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*
Laboratory of Immunology, National Heart, Lung, and Blood Institute, and
Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In general, the clonal expansion of hemopoietic cell lines and lymphocytes is associated with transient activation of telomerase, an RNA-dependent DNA polymerase (12). This enzyme maintains the integrity of chromosomes in dividing cells by placing additional telomeric DNA segments (TTAGGG in vertebrates) on the ends of chromosomal DNA and thus minimizing attrition of the terminal telomeres during DNA replication (13). In vertebrates, telomerase consists of a protein catalytic subunit, telomerase reverse transcriptase (TERT), the RNA template (TR), and associated proteins, such as telomerase-associated protein-1. Telomerase activity, which is low in nonreplicating somatic cells and high in germinal cells and tumor cell lines (reviewed in Ref. 14), is transiently activated in stimulated myeloid and lymphoid progenitor cells (reviewed in Refs. 12, 14 , and 15). Thus, stimulation of human bone marrow and blood stem cells (16, 17) or quiescent T and B cells with stimulants that induce cell proliferation (reviewed in Refs. 15 and 18) leads to rapid increase in telomerase activity early in the G1 phase of the cell cycle (18). The increase is short-lived and is thought to facilitate the limited clonal expansion of these cells by delaying telomere shortening.
Little is known about the regulation of telomerase expression or activity (reviewed in Ref. 19), although direct activation of TERT transcription by c-Myc has been reported (20). Telomerase TR subunits are expressed in most tissue cells, whereas expression of TERT is generally restricted to replicating cells through largely undefined mechanisms (19). The increase in telomerase activity in stimulated B cells is associated with increased expression of TERT (21), although a modest increase in TR expression is also apparent in stimulated T cells (22, 23, 24). In addition, telomerase may be regulated directly through its state of phosphorylation (reviewed in Ref. 19). Studies with pharmacologic agents in tumor cell systems suggest that dephosphorylation of TERT by protein phosphatase 2A results in loss of enzyme activity, and its rephosphorylation by protein kinase C (25) or Akt (protein kinase B) restores activity (26).
As an extension of previous work characterizing the growth and maturation of human mast cells from progenitor cells (1), we now show that, as in other hemopoietic cell lines, telomerase activity is transiently induced during proliferation of human peripheral blood CD34+ and CD34+/CD117+/CD13+ progenitor cells. This induction occurs well before the appearance of mature mast cells, but requires the presence of SCF in addition to IL-3 or IL-6. We have identified potential signals that lead to this induction, but we have also found that the same signals do not contribute to the constitutively high telomerase activity normally present in tumor mast cell lines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials were obtained from the following sources: SB 202190, PD 98059, 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 cyclohexamide from Sigma (St. Louis, MO); hexamethylene bisacetamide (HMBA) from Aldrich (Metuchen, NJ); annexin V-PE and 7-amino-actinomycin D from PharMingen (San Diego, CA); telomerase PCR ELISA kit from Roche Molecular Biochemicals (Indianapolis, IN); StemPro-34 SFM from Life Technologies (Gaithersburg, MD); other reagents for culture of human and tumor cell lines from Life Technologies or Biofluids (Rockville, MD); cell lines from American Type Culture Collection (Manassas, VA) or from ongoing cultures of RBL-2H3 and HMC-1 (originally from J. H. Butterfield, Department of Internal Medicine, Mayo Clinic, Rochester, MN) cells in the laboratory; recombinant human (rh) SCF, IL-6, and IL-3 from PeproTech (Rocky Hill, NJ); recombinant mouse IL-3 from R&D Systems (Minneapolis, MN); chimeric Fc-specific anti-4-hydroxy-3-nitrophenylacetyl-IgE from Serotec (Raleigh, NC); and 4-hydroxy-3-nitrophenylacetyl-BSA from Biosearch Technologies (Novoto, CA). Stock solutions of kinase inhibitors were prepared in DMSO and diluted for use with medium to yield a final concentration 0.1% DMSO. Other reagents were dissolved in culture medium.
Immunoselection and FACS of CD34+ and CD34+/CD117+/CD13+ cells
PBMC were obtained by leukapheresis following G-CSF treatment of normal volunteers who had given informed consent. The techniques for purification of pluripotent CD34+ cells by using CD34+ affinity columns and sorting CD34+/CD117+/CD13+ progenitor cells from isolated or cultured CD34+/CD117+ cells by FACS were described previously (1). These cells were stored in liquid nitrogen until use.
