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Institute of Oncology (L.A.M.C., G.C.) and Institute of Urology (M.P.-M.), Policlinico, University Medical School; and Experimental Oncology and Molecular Endocrinology Units, Palermo Branch of the National Cancer Institute of Genoa (L.A.M.C., A.T., O.M.G.), Cancer Hospital Center, Palermo, Italy; Max Planck Institute for Experimental Endocrinology (M.M.), Hannover, Germany; and GSF- National Research Center, Institute of Mammalian Genetics (J.A.), Neuherberg, Germany; and the Department of Obstetrics and Gynecology, Healthpartners for Environment and Health, St. Paul-Ramsey Medical Center (C.H.B.), St. Paul, Minnesota 55101-2595
Address all correspondence and requests for reprints to: Dr. Luigi A. M. Castagnetta, Istituto di Oncologia, Università di Palermo, Via Marchese Ugo 56, 90141 Palermo, Italy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4Ad), estradiol
(E2), and estrone (E1). To date at least four
different human 17ßHSDs have been identified, their complementary
DNAs (cDNAs) cloned, and their amino acid sequences deduced. The
soluble 17ßHSD1, consisting of 327 amino acids, was originally
isolated from human placenta and performs the oxidation of
E2 at the same efficiency as the reduction of
E1 (3, 4). 17ßHSD2 is a microsomal enzyme of 387 amino
acids that slightly favors the oxidation over the reduction of either
androgen or estrogen and is expressed at high levels in the human
placenta (5, 6). The microsomal 17ßHSD3, which is exclusively
expressed in human testis, consists of 310 amino acids and is
responsible for the reduction of estrogens and androgens (7). Recently,
cDNAs encoding for human, mouse, and porcine 17ßHSD4 have been
identified (8, 9, 10, 11). They share 85% amino acid identity and metabolize
estrogens and androgens very efficiently, displaying a 400-fold
preference for steroid oxidation. A 3-kb messenger RNA (mRNA) codes for
peroxisomal 80-kDa (737 amino acids) protein, featuring domains that
are absent in the other 17ßHSDs. We have shown that both the 80-kDa
and its N-terminal 32-kDa (amino acids 1–323) fragment are able to
perform the dehydrogenase reaction not only with steroids at the C17
position, but also with 3-hydroxyacyl coenzyme A (12). Evidence for key steroid enzymes in human prostate tissues has been repeatedly reported (13, 14). However, little is known of the expression and function of the different 17ßHSD types in either normal or malignant prostate gland. Most previous studies have used crude extracts of homogenized tissues to compare the activities of steroid enzymes in normal, hyperplastic, and carcinomatous human prostate (15). Although this in vitro characterization of enzymes (as either purified or expressed proteins) may represent a versatile tool in understanding their potential physiological roles, in vivo conditions (including subcellular localization, pH, concentrations of cofactors and substrates, feedback mechanisms, etc.) may well drive the reactions with different kinetics and oxidation/reduction preferences. This is also reflected in the discrepancies often emerging between estimates of intratissue amounts of individual steroids and the extent of relevant enzyme activities, as measured by crude extract methods. After optimization of liquid chromatographic procedures, we established an original intact cell analysis that allows measurement of several enzyme activities of steroid metabolism in living cultured cells (16, 17, 18). This in vivo approach, while retaining cells in a physiological microenvironment, enables to use close to physiological amounts of steroid precursors and preserves conversion rates and direction of steroid metabolism, which could be otherwise disturbed by tissue homogenization or simplification to few enzymes in vitro.
In the present work, we have used both intact cell analysis and subcellular fractionation assays to inspect pathways of 17ß-oxidation of androgens and estrogens in human prostate cancer cell lines that feature distinct steroid receptor contents and responses to hormones; these are used as helpful model systems for either hormone-sensitive or refractory prostate cancer. We compared the metabolic conversion rates of cells in culture with the expression of mRNAs for the four types of 17ßHSD identified to date to verify which enzyme(s) is responsible for the observed reactions. The results of these studies are reported herein.
