Inhibition of Histone Deacetylase Activity Promotes Invasion of Human Cancer Cells through Activation of Urokinase Plasminogen Activator
2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês
10.1074/jbc.m705867200
ISSN1083-351X
AutoresSai Murali Krishna Pulukuri, Bharathi Gorantla, Jasti S. Rao,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoHistone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA. Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA. Tumor invasion and metastasis are the major characteristics of aggressive phenotypes of various human cancers, and therefore, the major causes of cancer deaths (1Steeg P.S. Nat. Rev. Cancer. 2003; 3: 55-63Crossref PubMed Scopus (434) Google Scholar). Cancer cells must acquire several properties to disseminate from the primary tumor, including the ability to degrade and migrate through the extracellular matrix, a process called invasion (2Mohanam S. Sawaya R. McCutcheon I. Ali-Osman F. Boyd D. Rao J.S. Cancer Res. 1993; 53: 4143-4147PubMed Google Scholar, 3Rao J.S. Nat. Rev. Cancer. 2003; 3: 489-501Crossref PubMed Scopus (714) Google Scholar). Invasion is one of the first steps in the metastatic cascade and is a strong indicator of tumor progression. Tumor invasion and metastasis are often associated with increased expression of extracellular matrix-degrading proteases, among which urokinase plasminogen activator (uPA) 2The abbreviations used are: uPA, urokinase plasminogen activator; HDAC, histone deacetylase; HDAI, histone deacetylase inhibitor; TSA, trichostatin A; NaB, sodium butyrate; SCR, scriptaid; 5-aza, 5-aza-2′-deoxycytidine; siRNA, small interfering RNA; shRNA, small hairpin RNA; RNAi, RNA interference; ChIP, chromatin immunoprecipitation; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CMV, cytomegalovirus. 2The abbreviations used are: uPA, urokinase plasminogen activator; HDAC, histone deacetylase; HDAI, histone deacetylase inhibitor; TSA, trichostatin A; NaB, sodium butyrate; SCR, scriptaid; 5-aza, 5-aza-2′-deoxycytidine; siRNA, small interfering RNA; shRNA, small hairpin RNA; RNAi, RNA interference; ChIP, chromatin immunoprecipitation; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CMV, cytomegalovirus. is of central importance (4Sidenius N. Blasi F. Cancer Metastasis Rev. 2003; 22: 205-222Crossref PubMed Scopus (344) Google Scholar, 5Yamamoto M. Sawaya R. Mohanam S. Bindal A.K. Bruner J.M. Oka K. Rao V.H. Tomonaga M. Nicolson G.L. Rao J.S. Cancer Res. 1994; 54: 3656-3661PubMed Google Scholar). Mounting evidence from laboratories suggests a role for uPA in the invasion of cancer cells as well as the risk for a relapse in cancer patients (6Dazzi C. Cariello A. Maioli P. Magi S. Rosti G. Giovanis P. Giovannini G. Lanzanova G. Marangolo M. Cancer Invest. 2003; 21: 208-216Crossref PubMed Scopus (10) Google Scholar, 7Hsu D.W. Efird J.T. Hedley-Whyte E.T. Am. J. Pathol. 1995; 147: 114-123PubMed Google Scholar, 8Miyake H. Hara I. Yamanaka K. Arakawa S. Kamidono S. Int. J. Oncol. 1999; 14: 535-541PubMed Google Scholar, 9Schweinitz A. Steinmetzer T. Banke I.J. Arlt M.J. Sturzebecher A. Schuster O. Geissler A. Giersiefen H. Zeslawska E. Jacob U. Kruger A. Sturzebecher J. J. Biol. Chem. 2004; 279: 33613-33622Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 10Yang J.L. Seetoo D. Wang Y. Ranson M. Berney C.R. Ham J.M. Russell P.J. Crowe P.J. Int. J. Cancer. 2000; 20: 431-439Crossref Scopus (102) Google Scholar).Tumor invasion is mediated by uPA through the conversion of plasminogen to plasmin, which degrades basement membranes (11Legrand C. Polette M. Tournier J.M. de Bentzmann S. Huet E. Monteau M. Birembaut P. Exp. Cell Res. 2001; 264: 326-336Crossref PubMed Scopus (96) Google Scholar, 12Stewart D.A. Cooper C.R. Sikes R.A. Reprod. Biol. Endocrinol. 