formation is not required for function.

Digital imaging improves upright stereotactic core biopsy of mammographic microcalcifications.

AIM: This comparative study was carried out to assess the effect of using digital images compared to conventional film-screen mammography on the accuracy of core biopsy of microcalcifications using upright stereotactic equipment. MATERIALS AND METHODS: The biopsy results from a consecutive series of 104 upright stereotactic 14-gauge core biopsies performed with conventional X-ray (Group A) were compared with 40 biopsies carried out using stereotaxis with digital imaging (Group B). In all cases specimen radiography was performed and analysed for the presence of calcifications. Pathological correlation was then carried out with needle and surgical histology. RESULTS: The use of digital add-on equipment increased the radiographic calcification retrieval rate from 55 to 85% (P < 0.005). The absolute sensitivity of core biopsy in pure ductal carcinoma in situ (DCIS) cases rose from 34 to 69% (P < 0.03), with the complete sensitivity increasing from 52 to 94% (P < 0.005). For DCIS with or without an invasive component the absolute sensitivity rose from 41 to 67% (P = 0.052), while the complete sensitivity was 59% before and 86% after the introduction of digital imaging (P < 0.04). CONCLUSION: Digital equipment improves the performance of upright stereotactic core biopsy of microcalcifications, giving a significantly increased success rate in accurately obtaining calcifications. This leads to an improvement in absolute and complete sensitivity of core biopsy when diagnosing DCIS.

The aromatic hydrocarbon receptor, transcription, and endocrine aspects of dioxin action.

The widespread and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin elicits adaptive and adverse biological responses by inducing changes in gene transcription. Some of dioxin's effects reflect disruption of endocrine homeostasis. The aromatic hydrocarbon receptor protein, together with its heterodimerization partner, the aromatic hydrocarbon receptor nuclear translocator protein, mediates dioxin action. There are notable similarities between the mechanism of dioxin action and the mechanisms of steroid/retinoid/thyroid hormone action. Studies of dioxin action may provide insights into the regulation of hormone-responsive genes and endocrine physiology.

Regulation of GLUT1 gene transcription by the serine/threonine kinase Akt1.

We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.

Induction of cytochrome P4501A1.

Cytochrome P4501A1 is a substrate-inducible microsomal enzyme that oxygenates polycyclic aromatic hydrocarbons, such as the carcinogen benzo(a)pyrene, as the initial step in their metabolic processing to water-soluble derivatives. Enzyme induction reflects increased transcription of the cognate CYP1A1 gene. The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin is the most potent known cytochrome P4501A1 inducer. Two regulatory proteins, the aromatic (aryl) hydrocarbon receptor (AhR) and the AhR nuclear translocator (Arnt), mediate induction. AhR and Arnt are prototypical members of the basic helix-loop-helix/Per-Arnt-Sim class of transcription factors. Mechanistic analyses of cytochrome P4501A1 induction provide insights into ligand-dependent mammalian gene expression, basic helix-loop-helix/Per-Arnt-Sim protein function, and dioxin action; such studies also impact public health issues concerned with molecular epidemiology, carcinogenesis, and risk assessment.

A comparison of 14 and 12 gauge needles for core biopsy of suspicious mammographic calcification.

This study was carried out to compare the efficacy of 14 vs 12 G needles in stereotactic core biopsy of mammographic calcification. A consecutive series of 100 impalpable mammographic calcifications, without an associated mass and requiring stereotactic core biopsy were randomly allocated to either 14 G or 12 G needle sampling. All biopsies were performed using an upright stereotactic digital unit (Senovision GE) and a Bard automated biopsy gun. Core biopsy results were categorized as either normal, benign, atypical ductal hyperplasia, suspicious of ductal carcinoma in situ (DCIS), DCIS or invasive cancer. The radiographic calcification retrieval rates, complete and absolute sensitivity for malignancy of DCIS and DCIS with an invasive focus were obtained by comparison of core results with surgical histology. Radiographic calcification retrieval was achieved in 86% when using 14 G and 12 G needles. The absolute sensitivity and complete sensitivity for diagnosing DCIS were the same with 12 G and 14 G needles (72% versus 71% and 93% versus 94%, respectively). The use of 12 G needles does not appear to confer benefit over the use of 14 G needles in the diagnosis of mammographic calcification.

Hypoxia-inducible mammalian gene expression analyzed in vivo at a TATA-driven promoter and at an initiator-driven promoter.