Cell culture and time-course studies
CD34+ cells and
CD34+/CD117+/CD13+
cells derived therefrom were plated at a concentration of 2 x 10
4 cells in 25-cm2 flasks in
serum-free medium. This medium consisted of StemPro-34 supplemented
with L-glutamine (2 mM), penicillin (100 U/ml),
streptomycin (100 µg/ml), rhIL-3 (30 ng/ml), rhIL-6 (100 ng/ml), and
rhSCF (100 ng/ml). Cultures were diluted (2-fold) weekly or twice
weekly in the same medium, except that rhIL-3 was omitted after the
first week of culture as described previously (1). This
protocol was altered for determination of growth rates and changes in
telomerase activity. Namely, for these experiments samples were removed
each day or every other day, and the cultures were diluted with fresh
medium to maintain a cell density of 
105
cells/ml. Recombinant 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 cultured as monolayers in suspension-MEM supplemented with
10% heat-inactivated FBS, penicillin (100 U/ml), streptomycin (100
µg/ml), and L-glutamine (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), streptomycin (100 µg/ml), and IL-3 (0.5 ng/ml). HMC-1 cells
(30) were grown in suspension in IMEM supplemented with
10% heat-inactivated FBS, L-glutamine (2 mM), penicillin
(100 U/ml), streptomycin (100 µg/ml), and 
-thioglycerol (1.2
mM).
All cultures were maintained in humidified atmosphere of 95% air and 5% CO2 at 37°C. The cells were incubated as suspensions or monolayers as indicated above. Cell suspensions were sampled directly, or in the case of 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 in cell number was expressed as the fold increase in cell population from the start of culture after correction for dilutions of culture during the course of the experiment. Cell viability was determined by trypan blue exclusion. For the determination of apoptotic cells, cells were washed in ice-cold PBS and stained with annexin-V-PE to detect apoptotic cells and with 7-amino-actinomycin to detect necrotic cells according to the manufacturer’s product protocol (catalog no. 65875X; PharMingen). Samples of stained cells were 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 CD34+ cell-derived cultures of human mast cells and measurement of hexosaminidase release
Cells were collected at various stages of culture by centrifugation (Cytospin; Shandon, Pittsburgh, PA) onto slides and stained with 0.5% acidic toluidine blue (pH 1.0) or with a mixture of toluidine blue and light green as described by Kimura and coworkers (31). Cells were identified as mast cells by the presence of abundant numbers of metachromatically stained granules.
CD34+ cell-derived 8-wk cultures of mature mast
cells were collected as described above and incubated overnight with
chimeric human IgE directed against the Ag,
4-hydroxy-3-nitrophenylacetylated BSA. Cells were then washed three
times, resuspended at a density of 105 cells/ml
in HEPES-buffered medium (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.4 mM
Na2HPO4·7H2O,
5.6 mM glucose, 1.8 mM
CaCl2·2H2O, 1.3 mM
MgSO4·7H2O, and 0.025%
BSA, pH 7.4), and aliquoted into 96-well microtiter plates (90
µl/well). Cells were stimulated for 10 min with the designated
concentration of Ag for measurement of release of the granule marker,
-hexosaminidase, as previously described (32). Data are
expressed as a percentage of the intracellular -hexosaminidase
released into the medium.
Studies with inhibitors of protein kinases, cell cycle, and protein synthesis
CD34+ and CD34+/CD117+/CD13+ cells were plated in 25-cm2 flasks (4 x 104 cells/2 ml), and tumor mast cell lines were plated in 80-cm2 flasks (2 x 106 cells/10 ml) in the medium described above. Inhibitors or vehicle were 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 (3 x 105) were collected by centrifugation (3000 x g, 10 min, 4°C), washed once in ice-cold PBS, and resedimented by centrifugation (3000 x g, 10 min, 4°C). The pelleted cells were resuspended in 200 µl of lysis reagent as supplied in the kit and left on ice for 30 min. Cell lysates were centrifuged (6000 x g, 20 min, 4°C), and the supernatant extracts were stored at -80°C until use. The telomeric repeat amplification protocol was performed with extracts containing 1500 cell-equivalents for each reaction (30-min incubation and 30 cycles). For samples with high telomerase activity, a 15-min incubation and 25 cycles were used. The hybridization and ELISA procedures were performed according to the manufacturer’s protocols (Roche Molecular Biochemicals), and the absorbance of the final colored reaction product was measured in a microtiter plate reader at 450 nm. Each arbitrary unit was defined as absorbance per 1500 cell equivalents.
