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• W1986174976 abstract "Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). Because the truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells, we tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. Additionally, the catalytically inactive PTPROt acted as a trapping mutant that was also able to inhibit anchorage independence and facilitate apoptosis of K562 cells. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia. Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). Because the truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells, we tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. Additionally, the catalytically inactive PTPROt acted as a trapping mutant that was also able to inhibit anchorage independence and facilitate apoptosis of K562 cells. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia. Chronic myelogenous leukemia (CML), 3The abbreviations used are: CML, chronic myelogenous leukemia; BCR, breakpoint cluster region; AzaC, 5-azacytidine; GST, glutathione S-transferase; CPT, camptothecin; RT, reverse transcription; PTP, protein-tyrosine phosphatase; PTPRO, PTP receptor-type O; WT, wild type; CS, catalytic site mutant; STAT, signal transducers and activators of transcription; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CGI, CpG island.3The abbreviations used are: CML, chronic myelogenous leukemia; BCR, breakpoint cluster region; AzaC, 5-azacytidine; GST, glutathione S-transferase; CPT, camptothecin; RT, reverse transcription; PTP, protein-tyrosine phosphatase; PTPRO, PTP receptor-type O; WT, wild type; CS, catalytic site mutant; STAT, signal transducers and activators of transcription; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CGI, CpG island. a myeloproliferative disorder, accounts for more than half the cases of such disorders. The disease is characterized largely by the acquired genetic abnormality referred as Philadelphia chromosome (1Kurzrock R. Kantarjian H.M. Druker B.J. Talpaz M. Ann. Intern. Med. 2003; 138: 819-830Crossref PubMed Scopus (271) Google Scholar). Philadelphia chromosome is a short chromosome 22 arising from reciprocal translocations between the long arms of chromosomes 9 and 22. In this translocation the c-ABL gene located on 9q is translocated to the breakpoint cluster region (BCR) on 22q. Half of the CML patients that are negative for Philadelphia chromosome test positive for BCR/ABL fusion (1Kurzrock R. Kantarjian H.M. Druker B.J. Talpaz M. Ann. Intern. Med. 2003; 138: 819-830Crossref PubMed Scopus (271) Google Scholar). The change in conformation of the abl-tyrosine kinase due to fusion with bcr as well as the transphosphorylation of bcr/abl facilitated by bcr-mediated dimerization confers constitutive activity to this kinase (2McWhirter J.R. Galasso D.L. Wang J.Y. Mol. Cell. Biol. 1993; 13: 7587-7595Crossref PubMed Scopus (374) Google Scholar, 3Saglio G. Cilloni D. Cell. Mol. Life Sci. 2004; 61: 2897-2911Crossref PubMed Scopus (25) Google Scholar, 4Van Etten R.A. Leuk. Res. 2004; 28: 21-28Crossref PubMed Scopus (83) Google Scholar). The bcr/abl fusion protein is implicated in the etiology of CML (5Donato N.J. Talpaz M. Clin. Cancer Res. 2000; 6: 2965-2966PubMed Google Scholar, 6Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1923) Google Scholar) and has become an attractive molecular target for therapeutic intervention of CML and other Philadelphia positive leukemia such as acute lymphoblastic leukemia and rare cases of acute myelogenous leukemia. Therapeutic approaches to target this chimeric protein have focused on small molecule kinase inhibitors that led to the discovery of imatinib mesylate (also known as Gleevec, STI571, and CGP 57148), a potent and relatively selective kinase inhibitor (7Deininger M. Buchdunger E. Druker B.J. Blood. 