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Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2577-2585
HEMATOPOIESIS
From the Departments of Hematology/Oncology and Molecular Oncology,
Biomedical Research Center, Osaka University Medical School, Osaka, and
the Department of Biochemistry (I), Jikei University School of
Medicine, Tokyo, Japan.
The ubiquitin-proteasome pathway is responsible for selective
degradation of short-lived cellular proteins and is critical for the
regulation of many cellular processes. We previously showed that
ubiquitin (Ub) secreted from hairy cell leukemia cells had inhibitory
effects on clonogenic growth of normal hematopoietic progenitor cells.
In this study, we examined the effects of exogenous Ub on the growth
and survival of a series of human hematopoietic cells, including
myeloid cell lines (HL-60 and U937), a B-cell line (Daudi), and T-cell
lines (KT-3, MT-4, YTC-3, and MOLT-4). Exogenous Ub inhibited the
growth of various hematopoietic cell lines tested, especially of KT-3
and HL-60 cells. The growth-suppressive effects of Ub on KT-3 and HL-60
cells were almost completely abrogated by the proteasome inhibitor PSI
or MG132, suggesting the involvement of the proteasome pathway in this
process. Furthermore, exogenous Ub evoked severe apoptosis of KT-3 and
HL-60 cells through the activation of caspase-3. In interleukin-6
(IL-6)-dependent KT-3 cells, STAT3 was found to be conjugated by
exogenous biotinylated Ub and to be degraded in a proteasome-dependent
manner, whereas expression levels of STAT1, STAT5, or mitogen-activated
protein kinase were not affected. Moreover, IL-6-induced the
up-regulation of Bcl-2 and c-myc, and JunB was impaired in
Ub-treated KT-3 cells, suggesting that the anti-apoptotic and mitogenic
effects of IL-6 were disrupted by Ub. These results suggest that
extracellular Ub was incorporated into hematopoietic cells and mediated
their growth suppression and apoptosis through proteasome-dependent degradation of selective cellular proteins such as STAT3.
(Blood. 2000;95:2577-2585)
Hematopoiesis is regulated by a wide variety of
external stimuli, including those from hematopoietic growth factors. On
binding to cell-surface receptors, the growth factors activate multiple signaling molecules, resulting in the induction of specific target genes required for physiologic processes such as cell growth and survival (reviewed in Ihle1 and Darnell2).
Recently, several transcription factors such as STATs (signal
transducers and activators of transcription) and AP-1 have been
reported to be implicated as important mediators of growth signals from
membrane to nucleus, where they contribute to cell-cycle progression
through transcriptional regulation of cell-cycle regulatory genes,
including cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors.
Hematopoietic growth factors also exert anti-apoptotic effects through
the activation of Ras/mitogen-activated protein kinase (MAPK) pathways,
phosphatidylinositol 3'-kinase (PI3-K)/Akt pathways, or STATs
(reviewed in Miyajima et al3). In addition, Bcl-2 family
proteins behave as cell-death antagonists (eg, Bcl-2, Bcl-XL, and
Mcl-1) or agonists (eg, Bax, Bad, and Bak) (reviewed in
Reed4 and Tsuimoto5) and play crucial
roles in anti-apoptotic effects of hematopoietic growth factors. It was
reported that expression levels of Bcl-XL proteins were tightly
regulated by hematopoietic growth factors, and the overexpression of
Bcl-XL effectively protected IL-3-dependent myeloid cells from
factor-deprived apoptosis.6 Furthermore, IL-3-activated
Akt was shown to phosphorylate serine residues of BAD in a
PI-3K-dependent manner, thereby protecting the cells from intrinsic
death machinery.7,8
The expression levels of cell-cycle regulatory molecules and signal
transduction molecules are regulated at transcriptional and
posttranscriptional levels, and the ubiquitin (Ub)-proteasome system
plays the most important role in the latter step (reviewed in
Hochstrasser9 and Vershavsky10). Ub is a highly
conserved 76-amino acid polypeptide, and the Ub-proteasome-mediated
proteolysis is executed in a series of enzymatic reactions. First, Ub
is charged with adenosine triphosphate by Ub-activating enzyme (E1) and
then transferred to the substrate by a Ub-conjugating enzyme (E2). An
