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Departments of Urology (Q.Z., J.N.R., T.L.J., I.P., C.L.), Pathology (M.P., X.Y.), and Preventive Medicine (B.J.), Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611; and Laboratory of Cell Regulation and Carcinogenesis (S.-J.K.), National Cancer Institute, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Chung Lee, Ph.D., Northwestern University Medical School, 303 East Chicago Avenue, Tarry 16-733, Chicago, Illinois 60611. E-mail: c-lee7{at}northwestern.edu or Qiang Zhang, M.D., Ph.D., Department of Urology, Northwestern University, 303E, Chicago Avenue, Tarry 16-726, Chicago, Illinois 60611. E-mail: q-zhang2{at}northwestern.edu.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In the present study, we used a human prostate cancer cell line, LNCaP, to explore the potential role that methylation of CpG islands plays in altering sensitivity to TGF-ß in prostate cancer cells. LNCaP was established from a metastatic lesion of human prostate cancer to lymph nodes (10) and is insensitive to TGF-ß (11). Interestingly, different studies report seemingly conflicting information regarding the status of TGF-ß sensitivity in LNCaP cells. In our study, LNCaP cells showed a lack of expression of TßRI (12, 13); others reported a silenced TßRII (14). These variations in sensitivity to TGF-ß in LNCaP cells suggest that it is not likely to be caused by a genetic event. Therefore, we explored the methylation status in promoters of TßRI and TßRII in LNCaP cells. A normal prostate epithelial cell line, RWPE-1 (15), was used as a control. Finally, clinical specimens were used to verify these findings.
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RESULTS |
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shows changes in real-time PCR products for TßRI and TßRII in LNCaP and RWPE-1 cells with and without the treatment of 5-Aza. Treatment with 5-Aza increased the level of mRNA for both TßRI (Fig. 1A) and TßRII (Fig. 1B) approximately 20-fold in LNCaP cells; however, no significant change was observed in RWPE-1 cells after treatment with 5-Aza. This finding was confirmed by the result of Western blot analysis (Fig. 2, A and B). These changes in the expression of TßRI and TßRII after treatment with 5-Aza are consistent with the notion that promoter methylation repressed the expression of TßRI and TßRII in LNCaP cells.
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). An increased level of phosphorylated Smad-2 is associated with an increase in TGF-ß signaling (1), which could be accomplished by either an increase in TGF-ß ligand or an increase in its cognate receptors. Because treatment with 5-Aza did not result in an increase in TGF-ß1, it is likely that the increased level of phosphorylated Smad-2 resulted from increased TßRs.
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). Among these bands, the specific methylated band in TßRI and TßRII includes six and three potential methylation sites, respectively. Upon sequencing, the methylated sites were identified in cytosine positions –348, –356, –365, –231, –244, and –251 for the promoter of TßRI (Fig. 5A) and –140, +32 and +27 for the promoter of TßRII in LNCaP cells (Fig. 5B). The positive primer sets are listed in Table 1. As expected, after treatment with 5-Aza, methylation sites were not detected (Fig. 4B). No methylated site was identified in RWPE-1 cells for the promoter of either TßRI or TßRII. Finally, results of chromatin immunoprecipitation analysis (Fig. 6) demonstrated that DNA binding activity of AP-2 in the TßRI promoter and of Sp1 in the TßRII promoter in LNCaP cells was only evident after treatment with 5-Aza; in RWPE-1 cells, such binding was detected both before and after 5-Aza treatment.
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, in LNCaP cells, 5-Aza treatment resulted in a significant inhibition of cell proliferation, regardless of whether exogenous TGF-ß1 was added into the culture or not. There is no significant difference between treatment with both 5-Aza and TGF-ß1 or with 5-Aza alone (P > 0.05), suggesting that endogenously produced TGF-ß in LNCaP cells (16) was sufficient to maximally inhibit cell proliferation. In RWPE-1 cells (Fig. 7B), exogenous TGF-ß1 was able to inhibit cell proliferation and treatment with 5-Aza did not significantly affect the degree of cell proliferation. This observation also suggested that treating these cells with 5-Aza alone did not result in an inhibition of cell proliferation in the present study.
