Endocrinology, Vol 129, 1644-1652, Copyright © 1991 by Endocrine Society

ARTICLES

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.

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