Differences in phosphorylation of the IL-2R associatedJAK/STAT proteins between HTLV-I (+ ), IL-2-independent and
IL-2-dependent cell lines and uncultured leukemic cells frompatients with adult T-cell lymphoma/leukemia�
Qian Zhang a, Benhur Lee a, Magda Korecka a, Gong Li c, Charles Weyland b,Steven Eck a, Antoine Gessain d, Naochimi Arima e, Stuart R. Lessin c,
Leslie M. Shaw a, Selina Luger b, Malek Kamoun a, Mariusz A. Wasik a,*a Department of Pathology and Laboratory Medicine, Uni6ersity of Pennsyl6ania Center, 7.106 Founders Bldg., 3400 Spruce Str. Philadelphia,
PA 19104, USAb Department of Hematology-Oncology, Uni6ersity of Pennsyl6ania Center, 7.106 Founders Bldg., 3400 Spruce Str. Philadelphia, PA 19104, USA
c Department of Dermatology, Uni6ersity of Pennsyl6ania Center, 7.106 Founders Bldg., 3400 Spruce Str. Philadelphia, PA 19104, USAd Institut Pasteur, Paris, France
Keywords: IL-2R signaling; Malignant T cells; JAK5 kinase; STAT 5 protein; SHP-1 phosphatase
1. Introduction
IL-2 is a key cytokine involved in proliferation anddifferentiation of T lymphocytes and other cells of theimmune system. IL-2 signaling involves dimerization ofthe b chain and common g chain (gc) of the IL-2
receptor (IL-2R) [1]. In addition to IL-2R, the gc is acomponent of several other cytokine receptors; it canco-dimerize with cytokine-specific chains to transducesignals mediated by IL-4, IL-7, IL-9, and IL-15 [2–5].Signaling by cytokine receptors involves sequential acti-vation of the Janus-family tyrosine kineses (Jaks) andsignal transducer and activator of transcription proteins(STATs) (reviewed in [6]). Binding of IL-2 to the IL-2Rresults in activation of Jakl and Jak3 kineses andtyrosine phosphorylation of several substrates, includ-
� Part of this work was presented at the XXXVIII Annual Meetingof the American Society of Hematology.
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ing Jakl and Jak3 themselves, as well as the IL-2R b
and gc chains [7]. The phosphorylated IL-2R chainsrecruit proteins such as STAT5 [8,9] and, in phyto-hemagglutinin preactivated T-cell blasts, STAT3 [8,10].The STATs, upon phosphorylation, presumably by theJaks, translocate into the nucleus and bind to DNA toinitiate transcription of the IL-2 responsive genes. In-volvement of Jak3 is crucial for transduction of signalsmediated by rc because mutations of Jak3 result insevere immunodeficiency in patients [11,12] and mice[13,14] which mimics the immunodeficiency seen inmutations of the gc itself [15–18]. The immunodefi-ciency in the Jak3-deficient mice can be reversed bytransfection of the hematopoietic [19] or embryonic [20]cells with functional, wild-type Jak3. Stat 5 appearsalso critical for activation of normal, postthymic Tcells, because mature T cells derived from mice deficientin both a and b isoforms of STAT5 failed to yieldproliferative response upon stimulation [21].
Adult T-cell lymphoma leukemia (ATLL) is a malig-nancy affecting mature T lymphocytes. In most cases,the malignant ATLL cells display the CD3+ , CD4+ ,CD8− , CD7− , and T-cell receptor (TCR) a/b(+ )phenotype. Characteristically, they express activationantigens such as HLA-DR and CD25 (IL2Ra chain)[22]. Numerous epidemiological and clinical studieshave established the association of HTLV-I with ATLLas well as other diseases including tropical spastic para-paresis/HTLV-I associated myelopathy (TSP/HAM)[23]. Depending on the clinical course, the extent of thedisease and the serum calcium level, ATLL can bedivided into four clinical subtypes: acute, chronic,lymphomatous, and smoldering [24]. In all these vari-ants, patients have serum antibodies to HTLV-I andclonal integration of one or few copies of the virus inthe DNA of the malignant cells [25,26]. Patients withdetectable monoclonal or oligoclonal populations andelevated PBMC counts are at increased risk of develop-ing an overt ATLL disease [27]. However, the lowfrequency of ATLL (4–5%) among HTLV-I infectedindividuals [28] and the long average time intervalbetween the occurrence of infection and the develop-ment of malignancy (20–30 years) indicate that addi-tional events are required for malignant transformationof T-cells.
Experiments with HTLV-I transformed T-cell linessuggested that the virus may induce basal constitutiveactivation of the IL-2R associated Jak/STAT pathway,and that this pathway may be involved in HTLV-I-me-diated T-cell transformation [29,30]. However, we pre-viously found that constitutive activation ofIL-2R-associated Jak/STAT signaling pathway also oc-curs in HTLV-I (− ) malignant cells from patients withcutaneous anaplastic large T-cell lymphoma (ALCL)[31]. This finding indicated that the constitutive activa-tion of IL-2R Jak/STAT pathway in transformed T
cells may not be due to to the HTLV-I infection. Toexplore further the putative role of HTLV-I infection inthe activation of this pathway, we examined severalHTLV-I (+ ) T-cell lines that differ in their IL-2 depen-dency for the basal and IL-2-induced phosphorylationof the Jak3, STAT3, and STAT5 proteins. Further-more, we also examined uncultured leukemic cells iso-lated directly from patients with ATLL. Our datademonstrate that the constitutive phosphorylation ofthe IL-2R associated Jak and STAT proteins is de-tectable only in the IL-2 independent HTLV-I (+ ) celllines. It is not seen in the IL-2 dependent HTLV-I (+ )lines and the leukemic ATLL cells. Both these celltypes, however, phosphorylate Jak3 and STAT5 inresponse to IL-2. Furthermore, most of the IL-2 inde-pendent cell lines, but none of the leukemic ATLL cells,lacked expression of SHP-1 which down-regulatesphosphorylation of Jak3. Finally, the HTLV-I (+ )lines, in contrast to the control, HTLV-I (− ) T-celllines showed resistance to the IL-2R-signaling in-hibitors rapamycin and its novel analog RAD. Implica-tions of these findings for pathogenesis of the HTLV-Iinfection and ATLL are discussed.
