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Androgen
receptor isoforms in human prostatic cancer tissue and LNCaP cell line
Shu-Jie
XIA1, Xiao-Da TANG2, Qing-Zheng MA1
1University
Department of Urology, Shandong Provincial Hospital
Jinan 250021, China
2University Department of Urology, Shanghai First People's
Hospital Shanghai,
200080, China
Asian J Androl 2001 Sep; 3: 223-225
Keywords:
LNCaP
cell line; receptors,androgen; phenotype; prostate
Abstract
Aim: To investigate the androgen receptor (AR) isoform expressions in human prostatic cancer tissue and LNCaP cell line. Methods: With high resolution isoelectric focusing (IEF) method we demonstrated the different expressions of AR isoforms in human prostatic cancer tissues and LNCaP cell line. Results: Data were obtained from three prostatic cancer specimens and the LNCaP cell line. Three typesof AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. For the 3 prostatic cancer specimens, 1 sample showed all the three types of AR isoforms, the second specimen expressed at 6.5 and 6.0, and the third failed to show any type of isoforms. The LNCaP cell line expressed all the three AR isoforms. Binding of 3H-dihydrotestosterone (3 H-DHT) to these three isoforms was inhibited by the addition of l00-fold excess of DHT or testosterone, while not by progesterone, oestradiol and diethylstilboestrol. Conclusion: The expression of AR isoforms is different in different prostate cancer tissues, which may be related to the difference in the effect of anti-androgen therapy in different patients.
1
Introduction
Since
the publication of androgen receptor (AR) cDNA sequence from human prostate[1],
it has been generally accepted that AR is encoded by a single gene.
However,
there is evidence that the AR protein itself is heterogeneous[2,3].
A human
prostate
carcinoma cell line LNCaP expresses an AR that binds both testosterone (T)
and R1881, resu1ting in ligands capable of stimulating the growth of the
cell line
in the culture.Recently two distinct clones of AR cDNA were isolated from
the testes of the rainbow
trout and the Japanese eel[4,5] and two types of AR proteins
isolated
from the
fibroblasts of human genital skin[6]. In our previous paper[7],
it was demonstrated
that there were different AR isoform expressions in human benign prostatic
hyperplasia tissue and rat prostate. However, the AR isoforms in human prostate
cancer tissue and LNCaP cell line have not been reported so far. The present
study was designed to investigate the existence of different AR isoforms
in these
tissues. 2 Materials and methods
2.1 Tissue culture
Stock culture of the LNCaP cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and was maintained in RPMI-1640 medium containing l0 % (v/v) fetal calf serum (FCS) and 100 units/mL of penicillin. The cells were then harvested and stored in liquid nitrogen until processed.2.2 Tissue handling
Human prostatic cancer tissues were obtained at operation from patients undergoing prostatectomy. Each of the samples was divided into two portions, one snap-frozen in liquid nitrogen and the other used for pathological examination. The samples obtained were diagnosed as malignant tissues by a pathologist. All tissue processing was performed at 4 . The tissue was sliced and homogenized using a polytron homogeniser in glycerol phosphate buffer (10 % glycerol, 10 mmol/L phosphate,1.5 mmol/L EDTA and 5 mmol/L monothioglycerol, pH 7.4) containing l ng/mL each of the protease inhibitors, aprotinin and soybean trypsin inhibitor (both from Sigma, USA). The homogenate was centrifuged for l0 min at 800 g. The supernatant was further centrifuged for 60 min at 100,000 g and the final supernatant was used for receptor analysis.2.3 Dextran coated charcoal (DCC) assay of AR
AR was measured by the single saturating dose (SSD) assay. The cytosol (3 mg-protein/mL cytosol) was incubated at 4 for 24 h with 50 nmol/M tritiated DHT in the presence or absence of an l00-fold excess of unlabe1led DHT, testosterone, oestradiol or diethylstilbestrol. Free hormone was separated from bound by incubation with DCC buffer charcoal (0.5 %, w/v) and dextran T70 (0.05 %, w/v) (Sigma) at 4 , followed by centrifugation at 10,000 g for 5 min. An aliquot of the supernatant was counted in a liquid scintillation counter. Another aliquot was used for IEF analysis.2.4 Isoelectric focusing
The
IEF gels were cast in slabs of size 125260 mm and separation was conducted
along the short side of the polyacrylamide gels (2 mm thick), which contained
20 % (v/v) glycerol. A pH gradient was achieved using 1.5 % (w/v) ampholine
3.5-10.7 (LKB, Bromma, Sweden) and l % (w/v) ampholine 5-8. Gels were
photopolymerized at room temperature by means of a TR 26 polymerization
light, using riboflavin (0.004 % v/v) for at least 4 h. IEF was performed
in a cold room and temperature of the cooling water was kept constant
at 4 . Electrode
solution of 1 M NaOH (cathode)
and 1 M H2SO4 (anode) were used. Gels were pre-focused
for 45 min at 20 mA/20 W/l200 V.
