Reversal of the Hypomethylation Status of Urokinase (uPA) Promoter Blocks Breast Cancer Growth and Metastasis
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m401669200
ISSN1083-351X
AutoresPouya Pakneshan, Moshe Szyf, Robin Farias‐Eisner, Shafaat A. Rabbani,
Tópico(s)RNA modifications and cancer
ResumoMetastasis is a leading cause of mortality and morbidity in cancer. Urokinase (uPA), only expressed by the highly invasive cancer cells, has been implicated in invasion, metastases, and angiogenesis of several malignancies including breast cancer. Because uPA expression is strongly correlated with its hypomethylated state, we utilized the uPA gene in the highly invasive MDA-231 human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA promoter hypomethylation would result in its silencing and inhibition of metastasis. S-Adenosyl-l-methionine (AdoMet) has previously been shown to cause hypermethylation and inhibit demethylation. Treatment of MDA-231 cells with AdoMet, but not its unmethylated analogue S-adenosylhomocysteine, significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and metastasis in vivo. The effects of AdoMet on uPA expression were reversed by the demethylating agent 5′-azacytidine, supporting the conclusion that AdoMet effects are caused by hypermethylation. Knockdown of the methyl-binding protein 2 also causes a significant inhibition of uPA expression in vitro and tumor growth and metastasis in vivo. These treatments did not have any effects on estrogen receptor expression, suggesting that inhibition of hypomethylation will not affect genes already silenced by hypermethylation. These data are consistent with the hypothesis that hypomethylation of critical genes like uPA plays a causal role in metastasis. Inhibition of hypomethylation can thus be used as a novel therapeutic approach to silence the pro-metastatic gene uPA and block breast cancer progression into the aggressive and metastatic stages of the disease. Metastasis is a leading cause of mortality and morbidity in cancer. Urokinase (uPA), only expressed by the highly invasive cancer cells, has been implicated in invasion, metastases, and angiogenesis of several malignancies including breast cancer. Because uPA expression is strongly correlated with its hypomethylated state, we utilized the uPA gene in the highly invasive MDA-231 human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA promoter hypomethylation would result in its silencing and inhibition of metastasis. S-Adenosyl-l-methionine (AdoMet) has previously been shown to cause hypermethylation and inhibit demethylation. Treatment of MDA-231 cells with AdoMet, but not its unmethylated analogue S-adenosylhomocysteine, significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and metastasis in vivo. The effects of AdoMet on uPA expression were reversed by the demethylating agent 5′-azacytidine, supporting the conclusion that AdoMet effects are caused by hypermethylation. Knockdown of the methyl-binding protein 2 also causes a significant inhibition of uPA expression in vitro and tumor growth and metastasis in vivo. These treatments did not have any effects on estrogen receptor expression, suggesting that inhibition of hypomethylation will not affect genes already silenced by hypermethylation. These data are consistent with the hypothesis that hypomethylation of critical genes like uPA plays a causal role in metastasis. Inhibition of hypomethylation can thus be used as a novel therapeutic approach to silence the pro-metastatic gene uPA and block breast cancer progression into the aggressive and metastatic stages of the disease. Metastasis of tumor cells to different organs is the leading cause of cancer-associated morbidity and mortality in patients with breast cancer (1Rabbani S.A. Mazar A.P. Surg. Oncol. Clin. N. Am. 2001; 10: 393-415Google Scholar, 2Rabbani S.A. Xing R.H. Int. J. Oncol. 