Endocrinology, Vol 129, 1644-1652, Copyright © 1991 by Endocrine Society
Population density alters the responsiveness of GH4C1 pituitary tumor cells to 17 beta-estradiol
JD Shull
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
In this study we have examined the effect of population density on the
ability of 17 beta-estradiol (E2) to induce PRL mRNA, DNA synthesis, and
progesterone receptor in GH4C1 pituitary tumor cells. These parameters were
examined at three subconfluent population densities that varied over a
4-fold range. The culture medium was changed daily in these experiments to
reduce the possibility of nutrient depletion, medium toxification, or E2
metabolism. At the low population density, a 5-day treatment with E2 at a
concentration of 1.0 nM resulted in an 8.1- fold increase in the level of
PRL mRNA, as measured by the cytosolic dot blot procedure. At the
intermediate density, E2 induced PRL mRNA 2.4-fold. At the high density,
the level of PRL mRNA was reduced by 50% in response to 5 days of treatment
with E2. The cytosolic dot blot procedure would reflect changes in the
level of PRL mRNA per cell as well as changes in the number of cells per
culture. Therefore, the level of beta-actin mRNA was also measured,
assuming that it would remain constant on a per cell basis. When the level
of PRL mRNA was normalized to the level of beta-actin mRNA in the same
cytosols, E2 increased PRL mRNA 2.6-fold in the low density cultures and
1.9-fold in the intermediate density cultures, and had no effect on the
level of PRL mRNA in the high density cultures. The effect of population
density on the induction of GH4C1 cell proliferation by E2 was examined
directly by measuring cellular DNA, thymidine incorporation by whole cells,
and deoxythymidine triphosphate (dTTP) incorporation by isolated nuclei. At
the low density, E2 initially stimulated GH4C1 cell proliferation, as
evidenced by an increased rate of dTTP incorporation. However, this
stimulatory effect was lost by day 5 of treatment, as the population
density of the E2-treated low density cultures increased. At the high
density, the inhibitory effect of E2 on dTTP incorporation was observed by
day 2 of treatment and thereafter become more pronounced. These stimulatory
and inhibitory effects of E2 were also revealed when the level of cellular
DNA was measured. In contrast to the effects of population density on the
induction of PRL mRNA and cell proliferation, an increase in density had no
observable effect on the induction of progesterone receptor by E2. These
data illustrate the critical importance of the culture environment in
studies examining estrogen action in vitro.
