Syntheses of Cross-Linking Agents, Molecular Modeling of Oligonucleotides and Polypeptide-Ion Complexes

Each section of this thesis will be subdivided into three parts encompassing all of the research in which I have been involved during the past three years. These will be referred to under the headings "Syntheses:' "Molecular Modeling," and "Cross-linking Efficiencies." Each of these subdivisions may have divisions within them when necessary in order to fully detail the research.

Epichlorohydrin Cross-Linking of Synthetic DNA Oligomers

Epichlorohydrin (ECH), an important chemical in the synthetic polymer industry, is a bifunctional alkylating agent with the potential to form DNA interstrand crosslinks. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown to undergo reaction with DNA in vivo and in vitro. We are using denaturing polyacrylamide gel electrophoresis to assess cross-linking of synthetic DNA oligomers by both ECH and the related compound, epibromohydrin (EBH). Both epihalohydrins produce a low-mobility band on denaturing gels consistent with an interstrand cross-link. Moreover, the efficiencies, sequence preferences, reaction kinetics, and pH dependence differ for the two compounds, suggesting different mechanisms of reaction. Understanding these alkylation reactions may help explain the role of the epihalohydrins in cancer development.

Changes in Apoptotic Gene Expression Induced by DNA Cross-Linkers

The Millard Research Laboratory is interested in the cytotoxic mechanisms of the bifunctional alkylators diepoxybutane (DEB), epichlorohydrin (ECH), and (1-chloroethenyl) oxirane (COX). Studies performed in the laboratory examine the dual nature of these DNA cross-linking compounds that can act as carcinogens or anti-cancer agents. The mechanisms through which these compounds induce cell death are explored in this study. Cells either undergo cell death due to necrosis or apoptosis. HL-60 cells were treated with varying concentrations of DEB, ECH, or COX. A caspase 3/7 assay was used to test for induction of apoptosis in the treated cells at varying incubation times. It was concluded that DEB induces apoptosis in HL-60 cells treated with 100 μM for 24 hours. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was then used to explore the changes in gene expression of various genes involved in apoptosis signaling. The results were inconclusive as to specific genes involved in DEB induced apoptosis, but the data does suggest that apoptosis is induced by a mitochondrial-mediated apoptosis signaling pathway.

Cytotoxicity of Diepoxybutane and Epichlorohydrin in Relation to Stages of the Cell Cycle

Work conducted in the Millard Biochemistry Research Laboratory examines the dual nature of molecules as carcinogens and anti-tumor agents through the molecular mechanisms of duplex DNA damage by bifunctional alkylating agents. Diepoxybutane (DEB) and epichlorohydrin (ECH) are polar molecules that form covalent DNA interstrand lesions by cross-linking the N7 position of deoxyguanosine residues. A recent experiment indicated that ECH preferentially targets nuclear DNA over mitochondrial DNA, whereas DEB shows similar rates of lesion formation for both loci. It was concluded that preferential targeting of nuclear DNA results from relatively poor uptake of ECH across the mitochondrial membrane. The objective of my honors research was to determine if the cytotoxicities of DEB and ECH vary according to the presence of the nuclear envelope in 6C2 chicken erythro-progenitor cells. The cytotoxicity of DEB and ECH was compared between cells randomly distributed throughout the cell cycle (Go/G, and S » G2/M) and cells enriched in G2/M stages. Results indicated that ECH is more cytotoxic than DEB in both unsynchronized control 6C2 cells and synchronized 6C2 cells enriched in G2/M stages of the cell cycle. Treatment with either bifunctional alkylating agent induced greater cytotoxicity in 6C2 cells enriched in G2/M stages than in unsynchronized control 6C2 cells, suggesting that the presence of the nuclear envelope-or any plasma membrane-may inhibit the reactivity of DEB and ECH.