Insensitivity to Transforming Growth Factor-ß Results from Promoter Methylation of Cognate Receptors in Human Prostate Cancer Cells (LNCaP)
Qiang Zhang,
Jonathan N. Rubenstein,
Thomas L. Jang,
Michael Pins,
Borko Javonovic,
Ximing Yang,
Seong-Jin Kim,
Irwin Park and
Chung Lee
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.
 |
ABSTRACT
|
---|
Prostate cancers often develop insensitivity to TGF-ß to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-ß receptors (TßRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-ß. Sensitivity to TGF-ß was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TßRI), and type II (TßRII) TGF-ß receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-ß. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TßRI and 33 sites in TßRII using methylation-specific PCR and sequence analysis. There were six methylation sites (365, 356, 348, 251, 244, 231) in the promoter of TßRI. The 244 site was located in an activator protein (AP)-2 box. There were three methylated sites (140, +27, +32) in the TßRII promoter and the 140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TßRI promoter and of Sp1 in the TßRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TßRI or TßRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TßRI (six sites) and TßRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TßRI and/or TßRII, especially those with high Gleasons scores. In those specimens showing a loss of TßR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-ß in some prostate cancer cells is due to promoter methylation in TßRs.
 |
INTRODUCTION
|
---|
TGF-ß IS A PLEIOTROPIC growth factor with multiple functions through its type I (TßRI) and type II (TßRII) receptors (1). It mediates cell proliferation, growth arrest, differentiation, and apoptosis (2, 3). In cancer cells, it is a potent growth inhibitor. A reduction or loss of expression of TßRs enables cancer cells to escape the growth inhibitory effect of TGF-ß and to gain a growth advantage. The mechanism of such a loss of sensitivity to TGF-ß remains unclear. In human prostate cancer, down-regulation of TßRs is a frequent event (4, 5, 6, 7, 8). This event becomes more pronounced with higher Gleasons scores and is associated with reduced survival (6). Loss of expression of TßRs may be attributed to defects in either genetic or epigenetic events. Within the epigenetic paradigm, methylation of CpG islands is a known cause of gene silencing (9).
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.
 |
RESULTS
|
---|
Up-Regulation of TßRs by 5-Aza Treatment in LNCaP Cells But Not in RWPE-1 Cells
Figure 1
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.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 1. Real-Time PCR Products for TßRI and TßRII from LNCaP and RWPE-1 Cells with and without the Treatment with 5-Aza
After treatment with 5-Aza, the expression of TßRI (A) and TßRII (B) in LNCaP increased approximately 20-fold, reaching a level similar to that for RWPE-1. In contrast, no significant changes were found in RWPE-1 cells with or without treatment with 5-Aza.***, P < 0.001.
|
|

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 2. Western Blot Analysis for TßRI and TßRII in Lysates of LNCaP and RWPE-1 Cells with and without 5-Aza Treatment
Results of Western blot analysis showed that expression of TßRI (A) and TßRII (B) was increased after treatment with 5-Aza in LNCaP cells but not in RWPE-1 cells. GAPDH was probed as a loading control.
|
|
Effect of 5-Aza on the Expression of TGF-ß1 and Phosphorylated Smad2 in LNCaP Cells
Results of Western blot analysis showed that levels of TGF-ß1 in conditioned media and in cell lysates did not change significantly before and after 5-Aza treatment in both LNCaP and RWPE-1 (data not shown). However, there was a significant increase in the level of phosphorylated Smad-2 after treatment with 5-Aza in LNCaP cells, but not in RWPE-1 cells (Fig. 3
). 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.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 3. Western Blot Analysis for Phosphorylated Smad-2
The level of phosphorylated Smad-2 (57 kDa) was significantly increased after treatment of LNCaP cells with 5-Aza but not in RWPE-1 cells. GAPDH (36 kDa) was probed as a loading control.
|
|
Identification of Methylation Sites of TßRI and TßRII Promoters
In an attempt to identify methylation sites for TßRI and TßRII promoters, we performed methylation-specific PCR in LNCaP and RWPE-1 cells. Eighty-six and 35 sets of primers were designed for the TßRI and TßRII promoters, respectively. These primers accounted for all potential methylated sites of CpG islands in these two genes. Corresponding to each potential site are three primers: methylated specific primer, unmethylated specific primer, and wild-type control primer. To date, two methylated specific bands were obtained from TßRI and TßRII of LNCaP cells, respectively (Fig. 4A
). 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.

