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,
From the * Laboratory of Pharmaceutics, Gifu
Pharmaceutical University, Gifu, Japan; and
Department of Nephro-Urologic Surgery and
Andrology, Mie University Graduate School of Medicine, Tsu, Mie, Japan.
| Correspondence to: Dr Kazuyuki Hirano, Laboratory of Pharmaceutics, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan (e-mail: hirano{at}gifu-pu.ac.jp).. |
| Received for publication February 5, 2007; accepted for publication March 30, 2007. |
 |
Abstract |
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Key words: Androgen sensitivity, PSA, limiting dilution
Clinically, low androgen sensitivity in prostate cancer is associated with a more malignant phenotype and is currently difficult to cure. Several mechanisms responsible for changes in androgen sensitivity have been suggested—for instance, 1) androgen-insensitive activation of the androgen receptor (AR) by mutations or altered levels of coactivators and 2) activation of alternative growth factor pathways (Taplin and Ho, 2001; So et al, 2005). Further understanding of the mechanisms underlying the transition to an androgen-insensitive state is essential to develop new efficient strategies in the future. To be able to investigate the differences between androgen-sensitive and -insensitive prostate cancer, a variety of human prostate cancer cell lines with differences in androgen sensitivity have been generated and characterized (Veldscholte et al, 1990; Wu et al, 1994; Zhau et al, 1996; Tepper et al, 2002).
The human prostatic LNCaP cell line is one of the few androgen-sensitive prostatic cell lines and is useful for investigating the molecular mechanisms responsible for the changes in androgen sensitivity. Several sublines with different androgen sensitivity were generated and characterized (Kokontis et al, 1994; Wu et al, 1994; Joly-Pharaboz et al, 1995; Onishi et al, 2001; Hara et al, 2003; Gustavsson et al, 2005; Kawada et al, 2006). For example, Onishi et al (2001) previously established androgen-insensitive LNCaP (AIDL) cells by maintaining LNCaP cells under hormone-depleted conditions over 2 years, which mimics hormone ablation therapy. AIDL cells exhibited much less androgen sensitivity and showed lower zinc and metallothionein levels than the parental LNCaP cells (Iguchi et al, 2004).
LNCaP cells are a heterogeneous cell population containing various clones with naturally occurring differences in androgen sensitivity caused by spontaneously arising changes (Horoszewicz et al, 1983; Wan et al, 2003). In this study, to obtain a low androgen-sensitive clone spontaneously generated in LNCaP cells, we performed a limiting dilution of LNCaP cells and obtained 2 sublines of LNCaP cells that differ in androgen sensitivity.
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Materials and Methods |
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Cell Culture
Human prostatic carcinoma LNCaP cells were obtained from American Type
Culture Collection (Rockville, Md) and cultured in RPMI-1640 medium containing
10% fetal calf serum (FCS) under a humidified atmosphere with 5%
CO2 at 37°C.
Limiting Dilution
Parental LNCaP cells were cloned twice by limiting dilution in 96-well
plates at a density of 0.1 cells per well, and a homogeneous cell population
was obtained. The cells were used between passages 10 and 25.
Cell Growth Assay
The cells (1 x 105 cells) were incubated for 48 hours and
96 hours, and the total cell number was counted with a hemocytometer at the
end of incubation.
Reverse Transcriptase–Polymerase Chain Reaction Analysis and Real-Time Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif),
and then first-strand complementary DNA was synthesized from 5 µg of total
RNA using SuperScript III (Invitrogen) as described previously
(Iguchi et al, 2004).
Polymerase chain reaction (PCR) was performed with specific primers as
previously reported (Iguchi et al,
2004). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served
as an internal RNA control to allow comparison of RNA levels among different
specimens. After PCR, the reaction products were resolved on 1.75% agarose
gels and visualized with ethidium bromide.
Real-time monitoring of reactions was performed using the iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, Calif) with the SYBR Premix Ex Taq (Takara Bio Inc, Otsu, Japan). At the end of the PCR, a dissociation curve analysis was performed to examine the specificity of the product. The GAPDH housekeeping gene was used for normalization of prostate-specific antigen (PSA) mRNA expression. The PCR was performed under the following conditions: 30 cycles of 15 seconds at 95°C, 30 seconds at 55°C, and 30 seconds at 72°C for PSA; 35 cycles of 10 seconds at 95°C and 20 seconds at 60°C for GAPDH. The primers used in this study were 5'- GAGGTCCACACACTGAAGTT-3' and 5'-CCTCCTGAAGAATCGATTCCT-3' for PSA and 5'-CCAGCAAGAGCACAAGAGGA-3' and 5'-GCAACTGTGAGGAGGGGAGA-3' for GAPDH.
