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• W2086883696 abstract "A large family of protein tyrosine phosphatases (PTPs) bidirectionally regulate intracellular signaling pathways by reversing agonistic or antagonistic phosphorylation events derived from the action of protein tyrosine kinases. Receptor-like PTPPTP-U2 is expressed during phorbol ester-induced differentiation of monoblastoid leukemia U937 cells. We found that the shorter isoform, PTP-U2S, was expressed at an earlier phase in the course of differentiation and the longer isoform, PTP-U2L, was induced at a later phase. In the presence of 12-O-tetradecanoylphorbol-13-acetate, ectopic expression of PTP-U2L in U937 cells enhanced several characteristics of terminally differentiated cells. Most striking was that PTP-U2L enhanced apoptosis of the differentiated cells, which was only partially inhibited by caspase inhibitor Z-Asp-CH2-DCB. The catalytically inactive mutant PTP-U2L(C → S) still retained the ability to enhance the differentiation but retained the ability to enhance the following apoptosis of the cells to a lesser extent. These data indicate a functional involvement of PTP-U2L in apoptosis subsequent to terminal differentiation of U937 cells. Since terminally differentiated blood cells often undergo apoptosis, the data also suggest that PTP-U2L might be involved in physiological turnover of hematopoietic cells in vivo. A large family of protein tyrosine phosphatases (PTPs) bidirectionally regulate intracellular signaling pathways by reversing agonistic or antagonistic phosphorylation events derived from the action of protein tyrosine kinases. Receptor-like PTPPTP-U2 is expressed during phorbol ester-induced differentiation of monoblastoid leukemia U937 cells. We found that the shorter isoform, PTP-U2S, was expressed at an earlier phase in the course of differentiation and the longer isoform, PTP-U2L, was induced at a later phase. In the presence of 12-O-tetradecanoylphorbol-13-acetate, ectopic expression of PTP-U2L in U937 cells enhanced several characteristics of terminally differentiated cells. Most striking was that PTP-U2L enhanced apoptosis of the differentiated cells, which was only partially inhibited by caspase inhibitor Z-Asp-CH2-DCB. The catalytically inactive mutant PTP-U2L(C → S) still retained the ability to enhance the differentiation but retained the ability to enhance the following apoptosis of the cells to a lesser extent. These data indicate a functional involvement of PTP-U2L in apoptosis subsequent to terminal differentiation of U937 cells. Since terminally differentiated blood cells often undergo apoptosis, the data also suggest that PTP-U2L might be involved in physiological turnover of hematopoietic cells in vivo. Protein tyrosine phosphatases (PTPs), 1The abbreviations used are: PTPprotein tyrosine phosphataseMAPKmitogen-activated protein kinaseTPA12-O-tetradecanoyl-phorbol-13-acetateZ-Asp-CH2-DCBbenzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzeneSH2Src homology domain 2PBSphosphate-buffered saline. as well as protein tyrosine kinases, regulate tyrosine phosphorylation, which plays a significant role in cellular proliferation, differentiation, and oncogenesis (1Hunter T. Cell. 1989; 58: 1013-1016Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 2Walton K.M. Dixon J.E. Annu. Rev. Biochem. 1993; 62: 101-120Crossref PubMed Scopus (415) Google Scholar). Since hyperphosphorylation of tyrosine by oncogenic tyrosine kinases often leads to transformation of cells (3Yarden Y. Ullrich A. Annu. Rev. Biochem. 1988; 57: 443-478Crossref PubMed Scopus (1391) Google Scholar), it has been originally postulated that PTPs, which reverse the phosphorylation reaction, might play a role in suppressing oncogenesis (1Hunter T. Cell. 1989; 58: 1013-1016Abstract Full Text PDF PubMed Scopus (255) Google Scholar). Recently, however, the situation has become more complicated because a number of cDNAs that encode the PTP gene have been isolated from various organisms, mainly by sequence similarity between the conserved regions of the catalytic domains (4Krueger N.X. Streuli M. Saito H. EMBO J. 1990; 9: 3241-3252Crossref PubMed Scopus (370) Google Scholar, 5Tian S.-S. Tsoulfas P. Zinn K. Cell. 1991; 67: 675-685Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 6Yi T. Cleveland J.L. Ihle J.N. Blood. 1991; 78: 2222-2228Crossref PubMed Google Scholar). Despite the similarities in their catalytic domains, these PTP isozymes exhibit diverse structures outside the catalytic domains, suggesting involvement of the PTPs in each isozyme-specific function. protein tyrosine phosphatase mitogen-activated protein kinase 12-O-tetradecanoyl-phorbol-13-acetate benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene Src homology domain 2 phosphate-buffered saline. In fact, recent studies have demonstrated specific functions of PTP isozymes in physiological events (see for review, Ref. 7Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). For example, even among structurally similar PTP isozymes, entirely distinct functions have been documented: SHP1 and SHP2 are both non-receptor PTPs with two tandem Src homology 2 (SH2) domains in their amino-terminal regions (8Adachi M. Fischer E.H. Ihle J. Imai K. Jirik F. Neel B. Pawson T. Shen S.