View larger version (78K):
[in this window]
[in a new window]
|
Fig. 4. Methylation-Specific PCR
A, Using methylation-specific PCR, we detected methylation in the promoter of TßRI and TßRII in LNCaP cells before treatment with 5-Aza. But no methylation site was detected in the corresponding CpG islands in RWPE-1 cells. B, After treatment with 5-Aza, no methylation site was detected in the promoters of TßRI and TßRII in both LNCaP and RWPE-1 cells. Each set of methylation-specific PCR uses three different of primers: W, wild-type control primer; M, methylated specific primer; UM, unmethylated specific primer.
|
|

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 5. Sequence Analysis of Methylated and Unmethylated Products from Methylation-Specific PCR
A, Results showed the 348, 356, 365, 231, 244, 251 (arrow point) methylated cytosine in TßRI promoter in LNCaP (Methylated status). No methylated cytosine in RWPE-1 promoter (Unmethylated status). B, Results showed the 140, +32, +27 (arrow point) methylated cytosine in TßRII promoter in LNCaP (Methylated status). No methylated cytosine in RWPE-1 promoter (unmethylated status).
|
|

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 6. Chromatin Immunoprecipitation Analysis
Chromatin immunoprecipitation analysis 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 with 5-Aza. The DNA binding activity of Sp1 (138 to 143) in TßRII and AP2 (237 to 245) in TßRI promoter in LNCaP cells as methylated sites could only be detected by treatment with 5-Aza, whereas another unmethylated Sp1 site (20 to 25) in the TßRII promoter in LNCaP cells and all Sp1 and AP-2 sites in RWPE-1 cells were detected both before and after 5-Aza treatment. For no specific mouse IgG antibody, no band was found (data not shown).
|
|
Inhibition of Proliferation in LNCaP Cells after Treatment with 5-Aza
LNCaP cells produce large amounts of TGF-ß (16). If cells are sensitive to the inhibitory effect of TGF-ß, their proliferation could be inhibited by either endogenous or exogenous TGF-ß1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the inhibitory effects of 5-Aza on the proliferation of LNCaP and RWPE-1. As shown in Fig. 7A
, 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.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 7. Proliferation Activity Assay of LNCaP and REWP-1 before and after Treatment of 5-Aza and TGF-ß1
A, Results of MTT assay showed that TGF-ß1 caused 13.4% (P < 0.05) inhibition in proliferation in LNCaP cells before 5-Aza treatment, but it resulted in 55.5% (P < 0.05) of inhibition after treatment with 5-Aza. 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 (16 ) was sufficient to affect a maximum degree of inhibition in cell proliferation. B, Results of MTT assay showed that TGF-ß1 caused 36.9% (P < 0.05) inhibition in proliferation in RWPE-1 cells before 5-Aza treatment. Treatment with TGF-ß1 did not significantly change the inhibition rate after treatment with 5-Aza (P > 0.05). There is no significant difference between treatment with both 5-Aza and TGF-ß1 or with 5-Aza alone (P > 0.05), again, suggesting that the endogenous TGF-ß (17 ) was sufficient to affect the maximum degree of inhibition in cell proliferation.
|
|
Promoter Methylation in Clinical Specimens
In a tissue microarray system with 158 specimens of human prostatic tissues, immunohistochemical staining was carried out (5). We selected those that were stained negative for either TßRI or TßRII for analysis of promoter methylation. Figure 8A
shows the proportion of prostate cancer specimens with loss of expression of either TßRI or TßRII among 158 specimens. Specimens with high Gleasons 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 Gleasons score, and four of seven (57%) cancer specimens with high Gleasons 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 Gleasons score, and four of seven (57%) of cancer specimens with high Gleasons score (Fig. 8B
). The methylation status in other sites of these promoters was not investigated.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 8. Status of TßR Expression and Promoter Methylation in Clinical Prostate Specimens
We studied 158 prostate specimens from 80 patients, which were obtained from the Prostate SPORE tissue bank located at Northwestern University Memorial Hospital. All benign and cancer specimens were obtained by resection or biopsy. Among the specimens, 41 were benign prostate tissue, 104 were low Gleason score (from 27) prostate cancer tissue, and 13 were high Gleason score (from 810) prostate cancer tissue. Briefly, the tissues were cut into 5-mm-thick slices, fixed in 10% neutral buffered formalin, and embedded in paraffin as whole mounts. Tissues were selected by a pathologist based on the presence of benign tissue (normal prostate, prostate intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, adenoma hyperplasia) and prostate cancer (low to intermediate Gleasons sum 27; high Gleasons sum 810). The benign prostate tissue and prostate cancer tissue microarrays were assembled from all of the above 158 specimens. For each specimen, the largest and/or highest Gleason cancer focus was identified and mapped on the whole-mount sections. Accordingly, 2-mm cores were punched out of the tissue slices and transferred to a recipient block. The tissue microarrays were built using a manual tissue arrayer (Beecher Instruments, Silver Spring, MD). A, Percent of specimens in each category showing negative staining for TßRI or TßRII. Immunohistochemical staining for TßRI and TßRII was performed according to a previous paper (5 ). *, P < 0.05; **, P < 0.005 denotes that the percentage was significantly different from that of other groups. B, Results of methylation-specific PCR on samples showing negative staining for either TßRI or TßRII after laser capture microdissection and DNA isolation. A total of 5,00010,000 cells were captured and DNA was isolated for methylation-specific PCR. For the TßRI promoter, one of three in the benign category, four of 12 in the low Gleason score category, and four of seven in the high Gleason score category were methylated. For the TßRII promoter, one of five in the benign category, four of 10 in the low Gleason score category, and four of seven in the high Gleason score category were methylated. UM, Unmethylated; M, methylated.