2. Materials and methods
2.1. Patients
A total of eight ATLL patients, two from the US andsix from Japan, were tested. Peripheral blood mononu-clear cells (PBMC) were obtained by Ficoll centrifuga-tion [31,32] from two patients diagnosed at theUniversity of Pennsylvania with ATLL based on clini-cal, histopathological, and immunophenotypic criteria.Both patients developed anti-HTLV-I antibodies as de-termined by Western Blot of serum proteins, peripheralwhite blood cell count greater than 50×103/ul with apredominant lymphocytosis (greater than 50%), abnor-mal pathognomonic cells with multi-lobulated, flower-like nuclei on peripheral blood smear, serum LDHgreater than 1.5 times upper limit of normal, andcorrected Ca2+ of greater than 14 mg/dl. The abovefindings fulfilled criteria for the acute form of ATLL[24]. Serum concentrations of soluble IL-2R were59 520 U/ml (patient c1) and 45 740 U/ml (patientc2) (normalB1000 U/ml) indicating high tumor bur-dens ([33–35]). Flow cytometry analysis revealed thatgreater than 95% of PBMC from these patients hadphenotypes consistent with ATLL cells (CD3+ ,CD4+ , CD7− , CD25+ , HLA DR+ ). Fig. 1 showsthe cells with characteristic flower-like nuclei andsalient flow cytometry data from patient c1. SixJapanese patients were diagnosed with ATLL (fiveacute and one chronic form) at the Kagoshima Univer-sity using the same criteria as described above. Ficoll-
Q. Zhang et al. / Leukemia Research 23 (1999) 373–384 375
isolated PBMC were cryopreserved in DMSO/FBScontaining medium and thawed shortly before beingused for experiments.
2.2. Cell lines
Two types of HTLV-I (+ ) cell lines were used:IL-2 independent cell lines derived mostly fromATLL patients and IL-2 dependent cell lines derivedfrom nonleukemic, TSP/HAM patients. The HTLV-I(+ ), IL-2 independent cell lines were ATL-2: CD4+CD8− T cells originally cultured in IL-2 fromPBMC of a patient with acute ATLL [36]; C91PL:cord blood T cell line established by co-culturing cordblood cells with known ATLL cells in the presence ofIL-2 [37]; C1OMJ2: established from HTLV-I infectedlymphocytes in a patient with ATLL [38]; andHUT102B: constitutive producer of HTLV-I derivedfrom the lymph node of a patient with HTLV-I, alsoinitially dependent on IL-2 [39,40]. HTLV-I (+ ), IL-2 dependent cell lines, Boul, Laf, and Cor, werederived from HTLV-I (+ ) non-leukemic patientswith TSP/HAM [41]. These cell lines required 50–100U/ml IL-2 for optimal growth and did not becomeIL-2 independent even after multiple passages. Forcontrols, five HTLV-I (− ) cell lines were used. TheSez4 line, kindly provided by T. Abrams, HahnemannUniversity was derived from a patient with aleukemic phase of cutaneous T-cell lymphoma (Sezary
Table 1HTLV-I status of the cell populations examined
Method of detec-HTLV statusCell typestion
Control cell linesYT (NK-like) PCR tax−
−Sez-4 PCR taxPCR taxPB-1 −
2A − PCR tax−2B PCR tax
IL-2 dependent cell linesBoul + PCR tax
+ Immunofluores-Lafcence
+Cor Immunofluores-cence
IL-2 independent cell linesATL-2 PCR tax+
+C91PL PCR tax+C10MJ2 PCR tax+HUT102 PCR tax
ATLL Patients (8) Positive serology*+
* Cells were also histopathologically and immunophenotypicallyconsistent with HTLV-I infected cells.
Syndrome) and bears close morphological, pheno-typic, and genotypic resemblance to the fresh tumorcells [42]. The Sez4 line requires IL-2 (50–100 U/ml)for continuous proliferation. The YT line [43], a hu-man NK cell line, was kindly provided by J. Yodoi,Kyoto University, Kyoto, Japan. PB-1, 2A, and 2BT-cell lines which were established from a patientwith a progressive cutaneous T-cell lymphoprolifera-tive disorder have been described in detail previously[31,44,45]. The PB-1 cell line was obtained at a rela-tively early stage of the patient’s cutaneous T-celllymphoma from neoplastic T-cells circulating in pe-ripheral blood. The 2A and 2B lines were establishedat a later, more aggressive stage from two separateskin nodules, which represented a high-grade, T-cellanaplastic large-cell lymphoma. All five control celllines were determined to be HTLV-I (− ) by PCRdetection of the HTLV tax gene (Table 1). All thecell lines were propagated in a complete RPMI 1640medium containing 10% FBS (Hyclone, Logan, UT),1% L-glutamine (M.A. Bioproducts, Walkersville,MD), and 1% penicillin/streptomycin/fungizone mix-ture (M.A. Bioproducts).
2.3. Flow cytometry
Flow cytometry immunophenotyping of PBMC wasperformed using a standard panel of T- and B-cellreactive mAbs including the ones which recognize ac-tivation antigens HLA-DR and CD25 (Becton-Dickinson).
Fig. 1. Left panel: a representative peripheral blood smear fromATLL patient c1 showing pathognomonic large cells with flower-like nuclei (Wright–Giemsa, magnification 1000× ). Right panel:flow cytometry analysis of the patients PBMC which shows that\95% cells exhibit a CD3+ , CD4+ , CD8− , CD7− , HLA-DR+and CD25+ phenotype consistent with ATLL. Representative dotplots are shown where \95% of cells are CD4+/CD25+andCD7− .
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2.4. IL-2, antibodies (Ab), and immunosuppressi6eagents
Recombinant human IL-2 was kindly provided by C.Reynolds, NCI, Frederick, MD. Rabbit polyclonal Absagainst JAK3, STAT3, STAT5, and SHP-1 were pur-chased from Santa Cruz Biotechnology (Santa Cruz,CA). Anti-phosphotyrosine 4G10 murine monoclonalAb was purchased from UBI (Lake Placid, NY). Perox-idase-conjugated donkey anti-mouse and goat anti-rab-bit Abs were obtained from Jackson Jackson ImmunoResearch (West Grove, PA). Rapamycin and SZS RADwere kindly provided by, respectively, Wyeth-Ayerst(Princeton, NJ) and Novartis Pharma (Basel,Switzerland).
2.5. PCR/Southern blot analysis
A total of 1.0 ug of genomic DNA was added to 50ml of standard buffer containing 1.5 mM MgC12, 1.25mM dNTP mix, 15 pmol of 3% and 5% primers, and 2.5U Taq polymerase (Perkin-Elmer, Norwalk, CT). Reac-tion mixtures were amplified for 30 cycles of denatura-tion at 94°C for 30 s, annealing at 55°C for 30 s, andextension at 72°C for 30 s. Oligonucleotide primers forconserved sequences of the HTLV-I/II tax gene, SK43and SK44 [46], synthesized by Research Genetics,Huntsville, AL, were used for amplification of HTLV-I/II tax gene sequences. Amplification products wereseparated on 2% agarose gels, blotted and probed with32P-labeled SK45 probe [46] from Research Genetics asdescribed before [47]. DNA from HTLV-II infected cellline (MoT) served as positive control.