-H. Thomas M. Ullrich A. Zhao Z. Cell. 1996; 85: 15Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). SHP1 negatively regulates hematopoietic signaling pathways (9D'Ambrosio D. Hippen K.L. Minskoff S.A. Mellman I. Pani G. Siminovitch K.A. Cambier J.C. Science. 1995; 268: 293-297Crossref PubMed Scopus (507) Google Scholar, 10Klingmüller U. Lorenz U. Cantley L.C. Neel B.J. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 11Plas D.R. Johnson R. Pingel J.T. Matthews R.J. Dalton M. Roy G. Chan A.C. Thomas M.L. Science. 1996; 272: 1173-1176Crossref PubMed Scopus (334) Google Scholar). At the pathological level, genetic failure in the shp1 locus leads to autoimmune disease with hyperproliferation of hematopoietic cells (12Tsui H.W. Siminovitch K.A. de Souza L. Tsui F.W.L. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (521) Google Scholar, 13Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (690) Google Scholar). On the other hand, SHP2 is required for mitogenic signal transduction from receptor tyrosine kinases to the Ras/mitogen-activated protein kinase (MAPK) pathway (14Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (348) Google Scholar, 15Xiao S. Rose D.W. Sasaoka T. Maegawa H. Burke Jr., T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar, 16Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 17Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar). These observations suggest that other PTP isozymes, even if they are structurally related through entire sequences, also have distinct functions although some redundancy might exist. Meanwhile, a novel PTP isozyme PTEN/MMAC1/TEP1 has been identified as a candidate tumor suppressor gene (18Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4310) Google Scholar, 19Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K.A. Lin H. Ligon A.H. Langford L.A. Baumgard M., L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H.F. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2524) Google Scholar, 20Li D.-M. Sun H. Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar), still suggesting a functional involvement of certain PTPs in anti-oncogenesis. Previous studies, including ours, suggest a functional involvement of PTPs in terminal differentiation of myeloid leukemia cells. Pharmacological differentiation of leukemia cells is often accompanied by an increase in cellular PTP activity (21Frank D.A. Sartorelli A.C. Cancer Res. 1988; 48: 4299-4306PubMed Google Scholar, 22Frank D.A. Sartorelli A.C. Cancer Res. 1988; 48: 52-58PubMed Google Scholar, 23Bütler T.M. Ziemiecki A. Friis R.R. Cancer Res. 1990; 50: 6323-6329PubMed Google Scholar, 24Seimiya H. Tsuruo T. Cell Growth & Differ. 1993; 4: 1033-1039PubMed Google Scholar). Meanwhile, such differentiation is often inhibited by sodium orthovanadate, a PTP inhibitor (25Zhao Z. Shen S.-H. Fischer E.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5007-5011Crossref PubMed Scopus (53) Google Scholar). Previously, we identified 13 PTP gene fragments from the differentiated monoblastoid leukemia U937 cells by employing the reverse transcription-polymerase chain reaction strategy (24Seimiya H. Tsuruo T. Cell Growth & Differ. 1993; 4: 1033-1039PubMed Google Scholar). Among them, gene expression for four isozymes, including PTP-U2(also known as GLEPP1/PTPφ) (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar, 27Thomas P.E. Wharram B.L. Goyal M. Wiggins J.E. Holzman L.B. Wiggins R.C. J. Biol. Chem. 1994; 269: 19953-19962Abstract Full Text PDF PubMed Google Scholar, 28Pixley F.J. Lee P.S.W. Dominguez M.G. Einstein D.B. Stanley E.R. J. Biol. Chem. 1995; 270: 27339-27347Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), PTP-MEG2 (29Gu M. Warshawsky I. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2980-2984Crossref PubMed Scopus (78) Google Scholar), P19-PTP (30den Hertog J. Pals C.E.G.M. Jonk L.J.C. Kruijer W. Biochem. Biophys. Res. Commun. 1992; 184: 1241-1249Crossref PubMed Scopus (26) Google Scholar), and PTP-U1/DEP-1/HPTPη (24Seimiya H. Tsuruo T. Cell Growth & Differ. 1993; 4: 1033-1039PubMed Google Scholar,31Östman A. Yang Q. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9680-9684Crossref PubMed Scopus (202) Google Scholar, 32Honda H. Inazawa J. Nishida J. Yazaki Y. Hirai H. Blood. 1994; 84: 4186-4194Crossref PubMed Google Scholar), is induced during monocytic and granulocytic differentiation of myeloid leukemia cells (33Seimiya H. Sawabe T. Toho M. Tsuruo T. Oncogene. 