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Polypyrimidine Tract-binding Protein (PTB) Differentially Affects Malignancy in a Cell Line-dependent Manner

2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês

10.1074/jbc.m803682200

ISSN

1083-351X

Autores

Chen Wang, John T. Norton, Supurna Ghosh, Julie Kim, Kazuo Fushimi, Jane Y. Wu, M. Sharon Stack, Sui Huang,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment. RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment. The polypyrimidine tract-binding protein (PTB), 3The abbreviations used are: PTB, polypyrimidine tract-binding protein; RRM, RNA recognition motif; siRNA, small interfering RNA; pol, polymerase; Br, bromo; RT, reverse transcription; PBS, phosphate-buffered saline; GFP, green fluorescent protein; oligo, oligonucleotide. 3The abbreviations used are: PTB, polypyrimidine tract-binding protein; RRM, RNA recognition motif; siRNA, small interfering RNA; pol, polymerase; Br, bromo; RT, reverse transcription; PBS, phosphate-buffered saline; GFP, green fluorescent protein; oligo, oligonucleotide. also termed heterogeneous nuclear ribonucleoprotein I, is a 57-kDa RNA-binding protein that binds preferentially to pyrimidinerich sequences (1Ghetti A. Pinol-Roma S. Michael W.M. Morandi C. Dreyfuss G. Nucleic Acids Res. 1992; 20: 3671-3678Crossref PubMed Scopus (271) Google Scholar, 2Gil A. Sharp P.A. Jamison S.F. 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Biol. 1997; 7: R705-R708Abstract Full Text Full Text PDF PubMed Google Scholar) and acts as a splicing repressor in alternative splicing of pre-mRNA (5Perez I. Lin C.H. McAfee J.G. Patton J.G. RNA (N. Y.). 1997; 3: 764-778PubMed Google Scholar, 7Perez I. McAfee J.G. Patton J.G. Biochemistry. 1997; 36: 11881-11890Crossref PubMed Scopus (147) Google Scholar, 8Wagner E.J. Garcia-Blanco M.A. Mol. Cell. Biol. 2001; 21: 3281-3288Crossref PubMed Scopus (304) Google Scholar, 9Wagner E.J. Garcia-Blanco M.A. Mol. Cell. 2002; 10: 943-949Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 10Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1970) Google Scholar, 11Robinson F. Smith C.W.J. J. Biol. Chem. 2006; 281: 800-806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 12Spellman R. Smith C.W.J. Trends Biochem. Sci. 2006; 31: 73-76Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). PTB is also involved in 3′ end polyadenylation of pre-mRNA (13Lou H. Gagel R.F. Berget S.M. Genes Dev. 1996; 10: 208-219Crossref PubMed Scopus (130) Google Scholar, 14Lou H. Helfman D.M. Gagel R.F. Berget S.M. Mol. Cell. Biol. 1999; 19: 78-85Crossref PubMed Scopus (130) Google Scholar, 15Castelo-Branco P. Furger A. Wollerton M. Smith C. Moreira A. Proudfoot N. Mol. Cell. Biol. 2004; 24: 4174-4183Crossref PubMed Scopus (136) Google Scholar) and is important for translational regulation of certain RNA transcripts through internal ribosome entry sites (16Hellen C.U. Pestova T.V. Litterst M. Wimmer E. J. Virol. 1994; 68: 941-950Crossref PubMed Google Scholar, 17Kaminski A. Hunt S.L. Patton J.G. Jackson R.J. RNA (N. Y.). 1995; 1: 924-938PubMed Google Scholar, 18Witherell G.W. Schultz-Witherell C.S. Wimmer E. Virology. 1995; 214: 660-663Crossref PubMed Scopus (31) Google Scholar, 19Schneider R. Agol V.I. Andino R. Bayard F. Cavener D.R. Chappell S.A. Chen J.J. Darlix J.L. Dasgupta A. Donze O. Duncan R. Elroy-Stein O. Farabaugh P.J. Filipowicz W. Gale Jr., M. et al.Mol. Cell. Biol. 2001; 21: 8238-8246Crossref PubMed Scopus (44) Google Scholar, 20Pickering B.M. Mitchell S.A. Evans J.R. Willis A.E. Nucleic Acids Res. 2003; 31: 639-646Crossref PubMed Scopus (69) Google Scholar). In addition, PTB shuttles between the nucleus and the cytoplasm (21Kamath R.V. Leary D.J. Huang S. Mol. Biol. Cell. 2001; 12: 3808-3820Crossref PubMed Scopus (58) Google Scholar), which is regulated through phosphorylation by 3′,5′-cAMP-dependent protein kinase (22Xie J. Lee J.-A. Kress T.L. Mowry K.L. Black D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8776-8781Crossref PubMed Scopus (151) Google Scholar). Alternative splicing is a process that allows multiple different proteins to be made from the same pre-mRNA by either including or excluding particular exons during pre-mRNA splicing. PTB plays a key role in alternative site selection for many gene products by acting as a splicing repressor that prevents the inclusion of target exons (11Robinson F. Smith C.W.J. J. Biol. Chem. 2006; 281: 800-806Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Gromak N. Matlin A.J. Cooper T.A. Smith C.W. RNA (N. Y.). 