Glucocorticoid binding and [3H]thymidine incorporation into DNA in embryonic chick cell cultures

Fodge, D.W.; Chao, W.R.; Johnson, H.L.

Cancer Research 38(10): 3391-3397


ISSN/ISBN: 0008-5472
PMID: 210936
Accession: 068524222

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Glucocorticoid binding was examined in normal and Rous sarcoma virus (RSV)-transformed embryonic chick cell cultures to determine its role in the control of cell growth. Cell cultures were prepared from the body carcasses of 10 day old embryonic White Leghorn chicks. Mixtures of charcoal-dextran were used to separate bound from free forms of the glucocorticoids. [3H]Thymidine incorporation into DNA was used as a monitor of growth. In the normal embryonic cell cultures, only the glucocorticoids at physiological doses inhibited [3H]thymidine incorporation into DNA. .beta.-Estradiol, progesterone, norethynodrel, testosterone, digitoxin, indomethacin and oxybutazolidine were inactive. None of the steroids or other compounds inhibited [3H]thymidine incorporation into DNA in RSV-transformed cultures. Several related steroid hormones were unable to compete for [3H]-dexamethasone binding sites in embryonic chick cells, revealing that only glucocorticoids are bound tightly in both normal and RSV-transformed cultures. The dissociation constant for [3H]dexamethasone binding in normal cultures was 0.18 nM, and it was 1.2 nM in RSV-transformed cultures. The maximum amounts of glucocorticoid bound were 31.7 and 34.8 femtomoles/mg protein in normal and RSV-transformed cultures, respectively. The binding of glucocorticoids in normal embryonic chick cell cultures varied only slightly with changes in cell density. In normal embryonic cultures maintained at one density and stimulated to grow with charcoal-treated serum, the maximum binding of [3H]dexamethasone was increased slightly, although [3H]thymidine incorporation rates were 12-fold higher than in unstimulated cultures. Untreated serum stimulated other cultures to incorporate considerable amounts of [3H]thymidine, but [3H]dexamethasone binding in those cultures decreased to 1/3 of that in the unstimulated control cultures. Progesterone competed successfully with [3H]dexamethasone for binding sites in normal and RSV-transformed cultures, but this required a 300-fold greater concentration. Similar amounts of progesterone also prevented the inhibition of [3H]thymidine incorporation into DNA that usually occurs when normal cultures are treated with 1 nM hydrocortisone.