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Departments of Medicine and Oncology, McGill University Health Centre, Montreal, Canada
2Correspondence: McGill University Health Centre, 687 Pine Ave. West, Room H4.67, Montreal, Quebec, Canada, H3A 1A1. E-mail: shafaat.rabbani{at}mcgill.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: urokinase plasminogen activator • CpG dinucleotides • MSP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Due to its ability to break down different components of the extracellular matrix (ECM), including laminin, fibronectin, and collagen, uPA has been implicated in the invasion, metastases, and angiogenesis of several malignancies, including prostate cancer (3)
.
Prostate carcinoma is a leading hormone-dependent malignancy associated with a high incidence of morbidity and mortality due to its ability to develop metastatic lesions in different organs (4)
. Initiating as a less aggressive hormone-responsive type, prostate cancer gradually progresses to a highly invasive hormone-insensitive phenotype associated with the loss of functional androgen receptors (AR) due to AR gene silencing, mutations in the AR gene, or interference in hormone receptor signaling pathways (5)
. Over time, cancer cells become refractory to any kind of hormonal treatment, one of the few therapeutic strategies currently available for treating patients with prostate cancer (6)
. uPA expression is increased in the plasma of patients with prostate cancer compared with those with benign prostatic hyperplasia (7)
. uPA levels are particularly high in patients with advanced hormone refractory stages of this disease (8)
. Previous studies of several tumor cells have demonstrated that uPA is expressed only by highly invasive cancer cells and is responsible for the invasive characteristics of these tumor cells (9
10
11
12)
. However, the mechanism that regulates the expression of uPA gene expression at different stages of tumor progression has not been fully elucidated.
Within the context of CpG dinucleotides, DNA methylation is a molecular mechanism that causes epigenetic changes in the chromatin structure without altering the DNA sequence (13)
. DNA methylation can cause transcriptional silencing of genes by inhibiting the binding of transcription factors to regulatory sequences or by binding of methyl-cytosine binding proteins. The methyl-cytosine binding protein complexes specifically bind to methylated DNA, recruiting histone deacetylases that cause deacetylation of adjacent histones, leading to chromatin condensation and transcriptional repression (14)
. Hypermethylation has been shown to be responsible for inactivation of several tumor suppressor genes, including Rb, p16, and VHL, as well as several tumor-promoting genes like E-cadherin, ER, AR, and inhibitors of angiogenic factors (15
16
17
18
19
20
21)
.
To define the involvement of DNA methylation and the resulting epigenetic regulation mechanisms in differential regulation of uPA gene expression in prostate cancer cells, we performed a detailed methylation analysis of the 5'-regulatory region of uPA gene. Methylation status of uPA promoter was analyzed in normal human prostate epithelial cells (PrEC) and in hormone-responsive (LNCaP) and hormone-insensitive (PC-3) prostate cancer cell lines representing the early and the late stages of the disease, respectively. Using the chemical demethylating agent 5'-azacytidine (5'-azaC), we determined the effect of this agent on uPA mRNA expression and prostate cancer cell invasion in vitro and on tumor growth in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analysis and RT-PCR
Cellular RNA isolated from PrEC, LNCaP, and PC-3 cells was electrophoresed on a 1.1% agarose-formaldehyde gel and transferred to a nylon membrane (Amersham-Pharmacia Biotech Ltd., Baie d’Urfé, Quebec, Canada), using standard protocols. The blots were hybridized with a 32P-labeled human uPA and 18S cDNA for 14 h at 65°C. Autoradiography of the blots was performed at –80°C using XAR film (Easton Kodak Co., Rochester, NY, USA). The level of uPA mRNA expression was quantified by densitometric scanning (Gel Doc, Bio-Rad, Mississauga, Ontario, Canada).
Two micrograms of total RNA was used for reverse transcription and amplification. The primers used for RT-PCR were designed so that there is an intron between the amplified regions to recognize any DNA contamination. The primers were (5'-ACATTCACTGGTGCAACTGC-3' and 3'-CAAGCGTGTCAGCGCTGTAG-5') for uPA; (5'-GTCCTGGATGAGGAACAGCA-3' and 3'-GTAGACGGCAGTTCAAGTGT-5') for AR; (5'-CCCTTCATTGACCTCAACTACATGGT-3' and 3'-GAGGGGCCATCCACAGTCTTCTG-5') for GAPDH. The reactions were carried out using standard protocols. The DNA was amplified under the following conditions: 95°C for 3 min, 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and the final extension of 72°C for 5 min.