2.6. Protein expression and phosphorylation
These assays were performed as described [31,48]. Inbrief, the cells (l0×106) were washed, exposed for 5min to medium or 500 U IL-2, lysed for 20 min in l mlice-cold lysis buffer (0.5% NP-40, 10 mM Tris–HC1(pH 7.4), 150 mM NaCl, 0.4 mM EDTA, 1 mMsodium orthovanadate, 0.5 mM PMSF, 10 mM NaF,and 3 ug/ml each of pepstatin, leupeptin, chymostatin,and aprotinin: Sigma). The lysates were centrifuged at15 000 rpm for 10 min. Next, the supernatants wereprecleared overnight at 4°C with protein A-sepharose(Sigma, St Louis, MO), incubated with the anti-Jak3,-STAT3, -STAT5, or SHP-1 Ab, and protein A-sep-harose, washed, boiled, suspended in reducing SDSloading buffer, separated on a 10% polyacrylamide-SDS gel, and transferred electrophoretically to hy-bridization transfer membranes. The membranes wereblocked with 2% bovine serum albumin in TBST buffer(l0 mM Tris–HCl (pH 7.4), 75 mM NaCl, l mMEDTA, 0.1% Tween 20) for at least 2 h at roomtemperature or overnight in a cold room. To detect
protein phosphorylation, the membranes were incu-bated with 4G10 Ab, washed, incubated with donkeyanti-mouse, peroxidase-conjugated Ab and washedagain. To detect protein expression the membraneswere incubated with the same Jak3, -STAT3, -STAT5,or -SHP-1 Abs which were used for precipitation. Blotswere developed using the ECL chemiluminescencereagents (Amersham Life Science, Arlington Heights,IL).
2.7. Proliferation assays
These tests were performed as described previously[31,32]. In brief, the cell lines or PHA-stimulatedPBMC were cultured for either 10 or 18 h in triplicateat 2×104 cells/well in the presence of various concen-trations of the immunosupressive drugs; rapamycin orRAD. After 14 h pulse with 0.5 mCi of [3H]thymidine,radioactivity of the cells was measured.
3. Results
3.1. Determination of the HTLV-I infection status
The HTLV-I (+ ) status of all cell populations usedwas determined by detection of viral tax gene in ge-nomic DNA, detection of viral gene products via im-munofluorescence, or in patients, by clinical, histopath-ologic and immunophenotypic criteria for ATLL incombination with serological evidence for HTLV-I in-fection (see Section 2.1 and Fig. 1). Table 1 summarizesthe HTLV-I status of the cell populations examined.Controls used included cutaneous T-cell lymphoma celllines: Sez4 (IL-2 dependent) and PB-1, 2A, and 2B (allIL-2 independent) and an NK cell line, YT. All controlcell lines were shown to be HTLV-I (− ) by the PCRassay for the HTLV-I tax gene.
3.2. Phosphorylation of IL-2R associated Jak/STATproteins in IL-2 independent T-cell lines
The few HTLV-I (+ ), IL-2 independent T-cell linestested to date have all been reported to display consti-tutive activation of the IL-2R associated Jak/STATpathway [26,27]. We explored the extent of these find-ings by analyzing four HTLV-I (+ ), IL-2 independentT-cell lines (ATL-2, C1OMJ2, C91PL, HUT 102), twoof which (ATL-2, C1OMJ2) have not been examined todate. Fig. 2A and Table 2 show that all the cell linesdemonstrate a strong basal, constitutive phosphoryla-tion of Jak3 with only the HUT102 cell line showing aslight augmentation in response to IL-2. STAT3 andSTAT5 are also strongly, constitutively phosphorylatedin the ATL-2 and C10MJ2 cell lines (Fig. 2B, C, andTable 2). Interestingly, constitutive phosphorylation of
Q. Zhang et al. / Leukemia Research 23 (1999) 373–384 377
the entire Jak3/STAT3/STAT5 pathway does not seemto be a universal feature even in these cell lines becausetwo of the lines (C91PL and HUT102B) did not exhibitany detectable, basal phosphorylation of STAT5 inrepeated experiments although they expressed theprotein and strongly phosphorylated STAT5 in re-sponse to IL-2 (Fig. 2C, Table 2). This finding impliesa dissociation between Jak3 and STAT5 phosphoryla-
tion and suggests that the signals transduced by Jak3may not necessarily always depend on phosphorylationof STAT5. In addition, one cell line, C91PL, showed alack of STAT3 phosphorylation in the presence orabsence of IL-2. This pattern of response is similar tothat seen in resting PBMC rather than mitogen pre-ac-tivated T-cells [8,26,28]. The control Sez4 malignantT-cell line which is HTLV-I (− ) and, noteworthy, IL-2
Fig. 2. Phosphorylation of proteins associated with IL-2R signal transduction pathway in HTLV-I positive, IL-2 independent cell lines (ATL-2,C9lPL, C1OMJ2, HUT102B) derived from leukemic patients without (− ) and with (+ ) stimulation by IL-2: (A) Jak3, (B) STAT3, and (C)STAT5. The cell lysates were immunoprecipitated with the anti-Jak3, -STAT3, and -STAT5 Ab, electrophoretically separated, transferred to amembrane and probed with an Ab (4G10) which recognizes phosphorylated but not non-phosphorylated tyrosines. Loading of equal samplevolumes was confirmed by the subsesquent immunoblotting with the Ab used for immunoprecipitation after removal of the bound 4G10 Ab.
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Table 2Tyrosine phosphorylation of Jak3, STAT3 and STAT5 in HTLV-I positive cell populations: IL-2-independent and dependent T-cell lines, andleukemic cells denved from ATLL patients
JAK3 STAT3 STAT5
+ −(IL-2)* − + ++−
IL-2 Independent(HTLV I+, leukemic)
++++ATL-2 ++ ++ ++ +C10MJ2 + ++
+−−C91PL −+ ++ + − +HUT102B + ++
IL-2 Dependent(HTLV I+, non-leukemic)
+−+Boul −/+− +−/+ + − +Laf − +
− ++Cor NDa−/+ ++ATLL patients
+−−c1–c2 −− +ND ND − +c3 − +
ND NDNDc4–c8 ND− +Controls(HTLV I-)Sez-4
+−+YT (NK-like) +− +PBMC†
+−PHA blasts** − +− +
a ND, not done.* Cells were exposed in vitro to 500 units of IL-2.** Normal PBMC tested after 5 days of stimulation with PHA.† PBMC from a healthy donor.
dependent, also showed a lack of STAT3 phosphoryla-tion even after stimulation with IL-2. These observa-tions indicate that STAT3 activation may not be crucialfor the transduction of IL-2R/Jak 3 mediated mitogenicsignals.
3.3. Phosphorylation of IL-2R associated Jak/STATproteins in IL-2 dependent T-cell lines
To determine if HTLV-I infection per se inducesconstitutive phosphorylation of the IL-2R associatedJak/STAT pathway, we analyzed several HTLV-I (+ )cell lines which are IL-2 dependent. To ensure that anybasal phosphorylation of Jak/STAT proteins was notdue to the prior exposure to IL-2, these cells wereincubated for 4–6 h without IL-2 before the assay. Incontrast to the results obtained with the HTLV-I (+ ),IL-2 independent cell lines, Jak3 was not constitutivelyphosphorylated in any of these lines (Boul, Laf, Cor)(Fig. 3A, Table 2). Only one of the lines, Cor, appearsto have a minimal level of basal phosphorylation in theabsence of IL-2. However, this phosphorylation de-creased to non-detectable levels when time of IL-2withdrawl was increased to 16 h (such prolonged IL-2removal did not affect the ability of cells to respond toIL-2; data not shown). These findings indicate that the
Jak3/STAT3/STAT5 phosphorylation in all threeHTLV-I (+ ), IL-2 dependent T-cell lines was triggeredby IL-2 and not HTLV-I infection. All cell lines phos-phorylated Jak3 in response to IL-2 (Fig. 3A, Table 2)which indicates that this pathway is functional andpresumably required for the IL2 mediated proliferationof these cells. STAT3 appears to have, under the testingcondition, a minimal level of basal constitutive phos-phorylation in two lines (Boul and Laf), which wasmarkedly augmented by exogenous IL-2 (Fig. 3B, Table2). The control, HTLV-I negative, sez4 T-cell line failedto phosphorylate STAT3 upon IL-2 stimulation.