element within the target protein termed a degron is recognized by E2 either alone or in combination with a ubiquitin ligase (E3). After recognition of the degron, the Ub-target protein
conjugates are formed through an isopeptide bond between the
carboxyl-terminal glysine of Ub and a lysine residue on the target
protein. Repeated rounds of ubiquitylation result in highly
ubiquitylated target protein, which is recognized and destroyed rapidly
by the 26S proteasome. At present, a number of molecules are known to
be the targets of the Ub-proteasome system as follows: cell-cycle regulatory molecules, E2F, cyclin D1, cyclin E, cyclin A, cyclin B,
CDC25B, p21WAF,1
p27Kip,1 and p53; signal transduction
molecules, c-jun, c-myc, STAT1, STAT3, SOCS1, NF Hairy cell leukemia (HCL) is a hematologic malignancy characterized by
a unique morphology of leukemic cells bearing hairy cytoplasmic
projections.11 A major clinical manifestation in patients
with HCL is marked pancytopenia. We previously hypothesized that HCL
cells might secrete a factor capable of inhibiting normal hematopoiesis. Supporting this hypothesis, the conditioned media of
hairy cells were found to contain an approximately 8-kd protein that
inhibits clonogenic growth of granulocyte-macrophage colony-forming units and erythroid colony-forming units in vitro.12
Subsequently, we purified the factor from the conditioned media of an
HCL cell line and found it to have the amino-terminal sequence
identical to that of Ub.13 Furthermore, we demonstrated
that the exogenous addition of purified Ub significantly inhibited
colony formation of normal myeloid and erythroid hematopoietic
progenitor cells.14 These results suggested that
extracellular Ub could affect the growth of hematopoietic cells.
However, the biologic significance and molecular mechanism of growth
inhibition by extracellular Ub have yet to be determined. In this
study, we examined the effects of exogenous Ub on various types of
human hematopoietic cells lines, especially on a human interleukin-6
(IL-6)-dependent cell line KT-3, because IL-6 receptor (gp130) is
known to mediate signaling critical for various aspects of
hematopoiesis. Treatment with Ub was found to result in growth
suppression of various hematopoietic cells through the induction of
apoptosis. The Ub-induced apoptosis was proteasome dependent, and, in
IL-6-dependent KT-3 cells, Ub-induced growth suppression and apoptosis
was accompanied by proteasome-dependent STAT3 degradation. These
results suggest that extracellular Ub may suppress normal hematopoiesis
through the selective degradation of intracellular proteins such as STAT3.
Reagents and antibodies
Cells and cultures
Radioimmunoassays for measurement of Ub concentration Ub concentration in the sera of HCL patients and the conditioned media of the cultured cells was measured by radioimmunoassays, as previously reported.16Cell proliferation assay To quantitate the proliferation of cultured cells, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma) rapid colorimetric assay was used as previously reported.17 In brief, triplicate aliquots of cells (3 × 104 cells) were resuspended in 100 µL ASF103 medium (Ajinomoto, Kawasaki, Japan) and cultured in 96-well flat-bottom microtiter plates for 48 hours at 37°C in the conditions as indicated. Normal peripheral blood mononuclear cells were stimulated with 5 µg/mL PHA with or without Ub. MTT (10 µL a 5-mg/mL solution in phosphate-buffered saline [PBS]) was added for the final 4 hours of culture, and 100 µL acid isopropanol (0.04 N HCl in isopropanol) was added to all wells and mixed. The optical density was then measured on the MicroELISA plate reader (Corona Electric, Ibaragi, Japan) with a test wavelength of 595 nm and a reference wavelength of 620 nm. This assay was found to give equivalent results obtained by 3H-thymidine incorporation or cell enumeration as described previously.17DNA content analysis The DNA content of cultured cells was analyzed by staining with propidium iodide (PI) as previously described.18 Briefly, 1 × 106 cells were washed with ice-cold PBS twice and fixed by 70% ethanol at 20°C for 30 minutes. The fixed
cells were incubated in 500 µL staining buffer (1 mg/mL RNase, 20 mg/mL PI, and 0.01% NP-40 in PBS) at 37°C for 10 minutes and then
analyzed on FACSort (Becton Dickinson, Oxnard, CA) with a program
Modfit LT2.0 (Becton Dickinson).