2003; 9: 443-456Crossref PubMed Scopus (88) Google Scholar, 24Rideau A.P. Gooding C. Simpson P.J. Monie T.P. Lorenz M. Huttelmaier S. Singer R.H. Matthews S. Curry S. Smith C.W.J. Nat. Struct. Mol. Biol. 2006; 13: 839-848Crossref PubMed Scopus (76) Google Scholar, 25Sauliere J. Sureau A. Expert-Bezancon A. Marie J. Mol. Cell. Biol. 2006; 26: 8755-8769Crossref PubMed Scopus (69) Google Scholar). Changes in alternative splicing sites have been previously correlated with malignant transformation (26Jin W. McCutcheon I.E. Fuller G.N. Huang E.S. Cote G.J. Cancer Res. 2000; 60: 1221-1224PubMed Google Scholar, 27Jin W. Bruno I.G. Xie T.-X. Sanger L.J. Cote G.J. Cancer Res. 2003; 63: 6154-6157PubMed Google Scholar, 28He X. Ee P.L. Coon J.S. Beck W.T. Clin. Cancer Res. 2004; 10: 4652-4660Crossref PubMed Scopus (89) Google Scholar, 29McCutcheon I.E. Hentschel S.J. Fuller G.N. Jin W. Cote G.J. Neuro-Oncol. 2004; 6: 9-14Crossref PubMed Scopus (40) Google Scholar), and the expression level of PTB has been found elevated in transformed cells. Such an elevation is responsible for the increases in fibroblast growth factor receptor-1α-exon skipping in glioblastoma multiforme tumors (26Jin W. McCutcheon I.E. Fuller G.N. Huang E.S. Cote G.J. Cancer Res. 2000; 60: 1221-1224PubMed Google Scholar). Increases in PTB expression are also associated with changes in alternative splicing of multidrug resistance protein 1, which contributes to the drug-resistant phenotype associated with many cancers (28He X. Ee P.L. Coon J.S. Beck W.T. Clin. Cancer Res. 2004; 10: 4652-4660Crossref PubMed Scopus (89) Google Scholar). In addition to PTB, changes in the expression levels of other factors involved in alternative splicing, such as SR proteins, have been found to impact the metastatic phenotype. A classic example demonstrated that a CD44 splice variant, CD44 v6, confers metastatic potential when expressed in nonmetastatic cells (30Gunthert U. Hofmann M. Rudy W. Reber S. Zoller M. Haussmann I. Matzku S. Wenzel A. Ponta H. Herrlich P. Cell. 1991; 65: 13-24Abstract Full Text PDF PubMed Scopus (1597) Google Scholar). Therefore, changes in alternative splicing dynamics in tumor cells likely modify gene expression, in which the expression of functionally altered proteins may directly contribute to the malignant phenotype (31Venables J.P. BioEssays. 2006; 28: 378-386Crossref PubMed Scopus (256) Google Scholar). Being a splice repressor, PTB may influence the transformed phenotype through changing alternative splicing patterns. PTB itself also undergoes alternative splicing and has three splicing isoforms. PTB1 is the smallest, whereas PTB2 and -4 have an additional 19 or 26 amino acids, respectively, between RRM 2 and 3 as a result of exon 9 inclusion (1Ghetti A. Pinol-Roma S. Michael W.M. Morandi C. Dreyfuss G. Nucleic Acids Res. 1992; 20: 3671-3678Crossref PubMed Scopus (271) Google Scholar, 2Gil A. Sharp P.A. Jamison S.F. Garcia-Blanco M.A. Genes Dev. 1991; 5: 1224-1236Crossref PubMed Scopus (224) Google Scholar). These isoforms are differentially effective in the alternative splicing of α-tropomyosin. PTB4 has strongest influence and PTB1 the weakest on exon 3 skipping in vivo and in vitro (32Wollerton M.C. Gooding C. Robinson F. Brown E.C. Jackson R.J. Smith C.W. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (115) Google Scholar). However, differential splicing efficiency of the individual PTB isoforms is not observed for all PTB substrates, as demonstrated by the equal efficiency of α-actinin exon skipping by all isoforms (32Wollerton M.C. Gooding C. Robinson F. Brown E.C. Jackson R.J. Smith C.W. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (115) Google Scholar). Therefore, the differential expression of PTB isoforms may enormously influence gene expression because of the large number of PTB substrates present in the cell and thus differentially affect cellular behavior based on the amount of certain PTB substrates that are expressed in a given cell type. Although several studies have demonstrated altered PTB expression in cancer cells (31Venables J.P. BioEssays. 