2004; 2: 2Crossref PubMed Scopus (105) Google Scholar). Additionally, binding of uPA with its receptor uPA receptor activates the Ras/extracellular signal-regulated kinase pathway, which in turn, leads to cell proliferation, migration, and invasion (13Aguirre-Ghiso J.A. Estrada Y. Liu D. Ossowski L. Cancer Res. 2003; 63: 1684-1695PubMed Google Scholar). Several studies using uPA inhibitors (9Schweinitz A. Steinmetzer T. Banke I.J. Arlt M.J. Sturzebecher A. Schuster O. Geissler A. Giersiefen H. Zeslawska E. Jacob U. Kruger A. Sturzebecher J. J. Biol. Chem. 2004; 279: 33613-33622Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) or uPA gene-silencing approaches (14Pulukuri S.M. Gondi C.S. Lakka S.S. Jutla A. Estes N. Gujrati M. Rao J.S. J. Biol. Chem. 2005; 280: 36529-36540Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 15Salvi A. Arici B. De Petro G. Barlati S. Mol. Cancer Ther. 2004; 3: 671-678PubMed Google Scholar) have confirmed the important role of uPA in the processes of tumor invasion and metastasis. Because uPA is crucial for invasion and metastasis, we are interested in understanding how its transcriptional activity is regulated by epigenetic mechanisms in human cancer cells.Epigenetic mechanisms play crucial roles in the regulation of gene expression by affecting chromatin accessibility. DNA methylation and histone modifications are two important epigenetic mediators of transcriptional repression (16Jaenisch R. Bird A. Nat. Genet. 2003; 33 S: 245-254Crossref Scopus (4582) Google Scholar, 17Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). A previous study showed that repression of uPA gene expression in breast cancer cells is associated with methylation of its promoter (18Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). This study further showed that the repression of uPA in prostate cancer cells is due in part to the presence of methylated cytosines throughout its promoter (19Pakneshan P. Xing R.H. Rabbani S.A. FASEB J. 2003; 17: 1081-1088Crossref PubMed Scopus (86) Google Scholar). We recently showed that uPA expression is triggered by promoter demethylation in prostate carcinomas and in metastatic prostate cells (20Pulukuri S.M. Estes N. Patel J. Rao J.S. Cancer Res. 2007; 67: 930-939Crossref PubMed Scopus (68) Google Scholar). However, the functional relevance of histone modifications in the regulation of the uPA gene expression is unknown.An increasing body of evidence indicates that changes in chromatin structure by histone modification appear to play an important role in the regulation of gene transcription. Acetylation of core histone unpacks the condensed chromatin and renders the target DNA accessible to transcriptional machinery, hence contributing to gene expression (21Jaskelioff M. Peterson C.L. Nat. Cell Biol. 2003; 5: 395-399Crossref PubMed Scopus (66) Google Scholar). In contrast, deacetylation of core histones increases chromatin condensation and prevents the binding between DNA and transcriptional factors, which lead to transcriptional silence (22Iizuka M. Smith M.M. Curr. Opin. Genet. Dev. 2003; 13: 154-160Crossref PubMed Scopus (261) Google Scholar, 23Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar). Histone acetyl transferases and histone deacetylases (HDACs) regulate the acetylation of histones and interact with components of the transcription machinery (24Cheung W.L. Briggs S.D. Allis C.D. Curr. Opin. Cell Biol. 2000; 12: 326-333Crossref PubMed Scopus (260) Google Scholar, 25Sengupta N. Seto E. J. Cell Biochem. 