Figure 2

Retinoic acid receptor (RAR)γ modulates tumor cell growth, differentiation and morphology in MMTV-Myc xenografts in FVB mice. A) Primary cells from MMTV-Myc tumors were isolated and cultured. They were then transfected with the control vector pSG5 or a vector tethered to shRARγ or pSG5-RARγ, shRARγ or pSG5-RARγ plasmids. Transfected cells (0.5 × 10⁶) were inoculated orthotopically into the abdominal mammary gland fat pad (gland number 4) of 10 FVB female mice, whereas their contralateral fat pad was inoculated with pSG5-transfected cells to serve as an internal control. Tumor volume shown is after 15 days of in vivo growth. Results shown are representative of two independent experiments. B) Immunoblotting of tumor cell lysates with anti-RARγ antibody showing the levels of RARγ in the shRARγ and pSG5-RARγ-transfected Myc cells. Immunohistochemical analysis of five sections per group of tumors derived from the shRARγ-transfected cells compared to the vector control cells (C and D). C) Expression of cell growth-arrest marker p27 (brown); space bar = 100 um. D) Expression of differentiation marker E-cadherin. Plasma membrane-associated E-cadherin was detected in the shRARγ tumors (open arrow); space bar = 20 um. E) Morphologic analysis of Myc cells cultured under three-dimensional conditions in Matrigel^® (4 wells/group); bar = 100 μm. The number of colonies exhibiting the invasive (t-test P < 0.0001, light-gray bars) and noninvasive (t-test P < 0.0001, dark-gray bars) phenotype was quantified. F) RARγ antagonist SR11253 inhibits Myc tumor cell proliferation in a dose-dependent manner. Proliferating Myc cells were treated with increasing concentrations of SR11253 as shown. G) Confocal immunofluorescence for p27 (cell arrest marker, red) and pRB (proliferation marker, green) in Myc cells transfected with the shRARγ construct and in vector alone-transfected control cells; bar = 50 μm. Panels E, F and G are representative of three independent experiments.