After
DCC extraction, aliquots (150 L) of the radioactive (3H-DHT
labelled) supernatants derived from SSD assay was loaded near the cathode.
The runs were carried out using a 3000 xi CC power supply (LKB, Sweden)
at 1200 v/20 mA/20 W constant power
for l .5 h. A mixture of nine natural proteins (Bio-Rad) was used for
pH calibration. After the run, the gels were cut into 2.5 mm slices and
each slice was incubated with 5mL scinti1lation cocktail (Ready-Solv,
Beckman, USA) for 24 h at room temperature and radioactivity assayed.
2.5 Protein determination
Proteins were determined by the method of Lowry[8] , using BSA as the standard.3
Results
Table 1. AR isoforms in human prostate cancer and LNCaP cells.
|
Samples |
Isoforms |
Frequency |
||
| Prostate
cancer |
6.5 |
6.0 |
5.3 |
1/3 |
| 6.5 |
6.0 |
-- |
1/3 |
|
| -- |
-- |
-- |
1/3 |
|
| LNCaP
cells |
6.5 |
6.0 |
5.3 |
1/1 |
4
Discussion
The
isoelectric points of the cytoplasmic androgen receptors obtained from
human prostatic cancer
were found to be acidic (pH 6.5, 6.0 and 5.3). Auf and Ghanadian[9]
using a synthetic ligand indicated that the pI of the androgen receptor
isolated from BPH samples was also acidic (6.2). BPH tissue homogenates
contain high levels of sex hormone binding globulin (SHBG) that makes
the androgen receptor assay difficult when DHT is used as the ligand.
However, our data indicates that the peak at pI 6.5 is not resulted from
steroid binding to SHBG, as both oestradiol and diethylstilbestrol could
not compete with labelled DHT for displacing binding
from this peak. Puddefoot et al[10] and Marsigliante
et al[11] observed a non-specific binding peak at 5.0.
It
was reported that androgen receptor is coded by a single gene[1,12,13].
The AR isoforms mentioned above seems to arise as a consequence of protein
modification or phosphorylation[3]. Recently Yamashita[4]
and Ikeuchi et al[5]
isolated two types of cDNA clones from rainbow trout and Japanese eel
testis and both of the cDNA could encode proteins. The translation of
mRNA transcribed from AR cDNAs yielded 94-, 76-kDa and other proteins
in a protein synthesis system of rabbit reticulocyte
lysate that could bind DNA and androgens with a high affinity[12].
The predicted molecular weight of the AR was approximately 90-95 kDa[12],
which was confirmed by SDS--PAGE analysis of photoaffinity labelled receptor[14].
We found there are three AR isoforms that should be necessary for the
function of AR. The androgen receptor heterogeneity observed in LNCaP
cells is not due to mutation, because the same micro-heterogeneity (110-112
kDa doublet) is also found after expression of wild type androgen receptor
protein in COS-l cells[3]. Our data clearly showed that there
were three isoforms in human prostatic cancer tissues and LNCaP cells.
The genetic basis for human prostate AR isoforms needs further studies.
In
humans, a rare group of males with a defect in 5-alpha reductase activity
have been noted to develop incomplete differentiation of the male external
genitalia and prostate gland, whilst retaining normal development of Wolffian
duct derivatives, which undergo masculinization at puberty. In these subjects,
the prostate remains rudimentary and BPH does not occur[15].
Furthermore studies on castrated male rats implanted with the Dunning
rat prostatic carcinoma R-3327 and treated with testosterone and a 5-reductase
inhibitor, finasteride (MK906), did not influence
tumor growth. This suggests that the R-3327 tumor responds to testosterone[16].
The AR can bind both T and DHT, but T cannot substitute for DHT to induce
development of the prostate gland and the male external genitalia. In
our studies both T and
DHT are able to bind all three isoforms, thus the differential tissue
response to T and DHT may be a consequence of the ability of tissues to
express different AR isoforms.
References
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Correspondence
to: Prof.
Shu-Jie XIA, University Department of Urology, Shanghai
First People's Hospital, 85 Wu Jin Rd. Shanghai 200080, China.
Tel:
+86-21-6324 0090 Ext 5511 Fax: +86-21-6324
0825
E-mail: xiashujie@yahoo.com
Received 2001-03-15
Accepted 2001-08-28