1995; 11: 2047-2054PubMed Google Scholar). 2H. Seimiya and T. Tsuruo, unpublished results. PTP-U2 is a receptor-like PTP with a single transmembrane domain and a single intracellular catalytic domain (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar). Physiologically, thePTP-U2 gene is expressed as tissue-specific isoforms, which are probably due to alternative splicing of the transcript. The extracellular domain of the longest isoform contains 14 putativeN-glycosylation sites and eight repeats of a fibronectin-type III-like motif. Especially, in U937 cells,PTP-U2 gene expression is greatly induced by phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which arrests growth and induces monocytic differentiation of U937 cells (24Seimiya H. Tsuruo T. Cell Growth & Differ. 1993; 4: 1033-1039PubMed Google Scholar). Furthermore, PTP-U2 gene expression is also induced or enhanced during various types of differentiation of myeloid and erythroid leukemia cells, including HL-60, HEL, and K562. Other myeloid leukemia THP-1 cells, which often differentiate spontaneously even without inducers, express a relatively high amount of PTP-U2transcript. On the contrary, TPA does not induce or enhancePTP-U2 gene expression in solid tumor cells that do not differentiate upon treatment with TPA (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar). Thus, we supposed that PTP-U2 would be functionally involved in drug-induced differentiation of U937 cells. However, the precise role of PTP-U2 in terminal differentiation has yet to be determined. In this study, we investigated the function of the longest PTP-U2 isoform (PTP-U2L) during TPA-induced differentiation and subsequent cell death of U937 cells. We found that PTP-U2L enhances the apoptotic event that follows TPA-induced differentiation of U937 cells. The physiological importance of PTP-U2L function is also discussed. Human monoblastoid leukemia U937 cells were grown as described previously (24Seimiya H. Tsuruo T. Cell Growth & Differ. 1993; 4: 1033-1039PubMed Google Scholar). A phorbol ester-resistant U937 variant, UT16, was established and characterized as described (33Seimiya H. Sawabe T. Toho M. Tsuruo T. Oncogene. 1995; 11: 2047-2054PubMed Google Scholar). Human embryonal kidney 293T cells were kindly provided by Dr. T. Suzuki (University of Tokyo, Tokyo, Japan). Cellular differentiation was induced by treatment with 5 ng/ml TPA (Sigma) and characteristic changes in cellular morphology, development of adherence to plastic substratum, and cell surface expression of CD14 or CD11b glycoproteins were monitored. Expression of CD14 or CD11b was determined by flow cytometry, as described previously (33Seimiya H. Sawabe T. Toho M. Tsuruo T. Oncogene. 1995; 11: 2047-2054PubMed Google Scholar). Cells were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in 0.5 ml of buffer E consisting of 50 mm HEPES, pH 7.0, 2 mm EDTA, 2 mm EGTA, 10 mm2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 2% Triton X-100 (34Pallen C.J. Tong P.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6996-7000Crossref PubMed Scopus (82) Google Scholar). Differentiated cells were harvested by scraping in buffer E after PBS washing. The lysate was incubated with occasional mixing at 4 °C for 1 h, and centrifuged (10,000 × g for 10 min at 4 °C) to obtain the solubilized fractions. The protein concentration in the extracts was determined using a Protein Assay System reagent (Bio-Rad). The samples (30 μg of protein) were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Schreicher & Schuell, Dassel, Germany). The membranes were soaked in 10% skim milk, PBS at room temperature for 1 h and further incubated for 1 h with an affinity-purified rabbit antibody raised against PTP-U2 carboxyl-terminal 15-amino acid residues (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar) or mouse anti-T cell PTP antibody (Oncogene Science, Uniondale, NY). The membranes were then washed with 0.1% Tween 20, 0.5% skim milk, PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin (Amersham International, Buckinghamshire, United Kingdom). After washing the membranes extensively with 0.1% Tween 20, PBS, the specific signals were detected on X-Omat AR films (Kodak, Rochester, NY) using an ECL detection system (Amersham Int.). Cellular lysates were prepared as described above. Portions of the lysates (240 μg of protein per triplicate assay) were gently stirred with rabbit anti-PTP-U2 antibody and protein A-Sepharose (Sigma) at 4 °C for 1 h. The immunocomplex was extensively washed with the PTP assay buffer (25 mm HEPES, pH 6, 5 mm EDTA, 10 mm2,3-dihydroxybutane-1,4-dithiol). PTP activity in the immunocomplex was measured against phosphotyrosyl substrates, END(pY)INASL (PS1) (35Daum G. Solca F. Diltz C.D. Zhao Z. Cool D.E. Fischer E.H. Anal. Biochem. 