1998; 12: 911-920Google Scholar). One of the key mediators of this process is urokinase (uPA), 1The abbreviations used are: uPA, urokinase; ER, estrogen receptor; AdoMet, S-adenosyl-L-methionine; SAH, S-adenosylhomocysteine; 5′-azaC, 5′-azacytidine; MSP, methylation-specific PCR; MBD2, methylated DNA-binding protein 2; MBD2-AS, human MBD2 antisense oligonucleotide; MBD2-RS, reverse sequence oligonucleotide; RT, reverse transcriptase; GFP, green fluorescent protein; CTL, control; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: uPA, urokinase; ER, estrogen receptor; AdoMet, S-adenosyl-L-methionine; SAH, S-adenosylhomocysteine; 5′-azaC, 5′-azacytidine; MSP, methylation-specific PCR; MBD2, methylated DNA-binding protein 2; MBD2-AS, human MBD2 antisense oligonucleotide; MBD2-RS, reverse sequence oligonucleotide; RT, reverse transcriptase; GFP, green fluorescent protein; CTL, control; MOPS, 4-morpholinepropanesulfonic acid. a member of the serine protease family that catalyzes the conversion of inactive zymogen plasminogen to its active form plasmin (1Rabbani S.A. Mazar A.P. Surg. Oncol. Clin. N. Am. 2001; 10: 393-415Google Scholar, 3Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar). When activated, plasmin degrades most components of the ECM, such as laminin, fibronectin, and collagen (3Andreasen P.A. Kjoller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Google Scholar). Produced by tumor cells and tumor surrounding stroma, uPA is involved in the process of tumor progression and has been implicated in the invasion, metastasis, and angiogenesis of several malignancies including breast cancer (1Rabbani S.A. Mazar A.P. Surg. Oncol. Clin. N. Am. 2001; 10: 393-415Google Scholar). Breast cancer is a hormone-dependent malignancy that is often initiated as a less aggressive hormone responsive type and gradually progresses to a highly invasive hormone-insensitive phenotype associated with mutations in estrogen receptor (ER) or interference with ER signaling (4Ottaviano Y.L. Issa J.P. Parl F.F. Smith H.S. Baylin S.B. Davidson N.E. Cancer Res. 1994; 54: 2552-2555Google Scholar). The molecular mechanism regulating this transition is poorly understood. Abnormal patterns of DNA methylation are hallmarks of most malignancies, including breast cancer (5Baylin S.B. Esteller M. Rountree M.R. Bachman K.E. Schuebel K. Herman J.G. Hum. Mol. Genet. 2001; 10: 687-692Google Scholar). Widespread global DNA hypomethylation accompanied by region-specific hypermethylation and increased levels of expression and activity of DNMT1 are associated with the malignant phenotype (5Baylin S.B. Esteller M. Rountree M.R. Bachman K.E. Schuebel K. Herman J.G. Hum. Mol. Genet. 2001; 10: 687-692Google Scholar). It is not clear whether hypomethylation plays a causal role in cancer and which of the processes involved in tumorgenesis are controlled by hypomethylation. We have previously shown that uPA is differentially expressed in the highly invasive hormone-insensitive human breast cancer cell line MDA-231, and that the silencing of uPA expression in the non-invasive hormone responsive MCF-7 cells is because of methylation of the uPA promoter (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar). Our data raised the possibility that the enhanced demethylation activity in MDA-231 cells is responsible for activation of genes required for metastasis such as uPA causing the highly invasive and metastatic characteristics of this breast cancer cell line (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar). We therefore test the hypothesis that hypomethylation plays a causal role in metastasis by using the expression of the pro-metastatic uPA gene in the highly invasive MDA-231 breast cancer cells as a model system. If hypomethylation is responsible for uPA activation and thus plays a role in metastasis, inhibition of demethylation should result in methylation and inactivation of uPA and reversal of metastasis. If this is true, this study can have novel therapeutic implications for inhibiting invasive breast cancer metastasis. Pharmacological administration of the methyl donor S-adenosyl-l-methionine (AdoMet), which is a safe and natural compound, has been previously proposed to increase DNA methylation (7van der Westhuyzen J. Nutr. Cancer. 