Autocrine and paracrine actions of breast tumor aromatase. A three-dimensional cell culture study involving aromatase transfected MCF-7 and T-47D cells.

Stable aromatase-expressing MCF-7 and T-47D cell lines (i.e. MCF-7aro and T-47Daro) have been prepared by aromatase cDNA transfection and G418 (neomycin) selection. MCF-7aro was further subjected to a clonal purification. Aromatase activity in the transfected MCF-7 and T-47D cell lines was determined to be 73 +/- 6 pmol/mg/h and 48 +/- 4 pmol/mg/h, respectively. It is thought that these cell lines express aromatase in a stable manner, as demonstrated by a steady expression of the enzyme during culture in the absence of G418. The growth of these cells could be stimulated by androgens (1-10 nM) as demonstrated through a spheroid culture method. The androgen-stimulated growth could be suppressed by 4-hydroxyandrostenedione (4-OHA) (0.01-0.1 mM) or tamoxifen (50 nM-1 microM). In order to test the hypothesis that tumor aromatase can affect breast tumor growth in a paracrine manner, we have carried out cell culture experiments by co-culturing MCF-7 cells with either MCF-7aro or T-47Daro cells. Testosterone (1 nM) increased cell growth to a similar degree for MCF-7/MCF-7aro co-culture (0.75 x 10(6) cells each type) as with MCF-7aro only (2- to 3-fold). In addition, the enzyme activities remained unchanged for MCF-7/MCF-7aro co-culture samples with and without androgen treatment, indicating that estrogen produced by transfected cells can also stimulate the growth of untransfected cells. The androgen response could be inhibited by an addition of 4-OHA (0.01-0.1 mM). For MCF-7/T-47Daro co-culture experiments, a clear induction of cell growth by androgen was observed, and the level of the increase was similar to that on T-47Daro only. However, for either culture with T-47D only or with MCF-7/T-47Daro co-culture, the aromatase activity was found to increase significantly after testosterone treatment. T-47Daro cells were not subjected to a clonal purification, and it is therefore thought that the androgen treatment may selectively stimulate the growth of high aromatase-expressing T-47Daro cells. These results indicate that estrogen synthesized by tumor aromatase can stimulate breast tumor growth in both an autocrine and a paracrine manner.