REGULATION OF ION CHANNEL EXPRESSION, TRAFFICKING, AND FUNCTION
Heather Jones, Ph.D.
This lab focuses on the regulation of ion channel expression, trafficking, and function. Current projects include the role of calcium-activated potassium channels in the differentiation and function of osteoclasts. Osteoclasts are cells which break down bone as part of the normal remodeling process; however, inhibition of these cells’ activity may be used to treat degenerative bone disease. Our project focuses on the molecular aspects of differentiation from precursor macrophages. When exposed to differentiation factors, the precursor cells become osteoclasts. The process by which these differentiation factors cause the cells to become osteoclasts is not known. We are looking at the role that calcium-activated potassium channels have in this process. This project uses an immortalized macrophage cell line (RAW264.7), which is differentiated in the lab using stimulating factors. Western blots are used to compare potassium channel expression between differentiated and undifferentiated cells. We also use cell staining techniques and light microscopy to visualize differentiated osteoclasts. For the experimental condition, we either activate or inhibit the potassium channels and look for the level of differentiated osteoclasts. We also test functionality of the osteoclasts using culture dishes layered with a bone matrix. We grow the cells on the matrix and osteoclast activity can be determined by the formation of pits on the bone matrix. Below is an example of a western blot showing potassium channel expression in the undifferentiated and treated RAW 264.7 cells.
Additionally, the lab also has a project examining the effects of nitric oxide, a potent vasodilator, on the expression of calcium-activated potassium channels. These ion channels are known to be activated during endothelium stimulation of vascular smooth muscle relaxation. This project examines a connection between the nitric oxide-mediated vasodilation and potassium channel expression. Nitric oxide, in addition to activating guanylyl cyclase to relax smooth muscle, is also a transcription factor that alters expression of cellular proteins. This project uses a cell culture method in which cell expressing the potassium channels are treated with common nitric oxide donors and synthesis inhibitors, followed by measuring the expression of the potassium channels. Initial results show an increase in expression of the channels with nitric oxide donors (SNP, GSNO) and a decrease in expression with the inhibitor (L-NAME; see see figure below). Further studies include quantification of the transcriptional changes with the nitric oxide treatments using RT-PCR. The results of this project may provide insight into the treatment of hypertension at the level of the endothelium and channel activity.
ROLES OF CARBONIC ANHYDRASE ISOZYMES IN HOMEOSTASIS AND METABOLISM
Richard Chegwidden, Ph.D.
This project’s focus involves studies of possible clinical applications of carbonic anhydrase inhibitors, especially with respect to cancer therapy, including the potential of carbonic anhydrase inhibitors in the treatment of renal cell carcinoma. The methods being used include cell culture and cell invasion techniques, and xenografts in immunodeficient mice.
EFFECTS OF LYCOPENE ON PROSTATE CANCER
Donald Linville, Ph.D.
Our procedure has been to incubate PC-3 cells in the presence of lycopene, at varying concentrations, and then to measure cell concentration using a metabolic dye. Our current results indicate a trend of lycopene-induced inhibition of cell growth with the greatest inhibition occurring at higher concentrations. However, our aim is to show inhibition at physiologically relevant concentrations. This goal may be achieved by double-pulse the cells with lower concentrations of lycopene, once at the outset and secondly halfway through the 24 hour incubation period. Since lycopene may have a limited effective half-life, it is possible that lycopene-induced inhibition of growth occurs in the initial incubation period, but may be obscured by cell growth occurring later when lycopene is no longer effective. Once we obtain reliable inhibition of PC-3 cells at physiological concentrations of lycopene, we can examine the effects of lycopene on the chemotaxis and metastatic phases of PC-3 prostate cancer cells.