Autocrine regulation of cell proliferation by estradiol and hydroxytamoxifen of transformed mouse Leydig cells in serum-free culture.
We have previously reported that the cloned cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor shows an estrogen-dependent enhancement of cell proliferation in medium supplemented with charcoal-dextran-stripped fetal bovine serum. To avoid the involvement of unknown factors present in the serum in the pathway for estrogen-dependent cell growth, the present study was designed to establish a serum-free culture system to which growth factors could be added. To this end, we subcloned B-1 cells from the parental tumor cell line. The proliferation of B-1 cells was markedly stimulated by the addition of 10(-11)-10(-8) M estradiol into the serum-free medium [Eagle's Minimum Essential Medium-Ham's F-12 (1:1, vol/vol) containing 0.2% (wt/vol) BSA]. Epidermal growth factor (0.1-50 ng/ml) or insulin (0.1-50 micrograms/ml) alone or in combination with 10(-8) M estradiol did not affect the proliferation rate of B-1 cells. In contrast, a greater than 10-fold molar excess of 4-hydroxytamoxifen blocked estradiol-induced cell proliferation, while 4-hydroxytamoxifen alone failed to show a stimulatory effect on cell multiplication. Additionally, the conditioned medium collected from estradiol-stimulated cells was found to contain a growth-promoting factor(s) whose activity was not antagonized by 4-hydroxytamoxifen. Nonstimulated cells secreted a significant but low level of the growth-promoting factor. Finally, B-1 cells were found to be estrogen dependent for cell proliferation in BALB/c mice. Their growth was markedly inhibited by the administration of tamoxifen to the host mice. These results indicate that the serum-free culture system presented here is suitable for studying the autocrine mechanisms of cell growth regulated by estrogens as well as triphenylethylene compounds.
Nishizawa
Y
Y
Third Department of Internal Medicine, Osaka University Hospital, Japan.
Sato
B
B
Miyashita
Y
Y
Tsukada
S
S
Hirose
T
T
Kishimoto
S
S
Matsumoto
K
K
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D003470:Culture Media; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D007984:Leydig Cell Tumor / Q000473:pathology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011960:Receptors, Estrogen / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D013736:Testicular Neoplasms / Q000473:pathology*
D003470:Culture Media; D004965:Estrogen Antagonists; D007328:Insulin; D011960:Receptors, Estrogen; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D004815:Epidermal Growth Factor