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Clinical Investigations |
James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center [M. E., K. M., P. D., M. A. S. M.]; and New York Presbyterian Hospital-Weill Medical College of Cornell University, [R. T. S.] New York, New York 10021
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and PV patients were treated with IFN-.
The MDS patients were treated with folinic acid, retinoid acid, or
supportive care only, and in RAEB and AML, patients were treated with
induction chemotherapy treatment. For direct comparison, both BM and PB
specimens were collected from eight patients. Patients with follow-up
samples collected for TRAP and TRF analysis were also subjected
to a clinical remission inquiry performing standard remission analyses
(BM morphology, histology, FACS, cytogenetics, etc.). Specimens were
also collected from 108 normal healthy donors [PB
(n = 78), CB (n = 21), and BM (n = 9)] for comparative
analysis. The study was performed in accordance with local
institution-approved regulations. Specimens from patients and donors
were collected and analyzed between April 1995 and April 1997.
Cell Source and Separation of CD34+ Cells and
Subsets.
BM and PB specimens from healthy donors and patients were obtained
after gaining informed written consent. CB, which was otherwise to be
discarded, was exempt from the consent process according to the policy
of the Institutional Review Board of Research Associates of the
New York University Medical Center. Heparinized MNC from healthy donors
and patients were separated by Ficoll-Paque (Pharmacia, Uppsala,
Sweden), CD34+ cells were selected with
immunomagnetic beads (Dynal, Oslo, Norway) (13)
and used
for suspension culture assay, progenitor assay (colony assay), TRAP,
and TRF analysis.
Suspension Culture Assay (Delta Culture).
CD34+ cells were cultured at 4 x 104 cells per ml in Iscove’s modified
Dulbecco’s medium plus 20% FCS supplemented with gentamicin and
monothioglycerol in the presence of a five-factor cytokine cocktail of
kit ligand (20 ng/ml), interleukin-3 (50 ng/ml), interleukin-6 (100
units/ml), erythropoietin (6 units/ml), and granulocyte
colony-stimulating factor (1000 units/ml; K36EG), as previously
reported (13)
. The CD34+ cell
recovery after 1 week of culture was similar for patient and healthy
donor specimens with a median of 2.21%. However, the highest
CD34+ cell yield (5.6%) was obtained from AML
samples; next were CML, PV, and CLL specimens with 1.7, 1.6 and 0.9%,
respectively. Cell viability was assessed using the trypan blue
exclusion assay. After 7 days of culture, cells were recovered, plated
at 2 x 103 cells/milliliter in
agarose for CFU assay, and repassaged at the starting concentration of
4 x 104 cells/milliliter. Weekly
passages and colony assays were performed for 3–4 weeks until no
further cellular expansion was observed.
Colony Assay.
CD34+ cells (1 x 103/ml) and ex vivo expanded
CD34+ cells (2 x 103–4 x 104/ml) were cultured in triplicate in Iscove’s
modified Dulbecco’s medium containing 0.36% agarose (FMC Bioproducts,
Rockland, ME), 20% FCS, and cytokines (K36EG) (13)
. After
14 days of incubation, CFU-granulocyte-macrophage was scored using an
inverted microscope.
TRAP Assay.
The assay which incorporates an internal PCR control (designated
telomerase substrate-nontelomerase) was performed as described
previously (5
, 26, 27, 28)
. In brief, two µg of protein
extract were assayed in reaction tubes containing 50 µl of the TRAP
reaction mixture. For each assay, a negative control and 0.1 amol of
the quantification standard olinucleotide R8 were used. The amount of
TA for each reaction was calculated as published (9
, 10
, 25, 26, 27, 28)
, expressing the final quantitation as TPG. One unit of
TPG was defined as 0.001 amol (or 600 molecules) of telomerase
substrate primers extended by telomerase present in the extract
with at least three telomeric repeats. The assay was in the linear
range from 0.001 amol (1 TPG) to 1 amol (1000 TPG) of R8. This range
extended over three logs of our target protein concentration (data not
shown). All results were determined from at least three to six
independent TRAP assays.
TRF Assay.
DNA isolation and TRF analyses were performed as described (3
, 4 , 13
, 25)
. In brief, genomic DNA was digested with MSP I and RSA
I (Boehringer Mannheim, Indianapolis, IN); electrophoresis was
performed in 0.5% agarose gels; and gels were depurinated, denatured,
neutralized, transferred to a nylon membrane using 20x SSC, dried for
1 h, and hybridized with a 5'-end labeled telomeric probe
(TTAGGG)3 (Genset, La Jolla, CA). Telomeric
smears were visualized by exposing the membranes to imaging plates with
mean and peak TRF lengths analyzed as reported (13
, 25
, 29 , 30)
.
