of March 8, 2014.This information is current as

with Tumor Mast-Cell Lines Cells: Comparisons+Peripheral Blood CD34

Development of Human Mast Cells from Induction of Telomerase Activity During

Dean D. Metcalfe and Michael A. BeavenGilfillan, Arnold S. Kirshenbaum, Jose Renan Cunha-Melo, Cristina Chaves-Dias, Thomas R. Hundley, Alasdair M.

http://www.jimmunol.org/content/166/11/66472001; 166:6647-6656; ;J Immunol 

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Induction of Telomerase Activity During Development ofHuman Mast Cells from Peripheral Blood CD341 Cells:Comparisons with Tumor Mast-Cell Lines1

Cristina Chaves-Dias,2* Thomas R. Hundley,* Alasdair M. Gilfillan, † Arnold S. Kirshenbaum,†

Jose Renan Cunha-Melo,3* Dean D. Metcalfe,† and Michael A. Beaven4*

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the ex-pression of telomerase activity in cultured human peripheral blood CD341 cells, and CD341/CD1171/CD131 progenitor mastcells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD341/CD1171/CD131

cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factorand either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearanceof mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38mitogen-activated protein kinase and phosphatidylinositol 3*-kinase, but once cell replication was underway telomerase activity,but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently hightelomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways inmast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction oftelomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas mainte-nance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication. The Journal ofImmunology,2001, 166: 6647–6656.

Progenitors of human mast cells (CD341/CD1171/CD131

cells) are derived from bone marrow CD341 pluripotentcells (1). The clonal expansion of the progenitor cells and

their maturation into mast cells requires stem cell factor (SCF),5

the ligand for the receptor Kit (CD117), in combination with othercytokines, such as IL-3 and IL-6 (reviewed in Refs. 2 and 3).Although the specific roles of these cytokines are still debated,they are used in various combinations for the development of ma-ture mast cells from CD341 cells from bone marrow and periph-eral blood. The signaling mechanisms involved in these processeshave not been defined in mast cell progenitors, but activation of theKit tyrosine kinase, phosphatidylinositol 39-kinase (PI 3-kinase),and c-Jun N-terminal kinase (JNK) via Src kinase appears to beessential for mast cell proliferation and maturation (4–6). Other-

wise, SCF is known to activate PI 3-kinase and all three mitogen-activated protein (MAP) kinases in mature mast cells (7) and otherhemopoietic cells (8, 9). In addition, SCF (9) as well as IL-3 (10)and IL-6 (11) activate the Janus kinase (JAK)/STAT pathways inhemopoietic cells. In the case of IL-3, activation of the JAK/STATand MAP kinase pathways results in the production of a variety oftranscription factors thought to be necessary for cell proliferationand differentiation (reviewed in Ref. 10).

In general, the clonal expansion of hemopoietic cell lines andlymphocytes is associated with transient activation of telomerase,an RNA-dependent DNA polymerase (12). This enzyme maintainsthe integrity of chromosomes in dividing cells by placing addi-tional telomeric DNA segments (TTAGGG in vertebrates) on theends of chromosomal DNA and thus minimizing attrition of theterminal telomeres during DNA replication (13). In vertebrates,telomerase consists of a protein catalytic subunit, telomerase re-verse transcriptase (TERT), the RNA template (TR), and associ-ated proteins, such as telomerase-associated protein-1. Telomeraseactivity, which is low in nonreplicating somatic cells and high ingerminal cells and tumor cell lines (reviewed in Ref. 14), is tran-siently activated in stimulated myeloid and lymphoid progenitorcells (reviewed in Refs. 12, 14, and 15). Thus, stimulation of hu-man bone marrow and blood stem cells (16, 17) or quiescent T andB cells with stimulants that induce cell proliferation (reviewed inRefs. 15 and 18) leads to rapid increase in telomerase activity earlyin the G1 phase of the cell cycle (18). The increase is short-livedand is thought to facilitate the limited clonal expansion of thesecells by delaying telomere shortening.

Little is known about the regulation of telomerase expression oractivity (reviewed in Ref. 19), although direct activation ofTERTtranscription by c-Myc has been reported (20). Telomerase TRsubunits are expressed in most tissue cells, whereas expression ofTERT is generally restricted to replicating cells through largely

*Laboratory of Immunology, National Heart, Lung, and Blood Institute, and†Labo-ratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892

