FIG. 8.

Progesterone receptor expression is blocked by a small inhibitory RNA for cyclin D1 in breast cancer cells in addition to the T47D cells used for the previous four figures, and decreases in cyclin D1 sensitize cell lines to both antiestrogen and antiprogesterone treatments. (A and B) Progesterone receptor expression responds to CCND1 knockdowns in several human breast cancer cell lines. (A) Immunoblot experiments were performed using the indicated gene products and 50 μg of lysates harvested 48 h after transfection as described for Fig. 4. Cyclin D1 levels differed between the cell lines, with those toward the left of panel A showing generally higher levels, as is consistent with the known results of 11q13 amplifications in ZR75-1 and CAMA cells. siCCND1 knocked down CCND1 protein levels in all cell lines tested. As we observed with respect to T47D cells, CCND1 knockdown decreased levels of PR isoform A in MCF7 and ZR75-1 cells. Similar losses of candidate CCND1-target genes were seen in parallel for IER3 and ETV5 in these cell lines. An actin loading control is shown. (B) The indicated human breast cancer cell lines were transfected with scramble (Sc) or siCCND1 (Si). Levels of isoform AB- and B-specific transcripts of the progesterone receptor were measured using equal amounts of reverse-transcribed cDNA via isoform-specific qRT-PCR. Isoform A levels were derived by subtracting B from AB levels. The means and standard errors for fold changes from scramble to siCCND1 transfectants for each of the indicated cell lines are indicated. (C) The 3′ UTR of the PR gene responds to cyclin D1 in MCF 7 cells. The pGL2P-UTR vector was transfected into MCF 7 cells together with a scramble control RNA (scr), siCCND1 (siD1), an empty vector control (vec), an expression plasmid for cyclin D1 (D1), and the plasmid expressing the K112E mutant of cyclin D1 (KE). Reporter gene expression measured as described above is plotted as the ratio of the firefly luciferase (FF) in the PR reporter to the CMV-renilla control reporter.