2006; 28: 378-386Crossref PubMed Scopus (256) Google Scholar, 33Faustino N.A. Cooper T.A. Genes Dev. 2003; 17: 419-437Crossref PubMed Scopus (983) Google Scholar), fundamental questions regarding the role of PTB in cancer cells remain unresolved. It is not clear whether increased PTB expression is a phenomenon common to cancers of all origins, or whether PTB isoform expression changes similarly among cancers from different origins, or whether increased PTB expression is important for the transformed phenotype. Recently, a study using siRNA knockdown technique demonstrated that PTB promotes the malignant phenotype in ovarian tumor cell lines (34He X. Pool M. Darcy K.M. Lim S.B. Auersperg N. Coon J.S. Beck W.T. Oncogene. 2007; 26: 4961-4968Crossref PubMed Scopus (102) Google Scholar). To further address these issues, we investigated the changes in PTB expression in cancer cells from multiple origins and compared the PTB isoform profiles in these cells. We examined the role of PTB in malignant transformation by knocking down PTB expression in cultured tumor and nontransformed cells. We found that PTB levels generally increase in cancer cells from a variety of tissues; however, the expression levels of the three individual PTB isoforms are heterogeneous among different cell types. PTB knockdown by siRNA significantly reduces the growth rate for both cancer and normal cell lines, and reduces anchorage-independent growth in tumor cells to a great extent than monolayer culture. In addition, PTB knockdown inhibits the invasive capacity of two cancer cell lines but increased invasion in another. However, overexpression of PTB in normal and immortalized cells does not increase proliferation or induce traits associated with transformation in vitro. Our findings suggest that PTB itself is not transforming but may support or interfere with malignancy depending on the specific cellular environment as it can promote transformed phenotypes in some cells while antagonizing them in others. Cell Culture and Tissue Specimens—HeLa (human cervical cancer), Wacar and Homa (normal human skin fibroblasts), HEK-293 (human embryonic kidney transformed with adenovirus 5 DNA), and NIH-3T3 (Mus musculus, fibroblasts) were maintained in Dulbecco's modified Eagle's medium. PC-3, PC-3M, PC-3M Pro4, and PC-3M LN4 prostate cancer cell lines were generous gifts from the laboratories of Dr. Zhou Wang and Dr. Chung Lee (Northwestern University) and cultured in RPMI 1640 medium. WI-38 normal lung fibroblasts were grown in minimal essential medium. All media were supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 100 units/ml penicillin and streptomycin unless otherwise noted. CG cells (human neuroblastoma), T84 cells (human colon carcinoma), and SAOS-2 (osteosarcoma) were cultured according to the protocols provided by ATCC Cultures™. Stably expressing NIH-3T3 cells were created by transfecting ∼2 × 106 cells with 2 μg of either PTB1-GFP vector (39Wang C. Politz J.C. Pederson T. Huang S. Mol. Biol. Cell. 2003; 14: 2425-2435Crossref PubMed Scopus (54) Google Scholar) or constitutively active K-Ras vector (courtesy of Dr. William Hahn-Addgene plasmid 9051) or 2 μg of each at the same time and then selecting with 500 μg/ml G-418 (PTB-GFP) and/or 5 μg/ml puromycin (H-Ras) for 2 weeks. The resulting stable, nonclonal cell lines were utilized for assays within 1 month of creation. Human endometrial tissue samples were obtained by surgical resection, trypsinized, and seeded in culture (Robert H. Lurie Comprehensive Cancer Center of Northwestern University). Histopathological examination allowed the samples to be classified as benign, grade-1, grade-2, or grade-3 endometrial tumors. All cell culture products were obtained from Invitrogen, and all other reagents mentioned under "Materials and Methods" were obtained from Sigma unless otherwise noted. Immunostaining—Cells were fixed with 4% paraformaldehyde in PBS for 10 min followed by 5 min of permeabilization with 0.5% w/v Triton X-100 in PBS at room temperature. Primary antibody was applied for 1 h, and cells were washed with PBS three times for 10 min. The primary antibody, SH54 (anti-PTB) (35Huang S. Deerinck T.J. Ellisman M.H. Spector D.L. J. Cell Biol. 1997; 137: 965-974Crossref PubMed Scopus (97) Google Scholar), was used at a 1:300 dilution in PBS, and secondary anti-mouse antibodies conjugated to fluorescein isothiocyanate or Texas Red were used at