Received for publication January 4, 2001. Accepted for publication March 28, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was performed at the National Institutes of Health while the principalauthor was supported by a predoctoral fellowship from Coordenacao de Aperfeicoa-mento de Pessoal de Nıvel Superior (CNPq/CAPES), Brazil.2 Current address: Departamento de Biologia Animal, Universidade Federal de Vi-cosa, 36571-000 Vicosa MG, Brazil.3 Current address: Faculdade de Medicina, Universidade Federal de Minas Gerais,Avenida Alfredo Balena 190, 30130-100 Belo Horizonte MG, Brazil.4 Address correspondence and reprint requests to Dr. Michael A. Beaven, Building10, Room 8N109, National Institutes of Health, Bethesda, MD 20892-1760. E-mailaddress: beaven@helix.nih.gov5 Abbreviations used in this paper: SCF, stem cell factor; PI 3-kinase, phosphatidyl-inositol 39-kinase; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; TERT, telomerase reverse transcriptase; TR, TERT RNA; HMBA,hexamethylene bisacetamide; rh, recombinant human.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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undefinedmechanisms (19). The increase in telomerase activityin stimulated B cells is associated with increased expression ofTERT (21), although a modest increase in TR expression is alsoapparent in stimulated T cells (22–24). In addition, telomerasemay be regulated directly through itsstate of phosphorylation(reviewed in Ref. 19). Studies with pharmacologic agents in tumorcell systems suggest that dephosphorylation of TERT by proteinphosphatase 2A results in loss of enzyme activity, and its rephos-phorylation by protein kinase C (25) or Akt (protein kinase B)restores activity (26).

As an extension of previous work characterizing the growth andmaturation of human mast cells from progenitor cells (1), we nowshow that, as in other hemopoietic cell lines, telomerase activity istransiently induced during proliferation of human peripheral bloodCD341 and CD341/CD1171/CD131 progenitor cells. This induc-tion occurs well before the appearance ofmature mast cells, butrequires the presence of SCF in addition to IL-3 or IL-6. We haveidentified potential signals that lead to this induction, but we have alsofound that the same signals do not contribute to the constitutively hightelomerase activity normally present in tumor mast cell lines.

Materials and MethodsMaterials

Materials were obtained from the following sources: SB 202190, PD98059, AG490, PP1, and wortmannin from Calbiochem (San Diego, CA);LY 294.002 from Alexis (Milwaukee, WI); PD1152440 from Bachem(Torrance, CA); quercetin, hydroxyurea, nocodazole, and cyclohexamidefrom Sigma (St. Louis, MO); hexamethylene bisacetamide (HMBA) fromAldrich (Metuchen, NJ); annexin V-PE and 7-amino-actinomycin D fromPharMingen (San Diego, CA); telomerase PCR ELISA kit from RocheMolecular Biochemicals (Indianapolis, IN); StemPro-34 SFM from LifeTechnologies (Gaithersburg, MD); other reagents for culture of human andtumor cell lines from Life Technologies or Biofluids (Rockville, MD); celllines from American Type Culture Collection (Manassas, VA) or fromongoing cultures of RBL-2H3 and HMC-1 (originally from J. H. Butter-field, Department of Internal Medicine, Mayo Clinic, Rochester, MN) cellsin the laboratory; recombinant human (rh) SCF, IL-6, and IL-3 from Pep-roTech (Rocky Hill, NJ); recombinant mouse IL-3 from R&D Systems(Minneapolis, MN); chimeric Fc-specific anti-4-hydroxy-3-nitrophenylac-etyl-IgE from Serotec (Raleigh, NC); and 4-hydroxy-3-nitrophenylacetyl-BSA from Biosearch Technologies (Novoto, CA). Stock solutions of ki-nase inhibitors were prepared in DMSO and diluted for use with mediumto yield a final concentration 0.1% DMSO. Other reagents were dissolvedin culture medium.

Immunoselection and FACS of CD341 and CD341/CD1171/CD131 cells

PBMC were obtained by leukapheresis following G-CSF treatment of nor-mal volunteers who had given informed consent. The techniques for puri-fication of pluripotent CD341 cells by using CD341 affinity columns andsorting CD341/CD1171/CD131 progenitor cells from isolated or culturedCD341/CD1171 cells by FACS were described previously (1). These cellswere stored in liquid nitrogen until use.

Cell culture and time-course studies

CD341 cells and CD341/CD1171/CD131 cells derived therefrom wereplated at a concentration of 23 10 4 cells in 25-cm2 flasks in serum-freemedium. This medium consisted of StemPro-34 supplemented withL-glu-tamine (2 mM), penicillin (100 U/ml), streptomycin (100mg/ml), rhIL-3(30 ng/ml), rhIL-6 (100 ng/ml), and rhSCF (100 ng/ml). Cultures werediluted (2-fold) weekly or twice weekly in the same medium, except thatrhIL-3 was omitted after the first week of culture as described previously(1). This protocol was altered for determination of growth rates andchanges in telomerase activity. Namely, for these experiments sampleswere removed each day or every other day, and the cultures were dilutedwith fresh medium to maintain a cell density of;105 cells/ml. Recombi-nant hIL-3 was omitted or added to the medium as indicated in the text.