2004; 93: 57-67Crossref PubMed Scopus (295) Google Scholar). Several studies have shown that the inhibition of HDACs can induce gene expression in non-expressing cells (26Cong Y.S. Bacchetti S. J. Biol. Chem. 2000; 275: 35665-35668Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 27Qiu P. Li L. Circ. Res. 2002; 90: 858-865Crossref PubMed Scopus (81) Google Scholar, 28Zhang X. Wharton W. Yuan Z. Tsai S.C. Olashaw N. Seto E. Mol. Cell Biol. 2004; 24: 5106-5118Crossref PubMed Scopus (75) Google Scholar, 29Zhao S. Venkatasubbarao K. Li S. Freeman J.W. Cancer Res. 2003; 63: 2624-2630PubMed Google Scholar).In this study, we examined human uPA mRNA, uPA promoter activity, and acetylation of histones associated with uPA in human cancer cells treated with inhibitors of HDACs. We found that HDAC inhibitors induce uPA expression and activity in human cancer cells, resulting in enhanced cancer cell invasion. Our results show that histone deacetylation plays a central role in the transcriptional regulation of the uPA gene in cancer cells and that use of HDAC inhibitors results in the epigenetic activation of uPA.EXPERIMENTAL PROCEDURESReagents—TSA, SCR, and 5-aza-2′-deoxycytidine (5-aza) were purchased from Sigma. TSA and SCR were dissolved in Me2SO; 5-aza was dissolved in phosphate-buffered saline. Sodium butyrate (NaB) solution was purchased from the Upstate Group, Inc. (Lake Placid, NY).Cell Lines and Culture Conditions—Human neuroblastoma cells (SK-N-BE and SK-N-AS) and human prostate cancer cells (LNCaP and PC3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). SF-3061 human meningioma cells were provided by Dr. Anita Lal (University of California, San Francisco, CA). LNCaP cells were cultured in RPMI medium supplemented with 2 mm l-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mm HEPES, and 1.0 mm sodium pyruvate (Invitrogen). PC3, SF-3061, SK-N-BE, and SK-N-AS cells were cultured in advanced Dulbecco's modified Eagle's medium. Both media contained 10% fetal bovine serum (GIBCO Invitrogen) and 5% penicillin/streptomycin. Cells were maintained in a 37 °C incubator with a 5% CO2 humidified atmosphere.Drug Treatments—Cells were seeded at a density of 1 × 106 cells/100-mm dish and allowed to attach over 24 h. To reactivate uPA, we carried out HDAC inhibition treatment by adding 100 nm trichostatin A to the culture medium for 8 h or by treating cells for 12 h in medium supplemented with 1 mm NaB or 2 μm SCR. We carried out demethylating treatments using 5-aza (0–25 μm) for 5 days, replacing the drug and medium 24 h after the beginning of the treatment. For the synergistic study, cells were first incubated with 25 μm 5-aza for 72 h at 37 °C, followed by 100 nm TSA for an additional 24 h. The treated cells were washed once with phosphate-buffered saline. Cells were allowed to recover for 24 h in drug-free medium in a 37 °C incubator with a 5% CO2 humidified atmosphere.RT-PCR Analysis—Cellular RNA was isolated from SK-N-BE, SK-N-AS, SF-3061, and LNCaP cell lines using the Qiagen RNeasy kit. RNA (1 μg) was treated with DNase (10 units/μgof RNA for 1 h) and used as a template for the RT reaction (20 μl). The RT reaction mix (Invitrogen) contained 1 μl (10 pm) of primers. The resultant cDNA was then used in PCR reactions and analyzed by gel electrophoresis. We used the following primers for PCR: uPA-sense, 5′-TGC GTC CTG GTC GTG AGC GA-3′, and uPA-antisense, 5′-CTA CAG CGC TGA CAC GCT TG-3′; GAPDH-sense, 5′-CGG AGT CAA CGG ATT TGG TCG TAT-3′, and GAPDH-antisense, 5′-AGC CTT CTC CAT GGT GGT GAA GAC-3′. PCR conditions were as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. The final extension was at 72 °C for 5 min. All reactions were performed in triplicate. No reverse transcriptase or no template served as the negative controls.Fibrin Zymography—The enzymatic activity and molecular weight of electrophoretically separated forms of uPA were determined in conditioned medium of human cancer cell lines SK-N-BE, SK-N-AS, SF-3061, and LNCaP by SDS-PAGE as described previously (30Mohanam S. Go Y. Sawaya R. Venkaiah B. Mohan P.M. Kouraklis G.P. Gokaslan Z.L. Lagos G.K. Rao J.S. Int. J. Oncol. 