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shows the proportion of prostate cancer specimens with loss of expression of either TßRI or TßRII among 158 specimens. Specimens with high Gleason’s scores showed a significantly greater incidence of loss of expression of TßRs. This observation confirmed our earlier observation (5). As reported for the LNCaP cells, we tested methylation status using the same primers, which showed positive methylation for LNCaP cells for these clinical specimens. Therefore, methylation-specific PCR was performed for six CpG sites in the TßRI promoter and three CpG sites in the TßRII promoter as described earlier. For the TßRI promoter, methylation was detected in one of three (33%) benign tissues, four of 12 (33%) prostate cancer specimens with low Gleason’s score, and four of seven (57%) cancer specimens with high Gleason’s score (Fig. 8B). For the TßRII promoter, methylation was detected in one of five (20%) benign tissues, four of 10 (40%) cancer specimens with low Gleason’s score, and four of seven (57%) of cancer specimens with high Gleason’s score (Fig. 8B). The methylation status in other sites of these promoters was not investigated.
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DISCUSSION |
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Results of sequence analysis after methylation-specific PCR have provided insights into the modulation of gene expression of TßRs in LNCaP cells. Both TßRI and TßRII do not contain the TATA box or CAAT box in the 5'-flanking region (17, 18). Therefore, putative binding sites for Sp1, AP-1, and AP-2 in the proximal-promoter regions are important in regulating gene expression. Methylation in these sites will repress the transcriptional potential of the target genes.
For the TßRI promoter, our study detected six methylation sites, which cluster between –231 and –365 from the transcription start site. One of the six methylation sites (–244) is located in the AP-2 binding site, which is known to modulate transcription (19). In support of this AP-2 site, results of our chromatin immunoprecipitation analysis detected binding of AP-2 only after LNCaP cells were treated with 5-Aza. There are four putative Sp1 binding sites on the TßRI promoter; however, in the present study, no methylation site was found on any of these sites. Because there was a minimal expression of TßRI in LNCaP cells and an increased TßRI expression after 5'-Aza treatment, these results suggested that these six methylation sites did not result in a complete silencing of TßRI expression in the present study. A recent report indicated that the use of 5'-Aza is an acceptable approach to study the status of promoter methylation (20).
For the TßRII, we detected three methylation sites (–140, +32, +27). There are two putative Sp1 binding sites, one of which locates in the strongest positive regulatory element (–274 to –137) and contains the (–140) methylation site. Again, results of our chromatin immunoprecipitation analysis demonstrated that SP1 binding to TßRII promoter was only detected after treatment of LNCaP cells with 5-Aza. The remaining two methylated sites (+32 and +27) are located in the other positive regulatory element (+2 to +50) (17). Of interest is that there are two negative regulatory elements (–1240 to –504 and –137 to –47) within the TßRII promoter. We did not detect any methylation sites in these two regions in the present study. Again, these three methylated sites are not sufficient to silence TßRII expression because there was 13.6% inhibition in LNCaP proliferation when treated with TGF-ß1, suggesting partial functioning of both TßRI and TßRII under conditions of the present study.
Based on the above discussion, it is likely that the observed down-regulation of TßRs in LNCaP cells is at least in part due to the process of promoter methylation. LNCaP cells are known to be insensitive to TGF-ß (11). We found six of 82 potential methylation sites in the promoter of TßRI and three of 33 potential sites in TßRII. LNCaP cells are known to display a biphasic growth response to androgen stimulation (21). At low doses (10–12 M), androgen has a stimulatory effect on LNCaP proliferation. However, at high doses (10–10 M), androgen induces growth inhibition. The latter event was mediated through an autocrine production of TGF-ß1, which inhibited LNCaP growth (13, 19). These data demonstrate that LNCaP cells are able to respond to TGF-ß1 under high concentrations of environmental androgen, suggesting restoration of functional receptors.