With respect to the tumor cell lines, RBL-2H3 cells (27) were culturedas monolayers in suspension-MEM supplemented with 10% heat-inacti-vated FBS, penicillin (100 U/ml), streptomycin (100mg/ml), andL-glu-tamine (2 mM) as previously described (28). IL-3-dependent MC-9 cells

(29) were maintained as suspensions in DMEM supplemented with 10%heat-inactivated FBS,L-glutamine (2 mM), penicillin (100 U/ml), strepto-mycin (100mg/ml), and IL-3 (0.5 ng/ml). HMC-1 cells (30) were grown insuspension in IMEM supplemented with 10% heat-inactivated FBS,L-glu-tamine (2 mM), penicillin (100 U/ml), streptomycin (100mg/ml), anda-thioglycerol (1.2 mM).

All cultures were maintained in humidified atmosphere of 95% air and5% CO2 at 37°C. The cells were incubated as suspensions or monolayersas indicated above. Cell suspensions were sampled directly, or in the caseof RBL-2H3 cells, cells were suspended by trypsinization before use (28).

Determination of cell count, viability, and apoptotic cells

The cells were harvested and counted in a hemocytometer. The increase incell number was expressed as the fold increase in cell population from thestart of culture after correction for dilutions of culture during the course ofthe experiment. Cell viability was determined by trypan blue exclusion. Forthe determination of apoptotic cells, cells were washed in ice-cold PBS andstained with annexin-V-PE to detect apoptotic cells and with 7-amino-actinomycin to detect necrotic cells according to the manufacturer’s prod-uct protocol (catalog no. 65875X; PharMingen). Samples of stained cellswere analyzed using flow cytometry (EPICS XL-MCL; Beckman Coulter,Hialeah, FL). Protein was assayed by use of the Bio-Rad assay kit(Hercules, CA).

Microscopic examination of CD341 cell-derived cultures ofhuman mast cells and measurement of hexosaminidase release

Cells were collected at various stages of culture by centrifugation (Cyto-spin; Shandon, Pittsburgh, PA) onto slides and stained with 0.5% acidictoluidine blue (pH 1.0) or with a mixture of toluidine blue and light greenas described by Kimura and coworkers (31). Cells were identified as mastcells by the presence of abundant numbers of metachromatically stainedgranules.

CD341 cell-derived 8-wk cultures of mature mast cells were collectedas described above and incubated overnight with chimeric human IgE di-rected against the Ag, 4-hydroxy-3-nitrophenylacetylated BSA. Cells werethen washed three times, resuspended at a density of 105 cells/ml inHEPES-buffered medium (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl,0.4 mM Na2HPO4z7H2O, 5.6 mM glucose, 1.8 mM CaCl2z2H2O, 1.3 mMMgSO4z7H2O, and 0.025% BSA, pH 7.4), and aliquoted into 96-well mi-crotiter plates (90ml/well). Cells were stimulated for 10 min with thedesignated concentration of Ag for measurement of release of the granulemarker, b-hexosaminidase, as previously described (32). Data are ex-pressed as a percentage of the intracellularb-hexosaminidase released intothe medium.

Studies with inhibitors of protein kinases, cell cycle, and proteinsynthesis

CD341 and CD341/CD1171/CD131 cells were plated in 25-cm2 flasks(4 3 104 cells/2 ml), and tumor mast cell lines were plated in 80-cm2 flasks(2 3 106 cells/10 ml) in the medium described above. Inhibitors or vehiclewere added at the times and concentrations indicated in the text.

Telomerase assay

Telomerase was assayed by use of a telomerase polymerase reaction/ELISA kit. Cells (33 105) were collected by centrifugation (30003 g, 10min, 4°C), washed once in ice-cold PBS, and resedimented by centrifuga-tion (30003 g, 10 min, 4°C). The pelleted cells were resuspended in 200ml of lysis reagent as supplied in the kit and left on ice for 30 min. Celllysates were centrifuged (60003 g, 20 min, 4°C), and the supernatantextracts were stored at280°C until use. The telomeric repeat amplificationprotocol was performed with extracts containing 1500 cell-equivalents foreach reaction (30-min incubation and 30 cycles). For samples with hightelomerase activity, a 15-min incubation and 25 cycles were used. Thehybridization and ELISA procedures were performed according to themanufacturer’s protocols (Roche Molecular Biochemicals), and the absor-bance of the final colored reaction product was measured in a microtiterplate reader at 450 nm. Each arbitrary unit was defined as absorbance per1500 cell equivalents.

Phosphorylation of p38 MAP kinase, Atk (protein kinase B), andactivating transcription factor-2

Whole cell lysates were subjected to SDS-PAGE (10% acrylamide gels)and immmunoblotted for the phosphorylated activatedforms of theseproteins with rabbit polyclonal Abs (1/500 and 1/1000 dilutions) againstdoubly phosphorylated (Thr180/Tyr182)p38 MAP kinase, phosphorylated

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(Ser473)Akt, and phosphorylated (Thr71)ATF-2. The bands were detected withthe Amersham chemiluminescence kit and quantitated by densitometric scan-ning (Personal Densitometer; Molecular Dynamics, Sunnyvale, CA).