2005; 105: 2640-2653Crossref PubMed Scopus (1065) Google Scholar). This molecule competes with ATP and, therefore, deprives bcr/abl of the phosphate source required for its kinase activity (1Kurzrock R. Kantarjian H.M. Druker B.J. Talpaz M. Ann. Intern. Med. 2003; 138: 819-830Crossref PubMed Scopus (271) Google Scholar, 8Druker B.J. Lydon N.B. J. Clin. Investig. 2000; 105: 3-7Crossref PubMed Scopus (808) Google Scholar). Treatment with Gleevec reduces proliferation and increases apoptosis of bcr/abl+ cells (9Ren R. Nat. Rev. Cancer. 2005; 5: 172-183Crossref PubMed Scopus (807) Google Scholar, 10Druker B.J. Tamura S. Buchdunger E. Ohno S. Segal G.M. Fanning S. Zimmermann J. Lydon N.B. Nat. Med. 1996; 2: 561-566Crossref PubMed Scopus (3155) Google Scholar). There are, however, drawbacks associated with Gleevec that include persistence of bcr/abl+ cells (residual disease) requiring continuous exposure to Gleevec and resistance due to mutations in bcr/abl that prevent drug binding (9Ren R. Nat. Rev. Cancer. 2005; 5: 172-183Crossref PubMed Scopus (807) Google Scholar, 11Bhatia R. Holtz M. Niu N. Gray R. Snyder D.S. Sawyers C.L. Arber D.A. Slovak M.L. Forman S.J. Blood. 2003; 101: 4701-4707Crossref PubMed Scopus (461) Google Scholar, 12Lowenberg B. N. Engl. J. Med. 2003; 349: 1399-1401Crossref PubMed Scopus (49) Google Scholar, 13Gorre M.E. Sawyers C.L. Curr. Opin. Hematol. 2002; 9: 303-307Crossref PubMed Scopus (102) Google Scholar). In addition to binding ATP, the transforming activity of bcr/abl requires phosphorylation of key tyrosine residues (14Meyn III, M.A. Wilson M.B. Abdi F.A. Fahey N. Schiavone A.P. Wu J. Hochrein J.M. Engen J.R. Smithgall T.E. J. Biol. Chem. 2006; 281: 30907-30916Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which could be targeted to control its activity and transformation potential. In this context it would be important to identify the protein-tyrosine phosphatase(s) that could potentially dephosphorylate and inactivate the bcr/abl-associated kinase. Furthermore, it is critical to determine whether the levels of these tyrosine phosphatases are reduced in CML, which could explain the constitutive activity of bcr/abl. Subsequently, understanding the mechanism of their suppression in bcr/abl-positive leukemia will facilitate the development of novel therapeutic strategies to treat this disease. To date only two tyrosine phosphatases, PTP1B and SHP1, are known to dephosphorylate and moderately inhibit the transformation potential of bcr/abl (15Liedtke M. Pandey P. Kumar S. Kharbanda S. Kufe D. and Oncogene. 1998; 17: 1889-1892Crossref PubMed Scopus (26) Google Scholar, 16LaMontagne Jr., K.R. Flint A.J. Franza Jr., B.R. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Crossref PubMed Scopus (102) Google Scholar). The expression of PTP1B is initially up-regulated in chronic phase of CML as a defense mechanism against the fusion protein. It is, however, presumed that the suppressive effect of PTP1B on bcr/abl is lost due to secondary mutations associated with blast crisis (16LaMontagne Jr., K.R. Flint A.J. Franza Jr., B.R. Pandergast A.M. Tonks N.K. Mol. Cell. Biol. 1998; 18: 2965-2975Crossref PubMed Scopus (102) Google Scholar). Similarly, the expression of SHP1 is down-regulated in CML (17Amin H.M. Hoshino K. Yang H. Lin Q. Lai R. Garcia-Manero G. J. Pathol. 