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ToxSci Advance Access originally published online on November 3, 2004
Toxicological Sciences 2005 83(2):257-263; doi:10.1093/toxsci/kfi025
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Toxicological Sciences vol. 83 no. 2 © Society of Toxicology 2005; all rights reserved.

Co-culture of Primary Human Mammary Fibroblasts and MCF-7 Cells as an In Vitro Breast Cancer Model

Marjoke Heneweer*,1, Martine Muusse*, Milou Dingemans*, Paul C. de Jong{dagger}, Martin van den Berg* and J. Thomas Sanderson*

* Institute for Risk Assessment Sciences (IRAS), Utrecht University, PO Box 80176, 3508 TD Utrecht, The Netherlands and {dagger} St. Antonius Hospital, PO Box 2500, 3430 EM Nieuwegein, The Netherlands

Received August 31, 2004; accepted November 1, 2004

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.

Key Words: co-culture; breast tumor promotion; aromatase inducers; human mammary fibroblasts; MCF-7 cells.


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