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Assessment of Androgen Receptor Function in Genital Skin Fibroblasts Using a Recombinant Adenovirus to Deliver an Androgen-Responsive Reporter Gene¹

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235; and the Department of Internal Medicine, University of Bonn, Bonn, Germany

Address all correspondence and requests for reprints to: Michael J. McPhaul, M.D., Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-8857.

	Abstract

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Mutations of the androgen receptor (AR) cause defects in virilization and can result in a spectrum of phenotypic abnormalities of male sexual development that includes patients with a completely female phenotype (complete testicular feminization) and individuals with less severe defects of virilization, such as Reifenstein syndrome. These phenotypes are not specific for mutations of the AR gene, however, and defects in other genes can also result in similar abnormalities of male development. For this reason, the diagnosis of an AR defect is laborious and requires data from endocrine studies, the family history, and in vitro binding experiments.

To assist in the evaluation of patients with possible AR defects, we previously employed the use of a recombinant adenovirus to deliver an androgen-responsive gene into fibroblast cultures to assay AR function in normal subjects and patients with complete forms of androgen resistance. Although these studies demonstrated measurable differences between these two groups of subjects, we did not assay samples from patients with partial defects of androgen action. In the current study, we have modified this method to examine AR function in three groups of patients with known or suspected defects of AR function: patients with Reifenstein syndrome, patients with spinobulbar muscular atrophy, and patients with severe forms of isolated hypospadias. When assayed using this method, the AR function of patients with Reifenstein syndrome was intermediate between that of normal control subjects and that of patients with complete testicular feminization. Using the parameters established by the aforementioned experiments, we found that defective AR function can be detected in fibroblasts established from patients with spinobulbar muscular atrophy and in some patients with severe forms of isolated hypospadias, including two with a normal AR gene sequence. These results suggest that this method may have some utility in screening samples to detect defects of AR function, particularly when viewed in the context of other AR assays results.

	Introduction

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VIRILIZATION of the urogenital tract is mediated by the androgens testosterone (T) and 5-dihydrotestosterone (DHT). Defects in the development of the normal male phenotype can be caused by alterations in the synthesis or metabolism of androgens or to defects in the androgen receptor (AR) gene (1). In a substantial proportion of patients with abnormalities of virilization (such as severe isolated hypospadias), no such defects have been identified (2).

The diagnosis of patients with defects of AR function in the larger population of individuals with abnormalities of male development presents a considerable challenge and requires the use of data from the family history, endocrine testing, and measurement of androgen binding in cultures of genital skin fibroblasts. Even with such information, a proportion of mutations in the DNA-binding domain of the AR receptor would not be detected.

The diagnosis of patients with abnormalities of the AR would be facilitated by the availability of a test that assessed the function of the AR expressed in fibroblasts cultured from individual patients. As androgen-responsive genes have not been characterized in fibroblasts, we have used a recombinant adenovirus to deliver a model androgen-responsive reporter gene [mouse mammary tumor virus (MMTV)-luciferase] into genital skin fibroblast cultures. Our previous studies demonstrated the utility of this assay in discriminating subjects with normal AR function from those with the complete defects of AR function (complete testicular feminization) (3). In the current study, we have applied this assay to measure AR function in genital skin fibroblast cultures from three groups of patients with known or potential defects of AR function: men with the Reifenstein phenotype, spinobulbar muscular atrophy (SBMA), and isolated severe hypospadias.

	Materials and Methods

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Patient population

The fibroblast cultures assayed in the current investigation were from four distinct groups. Patients with Reifenstein syndrome displayed endocrine and clinical profiles and a family history consistent with X-linkage. The genetic defects in 7 of the 11 individual strains studied have been identified (4, 5) (our unpublished observations). Patients with SBMA have a progressive degeneration of the spinal and bulbar motor neurons and clinical evidence of androgen resistance. These clinical features are not present at birth, but are first evident in adulthood and have been traced to an expansion of the triplet repeat (CAG) encoding the glutamine homopolymeric segment within the amino-terminus of the AR (6). The samples from patients with severe isolated hypospadias (perineal or penoscrotal) were established from individuals that were without endocrine features or family history suggestive of an AR defect (7). Normal control strains were established from samples of foreskin or genital skin fibroblast biopsies from patients with unrelated disorders, as outlined below.