2007; 212: 402-410Crossref PubMed Scopus (44) Google Scholar). Lack of knowledge of the mechanism of their inactivation prevents their application as potential therapeutic targets. Although an escort/phosphatase approach has been used to enhance the anti-transformation potential of SHP1 by increasing its affinity for bcr/abl (18Lim Y.M. Wong S. Lau G. Witte O.N. Colicelli J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12233-12238Crossref PubMed Scopus (40) Google Scholar), clinical application of this technique does not appear to be feasible. It is interesting that a Ser/Thr phosphatase PP2A whose activity is negatively regulated by bcr/abl-induced SET protein can also dephosphorylate bcr/abl by recruiting the tyrosine phosphatase SHP1 (19Neviani P. Santhanam R. Trotta R. Notari M. Blaser B.W. Liu S. Mao H. Chang J.S. Galietta A. Uttam A. Roy D.C. Valtieri M. Bruner-Klisovic R. Caligiuri M.A. Bloomfield C.D. Marcucci G. Perrotti D. Cancer Cell. 2005; 8: 355-368Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). Targeting bcr/abl by inhibiting SET to activate PP2A will also require concurrent expression of SHP1 for its tyrosine dephosphorylation function. It was, therefore, important to identify a bcr/abl-targeting tyrosine phosphatase that could be used as a clinical target for the existing drugs against CML or for the development of new clinically applicable strategies to treat CML. Protein-tyrosine phosphatase receptor-type O (PTPRO) is a transmembrane phosphatase with a large extracellular domain of fibronectin repeats and a single catalytic domain. We have previously identified this tyrosine phosphatase as a candidate tumor suppressor in a screen for genes hypermethylated in cancer (20Motiwala T. Jacob S.T. Prog. Nucleic Acids Res. Mol. Biol. 2006; 81: 297-329Crossref PubMed Scopus (36) Google Scholar, 21Motiwala T. Ghoshal K. Das A. Majumder S. Weichenhan D. Wu Y.Z. Holman K. James S.J. Jacob S.T. Plass C. Oncogene. 2003; 22: 6319-6331Crossref PubMed Scopus (104) Google Scholar, 22Jacob S.T. Motiwala T. Cancer Gene Ther. 2005; 12: 665-672Crossref PubMed Scopus (50) Google Scholar). Subsequently, we demonstrated its tumor suppressor characteristics in lung cancer (23Motiwala T. Kutay H. Ghoshal K. Bai S. Seimiya H. Tsuruo T. Suster S. Morrison C. Jacob S.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13844-13849Crossref PubMed Scopus (100) Google Scholar). The same gene encodes a truncated variant (PTPROt), a transmembrane protein lacking the large extracellular fibronectin domains (22Jacob S.T. Motiwala T. Cancer Gene Ther. 2005; 12: 665-672Crossref PubMed Scopus (50) Google Scholar, 24Aguiar R.C. Yakushijin Y. Kharbanda S. Tiwari S. Freeman G.J. Shipp M.A. Blood. 1999; 94: 2403-2413Crossref PubMed Google Scholar) which also exhibits tumor suppressor characteristics (25Chen L. Juszczynski P. Takeyama K. Aguiar R.C. Shipp M.A. Blood. 2006; 108: 3428-3433Crossref PubMed Scopus (79) Google Scholar, 26Motiwala T. Majumder S. Kutay H. Smith D.S. Neuberg D.S. Lucas D.M. Byrd J.C. Grever M. Jacob S.T. Clin. Cancer Res. 2007; 13: 3174-3181Crossref PubMed Scopus (56) Google Scholar). Here, we demonstrate that the truncated isoform that is specifically expressed in hematopoietic cells can dephosphorylate bcr/abl and inactivate its downstream signaling. Such inhibition is reflected in the reversal of transformed phenotype of bcr/abl+ K562 cells expressing the catalytically active PTPROt and their increased susceptibility to drug-induced apoptosis. Interestingly, the catalytically inactive mutant of PTPROt also exhibits some anti-transformation potential, probably by functioning as a trapping mutant. Furthermore, we show that PTPROt is suppressed by promoter methylation in these cells, and the DNA hypomethylating agent 5-azacytidine, used for treating myeloproliferative disorders, can relieve this suppression. Reagents—The antibodies used in this study are as follows: α-FLAG-M2 (Sigma), α-Tyr(P) mixture (4G10 from Millipore, Tyr(P)20 and Tyr(P)99 from Santa Cruz Biotechnology), α-c-abl (Santa Cruz Biotechnology), α-p27 (Abcam), Pathscan bcr/abl activity assay mixture (Cell signaling Technology), α-c-fos (Santa Cruz Biotechnology). Cell Culture and 5-Azacytidine Treatment—K562 cells purchased from ATCC were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum. Cells were treated with 7.5 μm 5-azacytidine (AzaC) (Sigma) for 24 h (for RT-PCR) or 5 μm for 96 h (for bisulfite sequencing). 