Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (1545 IAM) at which it is found in tissue culture media. Phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells with an affinity 0.001% that of estradiol (Kd = 2 x 10-S M). It stimulates the proliferation of estrogen receptor-positive MCF-7 breast cancer cells in a dose-dependent manner but has no effect on the growth of estrogen receptor-negative MDA-MB-231 breast cancer cells. At the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media, thereby reducing the degree to which exogenous estrogen is able to stimulate responses. The antiestrogens tamoxifen and hydroxytamoxifen inhibit cell proliferation below the control level only when cells are grown in the presence of phenol red; in the absence of phenol red, the antiestrogens do not suppress growth. The estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture.

Transforming growth factor-beta (TGFb)1 is thought to be implicated in breast cancer progression. However, data about the influence of TGFb1 on breast cancer development are conflicting. To clarify the clinical relevance of TGFb1, TGFb1 protein level has been measured by enzyme-immoassay in 193 breast tumour samples. We found that 94.3% of patients expressed TGFb1 with a range of 0 -684 pg mg À1 protein.In the overall population, an increase of tumoral TGFb1 was observed in premenopausal patients when compared to postmenopausal subgroup (P ¼ 0.0006). When patients were subdivided according to nodal status, TGFb1 was correlated to type-1 plasminogen activator inhibitor in the node-negative subgroup (P ¼ 0.040). Multivariate analysis revealed that, after lymph node status (P ¼ 0.0002) and urokinase-type plasminogen activator (P ¼ 0.004), TGFb1 was an independent prognostic marker for DFS (P ¼ 0.005) in the overall population. In the node-negative population, TGFb1 was the prominent prognostic factor (P ¼ 0.010). In the same population, Kaplan -Meier curves demonstrated that high TGFb1 level was correlated with a shorter diseasefree survival (P ¼ 0.020). These data suggest that the measurement of tumoral TGFb1 protein level, especially for node-negative patients, might help to identify a high-risk population early in tumour progression.

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