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. / Graduation date: 2004
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30025 |
| Date | 15 December 2003 |
| Creators | Hooven, Louisa Ada |
| Contributors | Baird, William M. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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