In our earlier studies, we reported a lack of TßRI expression in LNCaP cells (9); Guo and Kyprianous (13), on the other hand, reported a lack of TßRII expression. Currently, we observed a partial sensitivity to TGF-ß1 in LNCaP cells, which were recently obtained from the American Type Culture Collection (ATCC; Manassas, VA). It is now possible to reconcile these seemingly contradictory differences on the basis of promoter methylation under different experimental conditions. It is likely that under conditions of our earlier studies methylation occurred in more critical sites for TßRI than for TßRII, resulting in the loss of expression of TßRI. Likewise, in Guo’s study (13), their experimental conditions might favor methylation in TßRII, leading to a loss of TßRII. Cells lacking either one or both receptors are insensitive to TGF-ß and would become more aggressive than those that remained sensitive to TGF-ß. As cancer cells progress, it is likely that the selection process would favor the insensitive phenotype, which would eventually predominate.
The LNCaP cells used in the present study were freshly purchased from ATCC and experiments were conducted at the third passage. Although these cells still retain partial sensitivity to TGF-ß, expression of TßRs was significantly attenuated. Our earlier studies (12, 14) and those conducted by Guo and Kyprianou (13) were at 10–20 passages. It is known that LNCaP cells are able to shift to an aggressive phenotype with increasing passages (22). Furthermore, cells with down-regulated TßRs are more aggressive and are favorably selected. Therefore, it seems reasonable that LNCaP cells, at early passages, may retain some sensitivity to TGF-ß, whereas at late passages they become TGF-ß insensitive.
The present data support the notion that methylation of the promoter and silencing of the receptors contribute to the loss of TGF-ß sensitivity in prostate cancer. In previous studies, we reported a correlation between loss of TßRs expression and high tumor grade in human prostate cancer tissues. This loss of expression also contributed to the poor prognosis and worsening survival time in prostate cancer patients (5, 6). Using limited clinical specimens in the existing tissue microarray program within the prostate SPORE (specialized program of research excellence), we were able to demonstrate that among clinical specimens, some of the cases of the loss of expression of TßRs are due to methylation of the promoter in a pattern similar to that of LNCaP cells. We only tested six CpG sites in the TßRI promoter and three CpG sites in the TßRII promoter for these clinical specimens. It is likely the other CpG islands may also contain methylation in these clinical specimens showing a loss of TßRs expression. It is also possible that the lack of expression of TßRs can be the result of deacetylation and/or genetic mutation (23, 24, 25). Investigation of these mechanisms is beyond the scope of the present study.
In conclusion, we have demonstrated that a TGF-ß-insensitive human aggressive prostate cancer cell line, LNCaP, became sensitive to TGF-ß after the treatment of a demethylation agent, 5-Aza. Probing of TßRI and TßRII genes revealed the presence of methylation in their promoter regions. On the other hand, a benign prostatic epithelial line, RWPE-1, showed no significant change in sensitivity to TGF-ß and a lack of methylation in the CpG islands in the promoters of TßRI and TßRII. Furthermore, such a pattern of promoter methylation was also observed in clinical specimens of prostate cancer. These results have provided evidence to support the concept that the insensitivity to TGF-ß in human prostate cancer cells is, at least in part, due to methylation-mediated silencing in the expression of TßRs in human prostate cancer.
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MATERIALS AND METHODS |
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Nucleic Acid Isolation
RNA and DNA were collected and extracted from both 5-Aza-treated and untreated cells. Total cellular RNA was purified by using Trizol (Invitrogen, Carlsbad, CA) by a standard protocol. Total DNA was isolated from the cells using DNAzol (Invitrogen) according to manufacturer’s recommendation.