Statistics.
Comparisons among groups were made with standard statistical tests.
Results are expressed as median values except when stated otherwise.
Statistical significance of the data obtained was analyzed by the
Wilcoxon rank sum test and the Student t test.
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RESULTS |
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Table 1B
summarizes the clinical patient characteristics. The
numbers of male and female patients were comparable in groups as well
as in subgroups of patients. PB and BM specimens were collected at
diagnosis and during the course of the disease (Table 1B)
. A
total of 54 patients were analyzed at diagnosis, 47 during the course
of their disease, and 19 before and after ex vivo culture.
Patients from which specimens were obtained at diagnosis had received
no prior therapy, whereas those samples from patients obtained during
the course of their disease had received 
6 chemotherapy cycles in 34
cases and >6 chemotherapy cycles in 13 cases.
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summarizes the results obtained from healthy donors
(n = 108). Median TA was higher in
CD34+ cells than in MNC and higher in PB and BM
compared with CB cells (Table 2)
. TA in MNC and
CD34+ cells using PB and BM was low and decreased
in intensity with age (TAMNC = 4.21 - 0.0482 x A; and
TACD34+ = 8.2 - 0.065 x A; where TA is telomerase activity in TPG and A
is age in years), as previously reported (25)
. Because
Southern blot TRF analysis is resolved as a characteristic smear of
cellular DNA due to interchromosomal and intercellular heterogeneity,
both mean and peak TRF values were analyzed. Mean and peak TRFs in
CD34+ cells were similar with 7.8 and 8.1 kbp,
respectively (Table 2)
. Telomere lengths of CB
CD34+ cells were longer than from PB and BM, in
accordance with previous studies (2
, 13)
. Telomere length
in MNC cells declined with age (TL = 10.052 - 0.054 x A [r = -0.6; p < 0.05], where TL is the TRF in
kbp and A is age in years) (Fig. 1A)
. CD34+ cells were a median of 0.2
kbp longer than MNC with 8.7 versus 8.5 kbp TRFs,
respectively (n = 11; n = 6 CB, n = 5 PB); however, no
significant telomere length difference in CD34+
cells and MNC was observed (Fig. 1B)
. In ex vivo
culture, CB (n = 12), PB
(n = 20), and BM (n = 7) CD34+ cells were found to lose 1–2 kbp
over a 4-week period, with a comparable TRF loss in all three cell
sources (Table 2)
.
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summarizes TA and telomere length in leukemia and MPD patients as well
as in healthy donors. Increased TA and short telomeres were defined as
such when TA in patients was higher than normal [>(mean - 2 x SD)] and when their TRF lengths were
shorter than normal relative to their age-matched healthy donors
[i.e., <(mean - 2 x SD)].
Median TA in all patients was increased 7.7-fold, and median telomeres
were 1.2 kbp shorter compared with age-matched healthy donors
(p < 0.0001). Median TA in
CD34+ cells from PV and CLL was similar to normal
CD34+ cells, moderately increased in CML and MDS
(p < 0.05), and increased 18-fold in AML
specimens (Fig. 2A)
. Telomere length, particularly in AML
and CML, was significantly shorter (p < 0.01), decreased to a lesser extend in PV and MDS
(p < 0.05), and was not significantly
different for CLL patients who had similar telomere lengths as
age-matched healthy donors (Fig. 2B)
. Again, we found no
significant telomere length difference in CD34+
and MNC from CML patients; however, telomeres of
CD34+ cells were a median of 0.4 kbp longer than
those of MNC with 4.8 versus 4.4 kbp, respectively
(n = 8) (Fig. 2C)
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. In serial samples from AML patients taken at diagnosis
and after courses of induction chemotherapy (n = 4), telomerase decreased 14.8-fold after chemotherapy,
correlating with the reduction of leukemic cells and the attainment of
normal cells in BM and PB. In serial specimens of four MDS patients, TA
increased with disease progression. Particularly in one patient who
progressed from RA to RAEB, TA increased 11-fold, and in progression
from RAEB to overt AML, TA increased 18-fold (Fig. 3B)
. No significant difference in telomere lengths could be
demonstrated in patient specimens at diagnosis and follow-up, with 5.7
(range: 3.2–7.7) versus 5.6 kbp (range: 3.4–8.2),
respectively (p = 0.833). A trend toward
telomere shortening was observed in subgroups of patients with PV, CLL,
and CML, in which telomeres tended to shorten as the disease continued,
with 5.9 versus 5.7, 6.6 versus 6.1, and 5.1
versus 4.7 kbp, respectively. In contrast, longer telomeres
were found in AML and MDS patients after induction chemotherapy (6.6
and 6.1 kbp) compared with those found in diagnostic specimens
(5 and 5.9 kbp). Most likely this was due to the loss of the leukemic
clone (with shorter telomeres) and the emergence of normal
hematopoietic cells (with longer telomeres) after induction therapy
(Fig. 3C)
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TA and Telomere Length in Patients with Stable and Accelerated
Disease Course.