Boyden chamber invasion assay
Using two-compartment Boyden chambers (Transwell, Costar, Cambridge, MA, USA) and basement membrane Matrigel (Becton Dickinson Labware, Bedford, MA, USA), the invasive capacities of PrEC, LNCaP, PC-3, and 5'-azaC-treated LNCaP cells alone or in combination with anti human uPA IgG (American Diagnostica, Greenwich, CT, USA) were determined as described previously (11
, 22)
. The 8 µm pore polycarbonate filters were coated with basement membrane Matrigel (50 µg/filter) and used to analyze 5 x 104 cells in each chamber as described (22)
. The filters were then fixed for 30 min in 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphated buffer (pH 7.4) at room temperature, washed with PBS, stained with 1.5% toluidine blue, and mounted onto glass slides. Cells were examined under a light microscope. Under 400x magnification, 10 randomly selected fields were examined and the average number of cells invaded was calculated.
Nuclear runoff assay
Nuclear extracts were isolated from PrEC, LNCaP, and PC-3 cells and nuclear runoff assay was performed as described previously (11
, 23)
. After DNase I (150 units per reaction) and proteinase K (0.2 mg/mL) treatment for 30 min at 37°C, the newly synthesized RNAs were precipitated and used to hybridized with immobilized human uPA cDNA.
uPA enzyme activity assay
The enzymatic activity of uPA in cell conditioned medium of 5 x 106 LNCaP cells alone or after treatment with 5'-azaC was determined using Spectozyme UK (American Diagnostica), a synthetic chromogenic substrate of uPA. High molecular weight recombinant uPA (American Diagnostica) was used to obtain a standard curve and the direct uPA activity assay was performed as the manufacturers recommended. The photometric absorbance of the reaction mixtures at 405 nm was monitored after 30 min at room temperature in a Vmax plate reader (Molecular Devices, Sunnyvale, CA, USA).
Luciferase reporter assay
The -745 to +30 region of the uPA promoter (gift from Dr. Blasi, Milan, Italy) was inserted into a luciferase reporter vector pGL-3 basic (Promega, Madison, WI, USA) to obtain uPAP-luc plasmid as described previously (23)
. The uPAP-luc was methylated in vitro using mSssI, mHpaII, and mHhaI bacterial methylases and the methyl donor as recommended by the manufacturer (New England Biolabs, Mississauga, ON, Canada). Resistance to digest by methylation-sensitive HpaII and HhaI restriction enzymes then confirmed methylation of the uPAP-luc plasmid. The PGL-3 basic was used as a control. uPAP-luc and PGL-3 basic plasmids were each cotransfected with PSV-ß-gal (Promega) into PC-3 cells using LipofectAMINE (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Luciferase reporter activity was determined 48 h after transfection as described (23)
.
Southern blot analysis
Genomic DNA (10 µg) was digested with PstI, PstI/HpaII, or PstI/HhaI (8 units / µg of total DNA) for 18 h at 37°C, electrophoresed on a 0.8% agarose gel and transferred to a nylon membrane (Amersham-Pharmacia Biotech Ltd.). Filters were hybridized with a 32P-labeled 778 bp SmaI-AvrII promoter probe (gift from Dr. F. Blasi, Milan, Italy). All filters were incubated and washed as described previously (24)
.
Methylation specific PCR (MSP) and bisulfite sequencing
Sodium bisulfite treatment of the genomic DNA was performed as described earlier (25)
. MSP primers were designed to amplify the methylated (5'-AGCGTTGCGGAAGTACGCGG-3', 3'-AAACCCGCCCCGACGCCGCC-5'), unmethylated (5'-AGTGTTGTGGAAGTATGTGG-3', 3'-AAACCCACCCCAACACCACC-5'), or wild-type (5'-CAGGTGCATGGGAGGAAGCA-3', 3'-ATCTCAGGACCGCGGCACTC-5') sequence. Primers used for bisulfite sequencing were located at –320: 5'-TAGGTGTATGGGAGGAAGTA-3', and 109: 3'-CAGGAGGTTTTAGAGTTGGG-5' of the uPA promoter region. The amplification reaction was performed under the following conditions: 95°C for 3 min, 10 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 45 s, 20 cycles of 95°C for 30 s, 50°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 5 min. The PCR products were purified and subcloned into Topo-PCR TA cloning vectors according to the manufacturer’s instruction (Clonetics) or sent directly for DNA sequencing analysis (Bio S&T, Montreal, Quebec, Canada).
