Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene

Lorenz C. Hofbauer, Kevin C. Hicok, Marcy J. Schroeder, Steven A. Harris, John A. Robinson, Sundeep Khosla

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild- type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5α-dihydrotestosterone (5α-DHT) (10-8 M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 ± 73 (mean ± SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 ± 395 functional hARs/nucleus in hFO/AR-2 cells, and 3,987 ±+ 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5α-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5α- DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

Original languageEnglish (US)
Pages (from-to)542-551
Number of pages10
JournalJournal of Cellular Biochemistry
Volume66
Issue number4
DOIs
StatePublished - Sep 15 1997

Fingerprint

Androgen Receptors
Genes
Cells
Cell Line
Androgens
Chloramphenicol O-Acetyltransferase
Dihydrotestosterone
Osteoblasts
Response Elements
human AR protein
Messenger RNA
Androgen Receptor Antagonists
Polymerase chain reaction
RNA-Directed DNA Polymerase
Intercellular Signaling Peptides and Proteins
Bone
Complementary DNA

Keywords

  • Androgen receptor
  • Androgens
  • Bone cells
  • Dehydroepiandrosterone
  • Dihydrotestosterone
  • Hydroxyflutamide
  • Osteoblasts
  • Stable transfection

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene. / Hofbauer, Lorenz C.; Hicok, Kevin C.; Schroeder, Marcy J.; Harris, Steven A.; Robinson, John A.; Khosla, Sundeep.

In: Journal of Cellular Biochemistry, Vol. 66, No. 4, 15.09.1997, p. 542-551.

Research output: Contribution to journalArticle

Hofbauer, Lorenz C. ; Hicok, Kevin C. ; Schroeder, Marcy J. ; Harris, Steven A. ; Robinson, John A. ; Khosla, Sundeep. / Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene. In: Journal of Cellular Biochemistry. 1997 ; Vol. 66, No. 4. pp. 542-551.
@article{69ab388effba44e49da3dd67c32a78a8,
title = "Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene",
abstract = "Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild- type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5α-dihydrotestosterone (5α-DHT) (10-8 M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 ± 73 (mean ± SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 ± 395 functional hARs/nucleus in hFO/AR-2 cells, and 3,987 ±+ 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5α-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5α- DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.",
keywords = "Androgen receptor, Androgens, Bone cells, Dehydroepiandrosterone, Dihydrotestosterone, Hydroxyflutamide, Osteoblasts, Stable transfection",
author = "Hofbauer, {Lorenz C.} and Hicok, {Kevin C.} and Schroeder, {Marcy J.} and Harris, {Steven A.} and Robinson, {John A.} and Sundeep Khosla",
year = "1997",
month = "9",
day = "15",
doi = "10.1002/(SICI)1097-4644(19970915)66:4<542::AID-JCB13>3.0.CO;2-D",
language = "English (US)",
volume = "66",
pages = "542--551",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
publisher = "Wiley-Liss Inc.",
number = "4",

}

TY - JOUR

T1 - Development and characterization of a conditionally immortalized human osteoblastic cell line stably transfected with the human androgen receptor gene

AU - Hofbauer, Lorenz C.

AU - Hicok, Kevin C.

AU - Schroeder, Marcy J.

AU - Harris, Steven A.

AU - Robinson, John A.

AU - Khosla, Sundeep

PY - 1997/9/15

Y1 - 1997/9/15

N2 - Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild- type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5α-dihydrotestosterone (5α-DHT) (10-8 M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 ± 73 (mean ± SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 ± 395 functional hARs/nucleus in hFO/AR-2 cells, and 3,987 ±+ 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5α-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5α- DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

AB - Androgens have significant beneficial effects on the skeleton. However, studies on the effects of androgens on osteoblasts are limited due to the absence of appropriate model systems that combine completness of the osteoblastic phenotype, rapid proliferation rate, and stable expression of the androgen receptor (AR). Thus, we stably transfected the conditionally immortalized human fetal osteoblastic cell line (hFOB) with the human wild- type AR (hAR) cDNA. Compared to nontransfected hFOB cells, constitutive hAR mRNA expression in three independent hAR-transfected hFOB clones (hFOB/AR) was 15-fold higher in hFOB/AR-16, 62-fold higher in hFOB/AR-2, and 72-fold higher in hFOB/AR-6 cells, respectively, as assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Detectable constitutive levels of hAR mRNA by Northern blot analysis were present in hFOB/AR-2 and hFOB/AR-6 cells, but not in hFOB/AR-16 or hFOB cells, respectively. Treatment with 5α-dihydrotestosterone (5α-DHT) (10-8 M) for 24 h did not alter hAR mRNA steady state levels in the hFOB/AR cell lines. Nuclear binding studies demonstrated 152 ± 73 (mean ± SEM) functional hARs/nucleus in non-transfected hFOB cells, 3,940 ± 395 functional hARs/nucleus in hFO/AR-2 cells, and 3,987 ±+ 823 hARs/nucleus in hFOB/AR-6 cells, respectively. Treatment with 5α-DHT increased the expression of a transiently transfected androgen response element-chloramphenicol acetyltransferase (ARE-CAT) reporter construct in hFOB/AR-6 cells in a dose- and time-dependent manner; no such effect was observed in transiently transfected hFOB cells lacking exogenously transfected hARs. Moreover, 5α- DHT-induced ARE-CAT expression was inhibited by the selective androgen receptor antagonist, hydroxyflutamide. In summary, we have developed and characterized androgen-responsive osteoblastic cell lines derived from normal human fetal bone that express physiological levels of functional hARs. These cell lines should provide a suitable model for further studies on the effects of androgens on osteoblast function, including the identification of potential androgen-regulated growth factors and cytokines.

KW - Androgen receptor

KW - Androgens

KW - Bone cells

KW - Dehydroepiandrosterone

KW - Dihydrotestosterone

KW - Hydroxyflutamide

KW - Osteoblasts

KW - Stable transfection

UR - http://www.scopus.com/inward/record.url?scp=0030879496&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030879496&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1097-4644(19970915)66:4<542::AID-JCB13>3.0.CO;2-D

DO - 10.1002/(SICI)1097-4644(19970915)66:4<542::AID-JCB13>3.0.CO;2-D

M3 - Article

VL - 66

SP - 542

EP - 551

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 4

ER -