A radioimmunoassay for the regulatory allylic steroid, 3α-hydroxy-4-pregnen-20-one (3αHP)

Wiebe, John P.; Barr, Kevin; Buckingham, K.D.

doi:10.1016/0960-0760(91)90339-7

Cited by 10 publications

(15 citation statements)

References 23 publications

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“…It is tempting to speculate that factors in the mammary Breast nipple aspirate fluid samples (provided by Dr E. Sauter, University of Missouri, Coumbia, MO, USA), were extracted and steroids separated chromatographically and measured by RIAs by methods similar to those described (Wiebe et al 1991).…”

Section: Changes In Expression Of Progesteronemetabolizing Enzymesmentioning

confidence: 99%

Progesterone metabolites in breast cancer

Wiebe¹

2006

Endocr Relat Cancer

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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.

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Section: Changes In Expression Of Progesteronemetabolizing Enzymesmentioning

confidence: 99%

Progesterone metabolites in breast cancer

Wiebe¹

2006

Endocr Relat Cancer

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“…The aqueous phases were frozen at -70°C; the organic phases were decanted and washed with 1.0 ml of 0.05 N NaOH and then allowed to evaporate to dryness under nitrogen. Methanol (1.0 ml) was added to each tube; the tubes were stored at 4°C until processed (1-3 days) for 3aHP and progesterone measurements by RIA [16]. Ovaries were snap-frozen and then homogenized (one pair of ovaries per 1.0 ml of 70% methanol) using a Polytron Tissue Homogenizer (PT-10 generator at 23 000 rpm for 20 sec; Polysciences, Warrington, PA); the homogenate was centrifuged at 1500 x g for 10 min.…”

Section: Ria Of 3ahp and Progesteronementioning

confidence: 99%

“…Ovaries were snap-frozen and then homogenized (one pair of ovaries per 1.0 ml of 70% methanol) using a Polytron Tissue Homogenizer (PT-10 generator at 23 000 rpm for 20 sec; Polysciences, Warrington, PA); the homogenate was centrifuged at 1500 x g for 10 min. The supernatant was extracted with diethyl ether; the concentrated extract was split into 3HP and progesterone fractions, via HPLC fitted with a C18 column, and eluted with 72% aqueous methanol [16]. In addition, some extracts from media and serum were submitted routinely to HPLC fractionation prior to the RIAs; these determinations indicated that essentially no cross-reaction between 3otHP and progesterone was occurring when the determinations were made on unfractionated samples, confirming our previous findings [16] showing that cross-reaction with progesterone was < 1%.…”

Section: Ria Of 3ahp and Progesteronementioning

confidence: 99%

“…Serum, ovaries, and culture media were treated as described previously [16] with minor modifications. Briefly, radiolabeled [ 3 H]-3aHP or progesterone (1500 cpm/1.8 pg) was added to extraction tubes containing serum (0.2-0.8 ml) or medium from cultures (2.0 ml).…”

Section: Ria Of 3ahp and Progesteronementioning

confidence: 99%

“…In intact or gonadectomized male and female rats, 3tHP has been demonstrated to selectively suppress serum FSH levels [12]; in primary cultures of anterior pituitary cells from both male and female rats, 3HP inhibits LHRH-induced FSH release [13] by actions at the level of the gonadotrope membrane/Ca 2 + channels [14] as well as the protein kinase C cell signaling pathway and intracellular Ca 2+ mobilization [15]. Recently a specific RIA for 3HP was developed [16]; and since the biological activities mentioned above have been described, it was of interest to investigate 3aHP production in the ovary, specifically that by granulosa cells.…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of the Allylic Regulatory Steroid, 3α-Hydroxy-4-Pregnen-20-One, by Rat Granulosa Cells and its Regulation by Gonadotropins1

Wiebe

Gannes

Dallaire

1994

Biology of Reproduction

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The production of the allylic regulatory steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) in the rat ovary was examined and compared to progesterone levels through use of specific RIAs that had been validated by capillary gas chromatography-mass spectrometry (GC/MS). Results showed that serum levels of 3 alpha HP are comparable to levels of progesterone at all ages examined. In the 4-day cycling rat, serum levels of 3 alpha HP were highest during diestrus and lowest during proestrus and estrus, while serum FSH levels were highest during proestrus/estrus and lowest during diestrus. Hypophysectomy resulted in decreases in ovarian and serum 3 alpha HP. Treatment of hypophysectomized rats with eCG, but not hCG, increased ovarian and serum 3 alpha HP, while serum progesterone was elevated by treatment with hCG. Ovariectomy resulted in a 55-60% reduction in serum 3 alpha HP, indicating that ovaries are a substantial, but not exclusive, source of 3 alpha HP in serum. As further evidence, cultures of preparations consisting primarily of either granulosa cells or granulosa/theca "shells" produced 3 alpha HP in time-dependent amounts comparable to those of progesterone. Granulosa cells in culture showed a significant increase in accumulation of 3 alpha HP (and progesterone) due to treatment with FSH, but not LH. In contrast to the granulosa-only cell cultures, follicle shells consisting of theca and granulosa cells responded to either LH or FSH treatment with marked increases in 3 alpha HP; increases resulting from combined treatment (FSH + LH) were significantly greater than those due to each hormone alone, but the increases were not additives.(ABSTRACT TRUNCATED AT 250 WORDS)

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