No basal activation of STAT5 was noted in the threeHTLV-I (+ ) and the control HTLV-I (− ) Sez4 line,but all four lines phosphorylated strongly STAT5 afterexposure to IL-2 (Fig. 3C, Table 2).
3.4. Analysis of Jak3, STAT3, and STAT5phosphorylation in leukemic T cells deri6ed from ATLLpatients
Because HTLV-I (+ ) cell lines examined thus farshowed either constitutive or IL-2 inducible phosphory-lation of Jak3, STAT3, and/or STAT5, we were inter-ested in determining which of these two patterns ispresent in the uncultured, malignant T cells derived
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directly from ATLL patients. Results from eight ATLLpatients are summarized in Table 2 and the representa-tive data from patient c1 (see Section 2.1 and Fig. 1)are shown in Fig. 4. There was no evidence for basalphosphorylation of either Jak3 (0/8 pts), STAT3 (0/2pts) or STAT5 (0/3 pts) in the absence of IL-2. How-ever, phosphorylation of Jak3 and STAT5 occured inresponse to IL-2 in all patients tested. These findingsindicate that there is no basal constitutive activation ofJak3 and STAT5 in the leukemic ATLL cells tested butthese cells are able to activate the IL-2R Jak/STATpathway when exposed to IL-2. Interestingly, noSTAT3 phosphorylation could be observed in responseto IL-2 in the two patients tested. This pattern ofresponse resembles the one seen in the normal restingPBMC, rather than in mitogen-preactivated T-cellblasts, which do activate STAT3 upon stimulation withIL-2 [8,29,31].
Fig. 4. Phosphorylation of the IL-2R-associated Jak/STAT pathwayin freshly isolated leukemic cells from a representative ATLL patient(c1) before (+ ) or after (− ) stimulation with IL-2. The membrane-immobilized cell immunoprecipitates were separated, transferred, andprobed with the 4G10 Ab. Equal sample loading was confirmed byreblotting with the Ab used for immunoprecipitation (data not pre-sented).
3.5. Analysis of SHP-1 expression in HTLV-I (+ )T-cells
It has been shown that a protein tyrosine phos-phatase, SHP-1 (also known as PTP1C, SHP, andSHPTP1), binds to several different cytokine receptors[6] including IL-2R [49]. SHP-1 appears to act bydephosphorylation of Jak kineses [50]. Noteworthy,dysfunction of SHP-1 as seen in a natural (‘motheaten’)SHP-1 knock-out mice, resuts in a hyperplasia of theerythroid and lymphoid lineages [51,52]. Furthermore,it has been found that some HTLV-I (+ ) cell linesfailed to express SHP-1 protein [49]. Because theseHTLV-I (+ ) T-cell lines displayed constitutive activa-tion of the IL-2R-associated Jak/STAT pathway[29,49], this finding suggested a casual relationship be-tween the lack of SHP-1 expression and the constitutiveJak/STAT activation. To explore further this apparentrelationship, we examined SHP-1 expression in ourpanel of HTLV-I (+ ), IL-2 independent T-cell lineswhich displayed constitutive phosphorylation of Jak3,STAT5, and/or STAT3. Furthermore, we examinedSHP-1 expression in the previously uncultured leukemiccells from ATLL patients which, as described above,showed phosphorylation of Jak3 and STAT5 only afterstimulation with IL-2. As shown in Fig. 5, three out offour HTLV-I (+ ) T-cell lines showed lack of SHP-1protein expression. Only one line; HUT102B expressedSHP-1; this observation was made also by others [49].In contrast to the cell lines, uncultured leukemic cellsfrom three ATLL patients expressed SHP-1. Thesefindings provide additional evidence that there is aninversed correlation between the SHP-1 expression andconstitutive IL-2R Jak/STAT activation and supportthe hypothesis that there indeed may be the cause-and-effect relationship between these two events.
Fig. 3. Analysis of Jak/STAT phosphorylation in HTLV-I positiveIL-2 dependent cell lines (Boul, Cor, Laf) derived from non-leukemicpatients before (− ) or after (+ ) stimulation with IL-2: (A) Jak3, (B)STAT3, (C) STAT5. The cell immunoprecipitates obtained with theanti-Jak3, -STAT3, and -STAT5 Ab were electrophoretically sepa-rated, transferred to a membrane and probed with the 4G10 Ab.Loading of equal sample volumes was confirmed by reblotting withthe Ab used for immunoprecipitation (data not presented).
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3.6. Effect of immunosuppressi6e drugs on proliferationof HTLV-I (+ ) and HTLV-I (− ) T-cells
Our previous study [31] has demonstrated that celllines derived from HTLV-I (− ) cutaneous large T-celllymphoma which display IL-2 independent growth anda constitutive activation of the IL-2R Jak/STAT signal-ing pathway, are sensitive to an immunosuppressiveagent rapamycin. Rapamycin has been shown to act asan inhibitor of the IL-2R signaling by interfering withactivation of one of the down-stream signalingmolecules: p70 S6 kinase [53,54]. It was interesting,therefore, to test the effect of rapamycin on the HTLV-I (+ ) T-cell lines which also show an IL-2 indepen-dence and constitutive activation of the IL-2RJak/STAT pathway. In addition to rapamycin we usedits novel analog RAD. As can be seen, both rapamycin(Fig. 6A) and RAD (Fig. 6B) profoundly inhibitedgrowth of the HTLV-I (− ) T-cells. To exert their effectboth drugs required a relatively long incubation periodwith the target cells. Whereas 24 h exposure resulted inthe maximal inhibition of 40–80% for the variousHTLV-I−T-cell lines tested, 32 h exposure resulted in80–95% inhibition. In striking contrast, none of thethree HTLV-I (+ ) cell lines was markedly inhibited byeither rapamycin or RAD (Fig. 6C and D, respectively)at even the highest doses after the 32 h exposure(0–30% inhibition). Interestingly, addition of RAD,particularly at the shorter exposure time, resulted in amild (0–30%) augmentation rather than suppression ofthe proliferative rate. Taken together these data suggestthat there are important differences in mitogenic signal-ing events downstream of IL-2R between the examinedhere HTLV-I (+ ) and HTLV-I (− ) T-cells despitetheir similarity in constitutive activation of the IL-2RJak/STAT pathway.