Androgen-responsive reporter gene delivery via a recombinant adenovirus

The recombinant adenovirus contains the firefly luciferase gene under the control of the MMTV-long terminal repeat (8). This long term repeat contains sequences that confer responsiveness to the AR and other related members of the steroid receptor family (including the glucocorticoid receptor).

Fibroblast strains were plated in six-well plates (3.5-cm diameter wells) and cultured in MEM containing 10% FCS until confluency. Experiments were initiated by infection of the confluent monolayers (2 x 10⁵ cells/well) with approximately 10⁷ plaque-forming units of the recombinant adenovirus in MEM for 1 h at 37 C. After infection, the medium was replaced with fresh MEM containing 10% FCS, and the incubations were continued for 24 h. After this 24-h incubation, the medium was removed and replaced with MEM containing 5% charcoal-stripped serum alone or the hormone to be assayed. Incubations were conducted with 2 nmol/L mibolerone (Mb), 2 nmol/L DHT, or 4 nmol/L T. The hormone stimulation was continued for 72 h, at which time the cells were harvested and assayed to determine luciferase activity. As noted below, in some incubations the 5-reductase inhibitor finasteride was included at a final concentration of 0.1 µmol/L.

	Results

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Assays of AR function are abnormal in selected patients with Reifenstein syndrome

We have characterized the AR expressed in fibroblast strains established from a number of patients with Reifenstein syndrome and have identified amino acid substitutions in seven patients (4, 5) (unpublished observations). The mutations are localized to the AR hormone-binding and DNA-binding domains and have variable effects on ligand binding by the receptor (see Table 1).

In prior studies of mutant ARs using transfection assays (8), we noted that AR function could be restored for some mutant ARs by the use of potent, nonmetabolizable androgen and suggested that the use of such ligands to stimulate the AR in functional assays might obscure defects of the AR caused by instability of the AR-hormone complex. To test this possibility, assays were performed using the physiologic ligands DHT and T to stimulate receptor function. As depicted in Table 1, the strains that demonstrated abnormalities of AR function using Mb (1061, 691, 497, and 217) also demonstrated similar abnormalities using T or DHT as ligand. The levels of AR function observed in strains 38, 787, and 593 remained in the normal range and were not markedly reduced in cultures stimulated with DHT or T compared to those in cultures stimulated with Mb.

The amino acid substitution mutations identified in strains 787, 38, and 593 caused a rapid dissociation of ligand from the receptor. In addition, our previous studies demonstrated that in transfection assays the mutant AR predicted for one patient (patient 38) was more responsive to DHT than to T (9). This finding prompted us to repeat these experiments including finasteride to block the conversion of T to DHT in the genital skin fibroblast cultures. As shown in Table 1, this modification of our assay procedure had two effects. First, consistent with previous results, the inclusion of finasteride dramatically reduced the activity of the AR predicted for patient 38; finasteride had a similar effect on the activity of the mutant AR expressed in the 787 and 593 fibroblast strains. The second effect was a subtle decrease in activation of the reporter gene in normal fibroblast strains. In contrast to the marked changes noted in the 38, 787, and 593 cell lines stimulated with T in the presence of finasteride (>70% reduction), the effect on the activity of the AR in normal fibroblasts decreased by an average of 50% (range, 24–67%). An important point is that the level of AR activity measured in the normal fibroblast samples remained in the normal range (>10-fold stimulation), even when measured after stimulation with T in the presence of finasteride. All of the Reifenstein samples showed low levels of AR function when assayed with T in the presence of finasteride.

AR function is reduced in fibroblast strains established from patients with SBMA

Expansions of the glutamine repeat segment in the amino-terminus of the AR are associated with degeneration of anterior motor neurons and clinical signs of androgen resistance, including gynecomastia (Kennedy’s disease) (5). Transfection assays of complementary DNAs encoding ARs containing such expanded glutamine repeats have demonstrated that these mutant ARs possess a reduced capacity to activate androgen-responsive genes (9, 10, 11), and these deficits are even more profound when the levels of immunoreactive AR are assessed (11).

To assess whether the expected reduction of AR function could be detected in fibroblast strains established from patients with this disorder, we infected the monolayers with the adenovirus carrying the MMTV-luciferase reporter gene. As before, AR function was assessed after following stimulation with saturating doses of Mb. As shown in Table 2, in all instances the level of AR function was reduced compared to that in normal control fibroblasts. In most cases, the level of AR function was comparable to that in patients with Reifenstein syndrome, although in some (e.g. strain 814), the level of AR function was markedly reduced and approached the level observed in samples from patients with complete androgen resistance.