1993; 211: 50-54Crossref PubMed Scopus (57) Google Scholar) and DADE(pY)LIPQQG (PS2) (36Zhang Z.Y. Thieme-Sefler A.M. Maclean D. McNamara D.J. Dobrusin E.M. Sawyer T.K. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4446-4450Crossref PubMed Scopus (180) Google Scholar), by using the Tyrosine Phosphatase Assay System (Promega, Madison, WI), according to the instruction manual. The full-length cDNA forPTP-U2L (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar) was cloned into a mammalian expression vector pCR3 (Invitrogen, Carlsbad, CA), and the resulting plasmid was designated as pCR3/PTP-U2L. pCR3/PTP-U2L(C → S), the expression vector for the catalytically inactive mutant of PTP-U2L (point mutated at Cys1136 to Ser1136) was constructed with pCR3/PTP-U2L as a template and a Chameleon mutagenesis kit (Stratagene, La Jolla, CA), according to the instruction manual. DNA sequences of the constructs were confirmed by an ABI PRISM Dye Primer Cycle Sequencing Kit (Applied Biosystems, Chiba, Japan) with a Perkin-Elmer DNA thermal cycler and an ABI PRISM 310 Genetic Analyzer. Twenty micrograms of pCR3, pCR3/PTP-U2L, or pCR3/PTP-U2L(C → S) were transfected into 107 U937 cells by electroporation using a Gene-Pulser (Bio-Rad). After the recovery culture of 48 h, transfectants were selected by treatment with 0.8 mg/ml geneticin (Sigma), and the resistant cells were cloned by limiting dilution. Expression of PTP-U2L or PTP-U2L(C → S) protein was monitored by Western blot analysis, as described above, and the positive clones were used for subsequent experiments. Apoptosis was monitored through characteristic changes in cellular morphology, externalization of phosphatidylserine (37Koopman G. Reutelingsperger C.P.M. Kuijten G.A.M. Keehnen R.M.J. Pals S.T. van Oers M.H.J. Blood. 1994; 85: 1415-1420Crossref Google Scholar, 38Martin J.S. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Schie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2566) Google Scholar), and the appearance of chromosomal DNA fragmentation (39Gunji H. Kharbanda S. Kufe D. Cancer Res. 1991; 51: 741-743PubMed Google Scholar). Externalization of phosphatidylserine was detected by annexin V-fluorescein isothiocyanate (Kamiya Biomedical, Seattle, WA) staining, as described previously (40Naito M. Nagashima K. Mashima T. Tsuruo T. Blood. 1997; 89: 2060-2066Crossref PubMed Google Scholar). DNA fragmentation was measured, as described previously (41Seimiya H. Mashima T. Toho M. Tsuruo T. J. Biol. Chem. 1997; 272: 4631-4636Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). In brief, PBS-washed cells (1 × 106 cells) were resuspended in 20 μl of solution A (50 mm Tris-Cl, pH 8.0, 10 mm EDTA, 0.5 mg/ml proteinase K) and incubated at 50 °C for 90 min. Then, 10 μl of solution B (10 mm Tris-Cl, pH 7.6, 15 mm NaCl, 1 μg/ml ribonuclease A) was added and the mixture was further incubated at 50 °C for 90 min. Finally, DNAs in the reaction were resolved by agarose gel electrophoresis (2% agarose, TBE buffer). Propidium iodide staining of cellular DNA was carried out as follows: cells were washed with ice-cold PBS, resuspended in 70% ethanol for 30 min, and treated with 2 mg/ml ribonuclease A/PBS at 37 °C for 30 min. After washing in PBS, cells were resuspended in 50 μg/ml propidium iodide (Sigma), PBS, passed through a 70-μm nylon mesh filter, and analyzed by using a FACScan system (Beckton-Dickinson, Franklin Lakes, NJ). When U937 cells were continuously treated with 5 ng/ml TPA, they differentiate into monocytes and macrophages (42Lotem H. Sachs L. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5158-5162Crossref PubMed Scopus (290) Google Scholar, 43Hass R. Bartels H. Topley N. Hadam M. Kohler L. Goppelt-Strube M. Resch K. Eur. J. Cell Biol. 1989; 48: 282-293PubMed Google Scholar). As shown in Fig. 1 A, the cell surface expression of CD11b glycoprotein, one marker for monocytic differentiation, gradually increased during the course of differentiation. Similarly, CD14 glycoprotein, another marker, was also induced (data not shown). Meanwhile, adhesion of cells to a plastic substratum was one of the most drastic changes observed in the differentiated U937 cells. While the undifferentiated U937 cells were grown in suspension, the TPA-treated cells adhered to a culture dish and the percentage of the adherent cells increased up to 70% in a time-dependent manner (Fig. 1 B). To investigate the kinetics of PTP-U2 activation, we first monitored the PTP-U2 enzyme activity in U937 cells with an anti-PTP-U2 immunocomplex PTP assay (see “Materials and Methods”). As shown in Fig. 2 A, PTP-U2 enzyme activity against phosphotyrosyl substrate 1 (PS1; END(pY)INASL; derived from a highly conserved region of T cell PTP) (35Daum G. Solca F. Diltz C.D. Zhao Z. Cool D.E. Fischer E.H. Anal. Biochem. 