FIG. 8.
Dissociation of p21 induction from p27 induction in SAOS2 cells through the GR dimerization mutant. (A) Inhibition of cell proliferation by the GR dimerization-deficient mutant. SAOS2 clones stably expressing the GR dim mutant were generated as described in Materials and Methods. SAOS2-GR(+) and SAOS2-dim cells were seeded on day 0 into six-well plates (20,000 cells/well) and cultured in the absence or presence of 100 nM Dex. Total numbers of viable cells were determined on the indicated days. Note incomplete inhibition of cell proliferation by the dim mutant compared to the wt GR-expressing clone. Quantitation of cell counts reveals 76% growth inhibition for the dim mutant and 96% for the wt GR. Identical growth kinetics were demonstrated for three independent clones expressing the GR dim mutant. (B) Induction of p21 but not p27 expression by the dim mutant. SAOS2 cells expressing the wt GR or the dimerization-deficient mutant were cultured in the presence or absence of 100 nM Dex for 3 days, and whole-cell extracts were prepared and subjected to immunoblotting for GR, p27, p21, and ERK. Note the increase in p21 but not p27 protein in the dim-expressing cells. (C) Induction of the steady-state p21 mRNA level by the dim mutant. Hormone treatment, RNA isolation, and Northern hybridization were performed exactly as described for Fig. 7C. Potent Dex-dependent induction of p21 mRNA is observed in both SAOS2-GR(+) (wt GR) and SAOS2-dim (dim) cells.