We have analyzed protein-DNA interactions in vivo at transcriptional control elements for two hypoxia-inducible genes in mouse hepatoma cells. The promoter for the phosphoglycerate kinase 1 (PGK1) gene contains an initiator element, but no TATA sequence, whereas the promoter for the glucose transporter 1 (Glut1) gene contains a TATA element but no initiator sequence. Our findings reveal hypoxia-inducible, Arnt-dependent occupancy of DNA recognition sites for hypoxia-inducible factor 1 (HIF-1) upstream of both target genes. The conserved recognition motif among the five recognition sites is 5'-CGTG-3'. The PGK1 promoter exhibits constitutive occupancy of a binding site for an unknown protein(s); however, we detect no protein-DNA interaction at the initiator element, in either uninduced or induced cells. The Glut1 promoter also exhibits constitutive protein binding; in addition, the TATA element exhibits partial occupancy in uninduced cells and increased occupancy under hypoxic conditions. We find no evidence for hypoxia-induced changes in chromatin structure of either gene. Time-course analyses of the Glut1 gene reveal a temporal relationship between occupancy of HIF-1 sites and TATA element occupancy. Our findings suggest that the promoters for both hypoxia-responsive genes constitutively maintain an accessible chromatin configuration and that HIF-1 facilitates transcription by recruiting and/or stabilizing a transcription factor(s), such as TFIID, at both promoters.

A novel response to dioxin. Induction of ecto-ATPase gene expression.

We used differential display to discover a new gene that the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) regulates in mouse hepatoma cells. Its predicted amino acid sequence suggests that the gene encodes an ecto-ATPase that contains multiple glycosylation sites, conserved cysteine residues, and apyrase conserved regions. cDNA expression experiments in mouse hepatoma cells confirm that the new gene encodes an ecto-ATPase. Wild-type mouse hepatoma cells contain both constitutive and TCDD-inducible ecto-ATPase activity. Induction of ecto-ATPase gene expression by TCDD is direct and occurs at the transcriptional level. Studies in mutant hepatoma cells indicate that induction requires both the aromatic hydrocarbon receptor (AhR) and the AhR nuclear translocator (Arnt). Furthermore, induction requires AhR's transactivation domain, but not that of Arnt. Our findings reveal new aspects of dioxin's biological effects and TCDD-dependent gene regulation.

Down-regulation of major histocompatibility complex Q1b gene expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We analyzed mouse hepatoma cells using differential display to discover new genes that respond to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We identified a class I major histocompatibility complex (MHC) gene, which we designated as MHC Q1b, whose expression decreases in the presence of TCDD. TCDD-induced down-regulation of MHC Q1b requires both the aromatic hydrocarbon receptor and the aromatic hydrocarbon receptor nuclear translocator, transcription factors that up-regulate other genes in response to TCDD. Down-regulation of MHC Q1b by TCDD appears to involve both transcriptional and post-transcriptional regulatory events; the post-transcriptional destabilization of MHC Q1b mRNA is probably a secondary response to TCDD. Our findings reveal new mechanistic aspects of gene regulation by TCDD. In addition, our observations suggest a mechanism that might account for some of TCDD's immunotoxic effects.

Transactivation domains facilitate promoter occupancy for the dioxin-inducible CYP1A1 gene in vivo.

We have studied the transcriptional regulation of the dioxin-inducible mouse CYP1A1 gene in its native chromosomal setting. We analyzed the ability of aromatic hydrocarbon receptor (AhR) mutants and AhR chimeras to restore dioxin responsiveness to the CYP1A1 gene in AhR-defective mouse hepatoma cells. Our data reveal that transactivation domains in AhR's C-terminal half mediate occupancy of the nuclear factor 1 site and TATA box for the CYP1A1 promoter in vivo. Transactivation domains of VP16 and AhR nuclear translocator, but not Sp1, can substitute for AhR's C-terminal half in facilitating protein binding at the promoter. Our data also reveal an apparent linear relationship between promoter occupancy and CYP1A1 gene expression in chromatin. These findings provide new insights into the in vivo mechanism of transcriptional activation for an interesting mammalian gene.

A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

To identify new proteins involved in dioxin-dependent signal transduction and transcriptional regulation, we used a yeast two-hybrid system to identify proteins that interact with the Ah receptor (AhR). We cloned a mouse cDNA, which encodes a novel approximately 37-kDa protein that binds to AhR; we have designated the protein as Ah receptor-interacting protein (AIP). The amino acid sequence of mouse AIP exhibits homology with members of the FK506-binding protein family. AIP also contains three tetratricopeptide repeat (TPR) motifs; the TPR sequence is present in proteins required for cell cycle control and RNA synthesis and in steroid receptor-binding immunophilins. Coimmunoprecipitation experiments in mouse hepatoma cells reveal that AIP is cytoplasmic and associates with unliganded Ah receptor and with hsp90; 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment disrupts the AhR-AIP-hsp90 interaction. Overexpression of AIP augments the response of the CYP1A1 gene to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Our data suggest that AIP influences ligand receptivity and/or nuclear targeting of AhR.