Animal protocols
Six-wk-old male BALB/c (nu/nu) were obtained from Charles River, Inc. (St. Constant, Quebec, Canada). Before inoculation, LNCaP cells were treated with 25 µM 5'-azaC for 24 h, followed by continuous maintenance in regular growth media for 9 days. On day 10, treated and untreated LNCaP cells were washed with Hank’s balanced buffer; 3 x 106 cells were resuspended in 100 µL saline with 20% Matrigel (Becton Dickinson Labware, Mississauga, Ontario, Canada) and injected subcutaneously into the flank region of the hind limb of mice. Both control and experimental animals were monitored at weekly intervals for the development of tumors. At the end of the study, the animals were killed and primary tumors were removed for RNA and DNA isolation for further analysis.
Statistical analysis
Results are expressed as the mean ± SE of at least triplicate determinations and statistical comparisons are based on the Students t test ANOVA. A probability value of < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The cells were also evaluated for their ability to invade through Matrigel in a Boyden chamber invasion assay. PrEC cells failed to invade through Matrigel whereas LNCaP cells exhibited low invasive capacity. In contrast, PC-3 cells that expressed high levels of uPA mRNA exhibited high invasive capacity (Fig. 1B
).
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To determine the mechanism that suppresses uPA mRNA expression in hormone-responsive PrEC and LNCaP cells, transcription of uPA gene was directly examined by nuclear runoff assay. Only PC-3 cells that also expressed uPA mRNA showed uPA gene transcription. In contrast to PC-3 cells, both PrEC and LNCaP cells failed to show any uPA gene transcription, indicating that failure to express uPA mRNA by these cells is due to inhibition of gene transcription (Fig. 1C
). The presence of the intact uPA gene was determined by Southern blot analysis of total genomic DNA (Fig. 1D
). These results demonstrated that although the uPA gene is present in PrEC and LNCaP cells, it is silenced due to transcriptional inhibition of uPA expression.
Effects of 5'-azaC on uPA expression and invasive capacity of the LNCaP cells
LNCaP cells were treated with different concentrations of demethylating agent 5'-azaC (10–25 µM) for 10 days and uPA mRNA expression was determined by RT-PCR and Northern blot analysis. RT-PCR analysis of the total cellular RNA isolated from cells treated with 25 µM 5'-azaC showed that uPA mRNA expression starts after 6 days of treatment (Fig. 2
A). A dose-dependent induction of uPA mRNA expression was observed by Northern blot analysis after 10 days of treatment (Fig. 2B
). Treatment of LNCaP cells with 5'-azaC did not result in any change in AR mRNA levels as determined by RT-PCR (data not shown).
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Levels of uPA activity in the control and treated LNCaP cells were determined by a direct chromogenic activity assay based on the ability of uPA to cleaveSpectrozyme UK and measuring the amidolytic activity of the molecule. The conditioned medium of LNCaP and treated LNCaP cells were incubated with the Spectrozyme UK for 30 min at room temperature and the absorbance was measured at 405 nm. The results showed a significant increase in uPA activity in the LNCaP cells after treatment with 25 µM 5'-azaC for 10 days (Fig. 2C
). To determine whether there was any effect on invasiveness of these cells, the invasive capacity of the control and treated LNCaP cells was determined by Boyden chamber invasion assay. Induction of uPA expression in LNCaP cells treated with 25 µM 5'-azaC for 10 days resulted in a significant increase in the invasive capacity of these cells. Incubation of these cells with anti-uPA antibody blocked this acquired invasive capacity (Fig. 2D
). This provided further convincing evidence that this increase in tumor cell invasion after treatment with 5'-azaC is due to induction of uPA expression.
Effect of in vitro methylation on uPA promoter activity
Since we established a correlation between uPA promoter methylation and uPA gene transcription, we next examined the effect of in vitro methylation of uPA promoter on the promoter activity and subsequent gene transcription. A uPA promoter construct containing -745 to +30 region of the uPA promoter (uPAP-luc) was methylated with bacterial methylases in vitro and transfected into the uPA-expressing PC-3 cells. The pGL-3 plasmid lacking uPA promoter was used as a control. Luciferase reporter activity was determined 48 h after transfection. The unmethylated uPAP-luc plasmid exhibited significantly high promoter activity compared with the methylated uPAP-luc plasmids and the control plasmid (Fig. 3
).