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Materials and Methods |
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-32P]deoxy-CTP was obtained from Hartmann Analytic
(Braunschweig, Germany).
Cell culture
LNCaP.FGC (passage 19), DU145 (passage 59), and PC3 (passage 17)
human prostate cancer cells were obtained from the American Type
Culture Collection (Rockville, MD). For routine maintenance, cells were
grown in RPMI 1640 medium, supplemented with 10% FCS, 2 mM
L-glutamine, and antibiotics (100 IU/ml penicillin, 100
µg/ml streptomycin, and 0.25 µg/ml amphotericin B), at 37 C in a
humidified atmosphere of 5% CO2 in air. Cells were
periodically tested for mycoplasma contamination. For all experiments,
the passage number of cells in culture was kept in a narrow range
(LNCaP, 22–25; DU145, 63–67; PC3, 19–23).
Steroid metabolism
The methodological procedures used to assess patterns of steroid
metabolism in in vitro systems were extensively described
previously (19, 20, 21). Cells (0.5–2 x 106) were plated
onto 60-mm cell culture dishes and left undisturbed for 24–48 h. After
two washes in PBS-A (170 mM NaCl, 3.4 mM KCl,
and 2 mM Na2PO4, pH 7.2), the
medium was substituted with FCS-free, phenol red-free RPMI medium to
avoid interfering factors that might modify the metabolic ability of
the cells. After an additional 24 h, medium was replaced with the
same medium containing 1 nM radioactive T
([1,2,6,7-3H]T; SA, 92.1 Ci/mmol; DuPont de Nemours
Italiana, Milan, Italy) or E2
([6,7-3H]E2; SA, 48 Ci/mmol; DuPont)
used as precursors. Both steroids were periodically checked and
purified using HPLC before experimental use. After 24-h incubation,
medium was transferred to sterile plastic tubes (Costar, Cambridge, MA)
and stored at -80 C until analysis. For time-course experiments,
triplicate dishes (5 x 105 cells/dish) were incubated
in the presence of 1 nM labeled T for 30 min and 2, 8, and
24 h under exactly the same experimental conditions. Medium and
cells were thus processed as described below.
Extraction procedures
Extraction of both conjugate and free steroids was carried out,
as previously described (19, 20), from the incubation medium as it has
been shown to contain proportionally greater amounts of radioactive
metabolites than those present in the cells (22, 23). Briefly, medium
aliquots (1 ml) were transferred to scintillation vials to assess the
total radioactivity. Before any sample manipulation, all glassware was
coated with 4 µg of the same radioinert steroid to minimize
radioactivity losses. Extracts of either conjugate or hydrolyzed
steroids were finally dried and stored at -20 C until chromatographic
analysis.
Chromatographic analysis
The dried extracts were resuspended in 30 µl acetonitrile
(androgens) or in a mixture consisting of 10 µl acetonitrile, 10 µl
acetic acid (0.2 M), and 10 µl equilin (estrogens), with
the latter used as internal standard. Twenty microliters of the
resulting solution were used for HPLC analysis, while 5 µl were
measured in a ß-counter to quantify the radioactivity extracted for
each steroid fraction; the extraction efficiency was calculated as
reported previously (19). Extracted steroids were separated under
isocratic conditions on HPLC in the reverse phase mode and quantified
using an on-line Flo-One/Beta (500TR) three-channel radioactive
detector (Camberra Packard, Meriden, CT). All of the chromatographic
procedures used have been previously established and optimized in our
laboratories (18). Precursor degradation and formation of metabolic
products were expressed either as a percentage of the conversion rates
or as femtomoles per ml. Data were normalized for total radioactivity
and/or corrected for equal cell numbers when appropriate.