TUNEL assays TUNEL assays were performed with the In Site Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN). Briefly, cells were fixed in 4% paraformaldehyde in PBS for 30 minutes, transferred to permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate), and incubated on ice for 2 minutes. After washing with PBS, the cells were resuspended in TUNEL reaction mixture containing TdT enzyme and digoxigenin nucleotide. Incorporation of nucleotides into 3'-DNA fragmented ends was detected by flow cytometry.Annexin-V staining Cells were washed with RPMI 1640 twice and resuspended in 100 µL labeling solution containing avidin-annexin-V conjugates at room temperature for 30 minutes. The cells were rinsed and developed with fluorescein-conjugated avidin (Becton Dickinson) at 4°C for 30 minutes. The stained cells were analyzed by flow cytometry.Assays for caspase-3 activities Caspase-3 activities were measured with PhiPhiLux-G1D2 kit (OncoImmunin, College Park, MD). Briefly, cells were washed with PBS and resuspended in 50 µL substrate solution supplied by the manufacturer containing the caspase-3-specific substrate with amino acid sequence GDEVDGI. After 60-minute incubation in a 5% CO2 incubator at 37°C, the cells were suspended in 500 µL dilution buffer supplied by the manufacturer and subjected to flow cytometry. In this system, caspase-3 activities are measured by fluorescence derived from the cleaved substrate specific for caspase-3.In vitro biotinylation of ubiquitin Ub was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacture's protocol, and unreacted biotin was removed by NAP-10 column (Pharmacia Biotech, Uppsala, Sweden) as previously described.19Northern blot analysis Northern blot analysis was performed as previously reported.20 Briefly, total cellular RNA was isolated with Trisol reagent (Gibco BRL, Gaithersburg, MD). For Northern blot analysis, equal amounts of RNA (15 µg) were size fractionated by electrophoresis through 1% formaldehyde agarose gels. After blotting to the nylon membrane (GeneScreen Plus; NEN, Boston, MA), the filters were prehybridized and then hybridized with random 32P-labeled probe in rapid hybridization buffer (Amersham, Tokyo, Japan) for 2 hours at 65°C. The filters were washed and autoradiographed at70°C with 2 intensifying screens for 1 to 2 days.
Immunoprecipitation and immunoblotting Isolation of total cellular lysates, immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to the methods described previously.21 Briefly, the cultured cells were lysed in lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 1% NP-40, 10 mmol/L EDTA, and 100 mmol/L NaF) containing protease and phosphatase inhibitors. Insoluble material was removed by centrifugation at 10 000g for 20 minutes at 4°C. For immunoprecipitation, the lysates were precleared with protein-G Sepharose beads (Pharmacia Biotech) for 2 hours at 4°C. The precleared lysates were incubated with 1 µg anti-STAT3 polyclonal antibody followed by the addition of protein-G Sepharose beads (Pharmacia Biotech). For Western blotting, immunoprecipitated proteins or total cellular lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Bedford, MA). After blocking residual binding sites on the filter by incubation in TBS (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl) containing 1% gelatin (Bio-Rad Laboratories, Richmond, CA), immunoblotting was performed with an appropriate antibody. Immunoreactive proteins were visualized with an enhanced chemiluminescence (ECL) detection system (DuPont NEN, Boston, MA). To detect biotin-labeled Ub, the blotted membrane was incubated with ExtrAvidin peroxidase (Sigma) at room temperature for 1 hour and washed. Then biotin-labeled Ub was visualized with the ECL detection system. In some experiments, the filters were stripped and reprobed with the anti-STAT3 or anti-MAPK antibody to examine the amounts of proteins.Transient transfection into the cells 293T cells (1 × 106 cells) were seeded in a 60-mm dish, cultured for 24 hours, and cotransfected with 10 µg pMT123 (an expression vector of HA-tagged Ub, kindly provided from Dr D. Bohmann, Heidelberg, Germany)22 and 1 µg pMX-GFP (an expression vector of green fluorescence protein [GFP], kindly provided by Dr T. Kitamura, Tokyo University, Tokyo, Japan) by calcium phosphate coprecipitation method. Transfection into Ba/F3 cells was performed by electroporation method as previously described.21 Briefly, 1 × 107 cells were transfected with 30 µg pMT123 together with 30 µg pMX-GFP by electroporation (350V, 960 µFD) (Bio-Lad Laboratories, Richmond, CA). After 12 hours, transfected cells were washed, serum deprived, and cultured for 24 hours. Then transfection efficiencies were monitored by the GFP expression by flow cytometric analyses. Ub concentrations in the conditioned media were measured as described above.