1993; 211: 50-54Crossref PubMed Scopus (57) Google Scholar) and PS2 (DADE(pY)LIPQQG; corresponding to the autophosphorylation site of epidermal growth factor receptor) (36Zhang Z.Y. Thieme-Sefler A.M. Maclean D. McNamara D.J. Dobrusin E.M. Sawyer T.K. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4446-4450Crossref PubMed Scopus (180) Google Scholar) was substantially induced during differentiation, and this enzyme activation was parallel to the appearance of differentiation markers (Fig. 1). Maximal activation of the enzyme was observed at 72 h (10.5- and 4.5-fold activation against PS1 and PS2, respectively). A nonspecific PTP inhibitor, sodium orthovanadate (1 mm), efficiently inhibited the activity, indicating that the release of free phosphate from the phosphotyrosyl substrates was due to the catalytic activity of the PTP. The control immunocomplex precipitated without anti-PTP-U2 antibody did not exhibit any trace of PTP activity (data not shown). On the other hand, UT16 cells, TPA-resistant variant of U937 (33Seimiya H. Sawabe T. Toho M. Tsuruo T. Oncogene. 1995; 11: 2047-2054PubMed Google Scholar), did not respond to TPA with the PTP-U2 enzymatic activation. To determine whether enzyme activation of PTP-U2 during TPA-induced differentiation was due to enhanced expression of PTP-U2 protein, we analyzed the amounts of PTP-U2 protein using Western blot analysis. As shown in Fig.2 B, while expression of PTP-U2 protein was not detected after treatment with TPA for 0–6 h, the 70-kDa protein that we reported to be the shorter isoform of PTP-U2 (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar) was induced at 9–72 h. Interestingly, further incubation with TPA led to reduction of the shorter isoform expression and induction of a larger protein at 220 kDa. The size of this protein corresponded to that of the PTP-U2 larger isoform ectopically expressed in 293T and U937 cells (Fig. 2 B, right panel). In this report we refer to the 70- and 220-kDa isoforms as PTP-U2S and PTP-U2L, respectively. Employing the reverse transcriptase-polymerase chain reaction, we also detected gene expression of PTP-U2L in U937 cells treated with TPA for 96 h but not in the control cells (data not shown). On the other hand, the amount of T cell PTP protein, nonreceptor PTP isozyme, was down-regulated during the differentiation, indicating differential expression of PTPs in each isozyme-specific manner. In UT16 cells, neither PTP-U2S nor PTP-U2L were induced by treatment with TPA (data not shown). To elucidate the functional involvement of PTP-U2L in TPA-induced differentiation of U937 cells, we transfected the expression vector for the PTP-U2L gene (under control of a cytomegalovirus immediate-early promoter) into U937 cells. Resulting transfectants named as U21L2, U21L4, and U21L6 constitutively expressed PTP-U2L protein (the representative data are shown in Fig.3 A) and displayed normal growth rates, compared with parental U937 cells and mock transfectants (data not shown). Under normal growth conditions, we did not find significant morphological differences between the cell lines (Fig.4, A-D). Upon treatment with 5 ng/ml TPA for 72 h, however, all the PTP-U2L transfectants exhibited higher induction of CD14 glycoprotein than control transfectants, such as mock4 (p < 0.01 orp < 0.025, evaluated by an unpaired Student'st test, Fig. 3 B). Furthermore, the PTP-U2L transfectants exhibited higher sensitivity to TPA than the control cells (Fig. 3 C). For example, sensitivity of the PTP-U2L transfectants to 1 ng/ml TPA was about 6-fold higher than that of mock4.Figure 4Morphological changes in TPA-treated PTP-U2L transfectants. Cells were cultured with or without 5 ng/ml TPA for 72 h. A and E, U937; B andF, mock4; C and G, U21L2; Dand H, U21L6; A-D, control; E-H, 5 ng/ml TPA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There were also morphological differences between the TPA-treated control and PTP-U2L transfectants (Fig. 4, E-H). In the case of U937 and the mock transfectants, the differentiated cells adhered to the culture dish, but still retained their round shape and looked phase-bright under phase-contrast microscopy. On the other hand, there were more drastic changes in the PTP-U2L transfectants. These cells adhered to the culture dish to such an extent that we could not distinguish them from the other kinds of adherent cells. Some of them lost the globular shape and showed a flat and polygonal morphology. The borders of the cell-cell contact were also observed. More interestingly, cellular fragments, probably due to the formation of apoptotic bodies, were often observed in the culture of TPA-treated PTP-U2L transfectants. This finding suggests that hypersensitivity to TPA of PTP-U2L transfectants (Fig. 3 C), was due to enhanced susceptibility of the cells to TPA-induced apoptosis. To determine whether TPA could induce apoptosis of PTP-U2L