A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts

Charles Eil, Marc E Lippman, D. Lynn Loriaux

Research output: Contribution to journalArticle

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Abstract

A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 3 The following trivial names were used: dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), methyltrienolone (R1881, 17β-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17β-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5α-androstane-3α,17β-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2α-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione). (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10-9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.

Original languageEnglish
JournalSteroids
Volume35
Issue number4
DOIs
StatePublished - Jan 1 1980
Externally publishedYes

Fingerprint

Metribolone
Androgen Receptors
Fibroblasts
Trientine
Skin
Promegestone
Androstane-3,17-diol
R 2956
Cells
Cyproterone Acetate
Centrifugation
Biopsy
Cell Count
Dihydrotestosterone
Washing
Cell Line
Dexamethasone
Androgens
Sucrose
Monolayers

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Molecular Biology

Cite this

A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts. / Eil, Charles; Lippman, Marc E; Loriaux, D. Lynn.

In: Steroids, Vol. 35, No. 4, 01.01.1980.

Research output: Contribution to journalArticle

Eil, Charles ; Lippman, Marc E ; Loriaux, D. Lynn. / A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts. In: Steroids. 1980 ; Vol. 35, No. 4.
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abstract = "A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 3 The following trivial names were used: dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), methyltrienolone (R1881, 17β-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17β-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5α-androstane-3α,17β-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2α-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione). (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10-9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.",
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N2 - A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 3 The following trivial names were used: dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), methyltrienolone (R1881, 17β-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17β-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5α-androstane-3α,17β-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2α-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione). (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10-9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.

AB - A method is described for the determination of binding capacity (Ro) and dissociation constant (Kd) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [3H]dihydrotestosterone3 3 The following trivial names were used: dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), methyltrienolone (R1881, 17β-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17β-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5α-androstane-3α,17β-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2α-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11β, 17,21-trihydroxy-16α-methylpregna-1,4-diene-3, 20-dione). (DHT) at 22°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (Ro = 12,105 ± 4305 (S.D.) sites/cell; Kd = 1.0 ± 0.7 (S.D.) × 10-9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision.

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