1985; 7: 179-183Google Scholar, 8Simile M.M. Pascale R. De Miglio M.R. Nufris A. Daino L. Seddaiu M.A. Gaspa L. Feo F. Cancer Lett. 1994; 79: 9-16Google Scholar, 9Fuso A. Cavallaro R. Orru L. Buttarelli F. Scarpa S. FEBS Lett. 2001; 508: 337-340Google Scholar, 10Ross S.A. Ann. N. Y. Acad. Sci. 2003; 983: 197-207Google Scholar). AdoMet could act by either stimulating DNA methyltransferase activity or by inhibiting demethylation activity (11Detich N. Hamm S. Just G. Knox J.D. Szyf M. J. Biol. Chem. 2003; 278: 20812-20820Google Scholar). It has recently been shown that AdoMet inhibits active demethylation in human 293 cells and can prevent demethylation of ectopically methylated DNA resulting in hypermethylation (11Detich N. Hamm S. Just G. Knox J.D. Szyf M. J. Biol. Chem. 2003; 278: 20812-20820Google Scholar). We therefore used AdoMet to test our hypothesis that pharmacological inhibition of demethylation can result in inhibition of uPA expression as well as metastasis. In addition, recent studies suggest that methylated DNA-binding protein 2 (MBD2) is required for tumorgenesis (12Slack A. Bovenzi V. Bigey P. Ivanov M.A. Ramchandani S. Bhattacharya S. tenOever B. Lamrihi B. Scherman D. Szyf M. J. Gene Med. 2002; 4: 381-389Google Scholar, 13Sansom O.J. Berger J. Bishop S.M. Hendrich B. Bird A. Clarke A.R. Nat. Genet. 2003; 34: 145-147Google Scholar) but not required for normal cell growth (12Slack A. Bovenzi V. Bigey P. Ivanov M.A. Ramchandani S. Bhattacharya S. tenOever B. Lamrihi B. Scherman D. Szyf M. J. Gene Med. 2002; 4: 381-389Google Scholar, 13Sansom O.J. Berger J. Bishop S.M. Hendrich B. Bird A. Clarke A.R. Nat. Genet. 2003; 34: 145-147Google Scholar), suggesting that it targets unique processes in tumorgenesis. The high levels of MBD2 expressed in MDA-231 cells, its requirement for tumorgenesis, and its methylated DNA binding properties prompted us to determine whether MBD2 is involved in regulation of uPA, and whether pharmacological inhibition of MBD2 would result in silencing of uPA expression, its increased methylation, and inhibition of metastasis. In this study, we demonstrate that expression of uPA in the metastatic breast cancer cell line and its hypomethylated state could be reversed pharmacologically by agents that modulate methylation like AdoMet and an antisense inhibitor of MBD2. This study confirms that uPA plays a role in growth, invasion, and metastasis of breast cancer, and that uPA is activated by hypomethylation in tumor cells. Moreover, our data are consistent with the hypothesis that DNA hypomethylation plays a causal role in metastatic cancer and suggests that pharmacological reversal of hypomethylation can be used as a novel therapeutic approach for blocking breast cancer progression into the aggressive and metastatic stages of the disease. Cell Lines and Reagents—We obtained all cell lines from the American Type Culture Collection (ATCC; Manassas, VA). The MDA-231 human breast cancer cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mm l-glutamine, and 100 units/ml penicillin-streptomycin sulfate (Invitrogen). The BT549, and HS578T human breast cancer cell lines were maintained in Dulbecco's modified Eagle's medium with 5% fetal bovine serum, 2 mm l-glutamine, and 100 units/ml penicillin-streptomycin sulfate (Invitrogen). We treated the MDA-231 cells with AdoMet (New England Biolabs, Mississauga, Canada) and SAH (Sigma), added to the regular growth media under sterile conditions, for 3 or 6 days. The media was changed every second day during treatments. A cell growth curve analysis was performed on the control MDA-231 cells and cells treated with AdoMet or SAH. All cells, cultured at a density of 1.0 × 105 viable cells/plate in triplicates, were trypsinized and the number of viable cells determined by 0.4% trypan blue (Sigma) was counted daily for 6 days. The 5′-azacytidine (5′-azaC) was obtained from Sigma. The MDA-231 cells were first treated with 100 μm AdoMet for 6 days and then with 30 μm 5′-azaC for 3 days. In another set of experiments, the MDA-231 cells were treated with both 100 μm AdoMet and 30 μm 5′-azaC for 6 days. We then extracted the genomic DNA and cellular RNA using DNAzol and TRIzol, respectively (Invitrogen), following the manufacturer's instructions. Antisense Oligonucleotides—The MBD2 antisense (MBD2-AS) and the reverse sequence (MBD2-RS) oligonucleotide sequences are 5′-TCAACAGTATTTCCCAGGTA-3′ and 5′-ATGGACCCTTTATGACAACT-3′, respectively (14Campbell P.M. Bovenzi V. Szyf M. Carcinogenesis. 