RESEARCH:

Reactive oxygen species in macrophage differentiation and function.

Regulation of apoptotic pathways and expression of modulators of apoptosis during induced differentiation of promonocytic leukemia cells. Receptor-mediated apoptosis and cytotoxicity of cancer cells.

As a consequence of normal redox activity, aerobic organisms generate partially reduced oxygen species variously called oxygen free radicals, active oxygen, or reactive oxygen species (ROS). ROS are potentially cytotoxic because they can damage (oxidize) lipids, proteins, and nucleic acids. The importance of ROS in cellular physiology is underscored by the fact that all aerobic cells possess complex enzymatic and chemical mechanisms for detoxifying ROS that include the enzymes copper-zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. The totality of processes by which a cell maintains concentrations of ROS compatible with cellular function is known as redox buffering. Certain physiological insults such as hyperbaria, radiation, and redox-active drugs may, however, overwhelm, impair or otherwise alter the redox buffering capacity of a cell, thereby generating a prooxidant state that predisposes the cell to neoplastic transformation (cancer), necrosis, or apoptosis (programmed cell death). Paradoxically, ROS have been implicated in the regulation of gene expression related to both normal and pathological processes. Like nitric oxide and carbon monoxide, ROS appear to function both as second messengers and as cytotoxic agents.

My research has addressed the regulation of ROS-scavenging enzymes in various physiological processes such as differentiation, neoplasia, thermotolerance, and apoptosis in diverse organisms (Crithidia lucilliae thermophila, an insect hemoflagellate related to the trypanosomes, Artemia salina, the brine shrimp, and Nicotiana tabacum), as well as in cultured human myeloid leukemia cell lines (THP-1, HL-60, U937). Current efforts in my laboratory are aimed at elucidating the role of oxidants in the induced differentiation of THP-1 and U937 cells to macrophages. The THP-1 model offers a unique opportunity for investigating the regulation of gene expression related to the acquisition of the macrophage phenotype(s), as well as for understanding changes in susceptibility to apoptosis as a function of differentiation, cytokine stimulation, or cellular interactions. Again, the role of ROS in apoptosis is paradoxical: the anti-apoptotic protein bcl-2 has anti-oxidant properties, while superoxide, an oxidant, is anti-apoptotic for some cells. The ability to respond to a death signal is an important attribute of most normal cells, and when this ability is lost, either through mutation or functional inactivation (promoter methylation, abrogation by viral proteins, etc.), cancer may result. This conclusion is based on the observation that most anti-cancer drugs kill cells not by inhibiting proliferation (as was previously thought), but by promoting apoptosis. What emerges from these studies is that the susceptibility of a cell to an apoptotic signal is a function of its ability to modulate the signal, as well as the functional completeness of its central death machinery. These interactions are subtle, complex, and cell type-specific. Much has been accomplished in the field of apoptosis in a relatively short time; even more remains to be done. A special interest of my laboratory at this time is the role of Fas-mediated apoptosis in the killing of tumor cells or virus-infected cells by THP-1-derived macrophages as well as in the autocrine suicide (activation-induced apoptosis) of the macrophages themselves.

Laboratory techniques used in these investigations include mammalian cell culture, cell viability assays, assays for enzyme activity, protein determination, protein isolation, immunoprecipitation, Western blotting, TUNEL assays, DNA fragmentation assays, RNA purification, reverse transcription-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assays.

TEACHING:

Biochemical genetics, a lecture-laboratory course that explores the regulation of gene expression in relation to proliferation and differentiation as well as human genetic diseases including cancer. Emphasis is placed on the analysis of current periodical literature.

Techniques in Biochemistry (a team-taught, lecture-laboratory course)

Biology 101 and 102 (Coordinator and instructor, team-taught, lecture-laboratory, introductory biology courses).

RECENT PUBLICATIONS:

Han, Z., Hendrickson, E.A., Bremner, T. A., and Wyche, J.H. (1997). A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. Journal of Biological Chemistry 272, 13432-13436.

Bremner, T. A., D'Costa, N., Dickson, L. A., and Asseffa, A. (1996). A decrease in glucose 6-phosphate dehydrogenase activity and mRNA is an early event in phorbol ester-induced differentiation of THP-1 promonocytic leukemia cells. Life Sciences 58, 1015-1022.

Asseffa, A., Dickson, L. A., Mohla, S., and Bremner, T. A. (1993). Phorbol myristate acetate-differentiated THP-1 cells display increased levels of MHC class I and class II mRNA and interferon-(-inducible tumoricidal activity. Oncology Research 5, 11-18.

Emtage, M. A. and Bremner, T. A. (1993). Thermal regulation of active oxygen scavenging enzymes in Crithidia luciliae thermophila. Journal of Parasitology 79, 809-814.