Hypospadias is one of the most common of human congenital defects (12, 13). Because this abnormality is a prominent component of some partial androgen resistance phenotypes, it has been suggested that a proportion of patients with severe forms of isolated hypospadias (i.e. not clearly part of a familial condition) might be caused by mutations of the AR.

To address this question, we investigated the level of AR function in eight patients with severe forms of isolated hypospadias (6). As shown in Table 3, AR function was normal after stimulation with Mb in two strains, was reduced in two others, and was virtually absent in one. As indicated in Table 3, the strain with near-absent AR function (strain 931) was later discovered to harbor a mutation in the DNA-binding domain of the receptor (G566V). Our studies indicate that strains 938 and 939 do not have amino acid substitutions in the AR-coding region (2).

	Discussion

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Using assays of Mb-stimulated AR function, the Reifenstein patients comprise a heterogeneous group. In the first group, exemplified by patient 691, the level of AR function is intermediate between that measured in fibroblasts of subjects and that in fibroblasts of patients with complete testicular feminization. By contrast, using this same assay, fibroblasts from three other patients with Reifenstein syndrome (787, 38, and 593) demonstrate levels of AR function that are well within the normal range.

This result suggested either that the level of AR function measured in patients with Reifenstein syndrome had no direct relationship to the observed phenotype or that the assay method used in these experiments was somehow obscuring this relationship. Our previous studies in transfected cells (9) and the nature of the AR defect in the 787 and 38 fibroblast strains (accelerated dissociation rate) suggested that the latter might be the case.

For this reason, the assays of AR function were repeated for normal control subjects and selected Reifenstein patients using single additions of saturating doses of Mb, DHT, or T in both the presence and absence of the 5-reductase inhibitor, finasteride. As shown in Table 1, the inclusion of finasteride in the incubations had a discernible effect on the level of induction of the reporter gene in normal subjects. Although this effect was somewhat variable, average decreases of 50% (range, 24–67%) were observed for assays performed with T in the presence of finasteride compared to parallel assays performed in the absence of finasteride. By contrast, when similar assays were performed on the mutant ARs expressed in fibroblast strains 787, 38, and 593, decreases of over 70% were observed in the samples stimulated with T in the presence of finasteride compared to those in which no finasteride was included. Importantly, in these latter assays, the levels of AR function were well below the range established as representing normal AR function. These observations suggest that defective AR function can be detected in fibroblast strains from most patients with the Reifenstein phenotype, although modifications of our previously described assay were necessary to detect defects in patients that harbor hormone-binding domain mutations that cause isolated abnormalities of ligand dissociation from the mutant AR.

The decreases observed in AR function in fibroblasts from normal controls measured after stimulation with T in the presence of finasteride are probably caused by inhibition of the formation and action of DHT in the fibroblasts. Although not tested rigorously in these experiments, the variability in the magnitude of this change seems to correlate with the varying levels of 5-reductase measured in the different strains. Of interest, the decreases observed in the effects of finasteride on Mb- and DHT-stimulated AR function for strains 787 and 593 are inconsistent with the lack of effects seen in the normal strains. This suggests that finasteride may have a more direct effect on the function of these mutant receptors, one that is not evident in the assays of normal fibroblast strains.

The results of the assays of the SBMA and hypospadias patients are interesting for different reasons. Using the adenovirus reporter gene assay, the levels of AR function in the SBMA samples are substantially reduced. In patients 711 and 814, AR function is reduced to a level approaching that observed in patients with complete forms of androgen resistance. Although decreases in AR function have been found after transfection of complementary DNAs encoding mutant ARs with such expanded repeats (10, 11, 12), the depressed levels of AR function seen here are much greater that those reported to date and suggest that such transfection assays may underestimate the degree to which AR function is impaired in vivo. Of note, the normal levels of AR measured in these samples by binding and immunoblot assays do not support a role for reductions in the levels of AR alone in the pathogenesis of androgen resistance associated with SBMA (15).

The assays of AR function in patients with isolated hypospadias represent our first attempts to employ this assay to identify fibroblasts with abnormalities of AR function in a population with clinical features suggestive of defects in androgen action, but in whom supporting endocrine data or a suggestive family history are not available. The level of Mb-stimulated AR function appears normal in four strains and abnormal in three strains. Although an AR gene mutation has been identified in one strain that exhibits reduced AR function (strain 931; G566V), the AR gene sequence is normal in the other two. These findings suggest that the observed deficit of AR function present in the remaining two strains (938 and 939) may be caused by an abnormality not localized to the AR gene itself.

