J. Steroid Biochem. Molec. Biol. Vol.41, No. 3-8, pp. 665--669,1992

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THE

ANDROGEN

CONTAINS DOMAIN

RECEPTOR

A MUTATION WHICH

IN

IN THE

AFFECTS

CHARACTERISTICS

LNCaP

LIGAND

STEROID

AND

CELLS BINDING

BINDING

RESPONSE

TO

ANTIANDROGENS J. VELDSCHOLTE,l* C. A. BERREVOETS,1 C. RIS-STALPERS,1 G. G. J. M. KUIPER,1 G. JENSTER,1 J. TRAPMAN,2 A. O. BRINKMANN1and E. MULDERl Departments of tEndocrinologyand Reproduction and 2Pathology,Erasmus University Rotterdam, P.O. Box 1738, 300 DR Rotterdam, The Netherlands S u m m a r y - - T h e human prostate tumor cell line LNCaP contains an abnormal androgen

receptor system with broad steroid binding specificity. Progestagens, estradiol and several antiandrogens compete with androgens for binding to the androgen receptor in the cells to a higher extent than in other androgen sensitive systems. Optimal growth of LNCaP cells is observed after addition of the synthetic androgen R1881 (0.1 nM). In addition, estrogens, progestagens and several antiandrogens do not inhibit androgen responsive growth, but have striking growth stimulatory effects and increase EGF receptor level and acid phosphatase secretion. We have found that the androgen receptor in the LNCaP cells contains a single point mutation changing the sense of codon 868 (Thr to Ala) in the ligand binding domain. Expression vectors containing the normal or mutated androgen receptor sequence were transfected into COS or HeLa cells. Androgens, progestagens, estrogens and several antiandrogens bind the mutated androgen receptor protein and activate the expression of an androgen-regulated reporter gene (GRE-tk-CAT), indicating that the mutation directly affects both binding specificity and the induction of gene expression. Interestingly, the antiandrogen casodex showed antiandrogenic properties in growth studies of LNCaP cells and did not induce reporter gene activity in Hela cells transfected with the mutant receptor. The mutated androgen receptor of LNCaP cells is therefore a useful tool in the elucidation of different levels of action of steroids and antisteroids.

INTRODUCTION

Androgens exert their effects on target tissue through binding to the androgen receptor, followed by association of the hormone receptor complex with specific binding sites on DNA [1, 2]. The specificity of hormonal action is accomplished both by the specific recognition of the enhancer element by the DNA binding domain of the receptor and by the specificity of the hormone receptor interaction, determined by the steroid binding domain of the receptor[3]. The androgen receptor complex can either induce [4] or repress [5] expression of specific genes. LNCaP tumor cells derived from a metastatic lesion of a human prostatic carcinoma contain androgen receptors and respond to androgens Proceedings of the lOth International Symposium of the Journal o f Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.

*To whom correspondenceshould be addressed. SB 41/3-8~EE

with growth in cell culture [6, 7]. In addition, increase in growth rate is observed in the presence of low doses of estrogens[6,8] and progestagens [9], despite the absence of estrogen or progestagen receptors, shown previously with specific antibodies against these receptor proteins [7, 9]. In addition, several antiandrogens stimulate both cell growth and excretion of prostatic acid phosphatase [10, 11]. In this paper we review studies of the androgen receptor system of the LNCaP cells. This receptor contains a point mutation in the steroid binding domain, which influences binding specificity for and transcription activation by several steroids and antisteroids and provides a tool for the study of the mechanism of action of antiandrogens. GROWTH OF LNCaP CELLS: EFFECT OF DIFFERENT STEROIDS AND ANTIHORMONES

The native androgen receptor ligand dihydrotestosterone is rapidly metabolized by

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concentration (log M) Fig. I. Effects of various concentrations of the synthetic androgen methyltrienolone (R1881) and the antiandrogens cyproterone acetate, nilutamide ( a n a n d r o n R U 23908), hydroxyflutamide (flutamide-OH) and casodex (ICI 176,334) on growth of L N C a P cells after 6 days treatment with the indicated c o m p o u n d s . G r o w t h is expressed as mean D N A content per culture relative to control cultures.

LNCaP cells, therefore the synthetic, nonmetabolizable androgen R1881 (methyltrienolone) was used for the study of growth stimulation. In LNCaP cells the effects of R 1881 are mediated by the androgen receptor because progestagen receptors are absent in these cells [9]. R1881 stimulated growth maximally at a concentration of 0.1 nM (3-4-fold increase in DNA content per culture vs control cultures). At higher concentrations of R1881 growth stimulation is limited and even negative effects on cell growth were observed (Fig. 1). Although there is no obvious explanation for this inhibitory effect, it might be related to the relatively large number of androgen receptors in LNCaP cells (> 100,000 sites/cell). Inhibition of cell growth by steroid hormones has been observed in cells artificially transfected with large numbers of estrogen receptors [12]. In addition to androgens, progestagens (progesterone and R5020, a synthetic progestagen non-metabolizable in vitro) as well as estradiol can stimulate growth of LNCaP cells. The antiandrogen cyproterone acetate stimulated cell growth 2-fold at 10 nM, whereas at this concentration nilutamide (anandron) had no effect (Fig. 1 and Table 1). Cyproterone acetate, nilutamide and hydroxy-flutamide stimulated LNCaP cell proliferation at a concentration of 100nM to the same extent as 0.1 nM R1881. When 0.1 nM R1881 was added to the cell cultures in combination with 100 nM of these antiandrogens, growth stimulation was similar to that observed for R1881 or antiandrogens added alone. The antiandrogen casodex did not influence the growth rate in concentrations up

to 1 #M (Fig. 1). Casodex could however inhibit the effect of 0.1 nM R1881 on cell growth at a concentration of 1/~M (Fig. 2). A N D R O G E N R E C E P T O R B I N D I N G : C H A N G E S IN AFFINITY DUE TO MUTATED RECEPTOR