Phosphorylation of p38 MAP kinase, Atk (protein kinase B), and activating transcription factor-2
Whole cell lysates were subjected to SDS-PAGE (10% acrylamide gels) and immmunoblotted for the phosphorylated activated forms of these proteins with rabbit polyclonal Abs (1/500 and 1/1000 dilutions) against doubly phosphorylated (Thr180/Tyr182)p38 MAP kinase, phosphorylated (Ser473)Akt, and phosphorylated (Thr71)ATF-2. The bands were detected with the Amersham chemiluminescence kit and quantitated by densitometric scanning (Personal Densitometer; Molecular Dynamics, Sunnyvale, CA).
Expression of results
Unless indicated otherwise, data were expressed as the mean and SEM of mean values from three or more separate experiments. For individual experiments, three to six samples or cultures were assayed for each data point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
shows examples of changes in
telomerase activity in cultures of CD34+
pluripotent cells (Fig. 1, A and B) and
CD34+/CD117+/CD13+
progenitor cells (Fig. 1, A and C) that were
maintained under standard culture conditions (i.e., in the presence of
IL-3, IL-6 plus SCF for 7 days, and omission of IL-3 thereafter). In
CD34+ cells telomerase activity increased
markedly within 24 h and continued to increase for several days
reaching a maximum between days 3 and 6 (Fig. 1, A and
B). Telomerase activity then declined rapidly to near basal
levels by day 12 and remained at low or undetectable levels for the
duration of culture (up to 50 days).
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A) or directly from peripheral blood pluripotent cells
on day 0 (as in Fig. 1C). The decline in telomerase activity
was equally rapid whether cells were deprived of IL-3 on day 7 as
described above or were exposed to all three cytokines throughout the
experiment (data not shown). The initial increase in telomerase
activity appeared to precede cell division, which was not apparent
until 48 h or later (Fig. 1B). The maximal expansion of
the cell cultures coincided with the virtual disappearance of
telomerase activity (Fig. 1, B and C). We believe
that rapid loss of telomerase activity was due to loss of enzyme from
all or most cells rather than to dilution of a subpopulation of cells
with high telomerase activity, because purified
CD34+/CD117+/CD13+
cells give rise to almost pure populations (85%) of mast cells
(1). However, this could not be tested directly, as
expansion of colonies from individual
CD13+-sorted cells in culture was too slow to
yield sufficient cells for assay. At best, colonies of 300 cells were
achieved after 3 wk of culture when telomerase activity had long
declined to near basal levels.
After 50 days in culture, cells derived from both
CD34+ and
CD34+/CD117+/CD13+
sorting had acquired the characteristics of normal mast cells. When
sensitized with a chimeric IgE (see Materials and Methods),
they secreted in response to the IgE-specific Ag in a dose-dependent
manner, as determined by measurement of release of the granule marker,
hexosaminidase (Fig. 1D). Histologic examination of cultures
at different stages of growth revealed that the appearance of mature
mast cells occurred after the decline in telomerase activity. At 2 wk
of culture, when telomerase activity had returned to minimal levels
(see Fig. 1), mast cells were undetectable in cultures of
CD34+ cells by light microscopy, but by 4 wk,
partially mature mast cells had begun to appear (Fig. 2). By 8 wk and times thereafter,
cultures were predominantly mature mast cells (
95% viable mast
cells).
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The effects of cytokines on telomerase activity in
CD34+ cells were examined in the initial 2 days
of culture before the apparent increase in cell numbers or, in the
absence of growth factors, apoptosis (data not shown). IL-3, IL-6, or
SCF individually caused minimal stimulation of telomerase activity
compared with that induced by SCF in combination with IL-3 or IL-6
(Fig. 3). The combination of IL-3 and
IL-6 elicited only modest increases in telomerase activity, and in
total the experiments indicated that maximal stimulation of telomerase
activity required stimulation with SCF in combination with IL-3 and
IL-6. As noted earlier, the subsequent decline in telomerase activity
occurred in the presence of all three cytokines.