Intracellularly synthesized ubiquitin is secreted outside the cells In previous studies, we found that intracellular Ub secreted from HCL cells would inhibit normal hematopoiesis in patients with HCL.12-14 In this study, we initially examined whether intracellularly synthesized Ub could be secreted outside the cells. For this purpose, we transfected an expression vector of Ub along with that of GFP into a murine pro-B cell line, Ba/F3, and a human kidney cell line, 293T. After 12 hours, the transfected cells were washed and cultured in serum-deprived conditions for 24 hours. After 36 hours from the transfection, approximately 60% of Ba/F3 cells and 95% of 293T cells were found to be positive for GFP expression by flow cytometric analysis (data not shown). The supernatant obtained from Ub-transfected Ba/F3 and 293T cells was found to contain more increased levels of Ub than those obtained from corresponding mock-transfected or untreated cells (Ub concentration in the supernatant, Ub-transfected Ba/F3 80 ng/mL, mock-transfected Ba/F3 12.4 ng/mL, and untreated Ba/F3 10.7 ng/mL; Ub-transfused 293T 141 ng/mL, mock-transfected 293T 19.7 ng/mL, and untreated 293T 12.4 ng/mL). These results indicated that intracellularly synthesized Ub could be secreted outside the cells.Addition of exogenous ubiquitin leads to growth suppression of hematopoietic cells We next examined the effects of exogenously added Ub on the growth of 2 human hematopoietic cell lines, a human promyelocytic leukemia cell line HL-60 and a human IL-6-dependent T-cell line KT-3. As shown in Figure 1A, the treatment with Ub suppressed the factor-independent growth of HL-60 cells and the IL-6-dependent and -independent growth of KT-3 cells. The suppressive effect of Ub was dose dependent, with readily detectable activity at more than 10 µg/mL Ub and maximal activity at 100 µg/mL. We next examined the effects of Ub (100 µg/mL; we used this concentration in the following experiments) on the growth of various types of hematopoietic cells, including a human monocytic leukemia cell line U937, a human Burkitt's lymphoma cell line Daudi, human T-cell lines MT-4, YTC-3, and MOLT-4, and PHA-stimulated normal peripheral blood mononuclear cells. As shown in Figure 1B, the treatment with Ub induced growth suppression in all cell types tested, though a considerable difference was observed in the rate of growth suppression between target cells. To determine further whether proteolysis mediated by the Ub-proteasome pathway was involved in the growth suppression, we investigated the effects of proteasome inhibitors PSI and MG132 on the Ub-induced growth suppression of KT-3 cells (Figure 1C). The suppressive effect of Ub on IL-6-induced proliferation of KT-3 cells was rescued by the addition of either PSI or MG132 in a dose-dependent manner, suggesting the involvement of the proteasome pathway in the growth inhibition by Ub. To further define the roles of extracellular Ub and to deny the effects of toxic contaminants, we examined the effects of methylated Ub (Met-Ub) (Sigma) on Ub-induced growth suppression of KT-3 cells. Met-Ub can be ligated to the target proteins but cannot form polyubiquitin chains. Thus, it has been shown to act as an specific inhibitor of ubiquitin-dependent protein degradation in a previous article.23 KT-3 cells were preincubated with or without Met-Ub for 3 hours and subjected to culture with Ub. As shown in Figure 1C, the pretreatment with Met-Ub alone showed little effect on the growth of KT-3 cells, whereas it restored Ub-induced growth suppression nearly completely, suggesting that extracellular Ub would induce growth suppression in KT-3 cells through the polyubiquitin chain formation on target proteins as would intracellular Ub.
Ubiquitin induces apoptosis in HL-60 and KT-3 cells To elucidate the mechanism of Ub-induced growth suppression, HL-60 and KT-3 cells were cultured in serum-deprived conditions with or without Ub treatment, and cell viability was quantitated by trypan blue dye exclusion method at various times after the addition of exogenous Ub. Although viability of HL-60 cells was not significantly changed in the absence of Ub (Figure 2A, left panel), Ub treatment of HL-60 cells led to a decrease in the proportion of viable cells. When KT-3 cells were cultured in a serum-deprived medium containing rhIL-6, their viability gradually decreased with the lapse of time even in the absence of Ub (Figure 2A, right panel). However, the decrease in cell viability of KT-3 cells was more rapidly and remarkably induced by Ub treatment. In accord with these findings, DNA content analysis revealed that Ub treatment led to a more marked increase in the proportions of apoptotic cells in both cell types, which were detected as a subdiploid fraction by PI staining (% subdiploid fraction at 48 hours: HL-60, Ub 8% vs.
