ABSTRACT

17β-Hydroxysteroid dehydrogenase (17β-HSD) is present in multiple forms in human breast tissue. One soluble form, with a molecular weight of approximately 35 kDa, was purified to near homogeneity from whole normal breast tissue. This form catalysed the oxidation of oestradiol and the reduction of oestrone, with NADP⁺ and NADPH as the preferred coenzymes. Three other soluble forms with higher molecular weights (in the range 50–80 kDa) were isolated. They catalysed the oxidation of oestradiol but not the reduction of oestrone, and all of them had properties very different from those of the low molecular weight enzyme.

Activities of 17β-HSD were measured in particulate and soluble fractions from normal breast adipose and non-adipose tissues, and from breast tumours obtained from post-menopausal women, in the oxidative direction with NAD⁺ and NADP⁺ as coenzymes and in the reductive direction with NADH and NADPH as coenzymes. Particulate fractions from tumours had much higher oxidative and reductive activities than those from normal tissues. Soluble fractions from tumours had higher oxidative activities than those from the normal tissues but similar reductive activities. The major soluble form of 17β-HSD in adipose tissue was the 35 kDa enzyme which had both oxidative and reductive activities. In contrast, the majority of the soluble activity in non-adipose tissue was due to enzymes, with molecular weights in the range 50–80 kDa, which had oxidative activity only. The soluble fractions of tumours, like those of non-adipose tissue, contained enzymes with molecular weights in the range 50–80 kDa. In addition, they contained a 35 kDa enzyme with properties different from those of the enzyme with the same molecular weight present in adipose tissue.

ABSTRACT

17β-Hydroxysteroid oxidoreductase, the enzyme that catalyses the interconversion of oestradiol and oestrone, is known to be present in human breast tissue. However, it is not known whether one or more forms of the enzyme is present. Homogenates of breast adipose tissue and breast glandular tissue were fractionated and fractions assayed in the oxidative direction with NAD⁺ and NADP⁺ as coenzymes, and in the reductive direction with NADH and NADPH as coenzymes. Ultracentrifugation of homogenates showed that there was membrane-bound activity and soluble activity. The soluble activity was due to a number of forms of the enzyme with different molecular weights, three in breast adipose tissue and two in breast glandular tissue, as shown by fractionation with (NH₄)₂SO₄ followed by chromatography on Sephadex G-200. The forms of the enzyme isolated differed in their affinities for substrates and coenzymes and in the relative rates at which they catalysed the oxidative and reductive reactions. Preliminary experiments with breast tumours showed that they also contained membrane-bound activity and more than one soluble form of the enzyme.

ABSTRACT

The GH₃ pituitary cell line has been used to investigate the role of the oestrogen receptor (ER) as a modulator of mitogenic signals in tumour cells in the absence of exogenous oestrogen. Using a chemically defined, serum- and oestrogen-free medium, we have demonstrated that the pure steroidal anti-oestrogens ICI 182780 and ICI 164384 are capable of blocking growth by more than 50% after 5 days of culture. Studies with conditioned medium have indicated that the basal growth is due to the secretion of autocrine growth stimulatory substances. Under serum- and oestrogen-free conditions, insulin and IGF-I increased the growth rate of these cells by twofold over a 5-day treatment period, and this effect was also blocked by the anti-oestrogens ICI 182780 and ICI 164384 (50% of maximum inhibition at 0·6 and 6 nM respectively). To explore the potential mechanism by which the ER apparently facilitates the growth factor effects under oestrogen-free conditions, GH₃ cells were transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element (ΔMTV-ERE-LUC). We have shown that as well as oestradiol (OE₂), insulin and IGF-I induce luciferase activity by between two- and sevenfold (four experiments), and these effects were completely blocked by ICI 182780. In contrast, growth factors and OE₂ were unable to induce luciferase expression when transfections were performed with a plasmid reporter lacking the oestrogen response element. The studies presented here strongly suggest that, in the absence of oestrogen, the ER in these pituitary tumour cells has a role in growth, as peptide factors are able to induce its conversion to a state which is capable of up-regulating the transcription of key growth-promoting genes.