Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
1999-06-21
2002-08-13
Priebe, Scott D. (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254210, C435S006120, C435S471000
Reexamination Certificate
active
06432692
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to detection and measurement of environmental pollutants. In particular, the invention provides highly sensitive biological assays for the detection of polycyclic aromatic hydrocarbons, dioxins, PCBs, and other substances which are agonists of the aryl hydrocarbon receptor.
BACKGROUND OF THE INVENTION
Several publications and patents are referenced in this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein.
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates a number of biological responses to planar aromatic hydrocarbons (PAHs). Chemicals which interact with the AHR include a variety of environmental contaminants, such as dioxins, PCBs, PBBs and benzo(a)pyrene, as well as natural products, such as flavones and carbazoles. One of the most potent agonists of the Ah-receptor is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or “dioxin”). TCDD is the prototype for a large family of highly toxic carcinogenic and teratogenic environmental contaminants (Poland A., Knutson, J. C., Ann. Rev. Pharmacol. Toxicol. 22:517-554, 1982). Members of this family include a number of halogenated dibenzo-p-dioxin, dibenzofuran, and biphenyl isomers, which induce a number of receptor-mediated toxic responses, including a severe wasting syndrome, epidermal hyperplasia and metaplasia, tumor promotion and thymic involution.
The AHR resides primarily in the cytosol, where it is associated with a dimer of the molecular chaperone, Hsp90. Upon binding an agonist, the AHR dissociates from Hsp90, translocates to the nucleus and dimerizes with a structurally related protein, ARNT. This complex interacts with enhancer elements upstream of target promoters and up-regulates the transcription of a variety of xenobiotic metabolizing enzymes (e.g., the Cyt P450 encoded by CYP1A1). The AHR and ARNT are both members of the basic helix-loop-helix-PAS superfamily. The helix-loop-helix domain serves as a dimerization surface for AHR and ARNT and also positions the basic &agr;-helix within the major groove of B-DNA to enable specific interactions with target enhancer elements. The PAS domain, a region of ~250 amino acids, functions as a dimerization surface, harbors a repressor region, and also contains regions required for binding agonist and forming interactions with Hsp90.
Recently, other proteins that interact with AHR have been identified. One such “Ah receptor-associated protein” is ARA9 (Carver & Bradfield, J. Biol. Chem. 272: 11452-11456, 1997), also referred to as AIP or XAP2 (Ma & Whitlock, J. Biol. Chem. 272: 8878-8884, 1997; Meyer et al., Mol. Cell. Biol. 18: 978-988, 1998). The 37 kDa ARA9 protein displays structural similarity to the glucocorticoid receptor-associated immunophilin FKBP52. In its amino-terminal half, ARA9 displays sequence identity to a region in FKBP52 known to harbor both peptidylprolyl cis-trans isomerase activity and a high affinity binding site for the immunosuppressant macrolide, FK506. The carboxyl terminal portion of ARA9 has amino acid sequence identity to a region in FKBP52 that harbors multiple TPR motifs (Carver & Bradfield, 1997, supra).
ARA9 has been reported to be involved in the AHR signaling pathway in vivo (Carver & Bradfield, 1997, supra). It has also been reported that overexpression of the protein in mouse hepatoma cells increases the response of that system to the AHR agonist, TCDD, by 2-3 fold.
Another type of protein that has been found to associate with the AHR is the tyrosine kinase encoded by the src oncogene. It has been reported that a c-Src protein kinase specifically associates with the AHR complexed with Hsp90, and upon ligand binding to AHR, the c-Src protein is activated and released from the complex (Enan & Matsamura, Biochem. Pharmacol. 52: 1599-1612, 1996). It has also been reported that tyrosine kinase signaling is needed to induce expression of AHR in 3T3 cells, and that a constitutively active tyrosine kinase, v-Src, is sufficient to confer high levels of AHR expression in the absence of serum (Vaziri et al., J. Biol. Chem. 271: 25921-25927, 1996).
Isolated nucleic acid molecules encoding AHR and ARNT have been used in a recombinant cellular assay system for the detection and measurement of AHR agonists (U.S. Pat. No. 5,650,283 to Bradfield et al., issued Jul. 22, 1997). Cells were engineered to express either ARNT-dependent or ARNT-independent AHR, capable of driving transcription of a reporter gene operably linked to a dioxin-responsive enhancer element (DRE), upon exposure of the cells to an AHR agonist.
The aforementioned cellular assay system provides an effective and rapid means for detecting the presence or amount of an AHR agonist in a test sample. Inasmuch as many such AHR agonists are serious environmental contaminants, the desirability of such an assay is apparent. It would be an advance in the art of environmental monitoring to improve upon such an assay system, e.g., by improving its sensitivity or responsiveness.
SUMMARY OF THE INVENTION
The present invention provides a cellular assay system for AHR agonists that comprises several features and advantages not found in currently available assay systems. These assays incorporate the use of additional proteins involved in the AHR signal transduction pathway, which, alone or in combination, result in an unexpectedly large increase in sensitivity and maximal response of AHR to agonists.
According to one aspect of the invention, a transgenic cell is provided that comprises at least one heterologous vector expressing AHR, ARNT and a protein selected from the group consisting of ARA9, ARA3, Src and any combination thereof. The cell further comprises a reporter gene operably linked to at least one dioxin responsive element, such that the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the AHR. The transgenic cell may be any appropriate procaryotic or eucaryotic cell. In a preferred embodiment, the transgenic cell is a yeast cell. In another embodiment, the transgenic cell is a vertebrate cell, most preferably a mammalian cell. In another embodiment, the transgenic cell is an insect cell.
According to another aspect of the invention, a transgenic cell is provided that comprises at least one heterologous vector expressing: (i) a chimeric AHR having its dimerization and DNA binding domain replaced with an analogous DNA binding domain from another protein capable of binding a DNA transcriptional activation element and activating transcription of a gene operably linked to the element; (ii) a protein selected from the group consisting of ARA9, ARA3, Src and any combination thereof; and (iii) a reporter gene operably linked to at least one DNA transcriptional activation element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the chimeric AHR. In a preferred embodiment, the transgenic cell is a yeast cell. In another embodiment, it is a vertebrate cell, preferably a mammalian cell. In another embodiment, it is an insect cell.
According to another aspect of the invention, a method is provided for determining if a test compound is an AHR agonist. In one embodiment, the method comprises: (a) providing a transgenic cell comprising at least one heterologous vector expressing AHR, ARNT and a protein selected from the group consisting of ARA9, ARA3, Src and any combination thereof, the cell further comprising a reporter gene operably linked to at least one dioxin responsive element, wherein the reporter gene expresses a detectable gene product as a result of an AHR agonist binding to the AHR; (b) preparing a culture of the transgenic cells; (c) incorporating the test compound into the cell culture under conditions permitting expression of the heterologous vectors, heterodimerization of the AHR and the ARNT, and binding of the heterodimer to the dioxin responsive element; and (d) measuring expression of the reporter gene by detecting t
Bradfield Christopher A.
Carver Lucy A.
Dunham Elizabeth E.
Kaushal Sumesh
Priebe Scott D.
Wisconsin Alumni Research Foundation
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