Patients with stable versus accelerated disease where
defined as a group with CP versus AP and BP disease and were
defined in MDS/AML as a patient group with RA/RAS (RA with
sideroblasts) versus RAEB/AML disease. In CLL, patients with
stage RAI I–III disease and patients with transformed
aggressive lymphocytic lymphoma (Richter’s syndrome) were
subdivided. TA was significantly lower in all patients with
stable compared with accelerated disease, with 2.8 TPG (range: 0–14.3)
compared with 21 TPG (range: 2–41.5), respectively
(p < 0.0001). This was also true for
subgroups of CLL, CML, and MDS/AML, with 1.9 versus 31.5,
2.3 versus 5, and 4.95 versus 15 TPG,
respectively. Telomeres in patients with stable disease were 5.9 kbp
(range: 4.2–8.6) compared with 5 kbp (range: 3.2–7.6) in patients
with progressive disease (p = 0.091), 6.5
versus 6.4 kbp in CLL, 5.6 versus 4.6 kbp in CML,
and 5.9 versus 5.1 kbp in AML/MDS.
TA and Telomeres in PB Compared with BM AML and MDS Specimens.
BM specimens obtained from AML and MDS patients at diagnosis had
slightly elevated TA (1.6-fold higher) and shorter telomeres (4.9 kbp)
compared with PB specimens (5.2 kbp) from the same patient. TA was
identical in BM and PB specimens from patients in complete remission,
displaying a low or undetectable TA with a median of 1.4 TPG. Telomeres
in remission patients (with a median age of 46 years) were 6.94 kbp in
BM and 6.9 in PB, which was 0.6 kbp shorter compared with age-matched
healthy donor controls.
TA, Telomere Dynamics, and Cell Expansion under ex
Vivo Culture Conditions.
Like TA kinetics observed in healthy donors (Fig. 4A)
, telomerase was up-regulated within 72 h of ex
vivo culture of CD34+ cells from CP-CML, PV,
and CLL patients (Fig. 4B)
. TA in these cultured cells
peaked after 1 week and decreased below detection after 2 weeks of
culture (Fig. 4A)
. In contrast, in AP/BP-CML and AML
patients, telomerase in CD34+ cells was
down-regulated after 1 week of expansion and decreased further
thereafter (Figs. 4, A and B
, and 5A)
.
Telomerase up-regulation in CP-CML, PV, and CLL patients was lower than
that observed in normal hematopoietic cells, whereas in AP/BP-CML and
AML, telomerase was elevated at baseline and highly exceeded levels
found in healthy donors (Fig. 5A)
. The expansion potential of CD34+
cells from patient samples was considerably decreased compared with
normal hematopoiesis (Fig. 5B)
. The cell expansion in
healthy donors in weeks 1 to 4 was 36-, 37-, 3.5-, and 1.2-fold,
respectively, whereas in patients, the expansion potential was
significantly reduced with 6.5-, 2.3-, 0.6-, and 0.2-fold,
respectively. The expansion potential was also reduced in each patient
subpopulation, as shown in Fig. 5B
. The cell
expansion potential of CML samples was higher compared with patients
with AML, PV, or CLL (Fig. 5B)
. Lower amounts of TA in weeks
2 to 4 of culture were associated with telomere erosion during ex
vivo culture. During 3 to 4 weeks of culture, a median telomeric
loss of 0.7 kbp was detected in patient samples compared with 1.1 kbp
in healthy donors (Fig. 5C)
. Higher TA in AML and AP/BP-CML
specimens correlated with a reduced telomere loss compared with lower
TA and a greater TRF loss in CLL and PV patients
(r = -0.61) (Fig. 5D)
.
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. As a result, total colony numbers generated after 3 to 4
weeks of culture were significantly lower in AML and AP/BP-CML compared
with those of CLL and PV patients (Fig. 6B)
. Compared with
healthy donors, the total cell and progenitor expansion were 1- to
5-log decreased in MPD, PV, and leukemia patients (CML, CLL, and AML)
(Fig. 6, C–F
). In normal donors, total cells
(starting with a seeding concentration of 4 x 104 cells/ml) were highly increased in weeks 1 to
4, reaching the maximum cell expansion in weeks 1 and 2 compared with
significantly lower numbers in leukemia (Fig. 6C)
. Total
colony numbers (Fig. 6D)
, fold cell increase (Fig. 6E)
, and the fold progenitor expansion (Fig. 6F)
were significantly diminished in leukemia patients.
