Glucocorticoids confer normal serum/growth factor-dependent growth regulation to Fu5 rat hepatoma cells in vitro. Sequential expression of cell cycle-regulated genes without changes in intracellular calcium or pH.
Glucocorticoid hormones induced a stringent dependence on serum for the in vitro proliferation of Fu5 rat hepatoma cells by suppressing the growth rate and final quiescent cell density. Treatment of dexamethasone-suppressed quiescent Fu5 with serum plus insulin caused a rapid reinitiation of cellular proliferation and DNA synthesis that peaked at 16 h. RNA dot blot analysis of this time course showed that the transcript levels for the proto-oncogenes c-fos, c-myc, and c-rasKi peaked at 0.5, 2, and 4 h, respectively, while expression of c-rasHa and ornithine decarboxylase transcripts rose steadily during 16 h. Microspectrofluorimetric measurements of cytosolic calcium (Ca2+i) with fura-2 showed that insulin and serum, alone or in combination, elicited no changes in Ca2+i over a 50-min time course, although ATP, which is not a mitogen, induced large increases in Ca2+i. Cytosolic pH, pHi, was also measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Insulin and serum, alone or in combination, did not cause pHi to increase in either 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pHi 7.17)- or HCO3/CO2 (pHi 7.19)- buffered media. Acid-loading of cells with NH4Cl indicated that both quiescent and proliferating Fu5 cells have equally active, amiloride-sensitive Na/H exchangers. Therefore, induction of DNA synthesis and proto-oncogene expression occurs in Fu5 epithelial tumor cells in the absence of any short term increases of pHi or Ca2+i.
Cook
P W
PW
Department of Physiology-Anatomy, University of California, Berkeley 94720.
Weintraub
W H
WH
Swanson
K T
KT
Machen
T E
TE
Firestone
G L
GL
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001769:Blood; D002118:Calcium / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D003470:Culture Media; D003907:Dexamethasone / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D011905:Genes, ras / Q000187:drug effects*; D005938:Glucocorticoids / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000473:pathology*; D015735:Mifepristone; D011519:Proto-Oncogenes / Q000187:drug effects*; D051381:Rats
D003470:Culture Media; D004963:Estrenes; D005938:Glucocorticoids; D015735:Mifepristone; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D002118:Calcium