These authors contributed equally to this article.
Isolation and Characterization of LNCaP Sublines Differing in Hormone Sensitivity
Article first published online: 2 JAN 2013
2007 American Society of Andrology
Journal of Andrology
Volume 28, Issue 5, pages 670–678, September-October 2007
How to Cite
Iguchi, K., Ishii, K., Nakano, T., Otsuka, T., Usui, S., Sugimura, Y. and Hirano, K. (2007), Isolation and Characterization of LNCaP Sublines Differing in Hormone Sensitivity. Journal of Andrology, 28: 670–678. doi: 10.2164/jandrol.107.002675
- Issue published online: 2 JAN 2013
- Article first published online: 2 JAN 2013
- Received for publication February 5, 2007; accepted for publication March 30, 2007
- Androgen sensitivity;
- limiting dilution
ABSTRACT: Prostate cancer is a heterogeneous disease with varying degrees of androgen sensitivity. In this study, we performed a limiting dilution of human prostate LNCaP cells, and isolated two sublines, LNCaP-E9 and LNCaP-G4, with differential hormone-sensitivity. Two LNCaP sublines were obtained by the limiting dilution method. The growth of E9 cells was decreased in the presence of androgens, while that of androgen-treated G4 cells was biphasic. Although the androgen receptor expression level in E9 cells was similar to that seen in G4 cells, the expression of PSA mRNA and protein was significantly lower in the E9 cells. Moreover, the androgen-based stimulation of PSA mRNA expression was less sensitive in E9 cells than G4 cells. Intracellular zinc level did not differ between E9 and G4 cells, but ZnT3 mRNA expression was significantly higher in the E9 cells. When the cells were grafted at the subrenal capsule, the number of CD31-positive vessels with a lumen was approximately 2.5 times higher than that in G4 tumors. LNCaP-E9 cells show lower androgen sensitivity than LNCaP-G4 cells. E9 and G4 cells would be helpful for understanding the biology of hormone-refractory prostate cancer.
Prostate cancer is a heterogeneous disease with various biologic behaviors, such as androgen sensitivity and response to therapy. Most prostate cancer cells, especially early prostate cancer cells, are sensitive to androgens, but during the disease's progression, a variety of cells differing in androgen sensitivity arise in the tumor cell population. The changes in androgen sensitivity are often caused 1) artificially as negative effects of hormone ablation therapy (ie, orchiectomy, luteinizing hormone—releasing hormone analog, and androgen blockade) and 2) by spontaneously arising variants (even before hormone ablation therapy is started) (Taplin and Ho, 2001; So et al, 2005).
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.
Materials and Methods
Dihydrotestosterone was purchased from Fluka (Buchs, Switzerland). Testosterone, β-estradiol, and progesterone were from Wako Pure Chemical Industries (Osaka, Japan). Synthetic androgen R1881 was purchased from NEN Life Science (Boston, Mass). All other chemicals were of analytical grade.
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.
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 × 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 × 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 × 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 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.
A total of 50 × 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 × a × a × b” (a = short axis, b = long axis). Values represent the means ± SD.
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 200× magnification. Values represent the means ± SD.
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.
Characteristics of E9 and G4 Cells Derived From LNCaP Cells
We subcloned prostate LNCaP cells by limiting dilution and obtained 2 sublines, low- androgen—sensitive LNCaP-E9 cells and high- androgen—sensitive LNCaP-G4 cells. The cell lines were both similar in morphology to the parental LNCaP cells, but G4 cells tended to be rounded and formed a botryoidal structure (Figure 1A). The growth of E9 cells was significantly greater than that of G4 cells (Figure 1D). PSA expression was markedly lower in E9 cells than in G4 cells and parental LNCaP cells although the AR expression level was almost similar among 3 sublines (Figure 1B and C). The secretion of PSA was significantly lower in E9 cells than in G4 cells (Figure 1E).
We then examined PSA mRNA expression in R1881-treated E9 and G4 cells using real-time reverse transcriptase (RT)–PCR. As shown in Figure 1F, the PSA mRNA expression was increased by treatment with a synthetic androgen R1881 in both E9 and G4 cells. The level of induction of PSA mRNA in G4 cells was significantly lower than that in E9 cells.
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.
Zinc Level and Zinc Transporter Expression in E9 Cells and G4 Cells
We previously have reported that androgen-insensitive AIDL cells exhibited lower zinc levels and higher mRNA levels of ZnT1 and ZnT3 than androgen-sensitive LNCaP cells (Iguchi et al, 2004). We then examined the zinc level and the mRNA expression of zinc transporters in E9 and G4 cells. As shown in Figure 3, there was no significant difference in zinc levels between them, but ZnT3 mRNA expression was about twofold higher in the low- androgen—sensitive E9 cells than in the androgen-sensitive G4 cells. Moreover, the expression of ZnT3 mRNA in E9 and G4 cells was decreased in a dose-dependent manner after R1881 treatment (Figure 3D). The expression of ZnT1, ZnT4, ZnT5, ZIP1, ZIP3, and Nramp2 showed no significant difference between E9 cells and G4 cells.
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).
To evaluate the effect of androgen-sensitivity on angiogenesis in vivo, mouse-specific CD31 staining was performed in zinc-fixed sections. The tumor vessel structure in E9 tumors was seen as more uniform appearance (Figure 6A). The number of vessels with a lumen in E9 tumors was approximately 2.5 times higher than that in G4 tumors (Figure 6B).
Human prostate LNCaP cells display phenotypic heterogeneity in culture (Horoszewicz et al, 1983; Wan et al, 2003). In the present study, we isolated 2 sublines of LNCaP cells, LNCaP-E9 cells and LNCaP-G4 cells, by limiting dilution and determined their characteristic features. E9 cells were less sensitive to androgen-mediated growth stimulation and PSA production than G4 cells. In addition, E9 cells were more malignant than G4 cells based on the data from the in vivo xenograft model.
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 α (TGFα) 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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