Expression of results

Unless indicated otherwise, data were expressed as the mean and SEM ofmean values from three or more separate experiments. For individual ex-periments, three to six samples or cultures were assayed for each datapoint.

ResultsTime course of stimulation of telomerase activity and cellproliferation by growth factors in CD341 and CD341/CD1171/CD131 cells

Fig. 1 shows examples of changes in telomerase activity in culturesof CD341 pluripotent cells (Fig. 1,A andB) and CD341/CD1171/CD131 progenitor cells (Fig. 1,A and C) that were maintainedunder standard culture conditions (i.e., in the presence of IL-3,IL-6 plus SCF for 7 days, and omission of IL-3 thereafter). InCD341 cells telomerase activity increased markedly within 24 hand continued to increase for several days reaching a maximumbetween days 3 and 6 (Fig. 1,A andB). Telomerase activity thendeclined rapidly to near basal levels by day 12 and remained at lowor undetectable levels for the duration of culture (up to 50 days).

A similar pattern was observed in selected CD341/CD1171/CD131 cells, with a rapid decline in activity whether cells wereisolated from 7-day-old cultures of CD341 cells (as in Fig. 1A) ordirectly from peripheral blood pluripotent cells on day 0 (as in Fig.1C). The decline in telomerase activity was equally rapid whethercells were deprived of IL-3 on day 7 as described above or wereexposed to all three cytokines throughout the experiment (data notshown). The initial increase in telomerase activity appeared to pre-cede cell division, which was not apparent until 48 h or later (Fig.1B). The maximal expansion of the cell cultures coincided with thevirtual disappearance of telomerase activity (Fig. 1,B andC). Webelieve that rapid loss of telomerase activity was due to loss ofenzyme from all or most cells rather than to dilution of a subpopu-lation of cells with high telomerase activity, because purifiedCD341/CD1171/CD131 cells give rise to almost pure populations(;85%) of mast cells (1). However, this could not be tested di-rectly, as expansion of colonies from individual CD131-sortedcells in culture was too slow to yield sufficient cells for assay. Atbest, colonies of 300 cells were achieved after 3 wk of culturewhen telomerase activity had long declined to near basal levels.

After 50 days in culture, cells derived from both CD341 andCD341/CD1171/CD131 sorting had acquired the characteristicsof normal mast cells. When sensitized with a chimeric IgE (see

FIGURE 1. Time course of changes in telom-erase activity in cultures of human pluripotentCD 341 and CD341/CD1171/CD131 cells andsecretory response of 8-wk cultures to Ag.A,Cells, collected from peripheral blood by leuka-pheresis, were selected by use of CD341 affinitycolumns and cultured in the presence of SCF,IL-3, and IL-6 for the periods indicated by thebars for assay of telomerase activity. In additionCD341/CD1171/CD131 cells were sorted fromseparate cultures on day 7 (arrow) and main-tained under the same conditions for measure-ment of telomerase activity. The data are themean of values from three or more experimentsfor the first 7 days of culture and two experi-ments for subsequent days.B andC, CD341 andCD341/CD1171/CD131 cells were collectedseparately from peripheral blood and grown inculture for the times indicated for measurementof telomerase activity and cell number. The foldincrease in cell number is shown after correctionfor dilutions during culture.D, Cells were col-lected from 8-wk cultures of CD341 cells, sen-sitized with Ag-specific human IgE, and stimu-lated with the indicated concentrations of Ag,4-hydroxy-3-nitrophenylacetylated BSA, for 30min for measurement of secretion of the granulemarker,b-hexosaminidase. The mean and SEMof values from three separate experiments areshown.

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Materials and Methods), they secreted in response to the IgE-spe-cific Ag in a dose-dependent manner, as determined by measure-ment of release of the granule marker, hexosaminidase (Fig. 1D).Histologic examination of cultures at different stages of growthrevealed that the appearance of mature mast cells occurred afterthe decline in telomerase activity. At 2 wk of culture, when te-lomerase activity had returned to minimal levels (see Fig. 1), mastcells were undetectable in cultures of CD341 cells by light mi-croscopy, but by 4 wk, partially mature mast cells had begun toappear (Fig. 2). By 8 wk and times thereafter, cultures were pre-dominantly mature mast cells ($95% viable mast cells).

IL-3 and IL-6 synergize stimulation of telomerase activity bySCF

The effects of cytokines on telomerase activity in CD341 cellswere examined in the initial 2 days of culture before the apparentincrease in cell numbers or, in the absence of growth factors, ap-optosis (data not shown). IL-3, IL-6, or SCF individually causedminimal stimulation of telomerase activity compared with that in-

duced by SCF in combination with IL-3 or IL-6 (Fig. 3). Thecombination of IL-3 and IL-6 elicited only modest increases intelomerase activity, and in total the experiments indicated thatmaximal stimulation of telomerase activity required stimulationwith SCF in combination with IL-3 and IL-6. As noted earlier, thesubsequent decline in telomerase activity occurred in the presenceof all three cytokines.