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DISCUSSION |
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Telomeres had progressively shortened, particularly in AML and CML, to a lesser extend in PV and MDS, and least in CLL CD34+ cells. Short telomeres in leukemia and MPD support the concept that there is progressive telomere erosion in patients with ongoing or active disease and that telomerase was turned on after extensive proliferation (25 , 31) . Assuming a bp loss of 50/cell division, average population doublings in CLL CD34+ cells (used as controls for normal stem cells) were low but increased 20-fold in PV and MDS and 45- to 50-fold in CML and AML. Telomere reduction was previously demonstrated in acute and chronic leukemia (22 , 25 , 31) , in MDS (33 , 34) , and in others such as aplastic anemia (35) , IDDM (insulin-dependent diabetes mellitus) (36) , and scleroderma (37) . Telomere shortening in disease states has been linked to chromosome abnormalities and disease progression, leading to increased proliferation, cell apoptosis, and/or immune destruction (12 , 22, 23, 24, 25 , 30 , 33 , 34) . Comparison of diagnostic and follow-up samples from leukemic patients revealed significantly increased TA in diagnostic specimens compared with specimens obtained after treatment initiation. This was true for all subgroups of patients except for MDS, most likely because only one MDS patient progressed to AML, whereas the others had a stable disease course. In AML patients, TA decreased after induction chemotherapy, which correlated with the disappearance of leukemic cells and with the attainment of remission and, conversely, whereby a substantial telomerase increase was observed with MDS progression to RAEB and AML. In general, we observed that TA was significantly lower and that telomeres were longer in patients with stable compared with accelerated disease. Telomeres in PV, CLL, and CML showed a trend of shortening from diagnosis to follow-up, whereas in AML and MDS patients, longer telomeres were found after induction chemotherapy, most likely due to the loss of the leukemic clone (with shorter telomeres) and the emergence of normal hematopoietic cells (with longer telomeres). Patients who received moderate doses of chemotherapy (<6 cycles) had significantly longer telomeres compared with those of patients exposed to >6 chemotherapy cycles, accounting for a telomere length difference of 1.4 kbp (or 28 years of premature aging). Telomeres in patients in complete remission were a median of 0.6 kbp shorter compared with age-matched healthy donors, accounting for 12 years of premature aging. Telomere shortening in hematopoietic cells has been observed after standard chemotherapy (25) , after intensified treatment protocols such as high-dose chemotherapy in autologous (38) and allogeneic transplantation (39 , 40) , in cells treated with cisplatin (41) , and after irradiation (42) . Telomere reduction associated with transplantation has been demonstrated to correlate inversely with the number of nucleated cells infused (40) , suggesting that the proliferative pressure is less intense with large progenitor cell support. Telomere erosion after high-dose chemotherapy protocols seems likely to occur as a consequence of strong proliferative stress, possibly accounting for hematopoietic abnormalities such as reduced hematopoietic and stromal cell compartments, impaired CFU and long-term culture-initiating cell (LTC-IC) capacity, poor response of progenitors to growth factors in vivo and ex vivo, and the high incidence of MDS that has been observed after autologous and allogeneic transplantation (43 , 44) .