transfectants, we analyzed DNA content of each clone during TPA-induced differentiation. Upon treatment with 5 ng/ml TPA for 48–96 h, mock4 cells arrested the cell cycle at the G0/G1 phase (Fig.5 A). Subsequent to this growth arrest, a trace of a subdiploid (apoptotic) fraction was observed at 192 h (day 8). Under the same culture conditions, U21L4 and U21L6 cells exhibited the G0/G1 arrest at 24 h (at least 24 h earlier than the mock4 cells). Furthermore, drastic accumulation of the subdiploid fraction was observed at 96 h. Like U21L4 and U21L6 cells, U21L2, but not mock1 and U937 cells, showed rapid accumulation of the G0/G1 fraction (data not shown) and a drastic shift of the population to the subdiploid fraction (Fig. 5 B). When a cell undergoes apoptosis, chromosomal DNA is degraded into nucleosome-size fragments, that is a most characteristic property of apoptotic cell death. We observed TPA-induced fragmentation of the chromosomal DNA in the PTP-U2L transfectants but not in the mock clones (Fig. 5 C). At the early phase of apoptosis, the plasma membrane phosphatidylserine is externalized to the cell surface (37Koopman G. Reutelingsperger C.P.M. Kuijten G.A.M. Keehnen R.M.J. Pals S.T. van Oers M.H.J. Blood. 1994; 85: 1415-1420Crossref Google Scholar, 38Martin J.S. Reutelingsperger C.P.M. McGahon A.J. Rader J.A. van Schie R.C.A.A. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2566) Google Scholar). Consistent to this event, TPA-treated PTP-U2L transfectants significantly externalized phosphatidylserine but the mock4 cells did not (Fig. 5 D). This also means that TPA-induced differentiation itself does not lead to externalization of phosphatidylserine. Taken together, these data indicate that PTP-U2L enhances TPA-induced cell death of U937 cells, and the mode of cell death seems to be apoptosis. Caspase family proteases (originally known as ICE-like proteases) are downstream regulators of apoptosis (44Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 45Kumar S. Harvey N.L. FEBS Lett. 1995; 375: 169-173Crossref PubMed Scopus (151) Google Scholar). We have previously reported that benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB), an aspartate-based inhibitor of caspases, inhibited apoptosis of U937 cells induced by various stimuli (46Mashima T. Naito M. Kataoka S. Kawai H. Tsuruo T. Biochem. Biophys. Res. Commun. 1995; 209: 907-915Crossref PubMed Scopus (162) Google Scholar). So, we examined the effect of Z-Asp-CH2-DCB on TPA-induced apoptosis of PTP-U2L transfectants. As shown in Fig.6, a topoisomerase I inhibitor, camptothecin, drastically induced apoptosis of U937 cells, but it was completely blocked in the presence of 50 μg/ml Z-Asp-CH2-DCB. On the other hand, the inhibitory effect of Z-Asp-CH2-DCB on TPA-induced apoptosis of PTP-U2L transfectants (U21L clones) was also detected, but it was more moderate than on the camptothecin-induced apoptosis. In the presence of Z-Asp-CH2-DCB, TPA-induced apoptosis of U21L clones was only 35% inhibited. To determine whether apoptosis enhancement by PTP-U2L required catalytic activity of the enzyme, we constructed a catalytically inactive mutant PTP-U2L(C → S) gene, in which the codon for an essential cysteine residue (TGC) in the conserved active-site HCxxGxxRS(T) motif (for review, see Ref. 47Denu J.M. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) was point mutated to that for serine (AGC). The mutant PTP-U2L(C → S) gene was transfected into U937 cells, and the resulting U2L/CS20 and U2L/CS21 were representative clones that stably expressed the mutant PTP-U2L(C → S) protein (Fig. 7 A). Under normal growth conditions, growth rates of PTP-U2L(C → S) transfectants were comparable to those of wild-type PTP-U2L and mock transfectants (data not shown). Intracellular amounts of the ectopic PTP-U2L protein in the mutant transfectants were often higher than those in wild-type transfectants; the representative result is shown in Fig. 7 A. Upon treatment with 5 ng/ml TPA for 72 h, U2L/CS20 and U2L/CS21 cells still differentiated into monocytic lineage and adhered to the culture dish. As shown in Fig. 8, the differentiated cells adhered extensively to the dish and showed flat and polygonal morphology, which was similar to the phenotypic changes observed in the TPA-treated wild-type PTP-U2L transfectants (Fig. 4). These observations indicate that PTP-U2L(C → S), despite the loss of its catalytic activity, retained its accelerative effect on TPA-induced differentiation of U937 cells. We further examined the susceptibility of U2L/CS20 and U2L/CS21 cells to TPA-induced apoptosis. As shown in Fig. 7 B, PTP-U2L(C → S) still enhanced apoptosis of the transfectant to some extent, as compared with mock4 cells, which differentiated and was strictly arrested at the G0/G1 phase of the cell cycle. However, the effect of PTP-U2L(C → S) on apoptosis