6.15 (32D-BCR/ABL) cells (27Koschmieder S. D'Alo F. Radomska H. Schoneich C. Chang J.S. Konopleva M. Kobayashi S. Levantini E. Suh N. Di Ruscio A. Voso M.T. Watt J.C. Santhanam R. Sargin B. Kantarjian H. Andreeff M. Sporn M.B. Perrotti D. Berdel W.E. Muller-Tidow C. Serve H. Tenen D.G. Blood. 2007; 110: 3695-3705Crossref PubMed Scopus (44) Google Scholar) were a generous gift from Dr. Danilo Perrotti (The Ohio State University). These cells were maintained in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum. Generation of K562 Cell Line Stably Expressing PTPROt—The coding region of PTPROt amplified from PTPU2L wild type (WT) and catalytic site mutant (CS; a generous gift of Dr. Hiroyuki Seimiya, (28Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar)) using the primers PTPt-EcoRI (5′-ATGAATTCCAATGGTTACAGAGATGA-3′) and PTP-R-Bam (5′-CTGGATCCCTTGCTAACATTCTCG-3′ (restriction sites underlined)) was cloned into the EcoRI/BamHI sites of p3XFLAG-CMV-14 (Sigma). Plasmid DNA of p3XFLAG-CMV-14 (Vector) or p3XFLAG-PTPROt (WT or CS) was transfected into K562 cells using Lipofectamine2000 (Invitrogen). After 48 h cells were selected with 500 μg/ml G418 for 7 days. The expression of PTPROt was monitored by Western blot using anti-FLAG M2 antibody. In Vitro Phosphatase Assay—Whole cell extract of K562 cells treated with 100 μm pervanadate for 30 min was prepared in lysis buffer B (20 mm Tris, pH 7.5, 100 mm NaCl, 1% Triton X-100, 10% glycerol, 5 mm iodoacetic acid, 1 mm sodium orthovanadate, and protease inhibitors). After incubation for 30 min on ice, dithiothreitol and EDTA were added to a final concentration of 10 and 1 mm, respectively, to inactivate the iodoacetic acid and vanadate followed by another 15 min of incubation on ice. The extract was then centrifuged at maximum speed to remove any cell debris. Phosphorylated bcr/abl was immunoprecipitated from this extract using anti-c-Abl antibody (Santa Cruz Biotechnology). After washing the immunoprecipitate with lysis buffer A (20 mm Tris, pH 7.5, 100 mm NaCl, 1% Triton X-100, 10% glycerol, 5 mm iodoacetic acid, 1 mm EDTA, and protease inhibitors), the protein A-agarose beads (with pulled-down bcr/abl) were equilibrated and suspended in assay buffer (9.375 mm HEPES, pH 7.4, 18.75 mm NaCl, 0.9375 mm EDTA, 1.875 mm dithiothreitol, and 125 μg/ml bovine serum albumin). The suspension was then divided into three equal parts to which purified GST proteins were added (GST alone as control or GST-PTPROt-WT or GST-PTPROt-CS). The assay mix was rocked for 30 min at 37 °C before separation of proteins on 8% SDS-PAGE. The proteins were transferred to nitrocellulose membrane and probed with anti-Tyr(P) mixture. The same blot was washed and reprobed with anti-c-abl antibody to normalize protein. DNA Replication Assay—K562 cells (vector control or PTPROt expressing) were serum-starved (0% fetal bovine serum) for 18 h. Equal numbers of serum-starved cells were allowed to grow in complete media containing [3H]thymidine (5 μCi) for 2 h followed by precipitation of DNA with 10% trichloroacetic acid. [3H]Thymidine incorporated into the DNA was measured in a scintillation counter. Cell Cycle Analysis—PTPROt-WT and CS-expressing and vector-transfected K562 cells were treated with nocodazole (1 μm) for 18 h to synchronize the cells at G2/M phase. After 18 h the cell cycle block was released, and the cells were allowed to grow in complete medium devoid of nocodazole. The cells harvested at the indicated time points were stained with propidium iodide and subjected to flow cytometric analysis. Soft Agar Assay—This assay was performed as described (23Motiwala T. Kutay H. Ghoshal K. Bai S. Seimiya H. Tsuruo T. Suster S. Morrison C. Jacob S.T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13844-13849Crossref