Graduate Thesis Or Dissertation
 

DNA binding and beyond : an investigation of proteins involved in PAH-induced carcinogesesis

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  • Exposure to polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has been determined to be a risk factor for various forms of human cancer. PAH DNA adducts have been shown to cause mutations, but carcinogenesis is also accompanied by alterations in gene expression. Inhibiting individual cytochrome P450s could clarify the interaction of P450s and other enzymes in the activation of polycyclic aromatic hydrocarbons to DNA binding intermediates. Phosphorodiamidate morpholino oligomers (PMOs), a class of antisense agents were targeted against cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1BI). No significant inhibition of enzyme activity or expression was observed with any PMO used as measured by ethoxyresorufin-O-deethylase (EROD) activity and immunoblots. It was demonstrated that BPDE may react with PMOs in vitro, and PMOs may be segregated in lysosomes, blocking their efficacy. Nonspecific effects by the PMO on CYP1A1 activity were observed. These observations indicate multiple confounding effects in the use of PMOs for this purpose. Many of the genes regulated by histone deacetylases are involved in proliferation, cell function, and differentiation, and HDAC inhibitors are of great interest in cancer research. To probe epigenetic regulation of CYP1A1, MCF-7 cells were treated with two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA). SARA and TSA increased EROD activity and in RT-PCR. SARA and TSA reduced B[a]P induced CYP1A1 and CYP1B1 mRNA levels. B[a]P DNA binding was not significantly altered by SAHA or TSA treatment. To assay global protein expression changes after treatment with PAR, MCF-7 cells were treated with B[a]P, DB{a,1]P, coal tar extract (SRM 1597) and diesel exhaust extract (SRM 1975), Proteins were separated by two-dimensional electrophoresis, and analyzed using PDQuest. Spots of interest were excised and identified by matrix assisted laser desorption/ionization time of flight time of flight mass spectroscopy. Alterations in expression of heat shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins were observed. Universally increased expression was observed for tubulin alpha and myosin light chain alkali, cyclophilin B, heterogeneous nuclear riboprotein B1, and alpha enolase. Additional proteins exhibiting change in expression included histone H2A.1, heat shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein.
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file details HoovenLouisaAda2004.pdf 2017-08-08 Pubblico Scaricare