Real-Time PCR
The relative levels of TßRI and TßRII expression were determined by real-time PCR. Total RNA was reverse-transcribed using 2 µg of RNA, random hexamers, deoxynucleotide triphosphates (Roche Molecular Biochemicals, Mannheim, Germany), and Superscript II reverse transcriptase (Invitrogen). The mixture was placed at room temperature for 10 min, 42 C for 45 min, and 90 C for 3 min and then rapidly cooled to 0 C. The cDNA thus generated was used for the real-time PCR as described below.
The real-time PCR was conducted in the presence of the DNA intercalating dye SYBR Green (Bio-Rad, Hercules, CA) and performed in Peltier Thermal Cycler (PTC-200) with CHROMO Continuous Fluorescence Detector 4 system (MJ Research Inc., Waltham, MA). An aliquot of 2 µl (100 ng) of cDNA was added to individual capillary tubes with deoxynucleotide triphosphate, Mg2+, SYBR Green, and relevant primers. Thirty-six cycles of PCR were programmed to ensure that the log-linear phase was reached. A PCR amplification profile was derived by recording the SYBR Green fluorescence intensity, which was in linear relation to the amount of formed PCR product. Standard curves were generated by Opticon Monitor 2 Software by plotting the PCR cycle number at which a reaction entered exponential amplification vs. the amount of input cDNA of each granzyme gene. The standard curves were then used to determine sample template concentration. Product of TßRI and TßRII were normalized by product of ß-actin. The ratio was used as the relative expression of TßRI and TßRII. Real-time PCR was amplified using the following conditions: one cycle of 95 C for 2 min, followed by 36 cycles of 95 C for 1 min, 60 C for 1 min, and 72 C for 1 min 30 sec, and one cycle of 72 C for 10 min. The PCR primers used were as follows: TßRI (GenBank accession nos. NM004612 and GI 4759225): sense, 5'-ATATCTGCCACAACCGCACTGTCA-3'; antisense, 5'-CAATGCTGTAAGCCTAGCTGCTCCA-3'. TßRII (GenBank accession nos. NM003242 and GI 23308726): sense, 5'-AGCAGAAGCTGAGTTCAACCTGGG-3'; antisense, 5'-CGAGATGTCATTTCCCAGAGCACC-3'. ß-Actin: (GenBank accession no. AF057040): sense, 5'-GTGGGGCGCCCCAGGCACCA-3'; antisense, 5'-CTTCCTTAATGTCACGCACGATTTC-3'. The PCR products were confirmed by sequencing.
Western Blot
Conditioned media (15 ml) was concentrated using the YM-3 Centriprep Centrifugal Filter Devices (CCFD, Millipore Corp., St. Louis, MO). The concentrated fraction was saved for Western blot probing for TGF-ß1. Cell lysates were prepared with the ice-cold modified RIPA buffer (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF). Protein concentration was determined by Spectra Max 190 (Molecular Devices, Sunnyvale, CA). Approximately 30 µg of total protein extract was loaded onto 10% acrylamide gel in Tris-HCl (Bio-Rad). Electrophoresis was carried out in Tris-glycine-SDS running buffer, and transferred to a polyvinylidene difluoride membrane in Tris-glycine buffer overnight at 4 C. Membranes were incubated with the following primary antibodies: TGF-ß1 (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), TßRI (1:100 dilution; Santa Cruz Biotechnology), TßRII (1:50 dilution, Upstate, Lake Placid, NY), Phospho-Smad2 (1:50 dilution; Upstate), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:300 dilution; Advanced ImmunoChemical, Long Beach, CA). Membranes were treated with 5% nonfat dry milk and were incubated with secondary antibodies as follows: antirabbit-IgG-horseradish peroxidase (HRP) (1:2000 dilution for TGF-ß1; Santa Cruz Biotechnology), antirabbit-IgG-HRP (1:1000 dilution for TßRI; Santa Cruz Biotechnology), and antirabbit-IgG-HRP (1:2000 dilution for TßRII; Upstate), and antimouse-IgG-HRP (1:2000 for GAPDH; Upstate). Incubation was carried out for 1 h at room temperature. Proteins of interest were detected with the enhanced chemiluminescence detection kit (Amersham Bioscience, Buckinghamshire, UK) and by exposure to Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY).