4. Discussion
Two previous reports have shown that HTLV-I (+ )T-cell lines have constitutively activated IL-2R-associ-ated Jak/STAT signal transduction pathway [29,30].This finding suggested that HTLV-I infection may acti-vate this signaling pathway. In addition, they impliedthat activation of the IL-2R-associated Jak/STAT path-way may be critical in the pathogenesis of ATLL. Thisconclusion was further supported by a recent report[55] which described a basal, apparently constitutiveactivation of this pathway in uncultured leukemic cellsfrom eight out of twelve ATLL patients. However, ourdata demonstrate clearly that HTLV-I infection per sedoes not result in a constitutive activation of the IL-2R-associated Jak/STAT pathway. Although all HTLV-I(+ ), IL-2 independent cell lines tested so far indeeddisplayed a strong, constitutive phosphorylation ofJak3, STAT3, and/or STAT5 (29, 31, Fig. 2, and Table2), the HTLV-I (+ ) T-cell lines which require IL-2 fortheir growth, showed phosphorylation of these proteinsonly after stimulation with IL-2 (Fig. 3 and Table 2).Withdrawal of IL-2 invariably resulted in the profounddecrease in the Jak3/STAT3/STAT5 phosphorylation,usually to the undetectable levels (Fig. 3, Table 1, datanot presented). Furthermore, in our experiments theIL-2R associated Jak/STAT pathway was also quies-cent in uncultured leukemic cells isolated directly fromall eight ATLL patients studied. This pathway was,however, fully functional in such leukemic cells asdemonstrated by strong phosphorylation of Jak3 andSTAT5 upon stimulation of the cells with IL-2. Inaddition to providing additional evidence that HTLV-Iinfection per se does not result in a basal constitutiveactivation of the IL-2R associated Jak/STAT signalingpathway, these data indicate that constitutive activationof this pathway may not be required for malignantT-cell transformation in at least some cases of ATLL.Because the leukemic ATLL cells responded to IL-2 invirtually all cases, it is likely that this cytokine plays arole in the pathogenesis of ATLL. Previous studies(reviewed in [56]) have shown that malignant ATLLcells derived from lymph nodes rather than peripheralblood, tend not only to respond but also produce IL-2.This IL-2 production which was not seen in the circu-lating leukemic cells, requires the presence ofmacrophages. These findings, combined with our data,suggest that IL-2 may indeed act as a growth factor formalignant ATLL cells in vivo. This may be true, how-ever, for lymph node based ATLL cells upon theirinteraction with macrophages or other types of acces-sory cells rather than for circulating leukemic cellswhich have little chance to interact efficiently with anyaccessory cells. The reasons for the discrepancy betweenour and the reported [55] data in regard to basalactivation of the IL-2R-associated Jak/STAT pathway
Fig. 5. Expression of SHP-1 phosphatase in HTLV-I (+ ) cell lines(ATL-2, C91PL, C1OMJ2, HUT102), uncultured leukemic ATLLcells (pts c1, 3, and 5), and control PBMC and PHA-prestimulatedT-cell blasts (PHA bl). The cell lysates were electophoretically sepa-rated, transfered, and probed with an anti-SHP-1 antibody. Equalsample loading was confirmed by reblotting with an anti-Jak3 Ab(data not presented).
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Fig. 6. Inhibition of proliferation of the HTLV-I (− ) (PB-1, 2A, and 2B) and HTLV-I (+ ) (HUT102B, ATL-2, and C1OMJ2) T-cell lines byimmunosupressive drugs rapamycin (RAPA) and RAD. The results are expressed as a percentage of the proliferative response in the presence ofan increasing amount of the drugs as compared to the medium alone.
in uncultured leukemic ATLL cells are unclear but maybe related to the relatively small group of patientstested (eight and 12, respectively) and/or differences inmethodology. Our previous study detected basal activa-tion of IL-2R associated pathway in leukemic cellsfrom ten out of 14 patients with Sezary syndrome [31].Overnight culture of the leukemic Sezary cells, withoutexogenous IL-2, reduced the basal phosphorylation ofJak3 and STAT5 to undetectable level which suggestedthat the basal activation of IL-2R Jak/STAT pathwaywas induced in the leukemic cells in vivo rather thantruly constitutive. It would be interesting to apply thesame experimental approach to the reported, appar-endy positive ATLL cases [55]. It is also unclear why allthe HTLV-I (+ ), IL-2 independent T-cell lines testedso far (29, 30, and Fig. 2 and Table 2) show a trulyconstitutive activation of the IL2R associated Jak/STAT signaling pathway independent of IL-2 or othercytokines ([51], Q. Zhang and M. Wasik, unpublisheddata). It is possible that the few established HTLV-I(+), IL-2 independent T-cell lines are derived fromvery advanced cases which are not representative for
most cases of ATLL. However, one can not excludethat the constitutive activation of the IL-2R Jak/STATsignaling pathway and IL-2 independence was acquiredin vitro, because many, if not all, of these cell lines wereexposed at one time to IL-2, particularly at the earlystage of line establishment ([36–40]).
In the light of our data additional oncogenic mecha-nisms in ATLL should also be considered. These in-clude constitutive stimulation by growth factors orcytokines different from IL-2, as well as mutations ofgenes controlling signal transduction and the cell cycle.Possible candidates include insulin-like growth factorreceptor [57], IL-15 [4], IL-10 [58], hepatocyte growthfactor (HCF) [59,60] and its receptor c-met [61,62]. Inthe light of our findings, IL-15 is an unlikely candidatebecause it binds to the same IL-2Rb and gc chains asIL-2 [4] and induces phosphorylation of the same Jak/STAT pathway. Similarly, IL-10 may also not be in-volved in the pathogenesis of ATLL because it inducesphosphorylation of STAT3 [63] which was not constitu-tively phosphorylated in the leukemic cells from ATLLpatients and the majority of the HTLV-I (+ ) cell lines
Q. Zhang et al. / Leukemia Research 23 (1999) 373–384382
we examined. Noteworthy, we found that severalHTLV-I (+ ) T-cell lines express both HCF and itsreceptor c-met which raises the possibility of an HCF-mediated autocrine stimulation in these cells ([55], M.Ratajczak and M. Wasik, unpublished data). However,the exact role, if any, of the HCF/c-met pathway aswell as the other factors and their receptors in thepathogenesis of HTLV-I infection and ATLL oncogen-esis remains to be determined. In regard to the geneticchanges, cytogenetic abnormalities involving deletion oftumor suppressor genes, p15 (MTS2) and p16(CDKN2/MTS1), have been reported in up to 40% ofATLL patients [64,65]. Significantly, occurrence of thep15/p16 deletion has been reported to be associatedwith the progression from the chronic to the acute formof ATLL [64] suggesting a casual relationship betweenthese two events.