Effect of protein kinase inhibitors on induction of telomeraseactivity and cell proliferation in CD341 cultures

A wide variety of kinase inhibitors were tested to identify potentialsignals for regulating telomerase activity and cell proliferation.Inhibitors were added on day 0 or 2 of culture, and the cultureswere examined 2 days thereafter (i.e., day 2 or 4). The inhibitorsincluded PD98059 (33), SB202190 (34), and wortmannin (35),inhibitors of the extracellular signal-regulated kinase-MAP kinasepathway, p38 MAP kinase, and PI 3-kinase, respectively (Fig. 4).Of these, SB202190 and wortmannin suppressed induction of te-lomerase activity if added at the start of culture (Fig. 4A, f), but

FIGURE 2. Maturation of mast cells in cul-tures of CD341 cells. Samples of cells removedfrom cultures at the indicated times were exam-ined histologically after staining cells with tolu-idine blue or (bottom right panel), the stain de-scribed by Kimura and coworkers (31).

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not when added at 2 days of culture (Fig. 4A, M) when the increasein telomerase was well underway and before cell proliferation hadcommenced (Fig. 4B). The initial suppression of telomerase activ-ity by SB202190 and wortmannin was dependent on concentrationof drug (Fig. 4,C andD). The initial induction of telomerase wasalso inhibited by another PI 3-kinase inhibitor, LY294002 (36)(data not shown). All drugs suppressed cell proliferation to varyingextents whether added on day 0 or 2 of culture (see Fig. 4B,M).These and the above results suggested that the increase in telom-erase activity was initially dependent on p38 MAP kinase andpossibly PI 3-kinase, but, once elevated, telomerase activity wasno longer affected by the inhibitors, even though cell proliferationwas markedly inhibited.

The activation of PI 3-kinase and p38 MAP kinase and the in-hibition of these kinases by wortmannin and SB202190, respec-tively, were confirmed by detection of phosphorylated (Ser473)Akt,a downstream marker of PI 3-kinase activity (37), the doubly phos-phorylated (Thr180/Tyr182)p38 MAP kinase (38), and phosphory-lated (Thr71)ATF-2 (38, 39), a substrate for both p38 MAP kinaseand JNK after 24 h (Fig. 5) and 72 h (data not shown) of culture.The presence of an additional retarded band of c-Jun suggestedthat JNK was activated as well, although phosphorylated JNK wasnot detectable by immunoblotting. The phosphorylation of p38MAP kinase and to some extent that of ATF-2 were reduced inSB202190-treated cells as was the phosphorylation of Akt in wort-mannin-treated cells. Paradoxically, wortmannin enhanced thephosphorylation of p38 MAP kinase (Fig. 5) (T. R. Hundley, un-published observations) consistent with the suggestion that PI 3-ki-nase may down-regulate this MAP kinase (40).

The above results indicated that p38 MAP kinase and PI 3-ki-nase were activated and were inhibited by the appropriate inhibi-tor, but also that JNK might be activated as well. SB202190, whichcan inhibit JNKb at high concentrations (41, 42), enhanced theexpression of c-Jun (Fig. 5). Therefore, these studies provided noclear indication of the role of JNK, if any, on telomerase induction.

High telomerase activity in tumor mast-cell lines: the effects ofinhibitors of the cell cycle and protein synthesis

Additional studies were conducted with cultured tumor mast celllines where cell numbers did not limit the scope of experiments.The cell lines examined included IL-3-independent rat RBL-2H3cells as well as IL-3-dependent mouse MC-9 cells and humanHMC-1 cells. All three cell lines had measurable telomerase ac-tivities. Although direct comparisons suggested that MC-9 cellsexpressed lower telomerase activity than other cell lines and 4-day

FIGURE 3. Increase in telomerase activity in CD341 pluripotent cellsin response to various growth factors. CD341 cells were cultured for 2 daysin the presence of 30 ng/ml IL-3, 100 ng/ml IL-6, 100 ng/ml SCF, orvarious combinations of these growth factors as indicated for measurementof telomerase activity. The data are the mean6 SEM of average valuesfrom three experiments.

FIGURE 4. Effects of kinase inhibitors on telomerase activity and cell proliferation in cultures of CD341 pluripotent cells. CD341 cells were grown inmedium that contained IL-3, IL-6, and SCF in the absence (C) or the presence of 25mM SB 202190 (SB), 50mM PD 98059 (PD), or 100 nM wortmannin(W; A andB) or in the presence of various concentrations of SB 202190 or wortmannin (C andD). Inhibitors were added either at the start (day 0) or after2 days of culture, and telomerase activity and increase in cell number were determined 48 h later.A andB, Inhibitors were present from days 0–2 (f) orfrom days 2–4 (M);C andD, inhibitors were present from days 0–2. Data are the mean6 SEM of average values from three or more separate experiments.