|
|
 |
DISCUSSION
|
---|
Results of the present study have demonstrated that expression of TßRs and sensitivity to TGF-ß are enhanced in LNCaP cells, but not in benign RWPE-1 cells, by the treatment with 5-Aza. This observation is supported by the identification of methylation sites in promoters of both TßRI and TßRII by methylation-specific PCR followed by nucleotide sequencing and chromatin immunoprecipitation assays. Furthermore, a significant number of cases of clinical specimens also showed a similar pattern of promoter methylation in TßRs. To the best of our knowledge, this report represents the first attempt to map potential methylation sites of the promoters of TßRs in prostate cancer cells. The current findings have added TßRI and TßRII to a growing list of tumor suppressor genes that are down-regulated or silenced by promoter methylation in cancer cells.
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 (1012 M), androgen has a stimulatory effect on LNCaP proliferation. However, at high doses (1010 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 Guos 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 1020 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.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
LNCaP and RWPE-1 cells were obtained from ATCC and cultured in RPMI-1640 medium (Invitrogen Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated FBS (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin. Treatment of these cells with 5-Aza was performed as previously reported (26). Cells were cultured in serum-free RPMI-1640 with 5-Aza (2 µg/ml; Sigma, St. Louis, MO) for 6 d. RNA, protein, and DNA were isolated from these cells. Real-time PCR, Western blot, and methylation-specific PCR were carried out.
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 manufacturers 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 Societys 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 manufacturers 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 Gleasons sum (Gleasons sum 2 to 7), and 13 had high Gleasons sum (Gleasons sum 810). 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 5,00010,000 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 Gleasons sum, and seven with high Gleasons 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 Gleasons sum and seven with high Gleasons sum).
 |
FOOTNOTES
|
---|
This study was supported in part, by grants from National Cancer Institute (CA 60553, CA 90386), the American Foundation for Urologic Disease (AFUD) (to Q.Z.), and Riba Urology Fellowship of Northwestern University (to J.N.R.).
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.
 |
REFERENCES
|
---|
- Massagué J 2000 How cells read TGF-ß signals. Nat Rev Mol Cell Biol 1:169178[CrossRef][Medline]
- Sintich SM, Lamm MLG, Sensibar JA, Lee C 1999 Transforming growth factor-ß1 induced proliferation of the prostate cancer cell line, TSU-Pr1: the role of platelet-derived growth factor. Endocrinology 140:34113415[Abstract/Free Full Text]
- Zhou W, Park I, Pins M, Kozlowski JM, Jovanovic B, Zhang J, Lee C, Ilio K 2003 Dual regulation of proliferation and growth arrest in prostatic stromal cells by transforming growth factor-ß1. Endocrinology 144:42804284[Abstract/Free Full Text]
- Steiner MS, Anthony CT, Metts J, Moses H 1995 Prostate cancer cells lose their sensitivity to TGFß1 growth inhibition with tumor progression. Urol Oncol 1:252262
- Kim IY, Ahn HJ, Zelner DJ, Shaw JW, Lang S, Kato M, Oefelein MG, Miyazono K, Kozlowski JM, Lee C 1996 Loss of expression of transforming growth factor-ß receptors type I and type II correlates with tumor grade in human prostate cancer tissues. Clin Cancer Res 2:12551261[Abstract]
- Kim IY, Ahn HJ, Lang S, Oefelein MG, Oyasu R, Kozlowski JM, Lee C 1998 Loss of expression of transforming growth factor-ß receptors is associated with poor prognosis in prostate cancer patients. Clin Cancer Res 4:16251630[Abstract]
- Williams RH, Stapleton MF, Yang G, Truong LD, Rogers E, Timme TL 1996 Reduced levels of transforming growth factor b receptor type II in human prostate cancer: an immunohistochemical study. Clin Cancer Res 2:635640[Abstract]
- Guo Y, Jacobs SC, Kyprianou N 1997 Down-regulation of protein and mRNA expression for transforming growth factor-ß (TGF-ß1) type I and type II receptors in human prostate cancer. Int J Cancer 71:573579[CrossRef][Medline]