Subcellular fractionation
Cell monolayers were washed twice with PBS-A. Cells were then
collected by centrifugation, suspended in 0.04 M potassium
phosphate buffer (pH 7.0) containing glycerol (20% vol/vol) and 10
mM EDTA, and gently homogenized by hand in a glass Dounce
homogenizer (Kontes Co., Vineland, NJ). Cell homogenates were
centrifuged at 1,000 x g for 10 min to remove cell
debris and then at 105,000 x g at 4 C for 40 min. The
resulting supernatant was saved as the cytosol. The pellet, designated
microsome, was suspended in 0.04 M potassium phosphate (pH
7.0) containing 10 mM EDTA but lacking glycerol. Both
fractions were stored at 4 C until analysis.
17ßHSD activity
17ßHSD activity was assayed under conditions that
differentiate among type 1, 2, 3, and 4 enzymes, as described
previously (24). Briefly, 10-µl aliquots of cytosols or microsomes
were combined with 10-µl aliquots of the reaction mixture to give 0.5
mM NAD, 1.0 µM
[3H]E2 or [3H]T, and 0.1
M bicine (pH 9.0) for the dehydrogenase assay and 0.5
mM NADH, 1.0 µM
[3H]E1 or
[3H]4Ad and 0.1 M HEPES (pH
7.2) for the measurement of reductase activity. For the determination
of apparent Km (Kmapp) and apparent
maximum velocity (Vmapp), 0.1 M HEPES, pH 7.2,
was used for both dehydrogenase and reductase assays. Reactions were
run at 37 C and stopped by transferring the reaction mixture to the
preadsorbent layer of a TLC plate (Silica Gel HL, Analtech, Newark,
DE). After the addition of 30 µl of an unlabeled carrier steroid (4.0
mg/ml) in ethanol, the spots were allowed to dry, and the plate was
developed in benzene-acetone (4:1, vol/vol). Substrate and product were
located by a light spraying with water. After drying, the spots were
scraped into 10 ml Ecolumn (ICN Research Products Division, Costa Mesa,
CA) for liquid scintillation counting. Specific activity (nanomoles per
mg protein/30 min) was calculated from the counts per min recovered in
product as a percentage of the total counts per min recovered in
substrate and product, as reported previously (25).
Protein determination
Protein content was measured by the method of Markwell et
al. (26). BSA was used as the standard.
Data analysis
The Kmapp and Vmapp values
were estimated according to the graphic method of Cornish-Bowden and
Eisenthal (27).
RNA blotting
Term placenta tissue was collected after normal delivery, and
testicular tissue was obtained from patients orchidectomized for
prostate cancer. Extraction of mRNA was performed directly from tissues
or PBS-washed cells (3 x 107) using
oligo(deoxythymidine) Dynabeads (Dynal, Hamburg, Germany) according to
the manufacturer’s instructions. Size fractionation of
polyadenylase-enriched RNA was achieved by electrophoresis through a
1% (wt/vol) agarose gel, followed by capillary blotting to a Hybond-N
membrane (Schleicher and Schuell, Dassel, Germany) (9).
Blotting procedures were performed in duplicate, using two different
sets of cells for any cell line. Loading of mRNA (10 µg from cell
lines, 3 µg from testes, and 5 µg from placenta) was checked using
ethidium bromide staining and subsequent UV visualization. The
membranes were prehybridized in a solution containing 100 µg/ml
denatured salmon sperm DNA, 50% formamide, 5 x Denhardt’s
solution, 0.3% SDS, and 6 x SSPE (0.1 x SSPE = 15
mM NaCl, 1 mM sodium phosphate, and 0.1
mM EDTA, pH 7.4; for 12 h at 42 C), and hybridization
(in the same solution except without Denhardt’s solution) was carried
out with -32P-labeled probes for 16 h at 42 C.