Preparation of Cell Lysate and Western Blot Analysis
Both G4 and E9 cells were cultured until 70%–80% confluent in 100-mm
dishes. The cell surface was washed with ice-cold phosphate-buffered saline
(PBS) and then lysed with the buffer containing PBS, 1% Nonidet P-40, 10 µM
4-(2-aminoethyl) benzensulfonyl fluoride (AEBSF), 0.8 µM aprotinin, 50
µM bestatin, 15 µM E-64, 20 µM leupeptin, and 10 µM pepstatin A
for 60 minutes on ice. The lysates were centrifuged at 10 000 x
g for 10 minutes, and the supernatants were collected. The protein in
the fractions was quantified using the Bio-Rad protein assay kit (Bio-Rad
Laboratories, Hercules, Calif), and 40 µg of the cell lysate was subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
with 12.5% polyacrylamide gels (Atto Corp, Tokyo, Japan). The proteins were
transferred to an Immobilon-P membrane (Millipore Corp, Billerica, Mass), and
the membrane was incubated with anti-AR antibody (N-20) (Santa Cruz
Biotechnology, Santa Cruz, Calif), anti-PSA antibody (DakoCytomation,
Glostrup, Denmark), and anti-actin antibody (AC-15) (Sigma-Aldrich, St Louis,
Mo). Detection was accomplished with an ECL detection system (Pierce
Biotechnology, Rockford, Ill).
Measurement of PSA in Conditioned Media
E9 and G4 cells were cultured in RPMI-1640 medium supplemented with 10% FCS
for 48 and 96 hours, and the media were subjected to an enzyme-linked
immunosorbent assay (ELISA; American Qualex, San Clemente, Calif) to determine
PSA expression. The amount of PSA in the conditioned medium was normalized to
cell numbers.
Hormonal Effect on the Cell Growth
Cells were cultured in phenol red–free RPMI-1640 medium supplemented
with 5% charcoal-stripped FCS. After 2 days, cells were seeded in 96-well
plates (Sumilon, Tokyo, Japan) at a density of 4 x 103 cells
per well in culture medium, incubated overnight, and treated with various
concentrations of hormones. After 72 hours, alamar blue solution (Wako) was
added and the fluorescence intensity was measured using a Cytofluor 2350.
Zinc Assay
Zinc concentration was measured as described previously using
2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol disodium salt
dihydrate (5-Br-PAPS; Dojindo Laboratory, Kumamoto, Japan)
(Iguchi et al, 2004). Briefly,
cells were collected and then treated with 10% trichloroacetic acid on ice for
15 minutes and centrifuged at 4°C for 10 minutes. The resulting
supernatant was incubated with 80 mM 5-Br-PAPS and 29 mM salicylaldoxime for
10 minutes at room temperature. The absorbance of the mixture was measured at
570 nm with a microplate reader.
Xenografting
A total of 50 x 104 cells of the LNCaP sublines G4 and E9
were prepared in 50 µL of neutralized type 1 rat tail collagen gel as
previously described (Hallowes et al,
1980). The cells were grafted at the subrenal capsule and
subcutaneous sites of 8-week-old male severe combined immunodeficient (SCID)
mice (CLEA Japan, Osaka, Japan) (Wang et
al, 2005). Mice were killed and the grafts harvested at 4 weeks
after grafting. The grafts were fixed in a 10% formalin neutral buffer
solution (Wako) for hematoxylin and eosin (H&E) and regular
immunohistochemical staining. For mouse-specific CD31 staining, zinc fixation
was performed at room temperature overnight (BD Bioscience, Franklin Lakes,
NJ). Finally, all grafts were embedded in paraffin.
To compare the growth of G4 and E9 tumors, tumor volumes were estimated by the following formula: "v = 0.5236 x a x a x b" (a = short axis, b = long axis). Values represent the means ± SD.
Immunohistochemistry
Sections (3 µm) were cut from representative paraffin-embedded samples.
For immunohistochemistry, sections were deparaffinized in Histo-Clear
(National Diagnostic, Atlanta, Ga) and rehydrated in a graded series of
ethanol concentrations. Endogenous peroxidase was blocked by 0.3% hydrogen
peroxide in methanol for 20 minutes. After extensive washing in tap water,
antigen retrieval was performed using 10 mM sodium citrate buffer, pH 6.0, for
AR and antigen unmasking solution (Vector Laboratories, Burlingame, Calif) for
PSA and E-cadherin immunostaining. After rinsing in PBS, the sections were
incubated with appropriate normal serum for at least 3 hours at room
temperature to block nonspecific binding. The sections were then incubated
with anti-AR antibody (N-20) (Santa Cruz), anti-PSA antibody (DakoCytomation),
anti-E-cadherin antibody (BD Biosciences), and anti-CD31 antibody (BD
Biosciences) at 4°C overnight. After incubation with primary antibody,
sections were incubated with appropriate biotinylated secondary anti-mouse,
anti-rabbit, or anti-rat immunoglobulin diluted with PBS for 30 minutes at
room temperature. The antigen-antibody reaction was visualized with the
Vectastain avidin-biotin complex (ABC) kit (Vector) using
3,3'-diaminobenzidine tetrahydrochloride as a substrate. The sections
were counterstained with H & E and examined under a light microscope.
