Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells.
BACKGROUND
Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells.


RESULTS
Cell pellets were collected and membrane protein was isolated to determine GIRK protein expression. GIRK protein was also analyzed by immuno-precipitation. GIRK protein was over-expressed in cells by transfection of GIRK plasmids. Gene expression studies were done by real-time RT-PCR. GIRK protein expression was identified in breast cancer cell lines. Expression of GIRK1 at the indicated molecular weight (MW) (62 kDa) was seen in cell lines MDA-MB-453 and ZR-75-1. In addition, GIRK1 expression was seen at a lower MW (40-42 kDa) in MDA-MB-361, MDA-MB-468, MCF-7, ZR-75-1, and MDA-MB-453 cell lines. To prove the lower MW protein was GIRK1, MDA-MB-453 cells were immuno-precipitated. GIRK2 expression was seen in MDA-MB-468, MCF-7, and ZR-75-1 and was variable in MDA-MB-453, while GIRK4 protein expression was seen in all six cell lines tested. This is the first report indicating GIRK protein expression in breast cancer cells. To determine functionality, MDA-MB-453 cells were stimulated with ethanol. Decreased GIRK1 protein expression levels were seen after treatment with 0.12% ethanol in MDA-MB-453 breast cancer cells. Serum-free media decreased GIRK protein expression, possibly due to lack of estrogen in the media. Transfection of GIRK1 or GIRK4 plasmids increased GIRK1 protein expression and decreased gene expression in MDA-MB-453 breast cancer cells.


CONCLUSIONS
Our data indicates that functional GIRK channels exist in breast cancer cells that are involved in cellular signaling.
Dhar
Madhu S
MS
Molecular Cancer Analysis Laboratory, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996-4542, USA. mdhar@utk.edu
Plummer
Howard K
HK
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D000431:Ethanol / Q000008:administration & dosage*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D015398:Signal Transduction / Q000187:drug effects
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D000431:Ethanol