2004; 25: 499-507Google Scholar). Synthesized by Integrated DNA Technologies (Coralville, IA), they both have a PS backbone, 2′ o-methyl modification of the ribose in the first four and last four bases, and are purified by ion exchange high performance liquid chromatography. We transfected these oligos into the MDA-231 cells using 10 μl/ml Lipofectin (Invitrogen) with different concentrations of the antisense oligos for different time points. At the end of the treatment, we isolated total cellular RNA and analyzed the levels of uPA and MBD2 mRNA expression by Northern blot analysis. Northern Blot and RT-PCR Analysis—We electrophoresed 20 μg of cellular RNA on a 1.1% agarose-formaldehyde gel in MOPS buffer and transferred to a nylon membrane (Amersham Biosciences), and then hybridized the blots with 32P-labeled human uPA, MBD2, and 18 S cDNA for 14 h at 65 °C. Autoradiography of the blots was carried out at -80 °C using X-AR film (Easton Kodak Co.). We quantified the levels of mRNA expression by densitometric scanning (Gel Doc, Bio-Rad). We used total RNA (2 μg) isolated from the primary tumors 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. We used three sets of primers to amplify uPA (5′-ACATTCACTGGTGCAACTGC-3′, 5′-CAAGCGTGTCAGCGCTGTAG-3′), MBD2 (5′-AGCGATGTCTACTACTTCAG-3′, 5′-AGATGTCTGCCATCAGTGCT-3′), and GAPDH (5′-CCCTTCATTGACCTCAACTACATGGT-3′ 5′-GAGGGGCCATCCACAGTCTTCTG-3′) for each RNA sample. The PCR were carried out using standard protocols and 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. We then analyzed the PCR products on a 1% agarose gel. Boyden Chamber Invasion Assay—Using two-compartment Boyden chambers (Transwell, Costar, Cambridge, MA) and basement membrane Matrigel (BD Biosciences), we examined the invasive capacities of the cells before and after treatment with AdoMet, MBD2-AS, or MBD2-RS oligos as previously described (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar, 15Guo Y. Higazi A.A. Arakelian A. Sachais B.S. Cines D. Goldfarb R.H. Jones T.R. Kwaan H. Mazar A.P. Rabbani S.A. FASEB J. 2000; 14: 1400-1410Google Scholar). We coated the 8-μm pore polycarbonate filters with basement membrane Matrigel (50 μg/filter) and analyzed 5 × 104 cells in each chamber as described (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar). We then fixed the filters for 30 min in 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m phosphate buffer (pH 7.4) at room temperature, and washed them with phosphate-buffered saline, and finally stained them with 1.5% toluidine blue and mounted them onto glass slides. Cells were examined under a light microscope. Under ×400 magnification, we examined 10 randomly selected fields and the average number of cells invaded was calculated. uPA Enzyme Activity Assay—We examined the enzymatic activity of uPA in cell-conditioned medium of the cells alone or after treatment with AdoMet, MBD2-AS, and MBD2-RS using Spectrozyme UK (American Diagnostica, Greenwich, CT), a synthetic chromogenic substrate of uPA (16Pakneshan P. Xing R.H. Rabbani S.A. FASEB J. 2003; 17: 1081-1088Google Scholar). We used high molecular weight recombinant uPA (American Diagnostica) to obtain a standard curve and the direct uPA activity assay was carried out as the manufacturers recommended. We used a Vmax plate reader (Molecular Devices, Sunnyvale, CA) to monitor the photometric absorbance of the reaction mixtures at 405 nm after 30 min incubation at room temperature. Western Blot Analysis—To examine changes in MBD2 levels in MDA-231 cells after treatment with AdoMet, MBD2-AS, and MBD2-RS, total nuclear extract was isolated from the untreated and treated MDA-231 cells as previously described (12Slack A. Bovenzi V. Bigey P. Ivanov M.A. Ramchandani S. Bhattacharya S. tenOever B. Lamrihi B. Scherman D. Szyf M. J. Gene Med. 2002; 4: 381-389Google Scholar). Equal amounts of proteins were analyzed on SDS-polyacrylamide gels and were then transferred to nitrocellulose membranes using standard protocols. Immunoblotting was performed using the sheep polyclonal MBD2 antibody purchased from Upstate Cell Signaling (Lake Placid, NY) and the β-actin antibody was purchased from Sigma. The secondary antibodies were purchased from Bio-Rad, and chemiluminescence was used for protein detection (PerkinElmer Life Sciences). Methylation Specific PCR (MSP)—We performed the sodium bisulfite treatment of the genomic DNA as previously described (16Pakneshan P. Xing R.H. Rabbani S.A. FASEB J. 2003; 17: 1081-1088Google Scholar, 17Herman J.G. Graff J.R. Myohanen S. Nelkin B.D. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9821-9826Google Scholar). We designed two sets of MSP primers to amplify the methylated (5′-AGCGTTGCGGAAGTACGCGG-3′, 3′-AAACCCGCCCCGACGCCGCC-5′), or unmethylated (5′-AGTGTTGTGGAAGTATGTGG-3′, 3′-AAACCCACCCCAACACCACC-5′) promoter sequence. We used these primers to amplify the uPA promoter 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 final extension of 72 °C for 5 min. Animal Protocols—For the animal study, we used MDA-231 cells transfected with the cDNA for green fluorescent protein (GFP). These cells exhibit no significant difference in their invasive capacity in the Matrigel invasion assay (15Guo Y. Higazi A.A. Arakelian A. Sachais B.S. Cines D. Goldfarb R.H. Jones T.R. Kwaan H. Mazar A.P. Rabbani S.A. FASEB J. 2000; 14: 1400-1410Google Scholar). We obtained 4–6-week-old female BALB/c (nu/nu) mice from Charles River, Inc. (St. Constant, Quebec, Canada). Before inoculation, MDA-231-GFP cells were treated with 100 μm AdoMet for 6 days, and transfected with 200 nm MBD2-AS and MBD2-RS for 72 h. At the end of the treatment, we inoculated 5 × 105 untreated (CTL) and treated MDA-231-GFP cells with 20% Matrigel (BD Biosciences) into the mammary fat pad of these female BALB/c nude mice. We determined the tumor volumes at timed intervals for a 10-week period after inoculation. At the end of the study, we sacrificed the animals by terminal bleeding and removed their lung, liver, spleen, kidney, and the primary tumors for further analysis. We examined 20 μl of the blood of each mouse, smeared on a glass slide, under the fluorescent microscope for the presence of GFP-labeled tumor cells in circulation. We sliced the lung, liver, spleen, and kidney to 1-mm thick slices of fresh tissue for direct examination under the fluorescent microscope for the presence of GFP-expressing tumor foci. We counted the number of GFP-expressing tumor foci per field of examination from 10 random sites of five different slides for each organ and calculated and graphed the average for each group. Using RNAqueous™-4PCR (Ambion), we extracted total RNA from the primary tumors for RT-PCR analysis to examine levels of uPA and MBD2 mRNA expression in these tumors. Immunohistochemistry—Paraffin-embedded tumor samples were cut into 5-μm thick sections for immunohistochemical analysis. Immunohistochemical staining for uPA was performed using the avidin-biotin-peroxidase complex method. Briefly, the sections were dewaxed in xylene, and rehydrated through a series of ethanol to water gradients. The sections were then incubated in 1% normal goat sera (Vector Laboratories Inc., Burlingame, CA) for 30 min at room temperature before treatment with the primary antibody (monoclonal antibody against human uPA from American Diagnostica) at 1:25 dilution overnight at 4 °C. Biotinylated goat anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA) was used as the secondary antibody at 1:200 for 30 min at room temperature. The slides were treated with Vectastain ABC-AP kit (Vector Laboratories Inc.) with dilution of 1:200 for 30 min at room temperature, and subsequently developed with Fast Red TR/Naphthol AS-MX phosphate (Sigma) containing 1 mm levamisole for 10–15 min. The slides were then counterstained with hematoxylin (Fisher) and mounted with Kaiser's glycerol jelly. All sections were washed three times for 10 min with Tris buffer (pH 7.6) after each step. For negative control sections, the primary antibody was omitted. A computer-assisted image analysis system was used to quantify the immunostaining. Images of stained sections were photographed with a Leica digital camera and processed using BioQuant