To evaluate angiogenesis in the grafts, mouse-specific CD31+ vessels with lumens were counted in 10 different areas at 200x magnification. Values represent the means ± SD.
Statistical Analysis
The significance of differences between 2 groups was calculated with
Student's t test, and the significance of differences between
multiple groups was assessed by 1-way analysis of variance followed by the
Dunnet test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Hormonal Regulation of Cell Growth and PSA Expression
The effect of various steroid hormones on the cell growth was examined by
alamar blue assay. The dose-response growth curves of E9 and G4 cells treated
with steroid hormones are shown in Figure
2. Androgen response of E9 cell growth was inhibited in a
dose-dependent manner, whereas that of G4 cell growth was a biphasic pattern
with a maximum at 0.1 nM dihydrotestosterone, 1 nM testosterone, and 1 nM
R1881. Estradiol and progesterone stimulated E9 and G4 cells in a
dose-response manner, but the sensitivity to the estrogens of E9 cells was
clearly lower than that of G4 cells.
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Tumorigenic Characteristics of Low-Androgen–Sensitive E9 Cells in Male SCID Mice
To compare the tumorigenicity of low-androgen–sensitive E9 cells
against androgen-sensitive G4 cells in vivo, the cells were grafted at the
subrenal capsule and subcutaneous sites. There was no significant difference
in tumor size between G4 and E9 tumors even at 4 weeks after grafting
(Figure 4). In terms of
recovery rates, subrenal capsule grafting (100% for both G4 and E9) was more
efficient than subcutaneous grafting (70% for G4 and 92% for E9)
(Figure 4). Particularly at
subcutaneous site, the recovery rate of E9 tumors was higher than that of G4
tumors. Both G4 and E9 cells gave rise to the formation of well-defined
globular tumors containing large blood-filled areas at both grafting sites
(Figure 5). AR was expressed in
both G4 and E9 tumors, whereas PSA immunostaining was strongly detected in
only G4 tumors (Figure 5). No
significant difference in E-cadherin expression between G4 and E9 tumors was
observed (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We found that E9 cells were less sensitive to androgen-related responses than G4 cells. The reason for the difference in androgen sensitivity between E9 and G4 cells is unclear. LNCaP C4-2 cells established by inoculation of LNCaP cells into castrated mice have been shown to exhibit androgen independency (Wu et al, 1994; Yeung et al, 2000). In these cells, androgen-insensitive recruitment of the Tip60 coactivator has been revealed and suspected to be one of the causes of their high basal PSA expression (Halkidou et al, 2003). LNCaP 104R cells generated by long-term androgen ablation in vitro have been shown to have androgen hypersensitivity because of increased AR expression (Kokontis et al, 1994). Because LNCaP-cxD2 cells from long-term casodex-treated LNCaP cells have a mutated AR, casodex interacts with the AR as an agonist (Hara et al, 2003). The expression level of AR in E9 cells was almost the same as that in G4 cells, suggesting that the difference in androgen sensitivity cannot be explained by the amount of AR expression. There might be a need to examine the AR cDNA sequence and the expression level of AR cofactors in E9 and G4 cells.
Less androgen-sensitive derivatives of LNCaP cells have been described (ie, C4-2, LNCaP-r, LNCaP-19, and AIDL) (Hasenson et al, 1985; Wu et al, 1994; Onishi et al, 2001; Gustavsson et al, 2005). C4-2 cells were generated in vivo by culturing LNCaP cells in castrated mice (Wu et al, 1994), and LNCaP-19 and AIDL cells were selected in vitro by culturing the cells in androgen-deprived medium (Onishi et al, 2001; Gustavsson et al, 2005). Thus, these cells were developed by chronic maintenance in a steroid-depleted environment, suggesting that the changes in the androgen sensitivity seen in these cells arose by artificial means, that is, by negative effects of hormone ablation therapy. Meanwhile, LNCaP-r cells were generated in normal medium (androgen present), suggesting that the changes in androgen sensitivity arose via spontaneous generation or preexisting variants in the parental population (Hasenson et al, 1985). In this study, we isolated E9 cells by limiting dilution under androgen-containing normal culture conditions. The growth responses to steroid hormones of E9 cells were similar to those of LNCaP-r cells. These results suggest that E9 and LNCaP-r cells are the same type of LNCaP derivative although E9 cells are clonal whereas LNCaP-r cells are not.