Induction of phosphoglycerate kinase 1 gene expression by hypoxia. Roles of Arnt and HIF1alpha.

To identify new dimerization partners for the aromatic hydrocarbon receptor nuclear translocator (Arnt), we used its N-terminal region (amino acids 1-470) as a target in a two-hybrid screening procedure, and we cloned the murine form of hypoxia-inducible factor 1alpha (HIF1alpha). Sequence comparisons reveal substantial identity between mouse and human HIF1alpha. Hypoxia induces a 10-fold accumulation of phosphoglycerate kinase 1 mRNA in wild type mouse hepatoma (Hepa 1c1c7) cells; the induction mechanism is Arnt dependent because induction does not occur in Arnt-defective cells. Furthermore, induction of phosphoglycerate kinase 1 mRNA requires Arnt's N-terminal region, which mediates DNA binding and heterodimerization; in contrast, induction does not require Arnt's C-terminal region, which mediates transactivation. We also show that a GAL4-HIF1alpha fusion protein transactivates a GAL4-dependent gene in the absence of Arnt, that HIF1alpha's transactivation capability is inducible by hypoxia, and that both hypoxia responsiveness and transactivation capability reside within the C-terminal 83 amino acids of HIF1alpha. Our findings generate new insights into the mechanism by which Arnt and HIF1alpha induce transcription in response to hypoxia.

Cytochromes P450 5: induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription.

The induction of microsomal cytochrome P4501A1 by polycyclic aromatic hydrocarbons represents an interesting response by which mammalian cells adapt to xenobiotic exposure. Enzyme induction reflects increased transcription of the corresponding CYP1A1 gene. Analyses of the induction mechanism using genetic, biochemical, and molecular biological approaches have revealed a novel transcriptional regulatory pathway that involves ligand-dependent heterodimerization between two basic helix-loop-helix proteins (the Ah receptor and Arnt), interaction of the heterodimer with a xenobiotic-responsive enhancer, transmission of the induction signal from the enhancer to the CYP1A1 promoter, and alterations in chromatin structure. Current techniques permit examination of the induction mechanism in intact cells and analyses of the CYP1A1 gene in its native chromosomal configuration. Such experiments generate new insights into the control of mammalian transcription that are of relatively broad interest.

The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin.

The aromatic hydrocarbon receptor (AhR) has been defined and characterized according to its ability to mediate biological responses to exogenous ligands, such as the synthetic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The natural ligand(s) for AhR is unknown, and we know relatively little about AhR function in the absence of TCDD. Here, we have exploited the availability of AhR-defective (AhR-D) mouse hepatoma (Hepa 1c1c7) cells to analyze AhR's effects under conditions in which TCDD is not present. Our results reveal that AhR-D cells exhibit a different morphology, decreased albumin synthesis, and a prolonged doubling time compared with wild-type cells. Introduction of AhR cDNA into AhR-D cells by stable transfection alters these characteristics such that the cells resemble wild-type cells. Conversely, introduction of antisense AhR cDNA into wild-type cells changes their phenotype such that they resemble AhR-D cells. Fluorescence microscopy reveals that AhR-D cells do not exhibit an increased rate of death. Flow cytometric and biochemical analyses imply that the slowed growth rate of AhR-D cells reflects prolongation of G1. Our findings reveal a potential link between AhR and the G1 phase of the Hepa 1c1c7 cell cycle. These effects of AhR occur in the absence of TCDD. We speculate that they represent responses to an endogenous AhR ligand in Hepa 1c1c7 cells.

DNA binding by the heterodimeric Ah receptor. Relationship to dioxin-induced CYP1A1 transcription in vivo.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the microsomal enzyme cytochrome P4501A1 by increasing the transcription rate of the CYP1A1 gene. Induction requires two basic helix-loop-helix proteins, the ligand-binding aromatic hydrocarbon receptor (AhR) and its heterodimerization partner, the AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer induces transcription by binding to dioxin-responsive elements (DREs) within an enhancer upstream of the CYP1A1 gene. The basic regions of AhR and Arnt are crucial for DRE binding. We have mutated these regions in order to analyze the relationship between DRE binding (determined in vitro using an electrophoretic mobility shift assay) and induction of CYP1A1 transcription (determined in vivo by genetic complementation of AhR-defective and Arnt-defective mouse hepatoma cells, using an RNase protection assay to measure mRNA accumulation). Our findings reveal the amino acids in the basic regions of AhR/Arnt that are important for both DRE binding and induction of transcription. This information provides biological background for the interpretation of structural (e.g. crystallographic) studies of the interactions between AhR/Arnt and the DRE. Our findings also indicate that the in vitro behavior of the mutants does not consistently predict their functional activity in vivo. Thus, genetic complementation constitutes an important and stringent test for analyzing the effects of mutations on AhR/Arnt function.