1999; 14: 169-174PubMed Google Scholar). Briefly, the SDS-PAGE gel contains acrylamide to which purified plasminogen and fibrinogen were substrates before polymerization. After polymerization, equal amounts of proteins in the samples were electrophoresed and the gel was washed and stained as described previously (30Mohanam S. Go Y. Sawaya R. Venkaiah B. Mohan P.M. Kouraklis G.P. Gokaslan Z.L. Lagos G.K. Rao J.S. Int. J. Oncol. 1999; 14: 169-174PubMed Google Scholar).Chromatin Immunoprecipitation Assay—ChIP assays were performed as per the manufacturer's instructions (catalog no. 17–295, Upstate Biotechnology, Lake Placid, NY). In brief, cells (∼1 × 106 cells/100 mm dish) were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 min at 37 °C. The cells were washed twice with ice-cold phosphate-buffered saline containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml pepstatin A), harvested, and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths below 1000 bp (amplitude 60%, 4 × 10 s, Fisher Sonic Dismembrator 60, Pittsburgh, PA). After pre-clearing the lysates, 4 μg of specific antibodies (anti-acetylated histone H3, anti-acetylated histone H4, anti-HDAC1, anti-HDAC3, and anti-HDAC7, Cell Signaling Technology Inc., Beverly, MA) were used to immunoprecipitate the protein-DNA complexes. Antibody controls were also included for each ChIP assay; no precipitation was observed. The antibody-protein complexes were collected using salmon sperm DNA-protein A-agarose slurry and washed several times as per the manufacturer's instructions. The immunocomplexes were eluted with 1% SDS and 0.1 m NaHCO3, and the cross-links were reversed by incubation at 65 °C for 4 h in the presence of 200 nm NaCl. The samples were treated with proteinase K for 1 h, and the DNA was purified by phenol/chloroform extraction and ethanol precipitation. The recovered DNA was resuspended in 30 μlof H2O and used as templates for PCR of uPA or β-actin gene promoters. The following primers were used for PCR: uPA promoter-sense, 5′-CAG GTG CAT GGG AGG AAG C-3′, and uPA promoter-antisense, 5′-AGG GGC GGC GCC GGG GCG G-3′; β-actin promoter-sense, 5′-CCA ACG CCA AAA CTC TCC C-3′, and β-actin promoter-antisense, 5′-AGC CAT AAA AGG CAA CTT TCG-3′. Initially, PCR was performed with different numbers of cycles or dilutions of input DNA to determine the linear range of the amplification; all results shown fall within this range. Following 30 cycles of amplification, PCR products were run on 2% agarose gels and analyzed by ethidium bromide staining.Restriction Enzyme Accessibility Assay—The nuclei of SK-N-BE, SK-N-AS, SF-3061, LNCaP, and PC3 cells were extracted according to published methods (31Gerber A.N. Klesert T.R. Bergstrom D.A. Tapscott S.J. Genes Dev. 1997; 11: 436-450Crossref PubMed Scopus (231) Google Scholar) and digested with restriction enzymes PvuII or PstI (New England Biolabs, Ipswich, MA). DNA was then extracted from the digested nuclei with the proteinase K/phenol procedure (32Cameron E.E. Bachman K.E. Myohanen S. Herman J.G. Baylin S.B. Nat. Genet. 1999; 21: 103-107Crossref PubMed Scopus (1669) Google Scholar). DNA from PvuII-digested nuclei was amplified by PCR with primers uPA-F1 (5′-CAG GTG CAT GGG AGG AAG CA-3′) and uPA-R (5′-GGC CAC CGG GAC TGC CCC AG-3′) and electrophoresed on a 2% agarose gel (Fig. 3A). The absence or presence of a 925-bp fragment indicates that PvuII is or is not accessible to the chromatin in the region of uPA promoter, respectively. To access the input of chromatin, another upstream primer, uPA-F2 (5′-TGC GTC CTG GTC GTG A-3′), was also mixed in the PCR (Fig. 3A). The amount of PvuII-digested chromatin was represented by the ratio of 925:241 bp. Similarly, DNA from