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 language | English (US) |
|---|---|
| Journal | Steroids |
| Volume | 35 |
| Issue number | 4 |
| DOIs | |
| State | Published - 1980 |
| Externally published | Yes |
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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, Donald (Lynn).
In: Steroids, Vol. 35, No. 4, 1980.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts
AU - Eil, Charles
AU - Lippman, Marc E.
AU - Loriaux, Donald (Lynn)
PY - 1980
Y1 - 1980
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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UR - http://www.scopus.com/inward/citedby.url?scp=0019304853&partnerID=8YFLogxK
U2 - 10.1016/0039-128X(80)90140-3
DO - 10.1016/0039-128X(80)90140-3
M3 - Article
C2 - 7376228
AN - SCOPUS:0019304853
VL - 35
JO - Steroids
JF - Steroids
SN - 0039-128X
IS - 4
ER -