From the Cancer Chemotherapy Center, Japanese
Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku,
Tokyo 170-8455, Japan and the § Institute of Molecular and
Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-0032, Japan
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ABSTRACT |
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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 PTP
PTP-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.
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INTRODUCTION |
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Protein tyrosine phosphatases (PTPs),1 as well as protein tyrosine kinases, regulate tyrosine phosphorylation, which plays a significant role in cellular proliferation, differentiation, and oncogenesis (1, 2). Since hyperphosphorylation of tyrosine by oncogenic tyrosine kinases often leads to transformation of cells (3), it has been originally postulated that PTPs, which reverse the phosphorylation reaction, might play a role in suppressing oncogenesis (1). 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 (4-6). 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.
In fact, recent studies have demonstrated specific functions of PTP isozymes in physiological events (see for review, Ref. 7). 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 (8). SHP1 negatively regulates hematopoietic signaling pathways (9-11). At the pathological level, genetic failure in the shp1 locus leads to autoimmune disease with hyperproliferation of hematopoietic cells (12, 13). On the other hand, SHP2 is required for mitogenic signal transduction from receptor tyrosine kinases to the Ras/mitogen-activated protein kinase (MAPK) pathway (14-17). 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 (18-20), 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 (21-24). Meanwhile, such
differentiation is often inhibited by sodium orthovanadate, a PTP
inhibitor (25). Previously, we identified 13 PTP gene fragments from
the differentiated monoblastoid leukemia U937 cells by employing the
reverse transcription-polymerase chain reaction strategy (24). Among
them, gene expression for four isozymes, including PTP-U2
(also known as GLEPP1/PTP) (26-28), PTP-MEG2 (29), P19-PTP (30), and PTP-U1/DEP-1/HPTP
(24,
31, 32), is induced during monocytic and granulocytic differentiation
of myeloid leukemia cells
(33).2
PTP-U2 is a receptor-like PTP with a single transmembrane domain and a single intracellular catalytic domain (26). Physiologically, the PTP-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 putative N-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 (24). 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-U2 transcript. On the contrary, TPA does not induce or enhance PTP-U2 gene expression in solid tumor cells that do not differentiate upon treatment with TPA (26). 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.
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MATERIALS AND METHODS |
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Cell Culture and Differentiation Induction-- Human monoblastoid leukemia U937 cells were grown as described previously (24). A phorbol ester-resistant U937 variant, UT16, was established and characterized as described (33). 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 (33).
Preparation of Cellular Lysates-- 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 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 2% Triton X-100 (34). 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).
Western Blot Analysis-- 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 (26) 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.).
PTP Enzyme Assay-- 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 mM 2,3-dihydroxybutane-1,4-dithiol). PTP activity in the immunocomplex was measured against phosphotyrosyl substrates, END(pY)INASL (PS1) (35) and DADE(pY)LIPQQG (PS2) (36), by using the Tyrosine Phosphatase Assay System (Promega, Madison, WI), according to the instruction manual.
Vector Constructions--
The full-length cDNA for
PTP-U2L (26) 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.
DNA Transfection--
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.
Measurement of Apoptotic Cells-- Apoptosis was monitored through characteristic changes in cellular morphology, externalization of phosphatidylserine (37, 38), and the appearance of chromosomal DNA fragmentation (39). Externalization of phosphatidylserine was detected by annexin V-fluorescein isothiocyanate (Kamiya Biomedical, Seattle, WA) staining, as described previously (40). DNA fragmentation was measured, as described previously (41). 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).
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RESULTS |
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PTP-U2 Enzyme Activation during TPA-induced Differentiation of U937 Cells-- When U937 cells were continuously treated with 5 ng/ml TPA, they differentiate into monocytes and macrophages (42, 43). As shown in Fig. 1A, 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. 1B).
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PTP-U2L Isoform Is Induced as the Late Event of TPA-induced Differentiation of U937 Cells-- 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. 2B, 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 (26) 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. 2B, 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).
Ectopic Expression of PTP-U2L Enhances the Growth Inhibitory Effect of TPA on U937 Cells-- 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. 3A) 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 or p < 0.025, evaluated by an unpaired Student's t test, Fig. 3B). Furthermore, the PTP-U2L transfectants exhibited higher sensitivity to TPA than the control cells (Fig. 3C). For example, sensitivity of the PTP-U2L transfectants to 1 ng/ml TPA was about 6-fold higher than that of mock4.
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Hypersensitivity to TPA of PTP-U2L Transfectants Is Associated with an Enhanced Rate of Apoptotic Cell Death-- 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. 5A). 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. 5B). 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. 5C).
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TPA-induced Apoptosis of PTP-U2L Transfectants Is Only Partially Inhibited by a Caspase Inhibitor-- Caspase family proteases (originally known as ICE-like proteases) are downstream regulators of apoptosis (44, 45). 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 (46). 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.
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PTP-U2L Catalytic Activity Is Required for Maximal Induction of
TPA-induced Apoptosis of U937 Cells--
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. 47) 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. 7A).
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. 7A.
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DISCUSSION |
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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 RPTP
, 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
(48-50). 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.
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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. 3B and 5A). 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 and 5), 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 (26). Among them are kidney and brain, in which podocyte and olfactory bulb exhibit dominant expression of PTP-U2L, respectively (27, 51). 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. 2B) 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 (52) and causes activation of the further downstream ERK/MAPK cascade (53), 1-oleoyl-2-acetylglycerol, another protein kinase C activator, does not induce differentiation of myeloid leukemia cells (54). 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. 7A). 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 (33), 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-type PTP-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 (7), 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 (26). In addition, more shorter transcripts than PTP-U2S have been reported as PTP, the
murine homolog of PTP-U2 (28). Quiescent macrophages express
PTP
, which is down-regulated when the cells are activated by
colony-stimulating factor-1 (28). 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. 2B). Given the
kinetics of PTP-U2S induction, one could speculate that the shorter
isoforms, such as PTP-U2S and PTP
, 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 (55). 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 (26), a region of interest because it is a common site of chromosomal abnormalities in such malignant proliferations as eosinophilia (56). 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.
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ACKNOWLEDGEMENT |
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We are grateful to Naomi Wada for skillful assistance with experiments.
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FOOTNOTES |
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* This work was supported in part by a special grant for Advanced Research on Cancer from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Tel.: 81-3-3918-0111; Fax: 81-3-3918-3716; E-mail, hseimiya{at}jfcr.or.jp.
The abbreviations used are: PTP, protein tyrosine phosphatase; MAPK, mitogen-activated protein kinase; TPA, 12-O-tetradecanoyl-phorbol-13-acetateZ-Asp-CH2-DCB, benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzeneSH2, Src homology domain 2PBS, phosphate-buffered saline.
2 H. Seimiya and T. Tsuruo, unpublished results.
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REFERENCES |
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