PubMed Scopus (100) Google Scholar). Tumor Growth in Nude Mice—Equal numbers (0.5 × 106) of K562 cells (Vector or PTPROt-WT) were injected subcutaneously into anterior and posterior sites on the back of athymic nude mice. These mice were then followed for tumor growth for 4 weeks. Once the tumor was visible, its size was measured using a slide caliper every 7 days. At the end of 4 weeks the tumor growth and tumor weight were recorded after sacrificing the animals. Annexin V/Propidium Iodide Staining—To analyze drug-induced apoptosis, K562 cells (Vector or PTPROt expressing) were treated with camptothecin (CPT, 10 μg/ml), a cytotoxic quinoline alkaloid, for 18 and 36 h. At the end of the treatment the cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences) following the manufacturer’s protocol and analyzed by flow cytometry. Generation of 6.15 Cells Transiently Expressing PTPROt—The cells were generated by retroviral infection as previously described (29Iervolino A. Santilli G. Trotta R. Guerzoni C. Cesi V. Bergamaschi A. Gambacorti-Passerini C. Calabretta B. Perrotti D. Mol. Cell. Biol. 2002; 22: 2255-2266Crossref PubMed Scopus (105) Google Scholar, 30Thomas E.K. Cancelas J.A. Chae H.D. Cox A.D. Keller P.J. Perrotti D. Neviani P. Druker B.J. Setchell K.D. Zheng Y. Harris C.E. Williams D.A. Cancer Cell. 2007; 12: 467-478Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Briefly, retroviral stock generated by transiently transfecting Phoenix cells with pRetroOn or pRetroOn-PTPROt (WT and CS) (26Motiwala T. Majumder S. Kutay H. Smith D.S. Neuberg D.S. Lucas D.M. Byrd J.C. Grever M. Jacob S.T. Clin. Cancer Res. 2007; 13: 3174-3181Crossref PubMed Scopus (56) Google Scholar) were used 48 h post-transfection for three rounds of spin-infection of 6.15 cells. The cells were harvested 24 h after the last infection for protein and metabolic assays. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide Assay—The assay was performed essentially as described previously (26Motiwala T. Majumder S. Kutay H. Smith D.S. Neuberg D.S. Lucas D.M. Byrd J.C. Grever M. Jacob S.T. Clin. Cancer Res. 2007; 13: 3174-3181Crossref PubMed Scopus (56) Google Scholar). For growth kinetics, the cells were seeded in 96-well plate at 3000 cells/100 μl in each well. For CPT treatment, 10,000 cells were seeded in each well of the 96-well plate. The cells were left untreated or treated with 10 μg/ml CPT for 7 h. The % apoptotic cells were calculated based on the loss of metabolic activity between 0 and 7 h of CPT treatment. RNA Isolation and RT-PCR—Total RNA was isolated following the guanidinium isothiocyanate-acid phenol method (31Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63167) Google Scholar). Reverse transcription of total RNA (3 μg) was carried out with random hexamers and murine Moloney leukemia virus reverse transcriptase (Applied Biosystems) according to the Gene-Amp RNA PCR kit (PerkinElmer Life Sciences) instructions. RT-PCR primers for PTPROt and details of the procedure are as described earlier (26Motiwala T. Majumder S. Kutay H. Smith D.S. Neuberg D.S. Lucas D.M. Byrd J.C. Grever M. Jacob S.T. Clin. Cancer Res. 2007; 13: 3174-3181Crossref PubMed Scopus (56) Google Scholar). The absence of DNA contamination was confirmed by negative RT-PCR for 18 S rRNA performed on RNA samples subjected to cDNA synthesis in the absence of reverse transcriptase. Expression of c-fos was measured by RT-PCR as well as real-time RT-PCR with SYBR Green chemistry using the primers human h Fos-RT-F (5′-GGGGCAAGGTGGAACAGTTATC-3′) and hFos-RT-R (5′-TTCAGCAGGTTGGCAATCTCGGTC-3′). Relative expression was calculated using the ΔCt method (32Schmittgen T.D. Livak K.J. Nat. Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (17381) Google Scholar). Plasmid Construction and Transient Transfection Assay—An ∼1300-bp fragment of the PTPROt promoter (from -1049 to +261) was cloned at the SmaI/BglII site of pGL3-Basic vector (Promega) to generate the promoter reporter vector PTPt-P-Luc. The promoter reporter construct was transfected into K562 cells along with the internal control pRL-TK using Lipofectamine2000 (Invitrogen). The cells were harvested 48 h post-transfection, and the luciferase activity was measured using the Dual Luciferase assay kit (Promega). The luciferase activity driven by the PTPROt promoter was normalized to that driven by thymidine kinase promoter (pRL-TK, internal control), and the data are expressed as -fold increase in promoter activity assigning a value of 1 to pGL3-Basic activity. Bcr/abl Is a Substrate of PTPROt in K562 Cells—K562 is a well studied chronic myelogenous leukemia cell line characterized by Philadelphia chromosome. These cells express the constitutively active tyrosine-phosphorylated 210-kDa fusion protein bcr/abl, which is thought to confer growth advantage. Identification of the protein-tyrosine phosphatase that dephosphorylates bcr/abl should, therefore, provide an important molecular target for therapy of bcr/abl positive CML or other bcr/abl+ leukemia such as acute lymphocytic leukemia. To determine whether PTPROt dephosphorylates this fusion protein, we generated K562 cells ectopically expressing both wild type (WT) PTPROt and its CS mutant as FLAG-tagged fusion proteins. Ectopic expression of PTPROt was monitored by Western blot analysis with anti-FLAG M2 antibody (Fig. 1a). The phosphorylation status of bcr/abl was assessed by immunoprecipitating bcr/abl from vector control as well as PTPROt (WT and CS)-expressing K562 cells followed by Western blotting with anti-phosphotyrosine antibody. The same blot was re-probed with anti-c-abl antibody to normalize total protein (Fig. 1b). Quantification of the data revealed significant reduction of phosphorylated bcr/abl in PTPROt-WT-expressing cells compared with PTPROt-CS-expressing and vector-transfected K562 cells (Fig. 1c). This observation suggests that bcr/abl is a potential substrate of PTPROt. To confirm that bcr/abl is a direct substrate of PTPROt, we used an in vitro phosphatase assay to demonstrate dephosphorylation of bcr/abl by PTPROt. For this purpose, bcr/abl was immunoprecipitated from whole cell extract of pervanadate-treated parental K562 cells followed by incubation with GST-tagged bacterially expressed PTPROt (WT and CS) under conditions optimal for the phosphatase function. The proteins were then separated on SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody. The same blot was washed and re-probed with anti-c-abl antibody to demonstrate comparable level of the protein in all lanes (Fig. 1d). The data demonstrate an ∼50% reduction in phosphorylation of bcr/abl upon incubation with PTPROt-WT but not when incubated with PTPROt-CS or GST alone (Fig. 1e). This observation confirms that PTPROt can indeed dephosphorylate bcr/abl in the absence of any other cellular proteins. Ectopic Expression of PTPROt Inhibits Growth and Clonogenic Survival of K562 Cells—To study whether dephosphorylation and potential inactivation of bcr/abl by PTPRO could reverse the transformation potential of K562 cells, we first studied its effect on cell growth by manual counting. There was a significant (p < 0.04) decrease in proliferation rate of the PTPROt-WT-expressing cells compared with the PTPROt-CS and vector-transfected cells (Fig. 2a). We also examined the effect of ectopic PTPROt on the replication potential of K562 cells by thymidine incorporation, which showed a 40% decrease in the PTPROt-WT-expressing cells relative to PTPROt-CS-expressing and vector-transfected cells (Fig. 2b). We next monitored the cell cycle profile of PTPROt-expressing cells to examine whether the delayed growth was due to cell cycle arrest. For this purpose, K562 cells were synchronized at G2-M phase with the microtubule-stabilizing agent nocodazole for 18 h. Cell cycle distribution was then analyzed using propidium iodide staining immediately after release (0 h) and after 14 h. As observed previously for DLBCL cells (24Aguiar R.C. Yakushijin Y. Kharbanda S. Tiwari S. Freeman G.J. Shipp M.A. Blood. 