Our data also indicate that phosphorylation of Jak3may not necessarily always be associated with phospho-rylation of STAT3 and/or STAT5. Accordingly,leukemic ATLL cells (Fig. 4) as well as C9lPL (Fig. 2)and Sez-4 (Fig. 3) cell lines failed to phosphorylateSTAT3 upon IL-2 stimulation. In this respect, theyresemble more resting, peripheral blood T-cells ratherthan PHA-prestimulated T-cell blasts. This lack ofSTAT3 phosphorylation is in agreement with the previ-ous studies which have shown that DNA synthesis andthe cell cycle proceeds even in the absence of STAT3tyrosine phosphorylation [66]. The dissociation of Jak3-from STAT5-phosphorylation as reported before [67]and seen here in the C91PL and HUT102B cell lines(Fig. 2), suggests that phosphorylation of STAT5 maynot always be required for the Jak3-mediated signaling,at least in the malignant T-cell lines. Recently, severalnovel proteins such as Akt [68], STAM [69,70], andPyk-2 [71] which transduce IL-2R-mediated mitogenicsignals have been identified. Their functional status inthe HTLV-I (+ ) T-cells is currently under investiga-tion. Our data which showed a striking difference be-tween the HTLV-I (+ ) and HTLV-I (− ) T-cell linesin their sensitivity to rapamycin and RAD, furtherstress the importance of molecules different from Jak3,STAT3, and STAT5 in the IL-2R-mediated mitogene-sis. The exact position of the known target molecule forrapamycin and RAD, p70 S6 kinase, in the IL-2Rsignaling pathway is not firmly established althoughAkt was recently identified as an upstream regulator ofthe p70 S6 kinase [68].
In summary, we have presented evidence that a basal,constitutive phosphorylation of the IL-2R associatedJak/STAT signaling pathway is not due to the HTLV-Iinfection and is not required for malignant T-cell trans-formation in at least some cases of ATLL. Further-more, our observation of dissociation of Jak3, STAT3,and STAT5 phosphorylation suggests that phosphory-lation of the STATs, particularly STAT3, may not
always be needed for transduction of IL-2 mediatedsignals. Finally, our data indicate the existence of majordifferences in T-cell immortalization mechanisms whichappear to involve target molecules of SHP-1 and ra-pamycin and RAD. Further studies are needed to un-derstand better the role of IL-2R signaling in thepathogenesis of HTLV-I infection and malignant trans-formation in ATLL and to determine an exact roleJak3, STAT5 and other proteins play in the IL-2R-me-diated signaling in both normal and malignant T-cells.
Acknowledgements
Supported in part by grants from the University ofPennsylvania Research Foundation (MAW), Novartis(MAW and LMS), and NIH (training grant to GL). BLis a William Pepper Fellow in the Department ofPathology and Laboratory Medicine, Hospital of theUniversity of Pennsylvania.
References
[1] Nelson BH, Lord JD, Greenberg PD. Cytoplasmic domains ofthe interleukin-2 receptor b and a chains mediate the signal forT-cell proliferation. Nature 1994;369:333–6.
[2] Leonard WJ, Noguchi M, Russell SM, McBride OW. Themolecular basis of X linked severe combined immunodeficiency:the role of the interleukin-2 receptor gamma chain as a commong chain, g c. Immunol Rev 1994;138:61–86.
[3] Noguchi M, Nakamura Y, Russell SM, Ziegler SF, Tsang M,Cao X, Leonard WJ. Interleukin-2 receptor gamma chain: afunctional component of the interleukin-7 receptor. Science1993;262:1877–80.
[4] Tagaya Y, Bamford RN, DeFilippis AP, Waldman TA. IL-15: apleiotropic cytokine with diverse receptor/signaling pathwayswhose expression is controlled at multiple levels. Immunity1996;4:329–36.
[5] Taniguchi T. Cytokine signalling through nonreceptor proteintyrosine kineses. Science 1995;268:251–5.
[6] Leonard WJ, O’Shea JJ. JAKS AND STATS: biological implica-tions. Ann Rev Immunol 1998;16:293–322.
[7] Witthuhn BA, Silvennoinen O, Miura O, Lai KS, Cwik C, LiuET, Ihle JN. Involvement of the Jak-3 Janus kinase in signallingby interleukins 2 and 4 in lymphoid and myeloid cells. Nature1994;370:153–7.
[8] Lin J-X, Migone T-S, Tsang M, Friedman M, Weatherbee JA,Zhou L, Yamauchi A, Bloom ET, Mietz J, John S, Leonard WJ.The role of shared receptor motifs and common STAT proteinsin the generation of cytokine pleiotrophy and redundancy byIL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 1995;2:331–9.
[9] Beadling C, Guschin D, Witthuhn BA, Ziemiecki A, Ihle JN,Kerr IM, Cantrell DA. Activation of JAK kineses and STATproteins by interleukin-2 and interferon-g, but not the T-cellantigen receptor, in human T lymphocytes. Embo J1994;13:5605–15.
[10] Nielsen M, Svejgaard A, Skov S, Odum N. Interleukin-2 inducestyrosine phosphorylation and nuclear translocation of STAT3 inhuman T lymphocytes. Eur J Immunol 1994;24:3082–6.
[11] Macchi P, Villa A, Gillani S, Sacco MG, Frattini A, Porta,Ugazio AG, Johnston JA, Candotti F, O’Shea JJ, Vezzoni P,
Q. Zhang et al. / Leukemia Research 23 (1999) 373–384 383
Notarangelo LD. Mutations of Jak-3 gene in patients withautosomal severe combined immune defiency (SCID). Nature1995;377:65–68.
[12] Russell SM, Tayebi N, Nakajima H, Riedy MC, Roberts JL,Aman MJ, Migone TS, Noguchi M, Markert ML, Buckley RH,O’Shea JJ, Leonard WJ. Mutation of Jak3 in a patient withSCID: essential role of Jak3 in lymphoid development. Science1995;270:797–800.
[13] Nosaka T, van Deursen JM, Tripp RA, Thierfelder WE, Wit-thuhn BA, McMickle AP, Dohrty PC, Grosveld GC, Ihle JN.Defective lymphoid development in mice lacking Jak3. Science1995;270:800–2.
[14] Thomis DC, Gurniak CB, Tivol E, Sharpe AH, Berg LJ. Defectsin B lymphocyte maturation and T lymphocyte activation inmice lacking Jak3. Science 1995;270:794–7.
[15] Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S,Modi WS, McBride OW, Leonard WJ. Interleukin-2 receptor g
chain mutation results in X-linked severe combined immunodefi-ciency in humans. Cell 1993;73:147–57.
[16] Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, RussellSM, et al. Defective lymphoid development in mice lackingexpression of the common cytokine receptor g chain. Immunity1995;2:223–38.
[17] DiSanto JP, Muller W, Guy-Grand D, Fischer A, Rajewsky K.Lymphoid development in mice with a targeted deletion of theinterleukin 2 receptor g chain. Proc Natl Acad Sci USA1995;92:377–81.
[18] Ohbo K, Suda T, Hashiyama M, Mantani A, Ikebe M,Miyakawa K, Moriyama M, Nakamura M, Katsuki M, Taka-hashi K, Yamamura K, Sugamura K. Modulation of hemato-poiesis in mice with a truncated mutant of the interleukin-2receptor chain. Blood 1996; 956–967.