a 1:200 dilution (Jackson ImmunoResearch Laboratories). Coverslips were analyzed with a Nikon Eclipse E800 microscope equipped with a SenSys cooled CCD camera (Photometrics). Images were captured using Metamorph image acquisition software (Universal Imaging). Protein Electrophoresis and Immunoblotting—Protein extracts were prepared by sonicating tissue or cells in RIPA buffer containing 1% Nonidet P-40, 1% deoxycholic acid, sodium salt, 0.1% SDS, 10 mm Tris-HCl, pH 7.4, and 150 mm NaCl. Protein concentrations were determined with the BCA protein assay kit (Pierce). Equal amounts of each protein sample were separated on 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Antibodies used for Western blot analysis were rabbit or mouse anti-PTB (SH54) at a 1:800 dilution, rabbit anti-actin (Sigma) at a 1:2000 dilution, mouse anti-GFP (BD Biosciences) at a 1:1000 dilution, rabbit anti-K-Ras (Santa Cruz Biotechnology) at a 1:500 dilution, and horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch) at a 1:10,000 dilution. SuperSignal West Pico Chemiluminescent Substrate (Pierce) detection reagents were used to detect immunoreactive bands. Northern Blotting—To determine the RNA expression level of PTB in different cell lines by Northern analysis, total RNAs were extracted from different cell lines with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNAs from different cell lines were loaded (5 μg/lane) and run on a 1% agarose gel and subsequently transferred onto GeneScreenPlus membranes (PerkinElmer Life Sciences) by capillary action with a high salt solution. Hybridization and washing conditions were standard as described previously (36Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 7.39-7.52, Cold Spring Harbor, NYGoogle Scholar). A 32P-labeled PTB probe was used to detect the expression level of RNA with labeled glyceraldehyde-3-phosphate dehydrogenase probe used as loading control. 32P was obtained from Amersham Biosciences. RT-PCR—RNA was converted to cDNA, and the DNA was amplified by PCR with a forward primer (5′-ACCAGCCTCAACGTCAAGTA) and a reverse primer (5′-GGGTTGAGGTTGCTGACCAG) in a single reaction. These primers were designed to include the alternatively spliced region of PTB so ratios of isoforms could be directly compared among cell lines. Reverse transcription was performed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) on total RNA obtained via TRIzol isolation from cell lines. PCR was carried out with 30 cycles at 95 °C for 30 s, 57 °C for 1 min, and 72 °C for 90 s. The PCR products were resolved on a 2% agarose gel. The intensity of the isoform bands were measured with Kodak MI software, which allowed for determination of isoform ratios. Alternative Splicing Efficiency Assay—A caspase 2 minigene was used as described previously (37Cote J. Dupuis S. Jiang Z. Wu J.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 938-943Crossref PubMed Scopus (52) Google Scholar). HEK-293 cells were plated onto 6-well culture plates at 60–70% confluence. After 24 h, 4 μg of GFP-tagged PTB expression vectors and 1 μg of reporter minigene were introduced into cells by a standard calcium phosphate precipitation protocol. Total RNA was purified with RNeasy mini kit (Qiagen) from 6-well culture plates 36 h after transfection. Alternative splicing products of the caspase 2 minigene were detected using RT-PCR in the presence of [32P]dCTP (GE Healthcare) as described previously (37Cote J. Dupuis S. Jiang Z. Wu J.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 938-943Crossref PubMed Scopus (52) Google Scholar, 38Zhang W.-J. Wu J.Y. Mol. Cell. Biol. 1996; 16: 5400-5408Crossref PubMed Scopus (83) Google Scholar). PCR products were fractionated with 6% polyacrylamide gel containing 1× TBE buffer and then detected and quantified using a PhosphorImager BAS-1800II (Fuji Film). RNA Interference—Double-stranded RNA was chemically synthesized, deprotected, and purified by Dharmacon Research, Inc. One strand of the double-stranded RNA was homologous to the PTB mRNA sequence 5′-UGACAAGAGCCGUGACUAC(dTdT)-3′. The scramble control siRNA was from Ambion, Inc. (Silencer® negative control 2 siRNA). Transfection of siRNA duplexes into various cell lines was conducted as described previously according to the manufacturer's instructions (Oligofectamine reagent, Invitrogen) (39Wang C. Politz J.C. Pederson