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A wide variety of kinase inhibitors were tested to identify
potential signals for regulating telomerase activity and cell
proliferation. Inhibitors were added on day 0 or 2 of culture, and the
cultures were examined 2 days thereafter (i.e., day 2 or 4). The
inhibitors included PD98059 (33), SB202190
(34), and wortmannin (35), inhibitors of the
extracellular signal-regulated kinase-MAP kinase pathway, p38 MAP
kinase, and PI 3-kinase, respectively (Fig. 4). Of these, SB202190 and wortmannin
suppressed induction of telomerase activity if added at the start of
culture (Fig. 4A, 
), but not when added at 2 days of
culture (Fig. 4A, 
) when the increase in telomerase was
well underway and before cell proliferation had commenced (Fig. 4B). The initial suppression of telomerase activity by
SB202190 and wortmannin was dependent on concentration of drug (Fig. 4, C and D). The initial induction of telomerase was
also inhibited by another PI 3-kinase inhibitor, LY294002
(36) (data not shown). All drugs suppressed cell
proliferation to varying extents whether added on day 0 or 2 of culture
(see Fig. 4B, ). These and the above results suggested
that the increase in telomerase activity was initially dependent on p38
MAP kinase and possibly PI 3-kinase, but, once elevated, telomerase
activity was no longer affected by the inhibitors, even though cell
proliferation was markedly inhibited.
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) and 72 h (data not shown) of
culture. The presence of an additional retarded band of c-Jun suggested
that JNK was activated as well, although phosphorylated JNK was not
detectable by immunoblotting. The phosphorylation of p38 MAP kinase and
to some extent that of ATF-2 were reduced in SB202190-treated cells as
was the phosphorylation of Akt in wortmannin-treated cells.
Paradoxically, wortmannin enhanced the phosphorylation of p38 MAP
kinase (Fig. 5) (T. R. Hundley, unpublished observations)
consistent with the suggestion that PI 3-kinase may down-regulate this
MAP kinase (40).
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at high concentrations (41, 42), enhanced the expression
of c-Jun (Fig. 5). Therefore, these studies provided no clear
indication of the role of JNK, if any, on telomerase induction. High telomerase activity in tumor mast-cell lines: the effects of inhibitors of the cell cycle and protein synthesis
Additional studies were conducted with cultured tumor mast cell
lines where cell numbers did not limit the scope of experiments. The
cell lines examined included IL-3-independent rat RBL-2H3 cells as well
as IL-3-dependent mouse MC-9 cells and human HMC-1 cells. All three
cell lines had measurable telomerase activities. Although direct
comparisons suggested that MC-9 cells expressed lower telomerase
activity than other cell lines and 4-day cultures of
CD34+ cells (Fig. 6A), it should be noted that
telomerase activity varied with different passages of cells and
experiments (compare Fig. 6 with subsequent figures).
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, B and C, show representative
experiments with 0.3 µM hydroxyurea (43), 50 nM
nocodazole (44), and 0.4 mM indomethacin
(45), which inhibit progression through the cell cycle in
S, M, and early G1 phases, respectively. All
three drugs blocked cell proliferation completely (data not shown)
without affecting telomerase activity. Modest decreases in telomerase
activity were observed occasionally as, for example, with nocodazole in
Fig. 6C. However, additional experiments with different
batches of cells indicated that such decreases were the exception even
when cells were exposed to these drugs for up to 72 h. Others have
noted no change in telomerase activity during the cell cycle of human
tumor cell lines except when agents caused cell toxicity
(46). Blockade of protein synthesis as well as cell
proliferation with cycloheximide also failed to reduce levels of
telomerase activity in RBL-2H3 and MC-9 cells after 24-h (Fig. 6D) or 48-h (data not shown) exposure to the drug.
