Fig 1.
Aromatase expression levels in ERα+ breast cancer cell lines.
A. mRNA levels of aromatase detected with RT-PCR in ERα+ breast cancer cell lines (ZR-75-1, ZR-75-1/Aro Cl.10, ZR-75-1/Aro-Clone10-TT1, T47D, CAMA-1 BT474). ZR: ZR-75-1 control cells stably transfected with an empty vector; Cl.10: a clone of ZR-75-1 cells stably transfected with an aromatase expression vector; TT1: cell line derived from a xenograft tumor formed by ZR-75-1/Aro Cl.10 cells in female nude mouse. B. Western immunoblotting of aromatase in various ERα+ cell lines. C. Aromatase enzyme activity in the extracts of ZR-75-1, ZR-75-1/Aro Cl.10, MCF-7 and MCF-7/Aro cell lines.
Fig 2.
Increased expression of aromatase in suspension culture.
Cells were plated at 1x106 per well in regular plates for adhesion culture for 24 hrs (24h/adh) or in ultra-low adhesion plates for suspension culture for 24 hrs (24h/sus) and 48 hrs (48h/sus). Cells were harvested for RNA isolation, which was used in real time RT-PCR for quantification of aromatase and actin mRNA. Aromatase mRNA levels presented are normalized by actin mRNA levels. The data represents mean±SEM from three replicate measurements. Fig. A shows the increased aromatase expression in suspension culture of MCF-7, Clone 10 and CAMA-1 cells by real time PCR while Fig. B shows the same by Western blot analysis in CAMA-1 cells.
Fig 3.
Increased expression of ERα in suspension culture.
Cells were plated in either regular culture plates for adhesion culture or in ultra-low adhesion plates for suspension culture. Their RNA and protein were extracted and used for real-time RT-PCR (Panel A) or immunoblotting (Panel B). The ERα mRNA levels presented are normalized by actin mRNA. The data in Panel A represents mean±SEM from three replicate measurements. Two-tailed student t-tests were performed to determine the significant difference between control and experimental data. GAPDH protein was blotted to indicate equal loading in Panel B. The density of the ERα band from 24h adherent culture was set as one unit after being normalized with the corresponding GAPDH band for each cell line.
Fig 4.
Aromatase-mediated inhibition of anoikis.
Cells were plated as duplicate in ultra-low attachment 6-well plate for suspension culture. The cells were treated without or with 10 nM testosterone or 1 M letrozole, or both for 16 hr. Cell lysates were then used for apoptosis measurement with an apoptosis detection kit (Roche). Absorbance(A405nm-A490nm) indicates relative apoptosis. The data are presented as mean±SEM of duplicate wells (*p<0.05). One way ANOVA were used to find the significant difference.
Fig 5.
Moderate aromatase expression rendered ZR-75-1 cells tumorigenic without estrogen supplementation.
A. Luc-GFP expressing ZR-75-1/Aro Cl.10 and TT1 cells (2x106) were inoculated into the inguinal mammary fat pads of 5-wk-old female nude mice. The tumor sizes were measured with a caliper in two dimensions. Tumor volumes were calculated with the equation V = (LxW2)/2, where L is length and W is width of a tumor. Values are mean±SEM of 10 tumors in 5 mice. B. The representative images (fluorescence on the left and bioluminescence on the right) C. The mice with growing tumors were divided into two groups and treated with vehicle or letrozole at 10 μg/mouse/day.
Fig 6.
Aromatase expression rendered non-tumorigenic ZR-75-1 cells bone metastatic.
The cells were inoculated through the left cardiac ventricle of female nude mice at 0.1x106 cells/mouse. Bone metastases of ZR-75-1/Aro Cl.10 cells in the mandible and tibiae/femora were detected with fluorescence (panel A) and bioluminescence (panel B) imaging. Representative images of two mice inoculated with the Cl.10 cells are presented, which were taken 5 week post-inoculation.