T. Huang S. Mol. Biol. Cell. 2003; 14: 2425-2435Crossref PubMed Scopus (54) Google Scholar). Cells were utilized for subsequent experiments 72 h post-transfection. In Vivo Br-UTP Incorporation—Seventy two hours after transfection with PTB siRNA, cells were rinsed once in Glycerol Buffer (20 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 25% glycerol, 0.5 mm EGTA, 0.5 mm phenylmethylsulfonyl fluoride) and permeabilized in the Glycerol Buffer with 50 μg/ml digitonin for 3 min. Cells were then incubated in transcription mixture (100 mm KCl, 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 0.5 mm EGTA, 25% glycerol, 2 mm ATP, 0.5 mm CTP, 0.5 mm GTP, 0.2 mm Br-UTP) at 37 °C for 5 min and then fixed in 4% (w/v) paraformaldehyde in PBS for 10 min. Fixed cells were double-labeled with anti-PTB at a 1:300 dilution and anti-BrdUrd that also recognizes Br-UTP (Sigma) at a 1:50 dilution and subsequently prepared as described above. Anchorage-independent Growth Assay—Seventy two hours after transfection, cells from the PTB siRNA and the control siRNA-treated dishes were trypsinized and counted using a hemocytometer. The same number of cells (about 5 × 104) from all experimental conditions were added into 2 ml of 1.5% (w/v) methylcellulose media and seeded onto 1% agarosecoated 35-mm Petri dishes and allowed to grow for 10 days. At this point, pictures were taken using phase microscopy to show the colony formation. Then the media containing the cells were removed from the dish, put in a 15-ml tube, vigorously pipetted and vortexed to break up the colonies, allowed to sit for 5 min, and then gently mixed as to suspend the cells homogeneously while avoiding air bubbles. The cell number relative to control was determined by measuring the scattering at 650 nm using a spectrophotometer (Beckman DU-64). Invasion Assay—Invasive activity was determined via the transwell Matrigel invasion (Boyden chamber) assay. Transwell inserts (0.8 μm; BD Biosciences) were coated with Matrigel (100 μg in 100 μl, for 1 h at room temperature), and coated inserts were then washed with PBS and used immediately. Seventy two hours after siRNA transfection, 2 × 105 cells from each experimental condition were added to the upper chamber in 500 μl of serum-free medium. Twenty four hours after incubation at 37 °C, the noninvading cells were removed from the upper chamber with a cotton swab, and invading cells adherent to the bottom of membrane were fixed and stained using a Diff-Quick staining kit (DADE AG). Invading cells were counted by tallying the number of cells in 10 random fields under a ×20 objective using an ocular micrometer. Data were expressed as average relative (compared with control) number of migrating cells in 10 fields from six experiments (40Ghosh S. Munshi H.G. Sen R. Linz-McGillem L.A. Goldman R.D. Lorch J. Green K.J. Jones J.C. Stack M.S. Cancer. 2002; 95: 2524-2533Crossref PubMed Scopus (28) Google Scholar). Plasminogen Activator Assay—Net plasminogen activator activity in conditioned media was quantified using a coupled assay to monitor plasminogen activation and the resulting plasmin hydrolysis of a colorimetric substrate (d-Val-Leu-Lys-p-nitroanilide; Sigma) as described previously (41Stack S. Gonzalez-Gronow M. Pizzo S.V. Biochemistry. 1990; 29: 4966-4970Crossref PubMed Scopus (69) Google Scholar, 42Ghosh S. Brown R. Jones J.C. Ellerbroek S.M. Stack M.S. J. Biol. Chem. 2000; 275: 23869-23876Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Transformed Cell Lines and Cancer Tissues Express Increased Levels of PTB—To evaluate the expression level of PTB in tumor cell lines and human tissue samples, we used both immunofluorescent staining and Western blotting. For the immunofluorescent staining, tumor and normal cells were immunolabeled in parallel, and the images were captured under the same image acquisition settings. The results show a significant increase in nuclear labeling intensity in tumor cells over that in normal cells as exemplified in PC-3M (a human prostate cancer cell line), HeLa (human cervical cancer), CG cells (human neuroblastoma), SAOS-2 (osteosarcoma), versus WI-38 (a normal human lung fibroblast cell line) (Fig. 1A). Many malignant cells also contain the perinucleolar compartment (Fig. 1A, arrowheads), a nuclear structure that is highly enriched with PTB (43Kopp K. Huang S. J. Cell Biochem. 