Alan J. Townsend
Experimental Therapeutics and Drug/Chemical Resistance
The process of natural selection has resulted in the evolution of a diverse array of defensive mechanisms that organisms can utilize to survive in a toxic environment. These include changes in the cellular accumulation or metabolism of toxic agents, or alterations in the structure or quantity of sensitive cellular target macromolecules. The research in my laboratory is aimed at defining the role of specific gene products (proteins) in determining the sensitivity of normal and tumor cells to mutagenic or cytotoxic agents, including both cancer chemotherapeutic drugs and environmental carcinogens. One current focus of my research is on enzymes of the glutathione S-transferase (GST) gene family, some of which can catalyze the detoxification of a broad spectrum of reactive electrophiles by conjugation with the sulfhydryl-containing tripeptide glutathione (GSH). The approach employed in this project is to transfer individual genes of interest, in this case one of several different GST isoenzymes, into cell lines which normally express little or none of the particular protein encoded by that gene. Stably transfected cell lines, which should be genetically identical except for overexpression of the transfected gene, can then be tested directly in comparison with the non-expressing parent cell line to see if increased expression of that gene confers any protective advantage against selected anticancer drugs, toxins or mutagens. A second project concerns the role of aldehyde dehydrogenases in resistance to damage by toxic aldehydes, particularly lipid aldehydes formed as a result of lipid peroxidation during oxidant stress. A similar transgenic modeling approach is employed in this project. A third project under development aims to understand the mechanism whereby production of prostaglandins by cyclooxygenase enzymes can induce programmed cell death under certain conditions, and thus may contribute to toxicity that results from oxidative stress.
The ultimate objective of this research is to identify and characterize cellular defense mechanisms which may be manipulated pharmacologically to therapeutic advantage, for chemoprevention or chemotherapy of cancer. Trainees in my laboratory may acquire expertise in enzyme kinetics, protein purification, analytical methods for proteins and nucleic acids, recombinant DNA techniques, gene cloning and transfer technology, drug metabolism, and cell culture.
Figure1. Overexpression of human GSTP-1 by transfection in T47D breast carcinoma cells results in protection against DNA damage and cytotoxicity. A) Covalent attachment of Benzo[a]pyrene diol-epoxide (BPDE) to cellular nucleic acids (DNA + RNA) is reduced in T47D. Cells (closed symbols); B) Lethality of Benzo[a]pyrene is also decreased in T47D cells.
The efficacy of therapeutic agents, as well as the toxicity of environmental chemicals is determined by their biological mechanisms of action at the cellular and physiological levels. These mechanisms may be augmented or reduced by modifying factors such as metabolism of the drug or toxin by cellular enzymes, uptake or removal of the compound by transporters, and the ability of cells to repair or bypass the affected cellular targets. Research in Dr. Suzy Torti’s lab has two major areas of focus. One project concerns how a novel iron binding compound kills cancer cells, and whether it can be used as a clinically effective chemotherapeutic drug. A second project in her lab is aimed at testing and understanding the mechanisms of new agents synthesized by Dr. Mark Welker in the WFU Chemistry Department that induce the cell to make enzymes that help prevent DNA damage and cancer by metabolic inactivation of reactive chemicals. Research in Dr. Alan Townsend’s lab utilizes genetically engineered cell lines as a tool to aid in understanding the functions and interactions of key enzymes that catalyze the activation and detoxification of drugs or carcinogens. One project in his lab examines the competition between activation of carcinogens by cytochrome P450 mixed-function oxidases and subsequent detoxification of the reactive products by glutathione S-transferases (GSTs). A second project revolves around the role of aldehyde dehydrogenases in protection against highly toxic aldehydes generated by oxidative damage to cellular lipids. Research in Dr. Charles Morrow’s lab is focused on the metabolic cooperation between detoxification by glutathione S-transferases and transmembrane transport “multidrug resistance proteins” (MRPs) that cause drug resistance in cancer cells. The expression of both the conjugation and transport pathways may have additive or synergistic protective effects, depending on the drug or toxin being studied and the specific isozymes of GST or MRP expressed in model cell lines by stable transfection. Together, the research in this section of the Biochemistry Department addresses a broad range of important questions regarding the biochemical pathways that govern the metabolism and cellular disposition of reactive chemicals and the resulting mechanisms of toxicity following environmental or therapeutic exposure of cells.
OTEO 562 (a novel chemopreventive agent)
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