Heneweer Marjoke, Muusse Martine, Dingemans Milou, de Jong Paul C, van den Berg Martin, Sanderson J Thomas
Institute for Risk Assessment Sciences , Utrecht University, PO Box 80176, 3508 TD Utrecht, The Netherlands.
Toxicol Sci. 2005 Feb;83(2):257-63. doi: 10.1093/toxsci/kfi025. Epub 2004 Nov 3.
Approximately 60% of all breast tumors are estrogen-responsive and chemicals that show estrogenic or anti-estrogenic properties are able to interact with breast tumor growth. In a breast tumor, adipose stromal cells (fibroblasts) surrounding the epithelial tumor contain the aromatase enzyme, which converts androgens into estrogens. Exposure to aromatase inducers can therefore lead to increased estrogen levels and possibly to accelerated breast tumor growth. Subsequently, breast tumor cells synthesize and secrete elevated levels of factors such as prostaglandin E2 (PGE2), interleukin-6 (IL-6), and IL-6 soluble receptor (IL-6sR), which in turn have the ability to stimulate aromatase gene transcription in fibroblasts, establishing a positive feedback loop. In this study, a technique that allows for culturing MCF-7 epithelial breast tumor cells and healthy primary human mammary fibroblasts together in one compartment was developed. To establish the positive feedback loop, the co-culture was exposed to estrogenic compounds. RNA was isolated and reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on the aromatase and pS2 genes. Exposure of the co-culture to estradiol (E2), diethylstilbestrol (DES), and bisphenol-A (BPA), resulted in a three- to seven-fold increase of pS2 transcription levels. Furthermore, pS2 transcription levels increased even more when the aromatase substrate testosterone (20 nM) was present in the co-culture medium. Exposure of the co-culture to the aromatase inducer dexamethasone (DEX) resulted in increased pS2 transcription levels, as well as increased aromatase transcription levels. Simultaneous exposure to DEX and the synthetic anti-estrogen ICI 182,780 almost completely blocked the pS2 response. The aromatase induction response was not altered by ICI 182,780 treatment. Simultaneous exposure to DEX and the non-steroidal aromatase inhibitor fadrozole, abolished the effect of the presence of testosterone in the co-culture medium, but did not result in pS2 gene transcription levels as low as seen after exposure to ICI 182,780. These observations indicate the presence of a positive feedback loop in our co-culture system. This co-culture provides a more sophisticated and sensitive system to detect direct and indirect estrogenic effects of compounds and their possible effects on breast tumor promotion.
大约60%的乳腺肿瘤对雌激素有反应，具有雌激素或抗雌激素特性的化学物质能够与乳腺肿瘤生长相互作用。在乳腺肿瘤中，上皮肿瘤周围的脂肪基质细胞（成纤维细胞）含有芳香化酶，该酶可将雄激素转化为雌激素。因此，接触芳香化酶诱导剂会导致雌激素水平升高，并可能加速乳腺肿瘤生长。随后，乳腺肿瘤细胞合成并分泌高水平的因子，如前列腺素E2（PGE2）、白细胞介素-6（IL-6）和IL-6可溶性受体（IL-6sR），这些因子反过来又能够刺激成纤维细胞中的芳香化酶基因转录，从而建立一个正反馈回路。在本研究中，开发了一种允许在一个隔室中共同培养MCF-7上皮性乳腺肿瘤细胞和健康的原代人乳腺成纤维细胞的技术。为了建立正反馈回路，将共培养物暴露于雌激素化合物中。分离RNA，并对芳香化酶和pS2基因进行逆转录聚合酶链反应（RT-PCR）。将共培养物暴露于雌二醇（E2）、己烯雌酚（DES）和双酚A（BPA）中，导致pS2转录水平增加三到七倍。此外，当共培养基中存在芳香化酶底物睾酮（20 nM）时，pS2转录水平增加得更多。将共培养物暴露于芳香化酶诱导剂地塞米松（DEX）中，导致pS2转录水平增加，同时芳香化酶转录水平也增加。同时暴露于DEX和合成抗雌激素ICI 182,780几乎完全阻断了pS2反应。ICI 182,780处理未改变芳香化酶诱导反应。同时暴露于DEX和非甾体芳香化酶抑制剂法倔唑，消除了共培养基中睾酮存在的影响，但未导致pS2基因转录水平低至暴露于ICI 182,780后所见的水平。这些观察结果表明我们的共培养系统中存在正反馈回路。这种共培养提供了一个更复杂、更敏感的系统，用于检测化合物的直接和间接雌激素效应及其对乳腺肿瘤促进的可能影响。