Binding specificity was measured by incubating cytosol of LNCaP cells and of the transplantable prostate tumor PC-EW [13] with [3H]R1881 in the presence of various concentrations of steroids and antiandrogens. Relative binding affinity values were calculated from competitive binding curves [14] as the ratio of concentration of unlabeled compound and concentration of R 1881 required to inhibit [3H]R1881 binding by 50% (Table 2). In LNCaP cells the affinities of the receptor for both progesterone and R5020 were much higher than in the prostate tumor PC-EW. Table 1. Stimulation of growth rate of LNCaP cells by different compounds Steroid Methyltrienolone Dihydrotestosterone Promegestone (R5020) Progesterone Estradiol Triamcinolone acetonide Cyproterone acetate Hydroxyflutamide Nilutamide Casodex Tamoxifen

Stimulation (fold) 3.5 3 3 3 3 1 3.5 3 3 1 1

Optimal concentration 0.1 nM I nM 1 nM 1 nM 10 nM --" 0.1 ~ M 0.1-1/.t M 0.1-1 # M --" a

Effect of different compounds on growth of LNCaP cells. The cells were treated for 6 days with various concentrations of the different compounds added to steroid free (charcoal treated) medium as described previously [1 I]. Medium was renewed after 3 days culture. The actual concentration of dihydrotestosteron¢ was lower than indicated due to considerable metabolism of this steroid.": range tested: 0.01-1000 nM.

Androgen receptor of LNCaP prostate cells 10

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Fig. 2. Effects o f casodex a n d h y d r o x y f l u t a m i d e alone or in c o m b i n a t i o n with 0.1 n M R1881 o n g r o w t h o f L N C a P cells. D N A c o n t e n t was d e t e r m i n e d after 6 d a y s culture w i t h the indicated c o m p o u n d s . M e a n a n d S D o f four m e a s u r e m e n t s are shown.

To establish whether a mutation in the androgen receptor gene of the LNCaP cells could be the cause of its abnormal properties, genomic DNA as well as androgen receptor cDNA were sequenced after amplification of the appropriate fragments by the polymerase chain reaction [15]. In the androgen receptor gene as well as the cDNA one point mutation was detected. This mutation (A to G) in codon 868 resulted in the transition of threonine into an alanine (in the C-terminal part of the steroid binding domain of the receptor). Expression vectors containing either the wild type sequence (pAR0) or the mutated sequence (pARL) were transiently expressed in COS cells. Competition experiments performed on the cytosols of these cells showed that the two receptors had similar affinities for androgenic compounds (dihydrotestosterone, R1881), but showed striking differences for a series of nonandrogenic compounds (Table 2). Especially Table 2. Relative binding affinities for normal and mutated androgen receptor Ligand

LNCaP

PC-EW

COS-L COS-N

Methyltrienolone (R1881) Dihydrotestosterone

100 88 17 8.4 2.4 4.3 1.0 ND ND < 0. I

100 83 0.3 0.3 0.2 ND = ND ND ND < 0.1

100 29 4 5 6 2.6 0.4 2.4 0.1 < 0.1

Progesterone

Promegestone (R5020) Estradiol Cyproterone acetate Nilutamide Hydroxyflutamide Casodex

Triamcinolone acetonide

100 33 0.4 0.5 1 1.4 0.1 0.4 0.3 < 0.1

Relative binding affinity (RBA) of different ligands for the androgen receptor in LNCaP cells, in PC-EW tumor and in COS cells transfected with normal (COS-N) or mutated receptor (COS-L). The RBA value of R1881 was set at 100. The PC-EW tumors, grown in nude mice, were kindly provided by Dr van Steenbrugge. =ND, not determined.

progestagens (progesterone, R5020) and estradiol were bound with high affinity. This result indicates that the mutation is responsible for the high affinity of the androgen receptor for these compounds in LNCaP cells. Several antiandrogens (except casodex) showed slightly increased affinities for the mutant receptor.

TRANSFECTION OF MUTATED RECEPTOR AND EXPRESSION OF A REPORTER GENE: EFFECT OF ANTIANDROGENS

To investigate whether the mutation described above was not only responsible for the altered binding characteristics of the receptor, but also for the stimulatory effects of nonandrogenic compounds on the growth rate of LNCaP cells, HeLa cells were co-transfected with pAR0 or pARL and an androgen responsive reporter gene. It has been shown that the progesterone/glucocorticoid responsive element (PRE/GRE) can also act as an androgen responsive element[3]. Therefore, the vector pG29Gtk-CAT (a gift from Dr Renkawitz [16]) was used for these studies. This reporter gene construct contains two hormone responsive elements at a distance of 29 bp. Androgens, but also progestagens (progesterone and R5020) and estradiol, could induce CAT activity in the cells transfected with pARL, whereas only androgens induced CAT activity in the cells containing the pAR0 construct at low ligand concentrations [17]. When antiandrogens were tested in this co-transfection system cyproterone acetate, nilutamide and hydroxy-flutamide stimulated CAT activity in the cells with the

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J. VELDSCHOLTEet al.

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