Browsing by Subject "PXR"
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Item Novel Functions for the Pregnane X Receptor include Regulation of mRNA Turnover and Involvement in Colon Cancer Progression(2011-10-21) Eagleton, Navada LorraineTo understand the mechanisms of transcriptional regulation of PXR, we performed yeast two-hybrid screenings to search for PXR-interacting proteins in a human liver cDNA library using the PXR ligand binding domain as the bait. More than one million independent clones were screened. One positive clone was a partial cDNA of CNOT2 (amino acid 183-540). CNOT2 is a component of CCR4-NOT that is a multi-subunit protein complex highly conserved from yeast to humans. Using a mammalian two-hybrid system in CV-1 cells and GST-pull down assays, we confirmed the direct interaction between PXR and CNOT2 and mapped the specific domains of association. In HepG2 cells, over expression of CNOT2 suppressed the PXR-regulated luciferase reporter gene activity. siRNA knockdown of CNOT2 potentiated PXR-transcriptional activity. These results strongly suggest that the CCR4-NOT complex is significantly involved in transcriptional regulation of PXR. The immuno-precipitated CNOT2 complex contained deadenylase activity as determined by an in vitro RNA decay assay. The presence of transfected PXR inhibited the cNOT2-associated deadenylase activity, as demonstrated by poly(A) tail PCR. Cellular localization of PXR and cNOT2 by immuno-fluorescence microscopy indicates that the interaction might occur within Cajal Bodies. Taken together, these results suggest that PXR regulates the mRNA turnover through direct interaction with the NOT2 component of the CCR4-NOT complex. PXR is also involved in colon cancer progression. Our results indicate that the evolutionarily conserved PXR protects organisms from carcinogenesis by inhibiting tumor growth as well as eliminating carcinogenic substances. Our laboratory proposes that pregnane X receptor has an important role in maintaining the balance of cells progressing through the cell cycle. In vitro and in vivo experiments demonstrate expression of PXR in colon cancer cells slows the progression of tumor formation. Colony growth of the PXR-transfected HT29 cells was suppressed in soft agar assay. In the xenograft assay, the tumor size formed in nude mice was significantly suppressed in HT29 cells stably transfected with PXR (310 mg /- 6.2 vs. 120 mg?6, p<0.01). The number of Ki-67 positive cells were significantly decreased in PXR-transfected HT29 xenograft tumor tissue compared vector-transfected HT29 controls (p<0.01) as determined by immuno-histochemistry suggesting that PXR inhibits proliferation of colon cancer cells. Results of flow cytometry analysis indicated that PXR-transfection in HT29 cells caused G0/G1 arrest. The growth inhibitory effects of PXR are likely mediated through the E2F/Rb-regulated check point since E2F1 nuclear expression was significantly inhibited by PXR over expression.Item Toxicity Analysis of Polycyclic Aromatic Hydrocarbon Mixtures(2010-01-16) Naspinski, Christine S.Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and are generated by many sources. Though the potential of PAH-rich mixtures to cause health effects has been known for almost a century, there are still unanswered questions about the levels of PAHs in the environment, the potential for human exposure to PAHs, the health effects associated with exposure, and how genetic susceptibility influences the extent of health effects in individuals. The first objective of this research was to quantify concentrations of PAHs in samples of settled house dust collected from homes in Azerbaijan, China, and Texas. The trends of PAH surface loadings and percentage of carcinogenic PAHs were China > Azerbaijan > Texas, indicating that the risk of health effects from exposure to PAHs in house dust is highest in the Chinese population and lowest in the Texas population. PAHs in China and Azerbaijan were derived mainly from combustion sources; Texas PAHs were derived from unburned fossil fuels such as petroleum. The second objective of this research was to investigate the effect of pregnane X receptor (PXR) on the genotoxicity of benzo[a]pyrene (BaP). BaP treatment resulted in significantly lower DNA adduct levels in PXR-transfected HepG2 cells than in parental HepG2 cells. Total GST enzymatic activity and mRNA levels of several metabolizing enyzmes were significantly higher in cells overexpressing PXR. These results suggest that PXR protects cells against DNA damage by PAHs such as BaP, possibly through a coordinated regulation of genes involved in xenobiotic metabolism. The third objective of this research was to investigate biomarkers of exposure in house mice (Mus musculus) exposed to PAH mixtures in situ. Mice and soil were collected near homes in Sumgayit and Khizi, Azerbaijan. Mean liver adduct levels were significantly higher in Khizi than in Sumgayit. Mean lung and kidney adduct levels were similar in the two regions. The DNA lesions detected may be a combination of environmentally-induced DNA adducts and naturally-occurring I-compounds. PAHs were present at background levels in soils from both Khizi and Sumgayit. It appears that health risks posed to rodents by soil-borne PAHs are low in these two areas.Item Transcriptional Regulation of Pregnane X Receptor by Protein Arginine Methyltransferase(2011-08-08) Xie, YingPregnane X receptor (PXR) is a ligand-dependent transcription factor that plays an important role in xenobiotic/drug metabolism. The ligand-receptor interaction transcriptionally activates phase I and phase II enzymes, and membrane-bound transporters in a coordinated manner and ultimately leads to detoxification and excretion of the ligands. One of the direct target genes is cytochrome P450 3A4 (CYP3A4) which is responsible for metabolism of over 50% of clinically used drugs. Understanding the regulation of PXR is important for treatment of disease and avoidance of untoward drug-drug interactions. In this research, we have used various biochemical and molecular approaches to investigate factors that regulate the transcriptional activity of PXR. We have stably transfected PXR into HepG2 human liver hepatoma cells. Using these PXR-HepG2 cells, we surveyed the histone methyltransferases that interact with PXR. Based on results from co-immunoprecipitation/methyltransferase, N-terminal peptide sequencing, GST-pulldown assays, we found that protein arginine methyltransferase 1 (PRMT1) is a predominant histone methyltransferase in HepG2 cells. Evidence from other laboratories suggests that histone methylation by PRMT1 sets the stage for subsequent histone modifications such as the acetylation of histone H4. These modifications are believed to be important for transcriptional and epigenetic regulation of gene expression. We hypothesize that PRMT1 plays a role in the epigenetic changes regulated by PXR. PRMT1-dependent histone methylation changes may be involved in epigenetic cell memory where prior exposure to certain agents may alter the chromatin (or priming the chromatin) with a "primed" state which alters the subsequent magnitude or duration of gene expression. In our study, we have found that pretreatment of PXR-HepG2 cells with DMSO greatly enhanced PXR-mediated activation of CYP3A4 upon rifampicin treatment. DMSO pretreatment altered histone modifications association with the promoter of the PXR-regulated gene (CYP3A4). Inhibition of histone methylation by PRMT1 either through RNAi or the methyltransferase inhibitor (Adox) abolished the priming effects. My research results strongly indicate that PRMT1 is involved in transcriptional regulation of PXR and may be involved in epigenetic memory of liver cells where prior exposure to agents changes the subsequent detoxification responses.