Apparently, telomerase was degraded slowly, at least in the absence of
protein synthesis. Further studies in mast cell tumor lines: effects of kinase inhibitors
Interestingly, telomerase activity in the tumor cell lines was not
suppressed by SB202190 and wortmannin or indeed by many other agents
that block known signaling pathways in mast cells. Examples of
experiments are shown in Fig. 7. The
effects of wortmannin, PD98059, and SB202190 on telomerase activity and
cell proliferation are shown for MC-9 cells (Fig. 7, A and
B). Similar results were obtained with RBL-2H3 cells (data
not shown). In both types of cells, SB202190 and wortmannin blocked
cell proliferation (i.e., no increase in cell number; Fig. 7B) without markedly reducing telomerase activity (Fig. 7A). Exposure to high concentrations of SB202190 (50 µM
instead of 25 µM as used in Fig. 7) caused significant loss of
telomerase activity (50%) in two of five experiments (data not
shown) and apoptosis (40% of the cell population) within
24 h.
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C), additional agents
tested included dexamethasone, an inhibitor of the MAP kinases in
RBL-2H3 cells (47); Go6796, an inhibitor of the
calcium-dependent isoforms of protein kinase C (48);
Ro31-7549, an inhibitor of calcium-dependent and independent
isoforms of protein kinase C (49); and PD 152440, a
farnesyltransferase inhibitor that suppresses the Ras/MAP kinase
pathway (50). The latter compound was tested because Ras
is present in its constitutively active GTP-bound form in RBL-2H3 cells
(47). None of these agents at high doses caused marked
decreases in telomerase activity in RBL-2H3 (Fig. 7C) or
MC-9 cells (data not shown) after 18 h. Decreases were not
observed after short term (2–6 h) or long term (up to 48 h)
exposure to the inhibitors (data not shown).
Other drugs found to be inactive (data not shown) over the short or
long term (2–48 h) included PP1 (10 µM), which disrupts SCF-mediated
signaling in hemopoietic cells and progenitors by blocking Src kinases
(51); piceatannol (10 µM), which selectively inhibits
Syk, as opposed to Src, kinases in mast cells (52, 53);
and AG490, a JAK2 inhibitor (54). Staurosporine (20 nM),
an inhibitor of cyclin-dependent kinases as well as protein kinase C
(55), blocked cell proliferation but failed to diminish
telomerase activity in RBL-2H3 and MC-9 cells. In addition, telomerase
was not decreased by depriving MC-9 cells of serum or IL-3, which also
led to arrest of cell proliferation (data not shown). Okadaic acid (300
nM), a protein phosphatase inhibitor (56), induced a
modest increase (35%) in telomerase activity in MC-9 cells within
2 h in one of three experiments.
The most notable finding of these studies was that high telomerase
activity was maintained even after prolonged arrest of cell
proliferation. This was most dramatically illustrated in studies with
RBL-2H3 cells, where telomerase activity remained unchanged (Fig. 7D) even after complete cessation of growth for 96 h in
the presence of quercetin (Fig. 7E). This compound inhibits
various protein kinases (57) and cell proliferation
(58).
Suppression of telomerase activity in tumor cell lines with HMBA
HMBA was the only compound of many tested that caused a
reproducible and substantial reduction of telomerase activity (Fig. 8A) along with cessation of
cell proliferation (Fig. 8B). The reduction in telomerase
activity was of slow onset and reached a maximum between 24- and 48-h
exposure to HMBA (data not shown). This compound was tested because of
its ability to induce terminal differentiation (59) and
loss of telomerase activity (60, 61) in tumor cell
lines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Analogous to mitogenically
stimulated lymphocytes and hemopoietic stem cells (12, 14, 15), the early induction of telomerase may facilitate clonal
expansion of
CD34+/CD117+/CD13+
cells before maturation into mast cells. This induction is dependent on
provision of SCF in combination with IL-3 and IL-6 (Fig. 3) and, in
turn, on intracellular signals mediated by p38 MAP kinase and PI
3-kinase. Inhibitors of these two enzymes suppress the initial increase
in telomerase activity, but they appear to have little effect once
telomerase activity is fully elevated. Both enzymes appear to be
activated early by growth factors in CD34+ cells
(Fig. 5) and remain activated for at least 3 days (data not shown).
The studies with inhibitors also suggested that induction
of telomerase was not dependent on extracellular signal-regulated
kinase, whereas cell proliferation was dependent to some extent on this
enzyme as well as on p38 MAP kinase and PI 3-kinase (Fig. 4B). Although these results do not conclusively establish
that cell proliferation and induction of telomerase require
different sets of signals, the suppression of cell proliferation by
these inhibitors suggest that telomerase activity is maintained
regardless of the proliferative activity of the cells (i.e., data for
day 4 in Fig. 4, A and B).