2005; 95: 217-225Crossref PubMed Scopus (25) Google Scholar). To quantify the expression of total PTB protein in cancer cell lines from various tissue origins, we performed Western blotting using the anti-PTB antibody SH54 (35Huang S. Deerinck T.J. Ellisman M.H. Spector D.L. J. Cell Biol. 1997; 137: 965-974Crossref PubMed Scopus (97) Google Scholar). The panel of human cancer cells examined (HeLa, CG, T84, SAOS-2, HEK-293, PC-3, PC-3M, PC-3M LN4, and PC-3M Pro4) was derived from a broad spectrum of tissue types and represents cells of varying degrees of malignancy. The normal cell lines evaluated were WI-38 and Homa, which are human fibroblasts. Western blotting demonstrates that the level of PTB protein is generally increased in transformed cell lines examined when compared with the normal cell lines (Fig. 2B). The increases in protein expression is consistent with the increased level of steady state PTB mRNA in tumor cells as measured by Northern blotting (Fig. 1B). Densitometry quantification of the PTB protein levels shows that most tumor cell lines express PTB at a level 2-fold, or greater, than normal cells (data not shown). To evaluate whether the increases in PTB levels are correlated with the degree of malignancy, we compared PTB expression in three prostate cancer cell lines of varying levels of malignancy with the parental PC-3 line. PC-3 cells were originally isolated from a human prostate cancer, and PC-3M was created by implanting a PC-3 xenograft tumor into a nude mouse, allowing distant metastases to form, and subsequently removing a metastatic lesion to culture (44Kozlowski J.M. Fidler I.J. Campbell D. Xu Z.L. Kaighn M.E. Hart I.R. Cancer Res. 1984; 44: 3522-3529PubMed Google Scholar). PC-3M LN4 is enriched with highly metastatic cells through four iterations of inoculating PC-3M cells into the mouse prostate and isolating the metastatic tumor cells. In contrast, PC-3M Pro4 is highly concentrated with nonmetastatic tumor cells through four iterations of inoculating PC-3M cells into mouse prostate and isolating tumor cells localized to the prostate (45Pettaway C.A. Pathak S. Greene G. Ramirez E. Wilson M.R. Killion J.J. Fidler I.J. Clin. Cancer Res. 1996; 2: 1627-1636PubMed Google Scholar). If PTB level is directly related to the metastatic capacity, we would expect PTB expression in the PC-3 panel of cell lines to correlate with their metastatic capacity; however, PTB is expressed at a higher level in the PC-3M cells compared with PC-3 cells, and there is little difference among the three PC-3M derivatives (Fig. 2B).FIGURE 2Differential expression patterns of PTB isoforms. A, schematic illustration of PTB isoforms and PCR primers used to detect all PTB splicing isoforms. B, Western blotting shows PTB levels are generally higher in transformed cell lines when compared with normal cell lines. The shortest isoform, PTB1, shows significant increases in all cancer cell lines tested compared with normal cells, in which PTB1 is often below the level of detection. The normal cell lines used were WI-38 (lung fibroblasts) and Homa (human skin fibroblasts). C, quantitative RT-PCR shows that the ratio of PTB1:PTB2 is substantially increased in some tumor cell lines but not in all.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To evaluate the expression of PTB in human tumor tissues, we examined the levels of PTB expression in freshly isolated normal endometrial and endometrial adenocarcinoma cells. PTB expression in cells isolated from five adenocarcinoma tissues of varying grades was compared with two normal endometrial tissue cells by Western blotting, and the results show the total level of PTB is increased in tumor tissues, which is consistent with the observations in cancer cell lines (Fig. 1C). However, there is no obvious correlation between the expression level of PTB and tumor grades as exemplified by the grade 3 tumor samples, in which one shows a substantial increase in PTB expression although the other is comparable with or slightly less than the grade 1 tumor (Fig. 1C). The lack of correlation between the levels of PTB and the severity of the disease are consistent with the findings from the prostate cancer PC-3 cell line derivatives (Fig. 2B). Together, these data demonstrate that PTB levels generally increase in cancer

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