Ub + 85%; KT-3, Ub 37% vs. Ub + 73%) (Figure 2B).
Treatment of ubiquitin leads to degradation of STAT3 in
IL-6-stimulated KT-3 cells
STAT3 is specifically degraded by extracellular ubiquitin
Exogenously added ubiquitin conjugates to and destroys STAT3 in a
proteasome-dependent manner
p21WAF1 and p27Kip1 are also degraded by
extracellular ubiquitin
IL-6-induced Bcl-2 expression was inhibited by ubiquitin treatment in KT-3 To clarify further the mechanisms underlying Ub-induced apoptosis of KT-3 cells, we examined changes in expression of apoptosis-regulating genes by Northern blot analysis (Figure 7A). After starvation of rhIL-6, KT-3 cells were stimulated with rhIL-6 in the presence or absence of Ub. After the addition of rhIL-6, expression of Bcl-2 mRNA was gradually induced at 4 to 12 hours in Ub-untreated KT-3 cells, whereas little or no induction was detected in Ub-treated cells. In contrast, expression of Bcl-XL mRNA was induced similarly in both cultures. Expression of Bcl-XS was not detectable in either culture. Bax, Bad, Bim, and Bak mRNA were expressed at basal levels in both cultures, and their expression was not influenced by rhIL-6 or Ub. Furthermore, consistent with data derived from Northern blot analysis, Western blot analysis demonstrated that Bcl-2 protein was induced by rhIL-6 in Ub-untreated KT-3 cells but not in Ub-treated cells (Figure 7B).
Ubiquitin-mediated degradation of cellular proteins plays a critical role in many cellular processes, including cell-cycle progression, signal transduction, transcriptional regulation, apoptosis, receptor down-regulation, and endocytosis.9,10 Because the ubiquitin-proteasome pathway is abundant and ubiquitous and participates in the degradation of cellular proteins located primarily in the cytosol and the nucleus, only a limited number of studies have been directed to the function of extracellular Ub. However, extracellular Ub secreted by activated T cells was shown to inhibit platelet activities.33 In addition, extracellular Ub was reported to suppress IgG production in lipopolysaccharide-stimulated splenocytes.34 Furthermore, we previously demonstrated that Ub secreted from hairy cells had an inhibitory effect on the growth of normal hematopoietic progenitor cells.12-14 In our preliminary experiments, serum Ub concentration in patients with HCL ranged from 0.08 to 1.7 µg/mL (0.44 ± 0.52 mg/mL, mean ± SD; n = 8), while those in normal controls were from 0.02 to 0.150 µg/mL (0.08 ± 0.02 µg/mL, mean ± SD; n = 64). In addition, Ub concentration in the conditioned media of an HCL-derived cell line,13 from which we purified Ub as an inhibitor of clonogenic growth of hematopoietic cells, was 0.34 µg/mL. To inhibit the growth of KT-3 and HL-60 cells in short-term cultures, Ub concentration was required to be raised more than 20 µg/mL, which is far higher than that detected in sera of patients with HCL; however, we previously found that extracellular Ub could inhibit the growth of hematopoietic cells from a concentration of 0.5 µg/mL in long-term (7 to 14 days) clonogenic assays.14 Moreover, it was speculated that Ub might be present at a higher concentration in confined spaces such as the spleen and bone marrow, in which HCL cells primarily proliferate, than in the serum. Therefore, we assumed that extracellular Ub could affect hematopoiesis in vivo of patients with HCL.
We thank Dr A. Yoshimura for providing us with SOCS-1 cDNA and Fujisaki Cell Center for providing KT-3.
Submitted July 6, 1999; accepted December 21, 1999.
Supported in part by grants from the Japanese Ministry of Education, Science, Sports and Culture, the Japanese Ministry of Health and Welfare, Senri Life Science Foundation, Uehara Memorial Foundation, Naito Foundation, and the Japan Medical Association.
Reprints: Yuzuru Kanakura, Department of Hematology and Oncology, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan; e-mail: kanakura{at}bldon.med.osaka-u.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
B,
I
-catenin, HIF-1, and HSF2 (reviewed in
Hochstrasser
.
chain, and a signal-transducing subunit, gp130, which is
shared by the receptors for ciliary neurotrophic factor, leukemia
inhibitory factor, oncostatin M, and cardiotropin 1 (reviewed by
Hirano
Japanese variant.
Leuk Lymphoma.
1997;25:373