In summary, we provide evidence for the utility of an adenovirus-based reporter gene assay in the identification of patients with partial forms of androgen resistance. In addition to identifying abnormalities of AR function in a range of patients with Reifenstein syndrome caused by amino acid substitutions in the DNA- and hormone-binding domains of the AR, this assay is able to detect the abnormalities of AR function present in patients with SBMA. Finally, our data suggest that abnormalities of AR function exist in a small proportion of patients with phenotypic abnormalities suggesting androgen resistance, such as severe hypospadias, and a normal AR gene sequence. Such individuals would be candidates to harbor defects in a gene(s) other than the AR that act to impair normal AR function.

	Footnotes

 
¹ This work was supported by March of Dimes Grant 6-FY-94–0213, NIH Grant DK-03892, a grant from the Perot Family Foundation, and Deutsche Forschungemeinschaft Grant SFB 351, A1.

Received September 6, 1996.

Revised February 13, 1997.

Accepted February 20, 1997.

	References

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1. Griffin JE, McPhaul MJ, Russell DW, Wilson JD. 1995 The androgen resistance syndromes: 5-reductase 2 deficiency, testicular feminization, and related disorders. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The metabolic and molecular basis of inherited disease, 7th ed. New York: McGraw-Hill; 2967–2998.
2. Állera A, Herbst MA, Griffin JE, Wilson JD, Schweikert H-U, McPhaul MJ. 1995 Mutations of the androgen receptor coding sequence are infrequent in patients with isolated hypospadias. J Clin Endocrinol Metab. 80:2697–2699.[Abstract]
3. McPhaul MJ, Deslypere J-P, Allman DR, Gerard RD. 1993 The adenovirus-mediated delivery of a reporter gene permits the assessment of androgen receptor function in genital skin fibroblast cultures. J Biol Chem. 268:26063–26066.[Abstract/Free Full Text]
4. McPhaul MJ, Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD. 1992 Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene. J Clin Invest. 90:2097–2101.
5. Weidemann W, Linck B, Haupt H, et al. Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene. In press.
6. LaSpada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. 1991 Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature. 352:77–79.[CrossRef][Medline]
7. Schweikert H-U, Schlüter M, Romalo G. 1989 Intracellular and nuclear binding of [³H]dihydrotestosterone in cultured genital skin fibroblasts of patients with severe hypospadias. J Clin Invest. 83:662–668.
8. Shih W, Mears T, Bradley DJ, Parandoosh Z, Weinberger C. 1991 An adenoviral vector system for functional identification of nuclear receptor ligands. Mol Endocrinol. 5:300–309.[Abstract]
9. Marcelli M, Zoppi S, Wilson CM, Griffin JE, McPhaul MJ. 1994 Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex. J Clin Invest. 94:1642–1650.
10. Mhatre AN, Trifiro MA, Kaufman M, et al. 1993 Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nat Genet. 5:184–188.[CrossRef][Medline]
11. Amato AA, Prior TW, Barohn RJ, Snyder P, Papp A, Mendell JR. 1993 Kennedy’s disease: a clinicopathologic correlation with mutations in the androgen receptor gene. Neurology. 43:791–794.[Abstract/Free Full Text]
12. Gao T, Marcelli M, McPhaul MJ. 1996 Transcriptional activation and transient expression of the human androgen receptor. J Steroid Biochem Mol Biol. 59:9–20.[CrossRef][Medline]
13. Böök JA. 1951 The incidence of congenital disease and defects in a south Swedish population. Acta Genet. 2:289–311.
14. Shapiro RN, Eddy W, Fizzgibbon J, O’Brien G. 1958 The incidence of congenital anomalies discovered in the neonatal period. Am J Surg. 96:396–400.[CrossRef][Medline]
15. Choong CS, Kemppainen JA, Zhou Z-X, Wilson EM. 1996 Reduced androgen receptor gene expression with first exon CAG repeat expansion. Mol Endocrinol. 10:1527–1535.[Abstract]
16. Griffin JE, Durrant JL. 1982 Qualitative receptor defects in families with androgen resistance: failure of stabilization of the fibroblast cytosol androgen receptor. J Clin Endocrinol Metab. 55:465–474.[Medline]

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