Dynamic changes in prolactin promoter activation in individual living lactotrophic cells.
The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene. Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5'-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mM luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20-100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20-100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response. In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.
Takasuka
N
N
Department of Medicine, University of Manchester, United Kingdom.
White
M R
MR
Wood
C D
CD
Robertson
W R
WR
Davis
J R
JR
D000818:Animals; D002450:Cell Communication; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D010902:Pituitary Gland / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology
D005576:Colforsin; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin