Regulation of endogenous gene expression by monotransregulators. MDA-MB-231 cells infected with the adenovirus bearing none (Ad5), ERα or ERα_EBD cDNA were treated without (data not shown) or with 10⁻⁹ M E2 for 48 h, while cells infected with theadenovirus bearing Ad5-PV or Ad5-PV_EBD were treated with vehicle. Total RNA was subjected to qPCR for the analysis of estrogen responsive gene expressions. Results, which are the mean ± SEM of three independent determinations, depict relative change in mRNA levels compared with those observed in cells infected with Ad5 in the absence of E2, which is set to 1. Asterisk (*) indicates significant change.

PV mimics the effects of E2-ERα on responses from BT-549 cells. (A & B) Synthesis of functional receptor proteins. BT-549 cells were infected with an adenovirus bearing none (Ad5) or a cDNA for ERα, ERα_EBD, PV, ERα_EBD at 80, 20, 20, 60 and 80 MOIs, respectively. The total MOI was adjusted to 80 MOI by supplementing with Ad5. (A) Extracts of infected BT-549 (25 μg) cells at 48 h after infection were subjected to WB using the horseradish peroxidase-conjugated monoclonal Flag antibody. Molecular mass in kDa is indicated. (B) Cell extracts were also subjected to EMSA in the presence (+) or absence (−) of a Flag antibody. ERE indicates the unbound and R-ERE depicts the receptor-bound radiolabeled ERE. F denotes the radiolabeled ERE only. (C, D) Infected cells in the presence (E2) or absence (−) of 10⁻⁹ M E2 for 6 d were subjected to (C) cell counting for cellular proliferation or (D) Annexin V assay at 96 h after infection for cellular death. The mean ± SEM, which represents three independent experiments, indicate percentage (%) change in cell number compared with those observed in cells infected with Ad5 in the absence of E2, which is set to 100. Asterisk (*) indicates significant change.

Effects of monotransregulators on tumor growth in xenografts. MDA-MB-231 cells were infected with Ad5 or Ad5-PV_EBD at 900 MOI for 48 h. A group of cells was also infected with Ad5-PV at 100 or 200 MOI supplemented with 800 or 700 MOI of Ad5, respectively. Infected cell suspensions (4 × 10⁶ cells/mouse) were subcutaneously inoculated into the left mammary fat pads of female athymic NCr-nu/nu mice (ten mice/group, with the exception of the Ad5-PV-100 group that contained nine mice). (A) The tumor size was measured with calipers weekly and the tumor volume was calculated. Tumor incidence for each group is shown in parenthesis. Points denote the mean ± SEM of tumor volume of each group. *^a denotes significant change (P < 0.041) compared with Ad5. *^b indicates significant change (P < 0.0161) compared with Ad5. (B) At wk 10, mice were killed. Tumors were removed and weighed. Bars are the mean ± SEM of tumor weight from each group. *^a denotes significant change (P < 0.008) compared with Ad5. *^b indicates significant change (P < 0.001) compared with Ad5.

Engineering of monotransregulators. (A) The activation domains (ADs) of p65 and VP16 proteins were genetically joined through multiple cloning sites (MCS) to the 5′ and 3′ ends, respectively, of the CDC-cDNA to generate the monotransregulator PV. The construct also contains sequences encoding the Flag and 6xHis epitopes at the amino-and carboxyl-termini, respectively. (B) MDA-MB-231 cells were infected with the parent recombinant adenovirus (Ad5) at MOI 900, a recombinant adenovirus bearing cDNA for ERα at 50, ERα_EBD at 150, PV at 200 and PV_EBD at 900 MOI. For all infections, the total MOI was adjusted to 900 by supplementing with the parent Ad5. Extracts (10 μg) of infected MDA-MB-231 cells at 48 h were subjected to Western blotting (WB) using a horseradish peroxidase-conjugated monoclonal Flag antibody. Molecular mass in kDa is indicated. NS denotes nonspecific protein detection. (C) Intracellular localization of receptor proteins was examined by immunocytochemistry (ICC). A fluorescein isothiocyanate-conjugated Flag antibody (FITC) localizes constructs to the nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI) at 48 h after infection. (D) Cell extracts (10 μg) at 48 h after infection also were subjected to electrophoretic mobility shift assay (EMSA) with (+) or without (−) a Flag antibody (Ab). ERE specifies unbound and R-ERE denotes receptor-bound radiolabeled ERE. F in panel B indicates the radiolabeled ERE only. In all experiments a representative result from three independent experiments is shown.

Effects of monotransregulators on cellular growth. (A) MDA-MB-231 cells infected with recombinant adenoviruses in the presence (+) or absence (−) of 10⁻⁹ M E2 for 48 h were subjected to fluorescence-activated cell sorting for the cell cycle analysis. Results depicted as the percentage of cells in G1, G2 and S phases are the mean ± SEM of three independent experiments. (B, C) Infected cells maintained in the presence (+) or absence (−) of 10⁻⁹ M E2 (E2) for 6 d were subjected to (B) cell counting or (C) MTT assay. The mean ± SEM, which depicts three independent experiments performed in duplicate, indicates percentage (%) change in cell number compared with those observed in cells infected with Ad5 in the absence of E2, which is set to 100. (D, E) MDA-MB-231 cells also were subjected to (D) Annexin V and (E) TUNEL assays at 96 h after infection for the effects of proteins on cellular death. Results, which are the mean ± SEM of three independent experiments, are summarized as percentage (%) change in the number of cells bound to Annexin V or as the number of cells that incorporated fluorescein isothiocyanate-conjugated dUTP into the fragmented DNA (TUNEL) obtained in comparison with the cells infected with Ad5 in the absence of E2, which is set to 100. Asterisk (*) indicates significant change.

Effects of receptor proteins on cellular motility. (A) Wound-healing assay. Infected MDA-MB-231 or BT-549 cells were incubated in the presence (+) or absence (−) of 10⁻⁹ M E2 as indicated for 48 h to allow cells to reach near confluence. A wound was then created and images were captured at 0 h and at 24 h intervals. Results, representing the mean ± SEM of three independent experiments performed in duplicate, summarize the wound closure at 96 h for MDA-MB-231 cells and 48 h for BT-549 cells relative to 0 h, which is set to 1. (B) Invasion assay. Cells were infected with recombinant adenoviruses with (+) or without (−) 10⁻⁹ M E2 for 48 h as depicted. Cells were collected and counted. The same number of cells from each group was then seeded on the upper section of the invasion chamber membrane with the corresponding treatment. After 24 h incubation, cells on the bottom of chamber membrane as an indication of invasiveness were stained and counted. Results, which represent the mean ± SEM of three independent experiments performed in duplicate, are relative changes compared with cells infected with Ad5 in the absence of E2, which is set to 1. Asterisk (*) indicates significant change.