Blots were sequentially hybridized with probes for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Clontech, Heidelberg,
Germany), four different types of 17ßHSD, and again for GAPDH. The
membranes were then washed to a final stringency of 0.1 x SSPE
containing 0.3% SDS at 42 C. The blots were exposed to x-ray X-Omat AR
film (Eastman Kodak, Rochester, NY) with an intensifying screen for
12 h after quantification of signals by a Fuji BAS 1000
phosphoimager. Before every hybridization the membranes were stripped
of bound radioactive DNA three times in 0.001 x SSPE-0.3% SDS
(wt/vol) at 80 C. The efficiency of stripping was checked by
phosphoimager. Signals for GAPDH remained unchanged after five
hybridizations.
Hybridization probes
Specific DNA probes for 17ßHSDs of approximately 600 bases
were PCR amplified using cDNA of 17ßHSD1 (provided by Dr. J. Simard),
17ßHSD2 and 17ßHSD3 (provided by Dr. S. Andersson), and 17ßHSD4
(10) as templates. The sequences of the primers used are given in the
Table 1
. Pfu DNA polymerase (Stratagene,
Heidelberg, Germany), an annealing temperature of 60 C, and 35 cycles
were used. To quantify the amounts of mRNA on the blots, a human GAPDH
probe (Clontech) consisting of a 1.1-kilobase (kb) EcoR
I/XhoI fragment of GAPDH cDNA was employed. The probes were
labeled with a random hexanucleotide Prime-It RmT Kit (Stratagene) and
had specific activities greater than 109 cpm/µg DNA.
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Results |
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The results obtained revealed that either androgen (T) or estrogen
(E2) oxidation was remarkably and consistently different in
the three cell lines studied (see Fig. 1). As far as T metabolism is concerned
(Table 2), PC3 cells showed large
precursor degradation (<2% unconverted T after 24 h) to yield
marked amounts (>70%) of 4Ad and its derivatives of
the 17-keto series, 5-androstenedione (5Adione), androsterone,
and epiandrosterone (in total, 
25%). In contrast, both LNCaP and
DU145 cells gave rise to limited T conversion rates into
4Ad, with the latter never exceeding 5% of all
radioactive androgens. Peculiarly, the formation of dihydrotestosterone
(DHT) and its derivatives 3- and 3ß-androstenediols
(3/3ß-diols) varied greatly in the three cell lines. In
time-course experiments, PC3 cells did not show measurable DHT or
3/3ß-diol production at any incubation time, whereas the
prevalence of the oxidative pathway leading to 4Ad
formation was confirmed (see Fig. 2A). In
fact, T was increasingly converted into 4Ad; an
appreciable amount (>8%) of this metabolite was found after only
30-min incubation. A proportional increase in 5Adione was also seen
at 8 and 24 h. Formation of 4Ad plus 5Adione was
inversely and significantly related to the proportion of metabolized T
(r = -0.9706; P < 0.03, by Spearman correlation
test). In contrast, the time course of T metabolism in both LNCaP and
DU145 cells revealed low T degradation rates (<8% and <18%,
respectively). Overall, 4Ad formation did not exceed
5%, whereas relatively greater amounts of both DHT and 3/3ß-diols
were found in DU145 cells (in total, nearly 15%) with respect to LNCaP
cells (only 2%; see Fig. 2, B and C).
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, oxidation of
E2 to E1 was strikingly greater in PC3 cells
(nearly 92% by 24 h) than in LNCaP (14%) or DU145 (7%) cells.
The proportion of undegraded precursor (E2) at 24 h ranged
from 81–94% in LNCaP and DU145 cells, whereas it remained below 4%
in PC3 cells. A small proportion of 16-hydroxy-E1 was
also seen in either LNCaP (2.4%) or PC3 (3.8%) cells, whereas small
amounts of 2-methoxy-E2 were found in LNCaP cells only.
Likewise for T, time-course experiments reinforced 24-h data on
estrogen metabolism; an increasingly greater extent of E2
oxidation into E1 was seen in PC3 cells over time (not
shown).