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cultures of CD341 cells (Fig. 6A), it should be noted that telom-erase activity varied with different passages of cells and experi-ments (compare Fig. 6 with subsequent figures).

Studies with inhibitors of the cell cycle suggested that the hightelomerase activity in tumor cell lines persisted throughout the cellcycle. Fig. 6,B andC, show representative experiments with 0.3mM hydroxyurea (43), 50 nM nocodazole (44), and 0.4 mM in-domethacin (45), which inhibit progression through the cell cycle

in S, M, and early G1 phases, respectively. All three drugs blockedcell proliferation completely (data not shown) without affectingtelomerase activity. Modest decreases in telomerase activity wereobserved occasionally as, for example, with nocodazole in Fig. 6C.However, additional experiments with different batches of cellsindicated that such decreases were the exception even when cellswere exposed to these drugs for up to 72 h. Others have noted nochange in telomerase activity during the cell cycle of human tumorcell lines except when agents caused cell toxicity (46). Blockade ofprotein synthesis as well as cell proliferation with cycloheximidealso failed to reduce levels of telomerase activity in RBL-2H3 andMC-9 cells after 24-h (Fig. 6D) or 48-h (data not shown) exposureto the drug. Apparently, telomerase was degraded slowly, at leastin the absence of protein synthesis.

Further studies in mast cell tumor lines: effects of kinaseinhibitors

Interestingly, telomerase activity in the tumor cell lines was notsuppressed by SB202190 and wortmannin or indeed by many otheragents that block known signaling pathways in mast cells. Exam-ples of experiments are shown in Fig. 7. The effects of wortman-nin, PD98059, and SB202190 on telomerase activity and cell pro-liferation are shown for MC-9 cells (Fig. 7,A and B). Similarresults were obtained with RBL-2H3 cells (data not shown). Inboth types of cells, SB202190 and wortmannin blocked cell pro-liferation (i.e., no increase in cell number; Fig. 7B) without mark-edly reducing telomerase activity (Fig. 7A). Exposure to high con-centrations of SB202190 ($50mM instead of 25mM as used inFig. 7) caused significant loss of telomerase activity (;50%) intwo of five experiments (data not shown) and apoptosis (;40% ofthe cell population) within 24 h.

FIGURE 6. Telomerase activities in tumor mast cell lines (RBL-2H3, MC-9, and HMC-1) persists after suppression of cell proliferation and proteinsynthesis.A, Telomerase activities were determined in 4-day cultures of the tumor cell lines and CD341 cells. All cultures contained the necessary growthfactors as described inMaterials and Methods.B—D, As in A, except that vehicle, 3 mM hydroxyurea, 25 ng/ml nocodazole, 0.4 mM indomethacin, or2 mM cycloheximide was added for the final 24 h of culture (2(f)/1 (M) indicates the absence or the presence of drug). These inhibitors arrested culturegrowth (data not shown). Data are the mean6 SEM of average values from three or more separate experiments (A andB) or combined values from twoidentical experiments (CandD).

FIGURE 5. Early activation of kinases in CD341 cells. Initial isolatesof cells from peripheral blood were cultured for 24 h in the presence ofvehicle (C), 25mM SB 202190, (SB), or 100 nM wortmannin (Wt). Cul-tures were examined by immunoblotting (seeMaterials and Methods) forphosphorylated (Ser473)Akt, doubly phosphorylated (Thr180/Tyr182)p38MAP kinase, and phosphorylated (Thr71)ATF-2. Immunoblots for Akt,ATF-2, and c-Jun proteins are also shown. Phosphorylated c-Jun and p38MAP kinase protein were not detectable (see text). The examples shownare representative of several experiments, and similar results were obtainedon days 2 and 3 of culture (data not shown).

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As shown for RBL-2H3 cells (Fig. 7C), additional agents testedincluded dexamethasone, an inhibitor of the MAP kinases in RBL-2H3 cells (47); Go6796, an inhibitor of the calcium-dependentisoforms of protein kinase C (48); Ro31-7549, an inhibitor ofcalcium-dependent and independent isoforms of protein kinase C(49); and PD 152440, a farnesyltransferase inhibitor that sup-presses the Ras/MAP kinase pathway (50). The latter compoundwas tested because Ras is present in its constitutively active GTP-bound form in RBL-2H3 cells (47). None of these agents at highdoses caused marked decreases in telomerase activity in RBL-2H3(Fig. 7C) or MC-9 cells (data not shown) after 18 h. Decreaseswere not observed after short term (2–6 h) or long term (up to48 h) exposure to the inhibitors (data not shown).

