Sun X Z, Zhou D, Chen S
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
J Steroid Biochem Mol Biol. 1997 Sep-Oct;63(1-3):29-36. doi: 10.1016/s0960-0760(97)00068-x.
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.
通过芳香化酶cDNA转染和G418（新霉素）筛选，制备了稳定表达芳香化酶的MCF-7和T-47D细胞系（即MCF-7aro和T-47Daro）。MCF-7aro进一步进行了克隆纯化。转染后的MCF-7和T-47D细胞系中的芳香化酶活性分别测定为73±6 pmol/mg/h和48±4 pmol/mg/h。据认为，这些细胞系以稳定的方式表达芳香化酶，这在无G418培养期间该酶的稳定表达中得到了证明。通过球体培养法证明，这些细胞的生长可被雄激素（1 - 10 nM）刺激。雄激素刺激的生长可被4-羟基雄烯二酮（4-OHA）（0.01 - 0.1 mM）或他莫昔芬（50 nM - 1 μM）抑制。为了检验肿瘤芳香化酶可通过旁分泌方式影响乳腺肿瘤生长这一假设，我们通过将MCF-7细胞与MCF-7aro或T-47Daro细胞共培养进行了细胞培养实验。睾酮（1 nM）使MCF-7/MCF-7aro共培养（每种类型0.75×10⁶个细胞）的细胞生长增加程度与仅MCF-7aro（2至3倍）相似。此外，有或无雄激素处理的MCF-7/MCF-7aro共培养样品的酶活性保持不变，表明转染细胞产生的雌激素也可刺激未转染细胞的生长。添加4-OHA（0.01 - 0.1 mM）可抑制雄激素反应。对于MCF-7/T-47Daro共培养实验，观察到雄激素明显诱导细胞生长，且增加水平与仅T-47Daro时相似。然而，对于仅T-4７D培养或MCF-7/T-47Daro共培养，发现睾酮处理后芳香化酶活性显著增加。T-47Daro细胞未进行克隆纯化，因此认为雄激素处理可能选择性刺激高表达芳香化酶的T-47Daro细胞的生长。这些结果表明，肿瘤芳香化酶合成的雌激素可通过自分泌和旁分泌方式刺激乳腺肿瘤生长。