[19] Bunting KD, Sangster MY, Ihle JN, Sorrentino BP. Restorationof lymphocyte function in Janus kinase 3-deficient mice byretroviral-mediated gene transfer. Nature Med 1998;4:58–64.
[20] Sohn SJ, Forbush KA, Nguyen N, Witthuhn B, Nosaka T, IhleJN, Perlmutter RM. Requirement for Jak3 in mature T-cells: itsrole in regulation of T-cell homeostasis. J Immunol 1998;160:2130–8.
[21] Teglund S, McKay C, Schuetz E, van Deursen JM, StravopodisD, Wang DM, Brown M, Bodner S, Grosveld G, Ihle JN. Stat5aand STAT5b proteins have essential and nonessential, or redun-dant, roles in cytokine responses. Cell 1998;93:841–50.
[22] Yamaguchi K, Takatsuki K. Adult T-cell leukemia-lymphoma.Clin Haematol 1993;6:899–915.
[23] Hollsberg P, Hafler DA. Pathogenesis of diseases induced byhuman lymphotrophic virus type I infection. New Eng J Med1993;328:1173–82.
[24] Shimoyama M. Diagnostic criteria and classification of clinicalsubtypes of adult T-cell leukemia-lymphoma. Br J Haematol1991;79:428–37.
[25] Yoshida M, Seiki M, Yamaguchi K, Takatsuki K. Monoclonalintegration of HTLV in all primary tumors of adult T-cellleukemia suggests causative role of HTLV in the disease. ProcNatl Acad Sci USA 1984;81:2534–7.
[26] Chen YA, Essex M. Antibody responses to different regions ofHTLV-I gp61 in asymptomatic carriers, ATL patients, andindividuals with TSP. In: Blattner WA, editor. Human Retro-viruses:HTLV. New Haven: Raven Press, 1990:447–60.
[27] Yamaguchi K, Kiyokawa T, Nakada K. Polyclonal integrationof HTLV-I proviral DNA in lymphocytes from HTLV-Iseropositive individuals: an intermediate state between thehealthy carrier state and smouldering ATL. Br J Haematol1988;68:169–74.
[28] Tokudome S, Tokunaga O, Shimamoto Y. Incidence of adultT-cell leukemiallymphoma among human T-lymphotrophic virustype I carrier in Saga, Japan. Cancer Res 1989;49:226–8.
[29] Migone TS, Lin JX, Cereseto A, Mulloy JC, O’Shea JJ, Fran-chini G, Leonard XWJ. Constitutively activated Jak-STAT path-way in T-cells transformed with HTLV I. Science1995;269:79–81.
[30] Xu X, Kang S-H, Heidenreich O, Okerholm M, O’Shea JJ,Nerenberg MI. Constitutive activation of different Jak tyrosinekineses in human T-cell leukemia. virus type 1 (HTLV-1) Taxprotein or virus-transformed cells. J Clin Invest 1995;96:1548–55.
[31] Zhang Q, Nowak I, Vonderheid EC, Rook AH, Kadin ME,Nowell PC, Shaw LM, Wasik MA. Activation of Jak/STATproteins involved in signal transduction pathway mediated byreceptor for interleukin 2 in malignant T lymphocytes derivedfrom cutaneous anaplastic large T-cell lymphoma and Sezarysyndrome. Proc Natl Acad Sci USA 1996;93:9148–53.
[32] Wasik MA, Morimoto C. Differential effects of cytokines onproliferative response of human CD4+ T-cell subsets stimulatedvia CD3-TCR complex. J Immunol 1990;144:3334–8.
[33] Kamihira S, Atogami S, Sohda H, Momita S, Yamada Y,Tomonaga M. Significance of soluble interleukin-2 receptor lev-els for evaluation of the progression of adult T-cell leukemia.Cancer 1994;73:2753–8.
[34] Srivastava MD, Srivastava A, Srivastava BI. Soluble inter-leukin–2 receptor, soluble CD8 and soluble intercellular adhe-sion molecule-1 levels in hematologic malignancies. LeukLymphoma 1994;12:241–51.
[35] Wasik MA, Vonderheid EC, Bigler RD, Marti R, Lessin S,Polansky M, Kadin ME. Increased serum concentration of ofthe soluble interleukin-2 receptor in cutaneous T-cell lymphomaclinical and prognostic implications. Arch Dermatol 1996;132:42–7.
[36] Maeda M, Shimuzu A, Ikuta K, Okamoto H, Kashihara M,Uchiyama T, Honjo T, Yodoi J. Origin of human T-lymphotrophic virus I-positive T-cell lines in adult. T-cellleukemia. Analysis of T-cell receptor gene rearrangement. J ExpMed 1985;162:2169–74.
[37] Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y,Shiraishi Y, Nagata K, Hinuma Y. Type C virus particles in acord T-cell line derived by co-cultivating normal human cordleukocytes and humna leukaemic T-cells. Nature 1981;294:770–1.
[38] Popovic M, Sarin PS, Robert-Gurnoff M, Kalyanaraman VS,Mann D, Minowada J, Gallo RC. Isolation and transmission ofhuman retrovirus (human T-cell leukemia virus). Science1983;219:856–9.
[39] Poiescz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD,Gallo RC. Detection and isolation of type C retrovirus particlesfrom fresh and cultured lymphocytes of a patient with cutaneousT-cell lymphoma. Proc Natl Acad Sci USA 1980;77:7415–9.
[40] Bunn PA Jr., Foss FM. T-cell lymphoma cell lines (HUT102 andHUT78) established at the National Cancer Institute: historyand importance to understanding the biology, clinical features,and therapy of cutaneous T-cell lymphomas (CTCL) and adultT-cell leukemia-lymphomas (ATLL). J Cell Biochem 1996-Suppl24: 12–23.
[41] Gessain A, Saal F, Giron M-L, Lasnaret J, Lagaye S, Gout O,De The G, Sigoux F, Peries J. Cell surface phenotype andhuman T lymphotropic virus type I antigen expression in 12T-cell lines derived from peripheral blood and cerebrospinal fluidof West Indian, Guyanese and African patients with tropicalspastic paraparesis. J Gen Virol 1990;71:333–41.
[42] Abrams JT, Lessin S, Ghosh SK, Ju W, Vonderheid EC, NowellP, Murphy G, Elfenbein B, DeFreitas E. A donal CD4-positiveT-cell line established from the blood of a patient with SezarySyndrome. J Invest Dermatol 1991;96:31–7.
[43] Yodoi J, Teshigawara K. TCGF (IL-2)-receptor inducing fac-tor(s). I. Regulation of 11-2 receptor on a natural killer-like cellline (YT cells). J Immuonol 1985;134:1623–30.
Q. Zhang et al. / Leukemia Research 23 (1999) 373–384384
[44] Wasik MA, Seldin DC, Butmarc JR, Gertz R, Marti R, Maslin-ski W, Kadin ME. Analysis of IL-2, IL-4 and their receptors inclonally-related cell lines derived from a patient with a progres-sive cutaneous T-cell lymphoproliferative disorder. LeukLymphoma 1996;23:125–36.