Bisulfite Treatment of Genomic DNA
Genomic DNA from LNCaP cells and RWPE-1 cells was modified with 3.0 mM sodium bisulfite (pH 5.0) (Sigma) and 0.5 mM hydroquinone (Sigma) for 16 h at 50 C (27). With this treatment, all cytosine residues would be converted to uracil in unmethylated DNA, but those that had been methylated (5-methylcytosine) would be resistant to this treatment and remained as cytosine. The reaction mixture was then purified with the Promega Wizard Clean-UP Kit (Madison, WI) and desulphanated with 0.3 M sodium hydroxide for 20 min at 40 C. DNA was then precipitated in cold ethanol, dissolved in H2O, and stored at –20 C.
Methylation-Specific PCR and Sequencing
Methylation-specific PCR primers were designed according to the sequence of the TßRI (GenBank accession nos. U51139 and GI 1255263) and TßRII promoters (GenBank accession nos. U37070 and GI 1110498). A total of 86 sets of primers (see the supplemental tables published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org), which cover all 82 potential methylation sites of CpG islands, were designed to distinguish methylated and unmethylated DNA of the TßRI promoter. Also, 35 sets of primers (see the supplemental tables) were designed for all 33 potential methylated sites of the TßRII promoter. Three different primers were prepared for each potential methylation site: methylated specific primer, unmethylated specific primer, and wild-type control primer. Methylation-specific PCR was carried out by using one set of three PCR primers at the same time, which could consistently detect 0.1% of methylated DNA (50 pg) in an otherwise unmethylated mixture. Bisulfite-converted DNA was PCR amplified using the appropriate primers. Each PCR mixture contained 100 µM deoxynucleotide triphosphates, 1 µM sense and antisense primers in 1x Taq buffer, and 1.25 U Taq DNA polymerase (Promega) in complex with 1.25 U Taq antibody (CLONTECH, Palo Alto, CA), MgCl2 6.7 mM. Each PCR run was conducted as follows: 95 C for 3 min, followed by 38 cycles of denaturation at 95 C for 1 min, annealing at the specified temperature listed in Table 1
for 45 sec, and finally, a 30-sec extension at 72 C. A final 10-min extension at 72 C completed each PCR program. PCR products were fractionated on 1% agarose gels, excised, and purified with the QIAGEN DNA extraction kit (QIAGEN, Valencia, CA) according to manufacturer’s recommendations. The purified PCR products were subjected to nucleotide sequence.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation analysis (28) was used to determine the DNA binding activity of Sp1 in TßRII promoter and AP-2 in TßRI promoter for the binding activity in LNCaP and RWPE-1 before and after treatment of 5-Aza. The DNA was isolated from the LNCaP and RWPE-1 cells by using DNAzol (Invitrogen) and the concentration determined. Anti-Sp1, Anti-AP-2 (Upstate) or a nonspecific mouse IgG (Santa Cruz) antibody was added at 4 C overnight with rotation. Immunoprecipitated complexes were collected by protein A/G plus agarose. Precipitants were sequentially washed with low-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], high-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl], and LiCl wash buffer [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)] once, followed by two washes with 1x TE. An aliquot of 250 µl elution buffer (1% SDS, 0.1 M NaHCO3) was added and was incubated at room temperature for 15 min with rotation, followed by 5 M NaCl to reverse the formaldehyde cross-linking by heating at 65 C for 4 h. After precipitation with ethanol, the pellets were resuspended and treated with proteinase K. DNA was recovered by phenol-chloroform extraction and ethanol precipitation. Pellets were resuspended in TE buffer and subjected to PCR amplification using forward and reverse primers:
Sp1 (unmethylated): 5'-ATGATTGGCAGCTACGAGAGAGCTA-3'
5'-ACTTCAACTCAGCGCTGCGGGGGAA-3'
Sp1 (methylated): 5'-TTTGTGAACTGTGTGCACTTAGTCA-3'
5'-GTTTCCTAGACCAGCCCCTCCGAGA-3'
AP-2: 5'-CCGGGGAGCGTGGGGCGTGGCCAGA-3'
5'-CCTCCCCGCCGCGAGCTGCCAA-3'
Finally, the PCR product was separated by agarose gel electrophoresis.