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The Kmapp and Vmapp values were
comparable, as were the Kmapp/Vmapp
ratios (Table 5). 17ßHSD activity with
E2 and T was detected in the other cell lines as well, but
at a lower level. Dehydrogenase specific activity for both
E2 and T was highest in the microsomal fraction from LNCaP
cells. The cytosolic and microsomal specific activities of DU145 cells
were comparable, being the lowest among the three cell lines.
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As shown in Fig. 3A, remarkable
expression levels of 17ßHSD2 mRNA were seen in PC3 cells, whereas no
signal for 17ßHSD2 was revealed in either LNCaP or DU145 cells; this
could not be due to uneven loading of mRNAs on the blots, as excluded
by either UV visualization of ethidium bromide-stained gels or
quantification at laser densitometry of GAPDH signals. In addition, all
three cell lines expressed comparable and significant amounts of a
2.9-kb mRNA species for 17ßHSD4. In the same experiments, substantial
amounts of 17ßHSD1 and 17ßHSD3 were detected in placenta and
testes, respectively. However, neither 17ßHSD1 nor 17ßHSD3 mRNAs
could be detected in any cell line, not even after exposure of blots
for 1 week (not shown). Duplicate experiments gave rise to the same
results.
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. As can be seen,
LNCaP cells expressed abundant 17ßHSD4 mRNA, whereas lower levels
were found in DU145 and, especially, PC3 cells. In the latter, the
amounts of 17ßHSD2 mRNA were nearly twice as high as those observed
for 17ßHSD4. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have investigated the expression and activity of 17ßHSDs in cultured human prostate cancer cells (PC3, LNCaP, and DU145). These cell lines have been previously characterized in our laboratories for their growth response to both androgen and estrogen as well as for their respective receptor contents. In particular, LNCaP cells contain both androgen and estrogen receptors; their proliferative activity is significantly stimulated by either steroid (32, 33). In contrast, androgen receptor-negative PC3 cells and DU145 cells fail to respond to androgens; this despite DU145 cells retain high affinity sites and apparently intact capacity for androgen binding (32).
In the present work we have seen remarkable expression of 17ßHSD2 in
androgen-nonresponsive PC3 cells, whereas no specific mRNA could be
detected in DU145 or in hormone-responsive LNCaP cells. On the other
hand, no specific transcript for 17ßHSD1 (1.3 or 2.3 kb) or 17ßHSD3
(1.3 kb) was found in any cell line, even after long exposure of
blots, thus confirming previous reports. The authenticity of these
observations is proven by strong parallel hybridization signals in
placenta (17ßHSD1) and testes (17ßHSD3). Although 17ßHSD1 and -2
could be detected in many tissues (6, 7, 31), 17ßHSD3 was observed
only in testis (5). High levels of 17ßHSD4 were seen in all three
cell lines studied; the amounts of mRNA were comparable to those we
previously observed in kidney and much greater than those in placenta
or normal prostate (10). Recent studies on 17ßHSD4 expression in
porcine testis have localized the enzyme in peroxisomes of Leydig cells
(34). As the expression levels of 17ßHSD4 are comparable in the three
cell lines, 17ßHSD2 is a likely candidate for the predominant
oxidative activity seen in PC3 cells. 17ßHSD4 may be of minor
importance in this respect, but it may account for the lesser extent of
E2 oxidation observed in both LNCaP and DU145 cells. As the
latter cell lines did not express detectable 17ßHSD2 mRNA, the
enzymatic activity responsible for T oxidation remains to be
identified.