Other drugs found to be inactive (data not shown) over the shortor long term (2–48 h) included PP1 (10mM), which disrupts SCF-mediated signaling in hemopoietic cells and progenitors by block-ing Src kinases (51); piceatannol (10mM), which selectively in-hibits Syk, as opposed to Src, kinases in mast cells (52, 53); andAG490, a JAK2 inhibitor (54). Staurosporine (20 nM), an inhibitorof cyclin-dependent kinases as well as protein kinase C (55,),blocked cell proliferation but failed to diminish telomerase activityin RBL-2H3 and MC-9 cells. In addition, telomerase was not de-creased by depriving MC-9 cells of serum or IL-3, which also ledto arrest of cell proliferation (data not shown). Okadaic acid (300nM), a protein phosphatase inhibitor (56), induced a modest in-crease (;35%) in telomerase activity in MC-9 cells within 2 h inone of three experiments.

The most notable finding of these studies was that high telom-erase activity was maintained even after prolonged arrest of cellproliferation. This was most dramatically illustrated in studies withRBL-2H3 cells, where telomerase activity remained unchanged(Fig. 7D) even after complete cessation of growth for 96 h in thepresence of quercetin (Fig. 7E). This compound inhibits variousprotein kinases (57) and cell proliferation (58).

Suppression of telomerase activity in tumor cell lines withHMBA

HMBA was the only compound of many tested that caused a re-producible and substantial reduction of telomerase activity (Fig.8A) along with cessation of cell proliferation (Fig. 8B). The re-duction in telomerase activity was of slow onset and reached amaximum between 24- and 48-h exposure to HMBA (data notshown). This compound was tested because of its ability to induceterminal differentiation (59) and loss of telomerase activity (60,61) in tumor cell lines.

DiscussionOur data show that the rapid and transient increase in telomeraseactivity in human CD341 pluripotent cells upon provision of mastcell growth factors extends to a subpopulation of CD341/CD1171/CD131 progenitor mast cells (1). The increase in telomerase ac-tivity probably precedes cell proliferation, although a further in-crease following the initial cell division is possible. Thesubsequent decline in telomerase activity occurs long before theappearance of mature mast cells (Fig. 2). Analogous to mitogeni-cally stimulated lymphocytes and hemopoietic stem cells (12, 14,15), the early induction of telomerase may facilitate clonal expan-sion of CD341/CD1171/CD131 cells before maturation into mastcells. This induction is dependent on provision of SCF in combi-nation with IL-3 and IL-6 (Fig. 3) and, in turn, on intracellularsignals mediated by p38 MAP kinase and PI 3-kinase. Inhibitors ofthese two enzymes suppress the initial increase in telomerase ac-tivity, but they appear to have little effect once telomerase activityis fully elevated. Both enzymes appear to be activated early bygrowth factors in CD341 cells (Fig. 5) and remain activated for atleast 3 days (data not shown).

The studies withinhibitors also suggested that induction of telom-erase was not dependent on extracellular signal-regulated kinase,

FIGURE 7. Expression of telomerase activity in tumor mast cell lines is unaffected by treatment with various kinase inhibitors. Cultures of the tumormast cell lines were incubated in the absence (control,f) or the presence (M) of 50mM PD98059, 25mM SB202190, 100 nM wortmannin, 100 nMdexamethasone, 5mM Go6796, 10mM Ro31-7549, or 100mM PD152440 for 24 h (A–C) or 20mM quercetin for 96 h (DandE) for determination oftelomerase activity and increase in cell number (fold increase in number of cells plated on day 0). Data are the mean6 SEM of average values from fiveor more separate experiments (AandB) or combined data from two separate experiments (C–E).

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whereas cell proliferation was dependent to some extent on thisenzyme as well as on p38 MAP kinase and PI 3-kinase (Fig. 4B).Although these results do not conclusively establish that cell prolif-eration and induction of telomerase require different sets of signals,the suppression of cell proliferation by these inhibitors suggest thattelomerase activity is maintained regardless of the proliferativeactivity of the cells (i.e., data for day 4 in Fig. 4,A andB).

As noted above, costimulation of cells with SCF and either IL-3or IL-6 is required for optimal induction of telomerase activity(Fig. 3). None of these cytokines individually nor the combinationof IL-3 and IL-6 appears to promote the necessary mix or strengthof signals for this induction. The overall pattern of responsesshown in Fig. 3 suggests that SCF either stimulates a unique andessential signaling pathway or synergizes IL-3- or IL-6-initiatedsignals for induction of telomerase. It is unclear which of these twoscenarios is correct in view of reports that SCF and IL-3 can ini-tiate convergent or divergent signaling pathways (8, 62–64) andthat SCF, IL-3, and IL-6 activate PI 3-kinase, p38 MAP kinase,and other common signaling pathways in various types of cells (8,9, 11, 65). The question of whether the cytokine-mediated signalsregulate the expression of TERT or the activity of pre-existingTERT could not be addressed because we failed to detect TERT byimmunoblotting techniques in the initial isolates of CD341/CD1171/CD131 cells (data not shown). Also, the effect of serumcould not be tested (our experiments were conducted in serum-freemedium) because we found that the presence of serum leads tovery low yields of immature mast cells that could not be appro-priately analyzed. As the present work demonstrated, the tumorcell lines proved to be inappropriate substitutes because of therefractoriness of the telomerase system to stimulants, inhibitors,and serum deprivation (see later).