As noted above, costimulation of cells with SCF and either IL-3 or IL-6
is required for optimal induction of telomerase activity (Fig. 3). None
of these cytokines individually nor the combination of IL-3 and IL-6
appears to promote the necessary mix or strength of signals for this
induction. The overall pattern of responses shown in Fig. 3 suggests
that SCF either stimulates a unique and essential signaling pathway or
synergizes IL-3- or IL-6-initiated signals for induction of telomerase.
It is unclear which of these two scenarios is correct in view of
reports that SCF and IL-3 can initiate convergent or divergent
signaling pathways (8, 62, 63, 64) and that 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 signals regulate the
expression of TERT or the activity of pre-existing TERT could not be
addressed because we failed to detect TERT by immunoblotting techniques
in the initial isolates of
CD34+/CD117+/CD13+
cells (data not shown). Also, the effect of serum could not be tested
(our experiments were conducted in serum-free medium) because we found
that the presence of serum leads to very low yields of immature mast
cells that could not be appropriately analyzed. As the present work
demonstrated, the tumor cell lines proved to be inappropriate
substitutes because of the refractoriness of the telomerase system to
stimulants, inhibitors, and serum deprivation (see later).
Previous studies with lymphocytes and hemopoietic progenitor cells provide analogies for the present studies. Both types of cells express low telomerase activity in the quiescent state. It is likely that induction of telomerase in activated lymphocytes is dependent on intracellular signals, in that wortmannin blocks activation of telomerase in Ag-stimulated B cells (66) as do inhibitors of protein kinase C (22) and tyrosine kinases (67) in activated T cells. In addition, phorbol esters in combination with calcium ionophore stimulate telomerase activity in T cells (22, 67, 68). Although these results suggest that telomerase activity is regulated by intracellular signals, it is unclear whether these signals are specifically linked to increased expression of telomerase, enzyme activation, or cell cycling events in general (15). With respect to hemopoietic cells, stimulation of these cells with IL-3 and IL-6 induces a transient increase in telomerase activity in early (i.e., CD34+) but not late (i.e., CD34-), progenitor cells (69). Optimal stimulation of telomerase activity in CD34+ bone marrow cells requires SCF in combination with other cytokines (17), whereas individual cytokines have weak stimulatory activity (22).
In contrast to CD34+ and
CD34+/CD117+/CD13+
cells, the tumor mast cell lines express high telomerase activity
regardless of their passage through the cell cycle or state of
proliferation (Figs. 6 and 7). Also, the enzyme appears to be highly
stable when proliferation is blocked by drugs. Nevertheless,
maintenance of high telomerase in proliferating cells would still
require continuous synthesis of components of the telomerase system.
However, this synthesis is not affected by a wide range of inhibitors
and appears to be uncoupled from ongoing signaling events in the tumor
cell lines as outlined below.
Kit in RBL-2H3 and HMC-1 cells possesses specific activating mutations and as a consequence is constitutively associated with PI 3-kinase (70) and oncogenic when introduced into normal hemopoietic cells (71). However, wortmannin, PP1, and other inhibitors that would be expected to disrupt Kit-mediated signaling pathways had no effect on telomerase activity in tumor cells (this paper). In addition to PI 3-kinase, Ras is constitutively activated in RBL-2H3 cells (47), but inhibitors of Ras-related events were equally ineffective in these cells. Even in MC-9 cells, whose proliferation is dependent on IL-3, removal of this cytokine and serum from the medium failed to reduce telomerase activity. Unless telomerase expression is continuously stimulated by an unidentified cryptic signal, the plausible alternative is that telomerase expression is maintained by the absence of an inhibitory activity such as phosphorylated p53 tumor suppressor protein (19). HMBA was the only compound found to suppress telomerase activity in the mast cell lines. This compound causes tumor cell lines to progressively accumulate in the G1 phase of the cell cycle and undergo terminal differentiation 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 of this compound is unclear (73), but its actions result in changes in the activities of various genes whose products are involved in cell division and differentiation, including down-regulation of c-myc transcriptional activity (reviewed in Ref. 72), which, as noted earlier, has been linked to expression of telomerase activity in various cell systems (20).
