Background: Recent evidence suggests that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that tumorous tissues have higher 5α-reductase (5αR) and lower 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities. The resulting higher levels of 5α-reduced progesterone metabolites such as 5α-pregnane-3,20-dione (5αP) in tumorous tissue promote cell proliferation and detachment, whereas the 4-pregnene metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αDHP), more prominent in normal tissue, have the opposite (anti-cancer-like) effects. The aim of this study was to determine if the differences in enzyme activities between tumorous and nontumorous breast tissues are associated with differences in progesterone metabolizing enzyme gene expression.

In the 70 years since progesterone (P) was identified in corpus luteum extracts, its metabolism has been examined extensively in many tissues and cell lines from numerous species. In addition to the reproductive tissues and adrenals, every other tissue that has been investigated appears to have one or more P-metabolizing enzyme, each of which is specific for a particular site on the P molecule. In the past, the actions of the P metabolizing enzymes generally have been equated to a means of reducing the P concentration in the tissue microenvironment, and the products have been dismissed as inactive waste metabolites. In human breast tissues and cell lines, the following P-metabolizing enzymes have been identified: 5a-reductase, 3a-hydroxysteroid oxidoreductase (3a-HSO), 3b-HSO, 20a-HSO, and 6a-hydroxylase. Rather than providing diverse pathways for inactivating and controlling the concentration of P in breast tissue microenvironments, it is proposed that the enzymes act directly on P to produce two types of autocrines/paracrines with opposing regulatory roles in breast cancer. Evidence is reviewed which shows that P is directly converted to the 4-pregnenes, 3a-hydroxy-4-pregnen-20-one (3a-dihydroprogesterone; 3aHP) and 20a-dihydro-progesterone (20aHP), by the actions of 3a-HSO and 20a-HSO respectively and to the 5a-pregnane, 5a-pregnane-3,20-dione(5a-dihydroprogesterone; 5aP), by the irreversible action of 5a-reductase. In vitro studies on a number of breast cell lines indicate that 3aHP promotes normalcy by downregulating cell proliferation and detachment, whereas 5aP promotes mitogenesis and metastasis by stimulating cell proliferation and detachment. The hormones bind to novel, separate, and specific plasma membrane-based receptors and influence opposing actions on mitosis, apoptosis, and cytoskeletal and adhesion plaque molecules via cell signaling pathways. In normal tissue, the ratio of 4-pregnenes:5a-pregnanes is high because of high P 3a-and 20a-HSO activities/expression and low P 5a-reductase activity/expression. In breast tumor tissue and tumorigenic cell lines, the ratio is reversed in favor of the 5a-pregnanes because of altered P-metabolizing enzyme activities/expression. The evidence suggests that the promotion of breast cancer is related to changes in in situ concentrations of cancerinhibiting and -promoting P metabolites. Current estrogen-based theories and therapies apply to only a fraction of all breast cancers; the majority (about two-thirds) of breast cancer cases are estrogeninsensitive and have lacked endocrine explanations. As the P metabolites, 5aP and 3aHP, have been shown to act with equal efficacy on all breast cell lines tested, regardless of their tumorigenicity, estrogen sensitivity, and estrogen receptor/progesterone receptor status, it is proposed that they offer a new hormonal basis for all forms of breast cancer. New diagnostic and therapeutic possibilities for breast cancer progression, control, regression, and prevention are suggested.

BackgroundRecent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5α-reduced metabolites (5α-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. Conversely, the activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5α-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive).MethodsFor the enzyme activity studies, either whole cells were incubated with [14C]progesterone for 2, 4, 8, and 24 hours, or the microsomal/cytosolic fraction was incubated for 15–60 minutes with [3H]progesterone, and the metabolites were identified and quantified. Semi-quantitative RT-PCR was employed to determine the relative levels of expression of 5αR type1 (SRD5A1), 5αR type 2 (SRD5A2), 20α-HSO (AKR1C1), 3α-HSO type 2 (AKR1C3), 3α-HSO type 3 (AKR1C2) and 3β-HSO (HSD3B1/HSD3B2) in the four cell lines using 18S rRNA as an internal control.ResultsThe relative 5α-reductase activity, when considered as a ratio of 5α-pregnanes/4-pregnenes, was 4.21 (± 0.49) for MCF-7 cells, 6.24 (± 1.14) for MDA-MB-231 cells, 4.62 (± 0.43) for T-47D cells and 0.65 (± 0.07) for MCF-10A cells, constituting approximately 6.5-fold, 9.6-fold and 7.1 fold higher conversion to 5α-pregnanes in the tumorigenic cells, respectively, than in the nontumorigenic MCF-10A cells. Conversely, the 20α-HSO and 3α-HSO activities were significantly higher (p < 0.001) in MCF-10A cells than in the other three cell types. In the MCF-10A cells, 20α-HSO activity was 8-14-fold higher and the 3α-HSO activity was 2.5-5.4-fold higher than in the other three cell types. The values of 5αR:20α-HSO ratios were 16.9 – 32.6-fold greater and the 5αR:3α-HSO ratios were 5.2 – 10.5-fold greater in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells. RT-PCR showed significantly higher expression of 5αR1 (p < 0.001), and lower expression of 20α-HSO (p < 0.001), 3α-HSO2 (p < 0.001), 3α-HSO3 (p < 0.001) in MCF-7, MDA-MB-231 and T-47D cells than in MCF-10A cells.ConclusionThe findings provide the first evidence that the 5αR activity (leading to the conversion of progesterone to the cancer promoting 5α-pregnanes) is significantly higher in the tumorigenic MCF-7, MDA-MB-231 and T-47D breast cell lines than in the nontumorigenic MCF-10A cell line. The higher 5αR activity coincides with significantly greater expression of 5αR1. On the other hand, the activities of 20α-HSO and 3α-HSO are higher in the MCF-10A cells than in MCF-7, MDA-MB-231 and T-47D cells; these differences in act...

A method is described for obtaining populations of viable Sertoli cells from rat testes. Minced whole testes from rats of 15 to 29 days of age are sequentially treated with collagenase and pancreatin. The resulting suspension of cells is sedimented through a sucrose density gradient. Preparations are produced consisting of from 60% to 82% Sertoli cells, an enrichment of 2 to 5 times the proportion of Sertoli cells in whole testes of these ages. The preparations are free of interstitial cells, are essentially free of peritubular cells and contain reduced numbers of germinal cells; the main contaminating cell types are spermatogonia and spermatocytes. The Sertoli cells are considered to be 95% viable by their ability to exclude trypan blue and by subsequent culturing in vitro. The entire procedure requires 3 h. Maintenance of the Sertoli-enriched fraction in modified Eagle's minimal essential medium temporarily at 41 C allows preparations of Sertoli cell monolayer cultures consisting of 95%-98% Sertoli cells within 3 days.

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