[45] Marti R, Wasik MA, Kadin ME. Constitutive and selectivesecretion of GM CSF by three different cell lines derived from asingle patient with a progressive cutaneous lymphoproliferativedisorder. Cytokine 1996;8:323–9.
[46] Kwok S, Kellogg D, Ehrlich G, Poiescz B, Bhagavati S, SninskyJJ. Characterization of a sequence of human T-cell leukemiavirus type I from a patient with chronic progressive myelopathy.J Inf Dis 1988;158:1193–7.
[47] Lessin SR, Rook AH, Rovera G. Molecular diagnosis of cuta-neous T-cell lymphoma polymerase chain amplification of T-cellantigen receptor b-gene rearrangements. J Invest Dermatol1991;96:299–302.
[48] Wasik MA, Nowak I, Zhang Q, Shaw LM. Suppression ofproliferation and phosphorylation of Jak3 and STAT5 in malig-nant T-cell lymphoma cells by derivatives of octylamino-unde-cyl-dimethylxanthine. Leuk Lymphoma 1998;28:551–60.
[49] Migone TS, Calacano NA. Recruitment of SH2-containingprotein tyrosine phosphatase SHP-1 to the interleukin 2 recep-tor; loss of SHP-1 expression in human T lymphotropic virustype I-transformed T-cells. Proc Natl Acad Sci USA1998;95:3845–50.
[50] Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HF.Specific recruitment of SH-PTP1 to the erythropooietin receptorcauses inactivation of Jak2 and terminaation of proliferativesignals. Cell 1995;80:729.
[51] Shultz LD, Schweitzer PA, Rajan TV, Yi T, Ihle JN, MatthewsRJ, Thomas ML, Beier DR. Mutations at the murine motheatenlocus are within the hematopoietic cell protein-tyrosine phos-phatase (Hcph) gene. Cell 1993;73:1445.
[52] Shultz LD, Rajan TV, Greiner DL. Severe defects in immunityand hematopoiesis caused by SHP-1 protein-tyrosine-phos-phatase deficiency. Trends Biotechnol 1997;15:302.
[53] Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J.PDGF- and insulin-dependent pp70S6K activation mediated byphosphatidylinositol-3-OH kinase. Nature 1994;370–81.
[54] Brown EJ, Beal PA, Keith CT, Chen J, Shin TB, Schreiber SL.Control of p70S6 kinase by kinase activity of FRAP in vivo.Nature 1995;377–441.
[55] Takemoto S, Mulloy JC, Cereseto A, Migone TS, Patel BKR,Matsuoka M, et al. Proliferation of adult T-cell leukemia/lymphoma cells is associated with the constitutive activation ofJAK/STAT proteins. Proc Natl Acad Sci USA 1997;94:13897–902.
[56] Arima N. Autonomous and interleukin-2-responsive growth ofleukemic cells in adult T-cell leukemia (ATL): a review of theclinical signifcance and molecular basis of ATL cell growth.Leuk Lymphoma 1997;26:479–89.
[57] Lal RB, Rudolph DL, Folks TM, Hooper WC. Over expressionof insulin-like growth factor receptor type-1 in T-cell lines in-fected with human T-lymphotropic virus types-I and-II.Leukemia Res 1993;17:31–5.
[58] Mori N, Gill PS, Mougdil T, Murakami S, Eto S, Prager D.Interleukin-10 gene expression in adult T-cell leukemia. Blood1996;88:1035–45.
[59] Ratajczak MZ, Marlicz W, Ratajczak J, Machalinski B, WasikMA, Carter A, Gewirtz AM. Effect of hepatocyte growth factor(HGF) on early human hematopoietic cell development. Brit JHaematol 1997;99:228–36.
[60] Koochekpour S, Jeffers M, Rulong S, Taylor G, Klineberg E,Hudson EA, Resau JH, Vande Woude GF. Met and hepatocytegrowth factor/scatter factor expression in human gliomas. Can-cer Res 1997;57:5391–8.
[61] Jeffers M, Schmidt L, Nakaigawa N, Webb CP, Weirich G,Kishida T, Zbar B, Vande Woude GF. Activating mutations forthe met tyrosine kinase receptor in human cancer. Proc NatlAcad Sci USA 1997;94:11445–50.
[62] Schmidt L, Junker K, Weirich G, Glenn G, Choyke P, LubenskyI, Zhuang Z, Jeffers M, Vande Woude G, Neumann H, WaltherM, Linehan WM, Zbar B. Two North American familieswith hereditary papillary renal carcinoma and identical novelmutations in the MET proto-oncogene. Cancer Res1998;58:1719–22.
[63] Finbloom DS, Winestock KD. IL-10 induces the tyrosine phos-phorylation of tyk2 and Jakl and the differential assembly ofSTAT 1 a and STAT3 complexes in human T-cells and mono-cytes. J Immunol 1995;155:1079–90.
[64] Hatta Y, Hirama T, Miller CW, Yamada Y, Tomonaga M,Koeffler HP. Homozygous deletions of the p15 (MTS2) and pl6(CDKN2/MTS 1) genes in adult T-cell leukemia. Blood1995;85:2699–704.
[65] Ogawa S, Hangaishi A, Miyawaki S, Hirosawa S, Miura Y,Takeyama K, et al. Loss of the cyclin-dependent kinase 4-in-hibitor (p16;MTS1) gene is frequent in and highly specific tolymphoid tumors in primary human hematopoietic malignancies.Blood 1995;86:1548–56.
[66] Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 familyof cytokines and gpl30. Blood 1995;86:1243–54.
[67] Fujii H, Nakagawa Y, Schindler U, Kawahara A, Mori H,Gouilleux F, Groner B, Ihle JN, Minami Y, Miyazaki T,Taniguchi T. Activation of Stat5 by interleukin 2 requires acarboxyl-terminal region of the interleukin 2 receptor beta chainbut is not essential for the proliferative signal transmission. ProcNatl Acad Sci USA 1995;92:5482–6.
[68] Ahmed NN, Grimes HL, Bellacosa A, Chan TO, Tsichlis PN.Transduction of interleukin-2 antiapoptotic and proliferativesignals via Akt protein kinase. Proc Natl Acad Sci USA1997;94:3627–32.
[69] Takeshita T, Arita T, Asao H, Tanaka N, Higuchi M, KurodaH, Kaneko K, Munakata H, Endo Y, Fujita T, Sugamura K.Cloning of a novel signaltransducing adapter molecule contain-ing an SH3 domain and ITAM. Biochem Biophys Res Comm1996;225:1035–41.
[70] Takeshita T, Arita T, Higuchi M, Asao H, Endo K, Kuroda H,Tanaka N, Murata K, Ishii N, Sugamura K. STAM, signaltransducing adaptor molecule, is associated with Janus kinesesand involved in signaling for cell growth and c-myc induction.Immunity 1997;6:449–57.
[71] Miyazaki T, Takaoka A, Nogueira L, Dikic I, Fujii H, TsujinoS, Mitani Y, Maeda M, Schlessinger J, Taniguchi T. Pyk2 is adownstream mediator of the IL-2 receptor-coupled Jak signalingpathway. Genes Dev 1998;12:770–5.