In summary, our studies suggest that, at least for the first cell cycle, the induction of telomerase activity in CD34+ cells was dependent, directly or indirectly, on growth factor-initiated signals. Shortly thereafter, the expressed telomerase activity becomes refractory to stimulatory signals. In both CD34+ and CD34+/CD117+/CD13+ cells, telomerase activity declines rapidly before maturation into mast cells. Tumor mast cell lines, in contrast, express high telomerase activity constitutively, and this activity is not subject to regulation by stimulatory processes that are thought to be essential for cell proliferation and mast cell functional responses. We are now attempting to express constitutively active telomerase in human mast cells or mast cell progenitors with the idea that high telomerase activity might maintain proliferation of mast cell progenitors (74, 75). However, we note that the present studies also suggest that telomerase activity can be disassociated from cell proliferation by the use of inhibitors and that expression of high telomerase activity by itself may not be sufficient for proliferation of these cells.
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Acknowledgments |
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Footnotes |
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2 Current address: Departamento de Biologia Animal, Universidade Federal de Viçosa, 36571-000 Viçosa 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, Building 10, Room 8N109, National Institutes of Health, Bethesda, MD 20892-1760. E-mail address: beaven{at}helix.nih.gov
5 Abbreviations used in this paper: SCF, stem cell factor; PI 3-kinase, phosphatidylinositol 3'-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.
Received for publication January 4, 2001. Accepted for publication March 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor I and stem cell factor receptor is differentially regulated by phosphatidylinositol 3-kinase and calcineurin in mouse bone marrow-derived mast cells. J. Immunol. 162:2087. in human breast cancer cells. J. Biol. Chem. 273:33436.-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.This article has been cited by other articles:
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  A. Ujike-Asai, A. Okada, Y. Du, M. Maruyama, X. Yuan, F. Ishikawa, Y. Motoo, K. Isobe, and H. Nakajima Large defects of type I allergic response in telomerase reverse transcriptase knockout mice J. Leukoc. Biol., August 1, 2007; 82(2): 429 - 435. [Abstract] [Full Text] [PDF]
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 N. M. Kushnir-Sukhov, A. M. Gilfillan, J. W. Coleman, J. M. Brown, S. Bruening, M. Toth, and D. D. Metcalfe 5-Hydroxytryptamine Induces Mast Cell Adhesion and Migration J. Immunol., November 1, 2006; 177(9): 6422 - 6432. [Abstract] [Full Text] [PDF]
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 S. Iwaki, C. Tkaczyk, A. B. Satterthwaite, K. Halcomb, M. A. Beaven, D. D. Metcalfe, and A. M. Gilfillan Btk Plays a Crucial Role in the Amplification of Fc{epsilon}RI-mediated Mast Cell Activation by Kit J. Biol. Chem., December 2, 2005; 280(48): 40261 - 40270. [Abstract] [Full Text] [PDF]
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 T. R. Hundley, A. M. Gilfillan, C. Tkaczyk, M. V. Andrade, D. D. Metcalfe, and M. A. Beaven Kit and Fc{epsilon}RI mediate unique and convergent signals for release of inflammatory mediators from human mast cells Blood, October 15, 2004; 104(8): 2410 - 2417. [Abstract] [Full Text] [PDF]
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C. Tkaczyk, V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D.-H. Nahm, D. D. Metcalfe, and A. M. Gilfillan NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc{epsilon}RI aggregation Blood, July 1, 2004; 104(1): 207 - 214. [Abstract] [Full Text] [PDF]
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C. Tkaczyk, M. A. Beaven, S. M. Brachman, D. D. Metcalfe, and A. M. Gilfillan The Phospholipase C{gamma}1-dependent Pathway of Fc{epsilon}RI-mediated Mast Cell Activation Is Regulated Independently of Phosphatidylinositol 3-Kinase J. Biol. Chem., November 28, 2003; 278(48): 48474 - 48484. [Abstract] [Full Text] [PDF]
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L. Ping, A. Asai, A. Okada, K. Isobe, and H. Nakajima Dramatic increase of telomerase activity during dendritic cell differentiation and maturation J. Leukoc. Biol., August 1, 2003; 74(2): 270 - 276. [Abstract] [Full Text] [PDF]
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N.-p. Weng Interplay between telomere length and telomerase in human leukocyte differentiation and aging J. Leukoc. Biol., December 1, 2001; 70(6): 861 - 867. [Abstract] [Full Text] [PDF]
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