1999; 94: 2403-2413Crossref PubMed Google Scholar), immediately after release from nocodazole block a greater number of PTPROt-WT-expressing cells were in G0/G1 phase compared with PTPROt-CS-expressing and vector-transfected cells (Fig. 2C, 0 h). Additionally, our data also showed that 14 h after release from cell cycle block a large population of PTPROt-WT cells were in G0/G1 phase with a consequently reduced number in S-phase. On the other hand, both PTPROt-CS-expressing and vector-transfected control cells exited the G0/G1 phase and were in S-phase (Fig. 2C, 14 h). Thus, reduced replication potential and prolonged G0/G1 phase of K562 cells expressing PTPROt-WT explains the reduced growth rate of these cells. CML is a disease of immature blast cell expansion within the bone marrow, which enter circulation and further proliferate. This behavior reflects in vitro anchorage-independent growth (33Schwartz M.A. J. Cell Biol. 1997; 139: 575-578Crossref PubMed Scopus (304) Google Scholar). We, therefore, investigated whether ectopic expression of PTPROt affects anchorage-independent growth of the K562 cells in soft agar. As expected, although vector-transfected K562 cells were able to form colonies in soft agar, K562 cells expressing PTPROt-WT were unable to survive and form colonies under the same conditions (Fig. 2d). Surprisingly, K562 cells expressing PTPROt-CS were also unable to form colonies in soft agar (Fig. 2d). These observations suggest that although inhibition of growth/proliferation by PTPROt requires phosphatase activity, reversal of anchorage independence could be independent of phosphatase activity or that PTPROt-WT and PTPROt-CS may be functioning differently to inhibit growth in soft agar. It was, therefore, imperative to study the mechanism of this differential regulation of growth and anchorage independence by PTPROt. PTPROt-WT and PTPROt-CS Probably Regulate Growth and Anchorage Independence of K562 Cells by Different Mechanisms—Bcr/abl oncogene can activate several signaling pathways that include Ras, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/Akt, and JAK/STAT pathways (4Van Etten R.A. Leuk. Res. 2004; 28: 21-28Crossref PubMed Scopus (83) Google Scholar). Although each of these pathways plays important roles in the transformation of K562 cells by bcr/abl (34Sonoyama J. Matsumura I. Ezoe S. Satoh Y. Zhang X. Kataoka Y. Takai E. Mizuki M. Machii T. Wakao H. Kanakura Y. J. Biol. Chem. 2002; 277: 8076-8082Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), they appear to function either independently or in co-operation with another signaling pathway to regulate different aspects of transformation, e.g. proliferation, apoptosis, and anchorage independence. It is, therefore, possible that two independent pathways are involved in the regulation of cell cycle and anchorage-independent growth of K562 cells by bcr/abl, and each of these is distinctly regulated by PTPROt. To explore this possibility we analyzed the potential regulatory mechanisms for these phenotypes. It appears that the Ras/Raf pathway is predominantly involved in cell cycle control by bcr/abl. Activated Raf-1 can activate Cdc25, which in turn can activate cyclin E/cyclin-dependent kinase 2 that favors G1 → S transition (35Steelman L.S. Pohnert S.C. Shelton J.G. Franklin R.A. Bertrand F.E. McCubrey J.A. Leukemia. 2004; 18: 189-218Crossref PubMed Scopus (575) Google Scholar) (see Fig. 3a). The kinase activity o" @default.
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