Figure 4

Overexpression of GP88 in AC1 cells and MCF-7CA cells confer letrozole resistance. A: GP88 overexpressing AC1 cells (AC1-P) cells were developed by stable transfection of GP88 cDNA expression vector as described in the method section. As control, AC1-EV cells were developed by transfecting empty vector (AC1-EV). For measuring anchorage-independent growth of stably transfected AC1-P cells, AC1-P cells were plated at a density of 5 × 10³ cells/well in a 12 wells plate and cultivated for 2 weeks with/without 200 nM letrozole, 25 nM AD, 300 ng/ml GP88 for the AC1 and CA1-EV in 5% ChX-FBS supplemented medium containing soft agar. Medium was changed every 3-4 days.B: Stimulation of anchorage independent growth of MCF-7-CA cells overexpressing GP88. The aromatase-overexpressing cells MCF-7CA were transfected with expression vector containing GP88 cDNA (CA-P) or with empty vector (CA-EV). Anchorage independent growth of stable transfectants was examined as described above.C: Comparison of aromatase enzymatic activity in AC1 and AC1-P cells. AC1 and AC1-P cells were plated in 6-well plates at a density of 1.5 × 10⁵ cells/well. Measurement of aromatase activity assay was performed following the protocol described in the method section.