Previous studies with lymphocytes and hemopoietic progenitorcells provide analogies for the present studies. Both types of cellsexpress low telomerase activity in the quiescent state. It is likelythat induction of telomerase in activated lymphocytes is dependenton intracellular signals, in that wortmannin blocks activation oftelomerase in Ag-stimulated B cells (66) as do inhibitors of proteinkinase C (22) and tyrosine kinases (67) in activated T cells. Inaddition, phorbol esters in combination with calcium ionophorestimulate telomerase activity in T cells (22, 67, 68). Although theseresults suggest that telomerase activity is regulated by intracellularsignals, it is unclear whether these signals are specifically linked toincreased expression of telomerase, enzyme activation, or cell cy-cling events in general (15). With respect to hemopoietic cells,stimulation of these cells with IL-3 and IL-6 induces a transientincrease in telomerase activity in early (i.e., CD341) but not late(i.e., CD342), progenitor cells (69). Optimal stimulation of telom-erase activity in CD341 bone marrow cells requires SCF in com-bination with other cytokines (17), whereas individual cytokineshave weak stimulatory activity (22).

In contrast to CD341 and CD341/CD1171/CD131 cells, thetumor mast cell lines express high telomerase activity regardless oftheir passage through the cell cycle or state of proliferation (Figs.6 and 7). Also, the enzyme appears to be highly stable when pro-liferation is blocked by drugs. Nevertheless, maintenance of hightelomerase in proliferating cells would still require continuous syn-thesis of components of the telomerase system. However, this syn-thesis is not affected by a wide range of inhibitors and appears tobe uncoupled from ongoing signaling events in the tumor cell linesas outlined below.

Kit in RBL-2H3 and HMC-1 cells possesses specific activatingmutations and as a consequence is constitutively associated with PI3-kinase (70) and oncogenic when introduced into normal hemo-poietic cells (71). However, wortmannin, PP1, and other inhibitorsthat would be expected to disrupt Kit-mediated signaling pathwayshad no effect on telomerase activity in tumor cells (this paper). Inaddition to PI 3-kinase, Ras is constitutively activated in RBL-2H3cells (47), but inhibitors of Ras-related events were equally inef-fective in these cells. Even in MC-9 cells, whose proliferation isdependent on IL-3, removal of this cytokine and serum from themedium failed to reduce telomerase activity. Unless telomeraseexpression is continuously stimulated by an unidentified crypticsignal, the plausible alternative is that telomerase expression ismaintained by the absence of an inhibitory activity such as phos-phorylated p53 tumor suppressor protein (19). HMBA was theonly compound found to suppress telomerase activity in the mastcell lines. This compound causes tumor cell lines to progressivelyaccumulate in the G1 phase of the cell cycle and undergo terminaldifferentiation or apoptosis (72). A decline in telomerase activity,which precedes the appearance of differentiated or apoptotic cells,has also been noted (60, 61). The primary mechanism of action ofthis compound is unclear (73), but its actions result in changes inthe activities of various genes whose products are involved in celldivision and differentiation, including down-regulation of c-myctranscriptional activity (reviewed in Ref. 72), which, as noted ear-lier, has been linked to expression of telomerase activity in variouscell systems (20).

In summary, our studies suggest that, at least for the first cellcycle, the induction of telomerase activity in CD341 cells wasdependent, directly or indirectly, on growth factor-initiated signals.Shortly thereafter, the expressed telomerase activity becomes re-fractory to stimulatory signals. In both CD341 and CD341/CD1171/CD131 cells, telomerase activity declines rapidly beforematuration into mast cells. Tumor mast cell lines, in contrast, ex-press high telomerase activity constitutively, and this activity is not

FIGURE 8. Suppression of telomerase activity and cell proliferation intumor cell lines by the tumor-differentiating agent, HMBA. Cultures of thetumor mast cell lines were incubated without (2,f) or with 5 mM HMBA(1, M) for 72 h for determination of telomerase activity and increase in cellnumber (fold increase in number of cells plated on day 0). Data are themean6 SEM values from five (RBL-2H3 cells) or three (HMC-1 cells)separate experiments.

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subject to regulation by stimulatory processes that are thought tobe essential for cell proliferation and mast cell functional re-sponses. We are now attempting to express constitutively activetelomerase in human mast cells or mast cell progenitors with theidea that high telomerase activity might maintain proliferation ofmast cell progenitors (74, 75). However, we note that the presentstudies also suggest that telomerase activity can be disassociatedfrom cell proliferation by the use of inhibitors and that expressionof high telomerase activity by itself may not be sufficient for pro-liferation of these cells.

AcknowledgmentsWe thank Stanislav Datskovskiy for his assistance with the measurement oftelomerase activity in some of these experiments and Martha Kirby for herexpert assistance in the analysis of apoptosis.

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