We and others have previously demonstrated TA in normal hematopoietic cells and that TA further increases in ex vivo culture which thereby prevents progressive telomere shortening despite high cell turnover (12 , 13) . We found that the kinetics of TA in CD34+ cells from CP-CML, PV, and CLL patients were similar to normal hematopoiesis, whereas high TA in AP/BP-CML and AML specimens was down-regulated. The amount of telomerase correlated with the cell expansion potential, in which elevated TA was associated with greater proliferative potential, whereas a decline in proliferation and increased cell apoptosis was observed with subsequent telomere erosion in weeks 2–4 of expansion when TA was low. C. Eaves and M. Dexter have convincingly demonstrated that leukemic cell lines or acute and chronic leukemia samples can be grown in suspension culture, colony-forming assay, LTC-IC assay, or severe combined immune-deficient (SCID) mice. In these systems, growth and maturation into specific lineages is observed and can be modulated by use of exogenous growth factors and differentiating agents (19, 20, 21) . Ex vivo culture of leukemic cells, as documented in AML and CML patients, previously allowed the identification and isolation of primitive normal stem cells, partial reconstitution of normal hematopoiesis, and loss of the malignant cell clone (19, 20, 21 , 45 , 46) . Telomerase down-regulation in our culture system seemed to demonstrate the phenomenon whereby, in chronic and acute leukemia, malignant and normal hematopoiesis coexist, and in many cases primitive hematopoietic cells proliferate normally, whereas the malignant clones die out ex vivo, presumably via terminal differentiation or apoptotic cell death. Leukemic cells may exhibit aberrant differentiation, altered susceptibility to apoptosis, and modified enzyme activity, as observed for telomerase. Apoptotic death and/or terminal differentiation of leukemic cells can be induced, with appropriate cytokines and/or use of retinoic acid in the case of promyelocytic leukemia (11 , 17 , 47) . During culture we observed that telomere loss inversely correlated with levels of telomerase: high TA in AML prevented excessive telomere shortening, was too low in chronic leukemia and MDS to completely prevent telomere loss, and was comparable with normal hematopoiesis in CLL CD34+ cells. Why the expansion potential of CLL progenitors was reduced even though TA and telomere loss were comparable with normal hematopoiesis seems only insufficiently explained by their advanced age and pretreatment rather than by the impaired biological features responsible for the perturbed ex vivo culture kinetics. Nevertheless, loss of TA and progressive telomere erosion in acute and chronic leukemia upon expansion may have demonstrated (a) the loss of the malignant phenotype (in favor of normal hematopoietic progenitors), (b) induction of terminal differentiation of leukemic cells, and/or (c) senescence and apoptosis of leukemic cells with critically short telomeres after further shortening during suspension culture. That telomere loss plays a crucial role for replicate cell senescence has been shown in telomerase knock-out mice, in which telomere loss led to depletion of male germ cells, diminished hematopoietic colony formation, impaired mitogen-induced proliferation of primary splenocytes, an increase in apoptosis, and (with critically short telomeres) cessation of proliferation (48 , 49) .
On the basis of these investigations, we conclude that TA (a) is up-regulated in freshly isolated acute and chronic leukemia cells, (b) correlates with acute versus chronic forms of leukemic disease and disease progression and correlates inversely with response to therapy, and (c) is down-regulated in acute leukemia during expansion, the latter phenomenon due to either partial differentiation of leukemic cells or leukemic cell apoptosis in culture. Telomere shortening in culture most likely occurs because telomerase levels are insufficient, particularly in chronic leukemic or MDS progenitors, to protect against telomere bp loss upon proliferation. Alternatively, telomere shortening may reflect the emergence of nonleukemic populations with low TA which is unable to prevent telomere shortening upon extensive proliferation (13) . Although telomerase may not be the only mechanism for maintaining chromosome ends and alternative lengthening of telomeres also functions to elongate telomeres (50) , TA seems vital for cell survival and organ homeostasis in most proliferating cells through maintenance of telomere structures during cell division. Novel telomerase inhibitors, as recently shown using a genetic approach (51) , should be tested in hematopoietic culture systems with use of normal and malignant hematopoietic cells because telomerase dynamics, cell proliferation, and telomere length changes can be determined.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant U9 CA 67842-01, the Gar
Reichman Fund of the Cancer Research Institute (New York), the Byrne
Fund (New York), and the Martin Himmel Fund (New York) (all to
M.A.S.M.). Support was also provided by the United Leukemia Fund,
Cancer Resaerch and Treatment Fund (New York) (to R.T.S.), and Grant
95/319/1-1 from Deutsche Forschungsgemeinschaft (Bonn) (to M.E.). 
2 To whom requests for reprints should be
addressed, at University of Freiburg, Department of
Hematology/Oncology, Hugstetterstr. 55, 79106 Freiburg, Germany. Phone:
49 0761 270 3406; Fax: 49 0761 270 3206; E-mail: engelhardtm{at}mm11.ukl.uni-freiburg.de
3 The abbreviations used are: TA, telomerase
activity; TRF, telomere restriction fragments; BM, bone marrow; PB,
peripheral blood; MPD, myelo-proliferative disease; TRAP, telomeric
repeat amplification protocol; kbp, kilobase pairs; bp, base pairs;
AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CLL,
chronic lymphocytic leukemia; PV, polycythemia vera; MDS,
myelodysplastic syndromes; CP, chronic phase; AP, accelerated phase;
BP, blastic phase; RA, refractory anemia; RAEB, RA with excess of
blasts; CB, cord blood; MNC, mononuclear cells; TPG, total product
generated; CFU, colony-forming unit.
Received 6/ 4/99. Accepted 12/ 2/99.
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