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Analysis of uPA promoter methylation status by Southern blot
Methylation-sensitive restriction enzyme analysis was used to determine the methylation status of the uPA promoter at the HpaII and HhaI restriction sites, where digestion of the promoter by these two enzymes is blocked when their respective recognition sites are methylated (Fig. 4
A). Genomic DNA isolated from PrEC, LNCaP, and PC-3 cell lines was digested with methylation-insensitive PstI restriction enzyme alone (lane 1) or with either methylation-sensitive HpaII (lanes 2) or HhaI (lane 3) restriction enzymes. The methylation status of these methylation-sensitive siteswas then analyzed by Southern blot analysis (Fig. 4B
). The HpaII and HhaI restriction sites were methylated in PrEC and LNCaP cells, thus preventing the digestion of the promoter and yielding restriction pattern identical to PstI digestion. In PC-3 cells, however, the uPA promoter was not methylated at these sites, resulting in digestion of the promoter into smaller fragments. These results indicate that in hormone-responsive PrEC and LNCaP cells the uPA promoter is hypermethylated, whereas in hormone-insensitive PC-3 cells the promoter is unmethylated. Treatment of LNCaP cells with 5-'azaC results in demethylation of uPA promoter and a restriction pattern similar to that seen in PC-3 cells (Fig. 4B
).
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Methylation-specific PCR (MSP) and bisulfite sequencing analysis of uPA promoter
We analyzed the methylation pattern of the uPA promoter in PrEC, LNCaP, and PC-3 cell lines by MSP. Using three sets of primers designed specifically to amplify the converted sequence within the uPA promoter upstream to the TATA box (Fig. 5
A), we distinguished between the methylated and unmethylated CpG sites based on their sequence differences after chemical modifications by sodium bisulfite treatment. One set of primers (u) anneal to the unmethylated DNA that underwent the chemical modification. Another set of primers (m) anneal to the methylated DNA that has undergone the chemical modification. The wild-type primers (w) used as a control anneal to any DNA that has not undergone a chemical modification. The results of MSP analysis of the genomic DNA confirmed methylation of CpG sites within the amplified region in the PrEC and LNCaP cells (Fig. 5B
). Treatment of LNCaP cells with 25 µM 5'-azaC for 10 days resulted in demethylation of this region (Fig. 5B
). Time course MSP analysis of the treated cells showed that demethylation of the uPA promoter occurs after 6 days of treatment with 5'-azaC (Fig. 5C
), compatible with the induction of uPA mRNA expression after treatment with 5'-azaC for 6 days (Fig. 2A
). This suggested that demethylation of the uPA promoter is the underlying molecular mechanism responsible for the induction of uPA mRNA expression.
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We used another set of primers to amplify and sequence a larger region of the uPA promoter. This region was selected due to its proximity to transcription start site where clusters of CpG dinucleotides and several transcription factor binding sites are located (26)
. The methylated cytosines were distinguished from the unmethylated cytosines within the sequenced fragment, since they were resistant to sodium bisulfite treatment and remained unchanged. Sequencing analysis showed that almost all potential methylation sites within the uPA promoter were methylated in PrEC and LNCaP cells, whereas none of these sites were methylated in the uPA expressing PC-3- and 5'-azaC-treated LNCaP cells (Fig. 6
).
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Effect of demethylation on uPA mRNA expression and tumor growth in vivo
To evaluate the effect of uPA induction in 5'-azaC-treated LNCaP cells on tumor progression, LNCaP cells and 5'-azaC-treated LNCaP cells were inoculated into the right flank of male BALB/c nude mice. After tumor cell inoculation, tumor volumes were determined at timed intervals for 9 wk. Results showed that animals inoculated with 5'-azaC-treated LNCaP cells developed tumors of significantly larger volume compared with the control group (Fig. 7
A). RT-PCR analysis of the total RNA isolated from these primary tumors showed an induction of uPA mRNA levels after treatment with 5'-azaC (Fig. 7B
). These results are compatible with the in vitro data where uPA mRNA expression was induced by 5'-azaC in LNCaP cells (Fig. 7B
).
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To determine whether demethylation of the uPA promoter is responsible for the induction of uPA mRNA in these tumors, methylation status of uPA promoter was determined by bisulfite sequencing of the genomic DNA isolated from the control and experimental tumors. Results showed that whereas uPA promoter continues to be methylated in tumors isolated from animals receiving wild-type LNCaP cells, it is fully demethylated in tumors from animals inoculated with LNCaP cells treated with 5'-azaC (Fig. 7C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 27)
. The 5'-regulatory regions of genes, often characterized by CpG islands located in their promoter region, are a target for methylation. There is evidence supporting the emerging role of DNA methylation in the regulation of gene transcription in human malignancies (28)
. Studies over the years have provided evidence for a global hypomethylation of the genome in tumor cells, accompanied by region-specific hypermethylation (28
, 29)
. In fact, hypomethylation of CpG islands has been shown to be responsible for overexpression of tumor-promoting genes and oncogenes, including ras and myc (21
, 30
31
32
33
34)
.