enhancement was considerably reduced, compared with wild-type PTP-U2L (p < 0.01, Fig. 7, B and C; see also Fig. 8, Cand D). This observation suggests that PTP-U2L catalytic activity is required for maximal enhancement of TPA-induced apoptosis by PTP-U2L. These findings were repeated with other PTP-U2L(C → S) transfectants with similar results (Fig. 7 C). PTP-U2L transfectants exhibited progressive adhesion to the culture dish (Fig. 4). PTP-U2L(C → S) transfectants also retained this ability (Fig. 8), suggesting that the extracellular domain of PTP-U2L, consisting of eight fibronectin type III-like repeats, directly functioned as an adhesion molecule in a catalytic activity-independent manner (Fig. 9). Similar to these observations, previous reports have revealed that type II receptor-like PTPs, RPTPμ and RPTPκ, which have extracellular domains consisting of four fibronectin type III-like repeats, a single Ig-like domain, and an NH2-terminal MAM domain, mediate homophilic cell-cell adhesion even without the cytoplasmic domain (48Brady-Kalnay S.M. Flint A.J. Tonks N.K. J. Cell Biol. 1993; 122: 961-972Crossref PubMed Scopus (242) Google Scholar, 49Sap J. Jiang Y.-P. Friedlander D. Grumet M. Schlessinger J. Mol. Cell. Biol. 1994; 14: 1-9Crossref PubMed Google Scholar, 50Gebbink M.F.B.G. Zondag G.C.M. Wubbolts R.W. Beijersbergen R.L. van Etten I. Moolenaar W.H. J. Biol. Chem. 1993; 268: 16101-16104Abstract Full Text PDF PubMed Google Scholar). Since the PTP-U2L transfectants were still in suspension in the absence of TPA, the extracellular domain of PTP-U2L would enhance cellular adhesion in cooperation with other molecules which expression was induced by TPA. Among other characteristics of TPA-induced differentiation, CD14 expression was enhanced and the G0/G1 arrest occurred earlier in the cell cycle of PTP-U2L transfectants than in that of mock transfectants (Figs. 3 B and 5 A). On the other hand, CD11b up-regulation by TPA was comparable with PTP-U2L and mock transfectants (data not shown). These observations indicate that there are parallel but distinct signaling pathways for TPA-induced differentiation and that PTP-U2L specifically participates in some of those pathways. While PTP-U2L enhanced TPA-induced apoptosis of U937 cells (Figs. 4 and5), it seems to be specific to terminal differentiation of leukemia cells or at least be limited to certain situations. First, there are normal tissues that constitutively express PTP-U2L (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar). Among them are kidney and brain, in which podocyte and olfactory bulb exhibit dominant expression of PTP-U2L, respectively (27Thomas P.E. Wharram B.L. Goyal M. Wiggins J.E. Holzman L.B. Wiggins R.C. J. Biol. Chem. 1994; 269: 19953-19962Abstract Full Text PDF PubMed Google Scholar, 51Yahagi Y. Tagawa M. Tomoda T. Shirasawa T. Neurosci. Lett. 1996; 211: 125-128Crossref PubMed Scopus (6) Google Scholar). Second, when 293T cells were transfected with the PTP-U2L gene, TPA sensitivity of the resulting transfectants was comparable to that of the mock cells.2 Third, PTP-U2L was induced during TPA-induced differentiation (Fig. 2 B) but not during camptothecin- or etoposide-induced apoptosis of U937 cells (data not shown). Apoptosis enhancement by PTP-U2L required exposure of the cells to TPA (Figs. 4 and 5). While TPA is a potent activator of protein kinase C (52Blumberg P.M. Cancer Res. 1988; 48: 1-8PubMed Google Scholar) and causes activation of the further downstream ERK/MAPK cascade (53Kharbanda S. Saleem A. Emoto Y. Stone R. Rapp U. Kufe D. J. Biol. Chem. 1994; 269: 872-878Abstract Full Text PDF PubMed Google Scholar), 1-oleoyl-2-acetylglycerol, another protein kinase C activator, does not induce differentiation of myeloid leukemia cells (54Kreutter D. Caldwell A.B. Morin M.J. J. Biol. Chem. 1985; 260: 5979-5984Abstract Full Text PDF PubMed Google Scholar). Consistently, 1-oleoyl-2-acetylglycerol did not induce apoptosis of the PTP-U2L transfectants at all despite ERK/MAPK activation (data not shown). These data suggest that apoptosis enhancement by PTP-U2L requires a differentiation-associated factor induced by TPA but not by 1-oleoyl-2-acetylglycerol (Fig. 9). The result in Fig. 6 suggests that Z-Asp-CH2-DCB-sensitive caspases are, in part, involved in the PTP-U2L-mediated apoptosis of differentiated U937 cells. Strictly speaking, the inhibitory effect of Z-Asp-CH2-DCB was only partial, and there could be other mechanisms of PTP-U2L-mediated apoptosis that are independent of Z-Asp-CH2-DCB-sensitive caspases. In fact, caspase-3 activation was significantly observed during camptothecin-induced apoptosis of U937 cells whereas that was only marginal during PTP-U2L-mediated apoptosis of the cells.2 PTP-U2L(C → S) exhibited an intermediate effect on TPA-induced apoptosis, as compared with mock and wild-type PTP-U2L (Figs. 7,B and C, and 8). These data suggest that maximal enhancement of apoptosis by PTP-U2L requires its catalytic activity. In that sense, it is still possible that overexpression of PTP-U2L itself could elicit apoptosis in U937 cells. In fact, the wild-type PTP-U2L transfectants generally expressed lower amounts of PTP-U2L protein than the mutant types did (Fig. 7 A). One explanation for this circumstance is that high producer clones for the wild-type PTP-U2L might die by apoptosis during the cloning procedures. Consistent with this idea, transfection experiments of wild-type PTP-U2L into TPA-resistant UT16 cells have not worked so far, whereas it has been easy to obtain the PTP-U2L(C → S)-transfected UT16 clones.2 As UT16 cells have been established by prolonged exposure of U937 cells to TPA (33Seimiya H. Sawabe T. Toho M. Tsuruo T. Oncogene. 1995; 11: 2047-2054PubMed Google Scholar), differentiation-associated factor(s), required for the PTP-U2L-mediated and its catalytic activity dependent apoptosis (see above), seem to have irreversibly up-regulated in UT16 cells. If this were the case, introducing the wild-typePTP-U2L gene would kill the cells by apoptosis. In the presence of a caspase inhibitor (Z-Asp-CH2-DCB), only 35% of the wild PTP-U2L-mediated apoptosis was inhibited (Fig. 6), whereas 53% of the C → S mutant-mediated apoptosis was inhibited (data not shown). These observations suggest that the PTP-U2L catalytic activity dependent apoptosis, which is abolished by the C → S mutation, represents the caspase-insensitive cell death. Meanwhile, it has been also suggested that PTP(C → S) mutants in some case might exhibit a dominant positive effect by trapping their substrates (7Tonks N.K. Neel B.G. Cell. 1996; 87: 365-368Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar), and determination of specific substrate(s) for PTP-U2L is our future interest to understand the downstream pathway of PTP-U2L-mediated signaling. PTP-U2 is expressed as at least three different isoforms in a tissue-specific manner (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar). In addition, more shorter transcripts than PTP-U2S have been reported as PTPφ, the murine homolog of PTP-U2 (28Pixley F.J. Lee P.S.W. Dominguez M.G. Einstein D.B. Stanley E.R. J. Biol. Chem. 1995; 270: 27339-27347Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Quiescent macrophages express PTPφ, which is down-regulated when the cells are activated by colony-stimulating factor-1 (28Pixley F.J. Lee P.S.W. Dominguez M.G. Einstein D.B. Stanley E.R. J. Biol. Chem. 1995; 270: 27339-27347Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). During the course of TPA-induced differentiation of U937 cells, PTP-U2S was induced as an early event whereas PTP-U2L was as a late event (Fig. 2 B). Given the kinetics of PTP-U2S induction, one could speculate that the shorter isoforms, such as PTP-U2S and PTPφ, might play a role in triggering commitment to differentiation or in maintaining differentiated phenotypes of the cells (see the scheme shown in Fig. 9). Determination of PTP-U2S whole cDNA structure would afford transfection experiments to elucidate the precise role of PTP-U2S. Terminally differentiated hematopoietic cells often die by apoptosis (55Martin S.J. Bradley J.G. Cotter T.G. Clin. Exp. Immunol. 1990; 79: 448-453Crossref PubMed Scopus (390) Google Scholar). Here we have demonstrated the first instance of PTP involvement in apoptosis subsequent to terminal differentiation of leukemia cells. The present data also suggest a functional involvement of PTP-U2L in the physiological events. For example, PTP-U2L might participate in turnover of peripheral myelocytes by eliminating them by apoptosis. If this is so and there are no functionally redundant PTP isozymes, failure in PTP-U2L function could have serious outcomes, such as hematopoietic disorders. The PTP-U2 gene is localized to chromosome 12p13.2-p13.3 (26Seimiya H. Sawabe T. Inazawa J. Tsuruo T. Oncogene. 1995; 10: 1731-1738PubMed Google Scholar), a region of interest because it is a common site of chromosomal abnormalities in such malignant proliferations as eosinophilia (56Keene P. Mendelow B. Pinto M.R. Bezwoda W. MacDougall L. Falkson G. Ruff P. Bernstein R. Br. J. Haematol. 1987; 67: 25-31Crossref PubMed Scopus (104) Google Scholar). Meanwhile, PTP-U2 is expressed as various isoforms in a tissue-specific manner. Thus, determination of the physiological role of PTP-U2 awaits the generation of PTP-U2 knock-out animals and cell lines. We are grateful to Naomi Wada for skillful assistance with experiments." @default.
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• W2086883696 title "Functional Involvement of PTP-U2L in Apoptosis Subsequent to Terminal Differentiation of Monoblastoid Leukemia Cells" @default.
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