PstI-digested nuclei was also amplified by PCR with primers uPA-F1, uPA-F2, and uPA-R. The absence or presence of a 925-bp product (compared with a control of 241 bp) indicates that PstI is or is not accessible to the chromatin, respectively.Promoter Activity Assay—A PCR product spanning nucleotide positions –562 to +83 of uPA promoter sequence (GenBank™ accession number X02419) was amplified using LNCaP genomic DNA and subsequently cloned into the pGL3 basic plasmid (Promega, Madison, WI). The uPA-luc construct and control null vector without the uPA promoter insert (pGL3) were transiently transfected into LNCaP cells using the FuGENE HD transfection method (Roche Applied Science) with a β-galactosidase plasmid for normalization. The cells were treated with 100 nm TSA for 8 h following transfection. The cells were harvested 24 h after TSA treatment, and promoter activities were determined using the luciferase assay system as recommended by Promega Corp.Matrigel Invasion Assay—We used 6.5 mm-diameter Transwell inserts (Costar, Cambridge, MA) with the 8-μm pore membranes coated with Matrigel (BD Biosciences, Bedford, MA) to assess the invasive potentials of human cancer cells before and after treatment with HDAC inhibitors. Cells were detached, washed twice in phosphate-buffered saline, and resuspended in serum-free advanced Dulbecco's modified Eagle's medium. A total of 5 × 105 cells in 0.2 ml was placed in the upper chamber of a Transwell, and the lower chamber was filled with 400 μl of advanced Dulbecco's modified Eagle's medium/10% fetal bovine serum. After a 24-h incubation period, the cells in the upper chamber that did not migrate were gently scraped away, and adherent cells present on the lower surface of the insert were stained with Hema-3 and photographed. To determine the importance of uPA in HDAI-induced invasion, LNCaP cells stably overexpressing uPA or vector-based shRNA against uPA were used in the Matrigel invasion assay along with each control cell. Cells were detached and subjected to HDAI-induced in vitro Matrigel invasion assay as described above. Construction and characteristics of the uPA shRNA vector have been previously described (20Pulukuri S.M. Estes N. Patel J. Rao J.S. Cancer Res. 2007; 67: 930-939Crossref PubMed Scopus (68) Google Scholar). For stable expression of uPA, LNCaP cells were transfected with the neomycin-selectable pCMV-uPA plasmid or with a control, neomycin-resistant expression vector pCMV. Stable transfection was performed using 5 μg/ml DNA and 10 μl/ml Lipofectin reagent (Invitrogen) following the manufacturer's protocol. The selection medium containing 1 mg/ml Geneticin (G418, Invitrogen) was added to the cells 72 h after transfection to select for neomycin-resistant transfectants.Nuclear Extract Preparation and Immunoblot Analysis—Nuclear extracts were prepared from control and TSA-treated SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells using a nuclear extraction kit from Panomics, Inc. (Redwood City, CA) as per the manufacturer's instructions. Equal amounts of nuclear extracts were resolved by SDS-PAGE and then blotted with rabbit anti-human HDAC1, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, and histone H3 (Cell Signaling Technology, Beverly, MA). Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were used for detection of immunoreactive proteins by chemiluminescence (Amersham Biosciences).Transfection of siRNAs—To silence HDAC1 expression by RNA interference, 150,000 cells per well were seeded in a 6-well plate at least 20 h before transfection. Either small interfering RNAs against HDAC1 or nonspecific control (siControl) were transfected using siLentFect transfection reagent (Bio-Rad) as per the manufacturer's instructions. Two days post-transfection, the nearly confluent cells were trypsinized, and the cells were used for fibrin zymography, RT-PCR analysis, ChIP assay, and Matrigel invasion assays. The sequences of siRNAs for HDAC1 gene knockdown were as follows: siHDAC1, 5′-TAA GGT TCT CAA ACA GTC G-3′; siHDAC2, 5′-TTT GAA GTT GGA AGA GTT C-3′; and siHDAC3, 5′-TTC AAT AAG GGC ACC TTT C-3′. "Smart pool" siRNAs that combined the above HDAC 1–3 siRNAs targeted against different regions of the HDAC1 mRNA sequence (NM_004964) were used for transfection to increase the knockdown effect.Densitometry—ImageJ software (National Institutes of Health) was used to quantify the band intensities. Data are represented as relative to the intensity of the indicated loading control.Statistical Analysis—Statistical comparisons were performed using analysis of variance for analysis of significance between different values using GraphPad Prism software (San Diego, CA). Values are expressed as mean ± S.D. from at least three separate experiments, and differences were considered significant at a p value of <0.05.RESULTSInhibition of Histone Deacetylation Activates uPA Expression—Besides DNA methylation, another epigenetic mechanism by which gene expression can be repressed involves deacetylation of chromosomal histones. Hypoacetylated chromatin is transcriptionally silent (23Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar). Inhibition of histone deacetylation can be accomplished by treatment with HDAIs such as TSA, NaB, and SCR (33Marks P.A. Rifkind R.A. Richon V.M. Breslow R. Clin. Cancer Res. 2001; 7: 759-760PubMed Google Scholar). To determine whether HDAI induces acetylation of histones and the concomitant induction of uPA expression, we treated uPA-silenced human cancer cell lines that originated from neuroblastoma (SK-N-BE and SK-N-AS), meningioma (SF-3061), and prostate (LNCaP) with 100 nm TSA for 8 h and performed immunoblot analysis on the nuclear extracts using antibodies to acetylated histones H3 and H4. Accumulation of acetylated histones was observed in TSA-treated human cancer cells (Fig. 1A). If histone deacetylation is associated with transcriptional repression, then histone acetylation following TSA treatment should lead to uPA expression. According to the RT-PCR results, treatment with TSA induced uPA mRNA expression in all four human cancer cell lines (Fig. 1B, top). Fibrin zymography for uPA activity supported the RT-PCR analyses (Fig. 1B, bottom). A similar trend was observed at both the activity and mRNA levels when these cells were treated with NaB or with SCR (Fig. 1, C and D). These results indicate that the effect of TSA on uPA expression can be extended to other HDAIs (i.e. NaB and SCR). They also suggest that the induction of uPA expression and activity by HDAIs was not confined to a single type of human cancer cell line model.FIGURE 1Inhibition of HDAC activity induces the acetylation of histones and concomitant uPA expression in human cancer cells. A, left, nuclear extracts were isolated from control and TSA-treated SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells, and immunoblot analysis was performed using anti-acetyl histone H3, anti-acetyl histone H4, and histone H3 antibodies. Histone H3 was utilized as a loading control. Right, densitometric analysis of immunoblots. Data are normalized to H3, averaged, and expressed as percentage of control (CTL = 1). B, left, uPA mRNA expression (top) and activity levels (bottom) in control and TSA-treated SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells were analyzed by RT-PCR and fibrin zymography, respectively. Right, densitometric analysis of RT-PCR gels. Data are normalized to GAPDH, averaged, and expressed as percentage of control (CTL = 1). C, left, uPA mRNA expression (top) and activity levels (bottom) in control and NaB-treated SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells were analyzed by RT-PCR and fibrin zymography, respectively. Right, densitometric analysis of RT-PCR gels as described in B. D, left, uPA mRNA expression (top) and activity levels (bottom) in control and SCR-treated SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells were analyzed by RT-PCR and fibrin zymography, respectively. Right, densitometric analysis of RT-PCR gels as described in B. (All results are representative of three separate experiments.