Use of the vertical rotor to facilitate estrogen and progesterone receptor analysis.

Hofman, L F; Moline, C; McGrath, G A; Barron, Edward J.

doi:10.1093/clinchem/24.9.1609

Cited by 4 publications

(1 citation statement)

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“…The supernatant was layered over a linear 20-30 %-sucrose gradient in TED buffer. These gradients were then centrifuged in a Sorvall TV 865 vertical rotor at 0°C for 3 h at 369,000 g (Jordan and Prestwich, 1977;Hofman et al, 1978). Seven-drop fractions were collected from the bottom of the tubes and the radioactivity was counted.…”

Section: Sucrose Gradient Analysismentioning

confidence: 99%

Androgen, estrogen and progestin binding sites in human leukemic cells

Danel

Martin²,

Escrich

et al. 1981

Intl Journal of Cancer

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Sex steroid binding capacity was investigated in malignant cells from 32 patients with acute non-lymphoblastic leukemia (25 patients with acute myeloid leukemia, 4 with subacute leukemia, 3 with chronic myeloid leukemia in blast crisis) and 30 patients with acute lymphoblastic leukemia. Specific binding of labelled steroids was characterized either by competition assay in cytosol fraction or by whole-cell incorporation. In some cases further characterization of the receptor complex was attempted by sucrose gradient centrifugation and gel filtration column. The results show the presence of specific binding sites for dexamethasone (22/32 in non ALL and 30/30 in ALL), for estrogens (11/15 in non-ALL and 5/12 in ALL), for progestins (8/25 in non-ALL and 5/13 in ALL) for androgens when R1881 was used as ligand (8/21 in non-ALL and 5/10 in ALL patients) but only 1/13 non-ALL patients and no ALL patients when labelled 5 alpha DHT was used. These results indicate that the blast cells from patients with acute leukemia contain specific proteins binding steroids with a high affinity. Our results for dexamethasone receptors are similar to those described in the literature in ALL and non-ALL.

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Section: Sucrose Gradient Analysismentioning

confidence: 99%

Androgen, estrogen and progestin binding sites in human leukemic cells

Danel

Martin²,

Escrich

et al. 1981

Intl Journal of Cancer

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Evaluation of preformed Percoll and reorientating sucrose density gradient centrifugation for the analytical subcellular fractionation of dog liver

Batt

Mann

1983

Research in Veterinary Science

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A Double-Labeling Assay for Simultaneous Estimation and Characterization of Estrogen and Progesterone Receptors using Radioiodinated Estradiol and Tritiated Org 2058

Ronchi

Granata

Brivio

et al. 1986

Tumori

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Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using 125I-E2 and 3H-Org 2058 as tracers. The interaction of 125I-E2 with ER and plasma proteins in comparison to 3H-E2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. 125I-E2 bound the same molecular forms displayed by 3H-E2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of 125I and 3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV < 8.0 %). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less purturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.

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Use of the vertical rotor to facilitate estrogen and progesterone receptor analysis.

Cited by 4 publications

References 0 publications

Androgen, estrogen and progestin binding sites in human leukemic cells

Androgen, estrogen and progestin binding sites in human leukemic cells

Evaluation of preformed Percoll and reorientating sucrose density gradient centrifugation for the analytical subcellular fractionation of dog liver

A Double-Labeling Assay for Simultaneous Estimation and Characterization of Estrogen and Progesterone Receptors using Radioiodinated Estradiol and Tritiated Org 2058

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