- Jones PA, Baylin SB 2002 The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415428[Medline]
- Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H, Chu TM, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic carcinoma. Cancer Res 43:18091818[Abstract]
- Wilding G, Zugmeier G, Knabbe C, Flanders K, Gelmann E 1989 Differential effects of transforming growth factor ß on human prostate cancer cell lines in vitro. Mol Cell Endocrinol 62:7987[CrossRef][Medline]
- Kim IY, Ahn HJ, Zelner DJ, Shaw JW, Sensibar JA, Kim J H, Kato M, Lee C 1996 Genetic change in transforming growth factor-ß (TGF-ß) receptor type I gene correlates with insensitivity to TGF-ß1 in human prostate cancer cells. Cancer Res 56:4448[Abstract]
- Kim IY, Zelner DJ, Sensibar JA, Ahn HJ, Park L, Lee C 1996 Modulation of sensitivity to transforming growth factor-ßl in an androgen responsive prostatic cancer cell line, LNCaP, by dihydrotestosterone. Exp Cell Res 222:103110[CrossRef][Medline]
- Guo Y, Kyprianou N 1998 Overexpression of transforming growth factor (TGF) ß1 type II receptor restores TGF-ß1 sensitivity and signaling in human prostate cancer cells. Cell Growth Differ 9:185193[Abstract]
- Bello D, Webber MM, Kleinman HK, Wartinger DD, Rhim JS 1997 Androgen responsive adult human prostate epithelial cell lines immortalized by human papillomavirus 18. Carcinogenesis 18:12151223[Abstract]
- Kim IY, Kim JH, Zelner DJ, Ahn HJ, Sensibar JA, Lee C 1996 Transforming growth factor-ß1 is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line, LNCaP. Endocrinology 137:991999[Abstract]
- Bae HW, Geiser AG, Kim DH, Chung MT, Burmester JK, Sporn MB, Roberts AB, Kim SJ 1995 Characterization of the promoter region of the human transforming growth factor-ß type II receptor gene. J Biol Chem 49:2946029468[CrossRef]
- Bloom BB, Humphries DE, Kuang PP, Fine A, Goldstein RH 1996 Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-ß receptor: regulation by TGF-ß. Biochim Biophys Acta 1312:243248[CrossRef][Medline]
- Zhou T, Chiang CM 2002 Sp1 and AP-2 regulate but do not constitute TATA-less human TAFII55 core promoter activity. Nucleic Acids Res 30:41454157[Abstract/Free Full Text]
- Gius D, Cui H, Bradbury M, Cook J, Smart DK, Zhao S, Young L, Brandenburg SA, Hu Y, Bisht KS, Ho AS, Mattson D, Sun L, Munson PJ, Chuang EY, Mitchell JB, Feinberg AP 2004 Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell 6:361371[CrossRef][Medline]
- Lee C, Sutkowski DM, Sensibar JA, Zelner D, Kim I, Amsel I, Shaw N, Prins GS, Kozlowski JM 1995 Regulation of proliferation and production of prostate specific antigen in androgen sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone. Endocrinology 136:796803[Abstract]
- Igawa T, Lin FF, Lee MS, Karan D, Batra SK, Lin MF 2002 Establishment and characterization of androgen-independent human prostate cancer LNCaP cell model. The Prostate 50:222235[CrossRef][Medline]
- Ammanamanchi S, Brattain MG 2004 Restoration of transforming growth factor-ß signaling through receptor RI induction by histone deacetylase activity inhibition in breast cancer cells. J Biol Chem 279:3262033265[Abstract/Free Full Text]
- Osada H, Tatematsu Y, Masuda A, Saito T, Sugiyama M, Yanagisawa K, Takahashi T 2001 Heterogeneous transforming growth factor (TGF)-ß unresponsiveness and loss of TGF-ß receptor type II expression caused by histone deacetylation in lung cancer cell lines. Cancer Res 61:83318339[Abstract/Free Full Text]
- Chen T, Triplett J, Dehner B, Hurst B, Colligan B, Pemberton J, Graff JR, Carter JH 2001 Transforming growth factor-ß receptor type I gene is frequently mutated in ovarian carcinomas. Cancer Res 61:46794682[Abstract/Free Full Text]
- Nguyen CT, Weisenberger DJ, Velicescu M, Gonzales FA, Lin JC, Liang G, Jones PA 2002 Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-Aza-2'-deoxycytidine. Cancer Res 62:64566461[Abstract/Free Full Text]
- Velicescu M, Weisenberger DJ, Gonzales FA, Tsai YC, Nguyen CT, Jones PA 2002 Cell division is required for de novo methylation of CpG islands in bladder cancer cells. Cancer Res 62:23782384[Abstract/Free Full Text]
- Tang QQ, Zhang JW, Daniel Lane M 2004 Sequential gene promoter interactions by C/EBPß, C/EBP
, and PPAR
during adipogenesis. Biochem Biophys Res Commun 318:213218[CrossRef][Medline]