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because it is known that uPA can be silenced by promoter DNA methylation (18Guo Y. Pakneshan P. Gladu J. Slack A. Szyf M. Rabbani S.A. J. Biol. Chem. 2002; 277: 41571-41579Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), we examined the effects of the DNA methylation inhibitor 5-aza on the re-activation of uPA in human cancer cell lines by RT-PCR. However, treatment with higher doses (10–25 μm) of 5-aza for 5 days individually or in combination with TSA did not restore or enhance the expression of uPA in all cell lines analyzed (data not shown).TSA Induces Accumulation of Acetylated Histones in Chromatin Associated with the uPA Gene—Previous studies have shown that TSA, as well as other HDAIs, induce the accumulation of acetylated histones in human cells (34Marks P. Rifkind R.A. Richon V.M. Breslow R. Miller T. Kelly W.K. Nat. Rev. Cancer. 2001; 1: 194-202Crossref PubMed Scopus (1680) Google Scholar, 35Munster P.N. Troso-Sandoval T. Rosen N. Rifkind R. Marks P.A. Richon V.M. Cancer Res. 2001; 61: 8492-8497PubMed Google Scholar, 36Richon V.M. Sandhoff T.W. Rifkind R.A. Marks P.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10014-10019Crossref PubMed Scopus (1008) Google Scholar, 37Sambucetti L.C. Fischer D.D. Zabludoff S. Kwon P.O. Chamberlin H. Trogani N. Xu H. Cohen D. J. Biol. Chem. 1999; 274: 34940-34947Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). ChIP analysis was used to examine the effect of HDAC inhibition on the acetylation of histones H3 and H4, which are associated with the uPA gene promoter. Chromatin fragments from human cancer cells cultured with or without TSA for 8 h were immunoprecipitated with antibodies to acetylated histones H3 or H4. DNA from the immunoprecipitates was isolated, and PCR was performed using uPA promoter primers (Fig. 2A). Acetylation of histones H3 and H4 associated with the uPA promoter region in all four human cancer cell lines was undetectable before TSA treatment. However, we observed remarkable increases in the acetylation of histones H3 and H4 in the promoter region of all four human cancer cell lines after treatment with TSA (100 nm, 8 h) (Fig. 2B). The accumulation of acetylated histones H3 and H4 confirmed that histone deacetylation was involved in the transcriptional repression of uPA. We also carried out PCR on the same set of immunoprecipitated DNA fractions for β-actin promoter as a control. The relative levels of acetylated histones H3 and H4 at β-actin promoter was similar in all TSA-treated and untreated cells.FIGURE 2TSA induces accumulation of acetylated histones H3 and H4 in chromatin associated with the uPA gene. A, schematic representation of the uPA promoter region and the location of primers used for PCR amplification in the ChIP assay. Bent arrow, transcriptional start site. B, chromatin fragments from SK-N-BE, SK-N-AS, SF-3061, and LNCaP cells cultured with (+) or without (–) TSA for 8 h were immunoprecipitated with antibody to acetylated (Ac) histones H3 and H4 or control normal rabbit serum (NRS). PCR primers for the uPA and β-actin promoters were used to amplify the DNA isolated from the immunoprecipitated chromatin as described under "E
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