image analysis software, version 6.50.10 (BioQuant Image Analysis Corp., Nashville, TN). The threshold was set by determining the positive staining of control sections and was used to automatically analyze all recorded images of all samples that were stained in the same session under identical conditions. The software calculated the area of immunohistochemical stained regions automatically in each microscopic field. Pixel counts of the immunoreaction product were calculated automatically and were given as total density of the integrated immunostaining over a given area, which reflected the relative amount of uPA detected by the antibodies. Statistical Analysis—Results are expressed as the mean ± S.E. of at least triplicate determinations and statistical comparisons are based on Student's t test and analysis of variance. A probability value of <0.05 was considered to be significant. Effect of AdoMet on uPA Expression and Activity in MDA-231 Cells—We have previously shown that the levels of uPA mRNA expression in breast cancer cell lines correlate with their state of methylation and that 5′-azacytidine, an inhibitor of DNA methylation, induces uPA in the non-metastatic breast cancer cell line MCF-7 (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar). Because uPA is unmethylated and highly expressed in the metastatic breast cancer cell line MDA-231 (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar), we used these cells as a model system to test the hypothesis that agents that inhibit demethylation would silence the expression of pro-metastatic genes such as uPA. The ubiquitous methyl donor of DNA methyltransferase reaction S-adenosyl-methionine or AdoMet was previously shown to reverse hypomethylation in cell lines and tumors in animals (9Fuso A. Cavallaro R. Orru L. Buttarelli F. Scarpa S. FEBS Lett. 2001; 508: 337-340Google Scholar) and was also recently shown to inhibit the active demethylation of ectopically methylated genes in HEK 293 cells (11Detich N. Hamm S. Just G. Knox J.D. Szyf M. J. Biol. Chem. 2003; 278: 20812-20820Google Scholar). We therefore treated MDA-231 cells with increasing concentrations of AdoMet from 25 to 100 μm for 3 and 6 days and determined the levels of expression of uPA and MBD2 mRNA. MBD2 was previously shown to be required for tumorigenesis but not normal cell growth in a number of human cancer cell lines and in mice (12Slack A. Bovenzi V. Bigey P. Ivanov M.A. Ramchandani S. Bhattacharya S. tenOever B. Lamrihi B. Scherman D. Szyf M. J. Gene Med. 2002; 4: 381-389Google Scholar, 13Sansom O.J. Berger J. Bishop S.M. Hendrich B. Bird A. Clarke A.R. Nat. Genet. 2003; 34: 145-147Google Scholar). These AdoMet treatments did not have any effect on ER expression (data not shown). This is not surprising because ER is silenced in MDA-231 cells by either mutations or DNA methylation, and inhibition of demethylation or hypermethylation should not change the state of methylation of a gene that is already methylated prior to the treatment. The results shown in Fig. 1A illustrate that while 3 days of treatment with AdoMet had no effect on uPA expression, 6 days treatment with AdoMet resulted in strong inhibition of both uPA and MBD2 mRNA expression in MDA-231 cells. A similar effect was not observed with the inactive analogue of AdoMet, SAH (Fig. 1B). To determine whether AdoMet and SAH treatments have any effects on cell viability and growth, the cell doubling time of the control MDA-231 cells and the cells treated with AdoMet and SAH was examined by a cell growth curve analysis. All cells, plated at a similar density of the viable cells per plate in triplicates, were trypsinized and the number of viable cells was counted daily throughout the 6-day treatment. The results of the analysis showed that AdoMet and SAH did not cause a statistically significant change in the MDA-231 cell doubling time throughout the treatments (Fig. 1C). This reduction in uPA mRNA expression by AdoMet was accompanied as expected with a reduction in uPA enzymatic activity in treated cells as determined using the Spectrozyme UK assay (Fig. 2A). We then determined whether reduction of uPA expression by AdoMet was also