MTT Cell Proliferation Assay
Proliferation of LNCaP cells and RWPE-1 cells was assessed with the MTT method by using a commercial cell proliferation kit (Roche Molecular Biochemicals) in 96-well plates (six wells/group). LNCaP cells and RWPE-1 cells were plated at a density of 1 x 103 cells/well in 96-well plates and allowed to grow in DMEM containing 10% FBS under standard tissue culture conditions. On d 2, 3, and 5, the medium was changed to serum-free DMEM containing 5-Aza (2 µg/ml, Sigma). The same number of cells was used in negative control cultures, which contained medium without 5-Aza. On d 7, the medium was changed to serum-free DMEM containing TGF-ß1 (10 ng/ml, R&D Systems, Minneapolis, MN). Negative control cultures were treated in the same manner without the addition of TGF-ß1. The medium was then changed 8 h later by adding DMEM without serum for MTT assay. An aliquot of 50 µl of MTT solution (2 mg/ml) was added to each well, and the plate was incubated at 37 C for 4 h. Dark blue formazan crystals formed by living cells were dissolved in 150 µl of dimethyl sulfoxide, and absorbance of individual wells at 545 nm was determined with a microplate reader (model 450; Bio-Rad). Absorbance values for cell-free wells were subtracted from all values. This experiment was repeated three times. All data represent the mean of 18 wells, which were subjected to statistical analysis.
Methylation-Specific PCR in Clinical Specimens
To determine whether promoter methylation occurs in a clinical setting, we used an existing tissue microarray of prostate cancer specimens, obtained from the Northwestern University prostate cancer SPORE tissue bank with the approval from the Institutional Review Board. The array contained 158 cores (2 mm in diameter) from 80 patients in two slides. Among the specimens, 41 were benign (normal prostate, benign prostatic hyperplasia and high-grade prostate intraepithelial neoplasia), 104 had low to intermediate Gleason’s sum (Gleason’s sum 2 to 7), and 13 had high Gleason’s sum (Gleason’s sum 8–10). Immunohistochemical staining for TßRI and TßRII was conducted as described (5). Samples showing lack of positive staining for TßRI or TßRII were subjected to laser capture microdissection (LCM, Leica LMD System, Nuhsbaum, McHenry, IL). About 5,000–10,000 cells from each core section were captured for isolation of DNA, from which methylation-specific PCR was carried out. The actual number of specimens selected for methylation-specific PCR for the TßRI promoter (six CpG sites) included three samples of benign prostate, 12 with low to intermediate Gleason’s sum, and seven with high Gleason’s sum. Specimens selected for methylation-specific PCR for the TßRII promoter (three CpG sites) included five samples of benign prostate and 17 samples of malignant prostate (10 with low to intermediate Gleason’s sum and seven with high Gleason’s sum).
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FOOTNOTES |
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First Published Online May 19, 2005
Abbreviations: AP, Activator protein; 5-Aza, 5-Aza-2'-deoxycytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; LCM, laser capture microdissection; LNCaP, human prostate cancer cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RWPE-1, normal prostate epithelial cell line; SPORE, specialized program of research excellence; TßR, TGF-ß receptor; TßRI, TGF-ß receptor type I; TßRII, TGF-ß receptor type II.
Received for publication February 22, 2005. Accepted for publication May 9, 2005.
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, and PPAR
during adipogenesis. Biochem Biophys Res Commun 318:213–218[CrossRef][Medline]This article has been cited by other articles:
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