Zinc is retained at a high concentration in the prostate gland, and zinc in prostate cancer decreases to the level detected in nonprostate tissues (Mawson and Fischer, 1952; Gyorkey et al, 1967). Furthermore, the zinc level in androgen-insensitive prostate cancer is much lower than that in androgen-sensitive prostate cancer (Shiina et al, 1996). Zinc in prostate cancer cells has been found to regulate cell growth and metastasis, possibly through the inhibition of enzymatic activities of various proteases and the induction of cell death (Costello et al, 1997; Iguchi et al, 1998; Ishii et al, 2001a,b). In this study, zinc and zinc transporter expression levels were examined, and it was found that the expression of ZnT3, abundant in brain and responsible for zinc transport into synaptic vesicles (Palmiter et al, 1996), was significantly higher in E9 cells than in G4 cells. The ZnT3 mRNA expression in both cells was decreased by the treatment with R1881. In addition, we have previously reported that less androgen-sensitive AIDL cells have significantly higher ZnT3 expression levels than parental LNCaP cells (Iguchi et al, 2004). The decrease in ZnT3 expression by R1881 treatment was seen in LNCaP cells but not in AIDL cells (Iguchi et al, 2004). From these observations, the regulation of ZnT3 expression would be under androgenic control in prostate cells, but it is currently unknown whether there is any physiologic or pathologic significance of ZnT3 in prostate cancer cells. The transfection of ZnT3 gene into those cells may demonstrate the relationship between ZnT3 levels and androgen response. It is interesting to examine how this affects androgen sensitivity. An interesting result, the regulation of ZnT3 expression by estrogen, has been reported (Lee et al, 2004).
An increase in angiogenesis was observed in low-androgen–sensitive E9
cells compared with high-androgen–sensitive G4 cells. The expression of
vascular endothelial growth factor ([VEGF] one of the most important
angiogenetic factors) in E9 cells in vitro was rather low compared with that
in G4 cells (E9, 221 ± 71 pg/106 cells per day; G4, 487
± 86 pg/106 cells per day; data not shown). In addition, no
secretion of significant amounts of basic fibroblast growth factor (bFGF) and
transforming growth factor 
(TGF) was detected by ELISA assay in
both cells (data not shown). A phenotype of increased angiogenesis has been
also observed in low-androgen–sensitive LNCaP-19 cells, which secrete
lower amounts of VEGF compared with the parental LNCaP cells
(Gustavsson et al, 2005).
Recently, angiogenin, an angiogenic factor, has been reported to be involved
in the angiogenesis of LNCaP subline
(Kawada et al, 2007). We
therefore examined the expression of angiogenin in E9 and G4 cells, but no
significant difference in the mRNA expression level was found by RT-PCR
analysis (data not shown). The VEGF and angiogenin expression levels in vivo
and the expression of its receptor (VEGFR, responsible for VEGF-mediated
signal transduction) in these cells were not determined in our study, and thus
further study might be necessary.
The growth of E9 cells was shown to be more rapid in vitro than that of G4 cells. Western blot analysis revealed that the phosphorylation level of Akt/protein kinase B (PKB) was markedly higher in E9 cells than G4 cells (data not shown). Akt signaling is well known to be involved in cell proliferation and antiapoptosis. Moreover, Akt is found to be an activator of AR signaling, leading to androgen-independent prostate cancer growth (Wen et al, 2000). Androgen-independent LNAI cells, established from xenograft tumors of LNCaP cells, have shown to exhibit an increased Akt phosphorylation (Graff et al, 2000). The reason for the different growth rate between E9 and G4 cells is obscure, but the difference of the level of Akt/PKB phospholylation might provide, at least in part, an explanation.
In conclusion, we have established 2 sublines of LNCaP cells, low-androgen–sensitive LNCaP-E9 cells and high-androgen–sensitive LNCaP-G4 cells. These cells will be useful for the investigation of human prostate cancer cell biology.
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Footnotes |
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These authors contributed equally to this article. 
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) cells (4 x 103 cells per well) were cultured
in phenol red–free RPMI-1640 medium supplemented with 5%
charcoal-stripped fetal calf serum containing the indicated concentration of
steroid hormone for 72 hours. After the incubation, cell growth was examined
by alamar blue assay. The growth for each cell line at 0 nM was taken as 100%.
Values represent the means ± SD from 10 incubations.
*P < .05, **P < .01,
corresponding untreated cells; 