accompanied by reduction in the invasive capacity of the cells using the Boyden chamber Matrigel invasion assay (Fig. 2B). This experiment demonstrates that AdoMet treatment inhibits the invasive capacity of the metastatic MDA-231 cells in parallel to its silencing of uPA. The reduction in MBD2 expression was confirmed by Western blot analysis as shown in Fig. 2C. We then tested the hypothesis that AdoMet silencing of uPA involves reversing its hypomethylated state by using MSP assay and measuring the state of methylation of CG sequences in the uPA promoter (Fig. 2D). AdoMet treatments resulted in hypermethylation of the uPA promoter because in AdoMet-treated cells an amplification product is observed only with primers that detect methylated CGs, whereas in control cells an amplification product is observed with primers that detect unmethylated CGs. We then examined whether the effects of AdoMet are mediated by DNA methylation or by other mechanisms through AdoMet. If the silencing of uPA expression by AdoMet is a consequence of increased DNA methylation, the effect should be reversed by the DNA demethylating agent 5′-azaC, which is in fact what was observed as shown in Fig. 2E. In summary, these experiments show that AdoMet treatment reverses the hypomethylated state of the uPA promoter in the MDA-231 cells resulting in silencing of this gene, and that this reversal of hypomethylation is associated with inhibition of the invasive capacity of these cells. These data are also consistent with the hypothesis that the hypomethylation of uPA in metastatic MDA-231 cells is responsible for its activation. To determine whether AdoMet exerts similar effects on uPA expression in other invasive human breast cancer cells or whether this effect is a peculiarity of the MDA-231 cells, we examined the effects of AdoMet treatments on BT549 and HS578T human breast cancer cells, which are among the ER-negative highly invasive human breast cancer cell lines, and are characterized by high levels of uPA expression and demethylation of uPA promoter (6Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Google Scholar). We treated these cells with 100 μm AdoMet and SAH for 6 days and determined uPA mRNA levels by Northern blot analysis (Fig. 3A). Treatment of these two cell lines with AdoMet resulted in significant reduction of uPA mRNA expression and its enzymatic activity (Fig. 3B). This reduction of uPA expression and activity was accompanied by significant reduction of the invasive capacity of the cells as determined by Boyden chamber Matrigel invasion assay (Fig. 3C). These experiments further confirmed our hypothesis that reversal of the hypomethylation state by AdoMet results in silencing of uPA gene expression and activity and thus inhibits the invasive capacity of the cancer cells. Effects of Antisense Knockdown of MBD2 on uPA Expression and Activity in MDA-231 Cells—Because MBD2 was previously shown to be required for tumorgenesis (12Slack A. Bovenzi V. Bigey P. Ivanov M.A. Ramchandani S. Bhattacharya S. tenOever B. Lamrihi B. Scherman D. Szyf M. J. Gene Med. 2002; 4: 381-389Google Scholar, 13Sansom O.J. Berger J. Bishop S.M. Hendrich B. Bird A. Clarke A.R. Nat. Genet. 2003; 34: 145-147Google Scholar), and because MBD2 mRNA expression is also inhibited by AdoMet treatment (Fig. 1), we determined whether knockdown of MBD2 would reverse the hypomethylated state and inhibit the expression of uPA and metastasis of MDA-231 cells similar to AdoMet. We used sequence-specific second generation gapmer 20-mer antisense oligonucleotides directed against human MBD2 mRNA (MBD2-AS) and its reverse control oligonucleotide (MBD2-RS) to block MBD2 expression. To directly examine the role of MBD2 in controlling uPA expression in MDA-231 cells, we treated MDA-231 cells with increasing concentrations of either MBD2-AS or MBD2-RS for 72 h. The results show that MBD2-AS treatment results in a dose-dependent inhibition of MBD2 mRNA in comparison with cells treated with MBD2-RS oligonucleotides. Knock down of MBD2 mRNA also results in a dose-dependent inhibition of uPA mRNA expression (Fig. 4A). Sevent
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