As a tumor-promoting gene, uPA has been implicated in the growth, invasion, metastases, and angiogenesis of several malignancies, including prostate cancer (3)
. However, the mechanism underlying the suppression of uPA gene expression in the less invasive cancer cells is not known. In this study, we have demonstrated that methylation of CpG sites within the uPA promoter is the molecular mechanism that can reversibly inactivate transcription of uPA gene. For these studies we have used normal human PrEC and two prostatic cancer cell lines: LNCaP cells representing the less aggressive hormone-responsive type and PC-3 cells representing the highly invasive hormone-insensitive type of prostate cancer. The absence of uPA mRNA expression and gene transcription in PrEC and LNCaP cells was confirmed and uPA expression was found to correlate with tumor cell invasive capacity. Since deletion of both alleles of a gene can result in silencing of gene expression, the presence of intact uPA gene in these tumor cells was verified, which demonstrated that the lack of uPA expression in PrEC and LNCaP cells is not via this mechanism.
To study the methylation status of the uPA promoter, we performed detailed methylation analysis of these cells by Southern blot, MSP, and bisulfite sequencing. Since methylation of the HpaII and HhaI restriction sites within the uPA promoter prevents digestion of the promoter by these enzymes, Southern blot analysis revealed the methylation status of the promoter. Results indicated that uPA promoter is hypermethylated in hormone-responsive PrEC and LNCaP cells and hypomethylated in hormone-insensitive PC-3 cells. To confirm that DNA methylation is in fact the regulatory mechanism that suppresses uPA gene expression in LNCaP cells, we examined the effect of 5'-azaC, a DNA methyltransferase inhibitor, on methylation status and expression of uPA. After treatment with 5'-azaC for 10 days, an induction of uPA mRNA expression and activity in LNCaP cells resulted in an increase in the invasive capacity of these cells. Specificity of these uPA mediated effects was shown by incubating these experimental LNCaP cells with an anti-uPA antibody that significantly decreased this acquired tumor cell invasive capacity. These results provided convincing evidence that induction of uPA expression is indeed responsible for the higher invasive capacity of these cells. Methylation of uPA promoter in a luciferase construct provided further evidence that methylation is the primary mechanism responsible for silencing of the uPA promoter activity. Genomic DNA isolated from these experimental LNCaP cells was therefore analyzed by restriction digestion with methylation-sensitive enzymes, indicating that uPA promoter becomes hypomethylated after treatment with 5'-azaC. Moreover, as MSP and RT-PCR analysis revealed, demethylation of the uPA promoter and induction of uPA mRNA expression both occur in LNCaP cells after 6 days of treatment with 5'-azaC. These simultaneous changes suggest that the resulting expression of uPA in LNCaP cells treated with 5'-azaC is due to demethylation of uPA promoter.
Further methylation analysis of the uPA promoter in PrEC, LNCaP, and PC-3 cells by MSP and bisulfite sequencing confirmed the aberrant methylation of uPA promoter in AR positive hormone-responsive PrEC and LNCaP cells. In addition, demethylation of uPA promoter resulted in an increased tumor volume and induction of uPA mRNA expression in vivo.
These results collectively provided convincing evidence for cytosine methylation as a unique molecular mechanism involved in transcriptional regulation of uPA gene expression. This is associated with higher invasive phenotype of these tumor cells, allowing the production of this critical protease to be differentially regulated in different stages of prostate cancer progression. In recent studies, we have shown that balanced activity of DNA methyltransferase and demethylase, the two key enzymes involved in DNA methylation, is required for regulating the expression of uPA by tumor cells (35
, 23)
. Novel approaches to selectively alter the expression of these key enzymes such as antisense oligonucleotides and chemical molecules may lead to inhibition of demethylation of uPA promoter and subsequent blockage of its gene transcription, thus resulting in the prevention of cancer progression to the highly invasive and metastatic stages. Indeed, similar molecular and biochemical approaches are now in clinical development to block cellular transformation and tumorigenesis (36
, 37)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 9, 2002. Accepted for publication February 3, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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