Expression of Drosophila Ca2+ permeable transient receptor potential-like channel protein in a prostate cancer cell line decreases cell survival.
The effects of expression of Drosophila melanoga ster Ca(2+) permeable transient receptor potential-like (TRPL) channels, under the control of the cytomegalovirus (CMV) or prostate cell-specific promoters, on cell survival and apoptosis in the androgen-sensitive LNCaP prostate cancer cell line were investigated. A prostate-specific antigen (PSA) promoter construct (designated PSAEn/PSAPr) composed of a 0.6 kb region of the promoter and a 1.45 kb region of the enhancer resulted in androgen-dependent and prostate-specific expression of a luciferase reporter gene in transiently transfected LNCaP cells. Expression of the enhanced green fluorescence protein-TRPL chimeric protein under the control of the CMV promoter was confirmed by Western blot. Whereas the majority of the expressed protein was located in the cytoplasmic space, confocal microscopy with the CD-9 protein as a plasma membrane marker demonstrated that approximately 10% of the expressed TRPL protein was located in a band in the plasma membrane. Using recombinant adenoviruses, expression of the TRPL protein was associated with an increase in both the initial and sustained rates of Ca(2+) inflow. Expression of TRPL under the control of the CMV promoter for 96 hours decreased cell number and increased the number of cells undergoing apoptosis by 23 and 27%, respectively. Apoptosis was inhibited by a caspase-3 inhibitor, Z-DEVD-fmk. It is concluded that, when heterologously expressed in LNCaP cells, the TRPL protein leads to a reduction in cell survival due, in part, to the induction of apoptosis. The effects of TRPL are likely caused by enhanced Na(+) and Ca(2+) inflow to the cells. This finding suggests a novel approach to modify the growth of prostate cancer cells that fail to undergo apoptosis following androgen ablation therapy.
Zhang
Lei
L
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia.
Brereton
Helen M
HM
Hahn
Michael
M
Froscio
Mario
M
Tilley
Wayne D
WD
Brown
Michael P
MP
Barritt
Greg J
GJ
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D003587:Cytomegalovirus / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D008164:Luminescent Proteins; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017430:Prostate-Specific Antigen / Q000235:genetics; D011471:Prostatic Neoplasms / Q000378:metabolism*; D014162:Transfection; D050051:Transient Receptor Potential Channels
D002148:Calmodulin-Binding Proteins; D029721:Drosophila Proteins; D007473:Ion Channels; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D049452:Green Fluorescent Proteins; D017430:Prostate-Specific Antigen; D002118:Calcium