From a metabolic standpoint, striking differences emerged in the extent
of 17ß-oxidation of both androgen (T) and estrogen (E2)
using intact cell analysis; the extent of reaction was, in fact,
significantly greater in PC3 cells, lower in LNCaP cells, and very low
DU145 cells. This evidence was reproducible and consistent for both T
and E2 metabolism. It is worth mentioning that the large
E2 oxidation seen in PC3 cells combines with the formation
of 16-hydroxy-E1, exactly as we have observed in other
hormone-unresponsive human cancer cell lines, in which production of
this estrogen derivative is much enhanced by FCS or its major component
albumin (35).
The assay conditions used in this study for estimating 17ßHSD activity on cell homogenates differentiate among the four 17ßHSD types on the basis of their differing substrate specificities and subcellular localization (28). 17ßHSD1 is a cytosolic enzyme with an E2/T activity ratio of approximately 100 (25). In contrast, 17ßHSD2 is a microsomal enzyme with an E2/T activity ratio of approximately 1 (5). For PC3 cells, the high level of microsomal activity compared with cytosol, the low E2/T activity ratio, and the approximately equal Kmapp values for E2 and T are consistent with the presence of type 2 17ßHSD. Although less than that of PC3 cells, a significant level of activity with E2 and T was detected in microsomes from LNCaP cells. The low E2/T activity ratio is suggestive of the presence of 17ßHSD2. However, the absence of type 2 17ßHSD mRNA and the detection of mRNA for the type 4 isoform suggest that this low ratio may be misleading. It is noteworthy in this regard that 17ßHSD4 is highly specific for E2, with little or no affinity for T (10). The presence of a high level of 17ßHSD4 mRNA in LNCaP and DU145 cells suggests that the microsomal dehydrogenase activity found with E2 may be due to the type 4 enzyme. A comparable level of microsomal activity with T in the absence of 17ßHSD2 mRNA raises the interesting possibility of the presence in these cancer cells of an as yet unidentified enzyme reactive with T.
To our knowledge, this is the first report on the expression of the four different 17ßHSD types in human prostate cancer cells. Furthermore, for the first time, in vivo measurement of 17ßHSD activity harmonizes with the amounts of relevant 17ßHSD transcripts, as demonstrated by Northern blot analysis. The apparent lack of both type 1 and 3 enzyme transcripts, precluding any reductive activity by 17ßHSD enzymes, deserves a deeper scrutiny, using more sensitive assays for specific mRNAs and proteins.
All of this evidence would imply that distinct 17ßHSDs may be differently regulated in cells with different sensitivities to sex steroids, eventually leading to a differential accumulation of biologically active hormones. Taking into account the multifarious response to estrogen we have observed in these prostate tumor cells (33, 36), this could be relevant to both growth and progression of human prostatic carcinoma. Further studies should ascertain whether a prevalent 17ß-oxidation driven by 17ßHSD2 and/or 17ßHSD4 associates with hormone refractory prostate cancer, in vivo.
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Acknowledgments |
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Footnotes |
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Received March 7, 1997.
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References |
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-hydroxysteroid dehydrogenase activity. J Biol
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 H. Le, J. T. Arnold, K. K. McFann, and M. R. Blackman DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E952 - E960. [Abstract] [Full Text] [PDF]
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M. El-Alfy, V. Luu-The, X.-F. Huang, L. Berger, F. Labrie, and G. Pelletier Localization of Type 5 17{beta}-Hydroxysteroid Dehydrogenase, 3{beta}-Hydroxysteroid Dehydrogenase, and Androgen Receptor in the Human Prostate by in Situ Hybridization and Immunocytochemistry Endocrinology, March 1, 1999; 140(3): 1481 - 1491. [Abstract] [Full Text]
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I. Dufort, P. Rheault, X.-F. Huang, P. Soucy, and V. Luu-The Characteristics of a Highly Labile Human Type 5 17{beta}-Hydroxysteroid Dehydrogenase Endocrinology, February 1, 1999; 140(2): 568 - 574. [Abstract] [Full Text]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, [3H]T; •,
, DHT; 
, 3
, 5
,
androsterone (A); 
, epiandrosterone (EpiA).