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HEMATOPOIESIS
From the Terry Fox Laboratory, British Columbia Cancer
Agency, and the Department of Medicine, University of British Columbia,
Vancouver, BC, Canada; the Abteilung Hämatologie, Onkologie und
Immunologie, Medizinische Universitätsklinik,
Tübingen, Germany; and the Hematology Branch, National Heart,
Lung and Blood Institute, National Institutes of Health, Bethesda, MD.
In most human cells, the average length of telomere repeats
at the ends of chromosomes provides indirect information about their
mitotic history. To study the turnover of stem cells in patients with
bone marrow failure syndromes, the telomere length in peripheral blood
granulocytes and lymphocytes from patients with aplastic anemia (AA,
n = 56) and hemolytic paroxysmal nocturnal hemoglobinuria (n = 6)
was analyzed relative to age-matched controls by means of fluorescence
in situ hybridization and flow cytometry. The telomere lengths
in granulocytes from patients with AA were found to be significantly
shorter than those in age-adjusted controls (P = .001).
However, surprisingly, telomere length in granulocytes from AA patients
who had recovered after immunosuppressive therapy did not differ
significantly from controls, whereas untreated patients and
nonresponders with persistent severe pancytopenia showed marked and
significant telomere shortening. These results support extensive
proliferation of hematopoietic stem cells in subgroups of AA patients.
Because normal individuals show significant variation in telomere
length, individual measurements in blood cells from AA patients may be
of limited value. Whether sequential telomere length measurements can
be used as a prognostic tool in this group of disorders remains to be clarified.
(Blood. 2001;97:895-900) Most acquired aplastic anemia (AA) is thought to
result from immune-mediated damage to the stem and progenitor cell
compartment. Pancytopenia, the hallmark of the disease, correlates well
with the decreased numbers of immature hematopoietic cells measured either as CD34+ cells or as colony-forming
cells.1-3 That the stem cell compartment is also affected
by the pathologic process has been inferred from the finding that very
primitive human hematopoietic cells measured in long-term
culture-initiating cell (LTC-IC) assays or cobblestone area-forming
cell assays are also reduced in number.4,5
Although some recovery of LTC-IC numbers may occur in patients
successfully treated with immunosuppression, in most patients a
significant deficit in stem cell numbers persists despite apparent
hematologic recovery4 (also, J.P.M. et al, unpublished
data, 1999). Similar findings have been reported by
others,1,5,6 and a sustained deficit in LTC-ICs has been
observed up to 5 years after bone marrow transplantation for AA
and hematologic malignancies.6-9 Possibly, reconstitution
of hematopoiesis from a relatively small number of stem cells does not
fully restore the size of the stem cell compartment.
Telomeres play an important role in maintaining chromosomal
integrity,10,11 and shortening of telomeres is associated
with genetic instability (for review, see de Lange12).
Several experimental findings suggest that telomere length measurements
can be used as a marker of stem cell turnover in vitro and in vivo. In
most normal human somatic cells, telomere repeats are gradually lost with replication and age13,14 owing to the inability of
conventional DNA polymerase to fully replicate the 3' end of DNA; this
is also known as the "end-replication problem."15,16
Human telomerase is a reverse transcriptase enzyme capable of
counteracting replicative telomere shortening by adding single-stranded
telomeric DNA to the 3' ends of chromosomes (for review, see
Greider17). Telomerase is constitutively expressed in
cells of the germ line and is present at variable levels in normal
hematopoietic progenitor cells, activated T cells, and germinal center
B lymphocytes as well as in the majority of human
cancers.18-20 Studies of telomerase knock-out mice have provided evidence in support of a functional link between replicative life span, genetic instability, and telomere length.21-23
In addition, overexpression of telomerase was reported to result in the
extension of telomeres and immortalization of normal human epithelial
cells and fibroblasts.24,25 Hematopoietic cells from
different stages in ontogeny differ in proliferative
potential,26 and these functional differences correlate
with differences in telomere length.27 Telomeres in
donor-derived hematopoietic cells from transplant recipients show
accelerated telomere shortening following allogeneic bone marrow
transplantation as compared with cells in the donor,28 with the degree of telomere shortening inversely correlated with the
number of infused stem cells.29
On the basis of these previous studies, the measurement of telomere
length seems a reasonable approach to the study of stem cell dynamics
in bone marrow failure syndromes of different etiology. Indeed, in a
recent study, this approach was used to study patients with AA, and a
significant shortening of telomeres in AA compared with
age-matched controls was reported.30 In patients with
persistent cytopenia, a correlation between telomere loss and disease
duration was found, whereas in patients whose blood counts became
normal after therapy, the rate of telomere loss was stabilized. No
differences in age-adjusted telomere length were found between patients
with active AA and patients who responded to immunosupressive
therapy.30 However, that study did not take into account
the telomere length in different leukocyte subpopulations, ie,
lymphocytes and granulocytes.
We recently introduced a novel technique for measurement of the average
telomere length in cells using fluorescence in situ hybridization
(FISH) with peptide nucleic acid (PNA) probes and flow cytometry
(flow-FISH).31 Using this technique, we have analyzed the
telomere length kinetics in subpopulations of unseparated peripheral
blood leukocytes (PBLs) in a large population of healthy donors.32 In this study, it was found that both
granulocytes and naive T lymphocytes show a rapid decline in telomere
length in early childhood and a much more gradual but steady decline thereafter, most likely reflecting the turnover of their common precursors: hematopoietic stem cells.
One of the attractive features of flow-FISH is the ability to measure
the telomere fluorescence in different cell types from the same
patient. With such comparisons, it is possible to compensate to some
extent for the pronounced heritable variation in telomere length observed in comparable cell types from normal individuals of the
same age.32,33 The value of this approach was recently illustrated in studies of normal T cells and malignant myeloid cells
from the blood of patients with chronic myeloid
leukemia.34 Here, we describe our initial studies of
telomere length dynamics in blood leukocyte subsets from patients with AA.
Patient characteristics
Treated AA patients were subdivided into those who improved after
therapy with antithymocyte globulin and cyclosporine (CsA) or
cyclophosphamide and CsA in combination (recAA group, n = 23). All
patients treated with these drugs had initially presented with severe
disease. Hematopoietic recovery was defined as substantial improvement
in at least 2 lineages, decrease in transfusion requirements or
transfusion-independence, and/or elevation of absolute neutrophil counts above 5 × 107/L. We also analyzed sAA patients
who had relapsed or who did not respond to initial therapy, all of whom
continued to fulfill the severity criteria (sAANR group, n = 19). In
one patient with recAA, we did not recover enough cells for analysis.
In another patient with recAA, as well as in 3 patients with sAANR,
gating on granulocytes was not possible because of insufficient numbers of granulocytes and/or failure to discriminate between granulocytes and
lymphocytes by flow cytometry after hybridization.
Results are indicated as the mean ± SEM unless indicated
otherwise. As controls, we used telomere fluorescence values after gating on the granulocyte or lymphocyte population of total PBLs obtained from healthy individuals, as described
previously.32
Sample preparation
Telomere fluorescence in situ hybridization and flow cytometry The average length of telomere repeats at chromosome ends in individual peripheral blood leukocytes was measured by flow-FISH as previously reported.32 Analysis of the PBLs of one patient with sAANR after gating on lymphocytes and granulocytes is shown in Figure 1. Briefly, after gating on diploid cells on the basis of staining with propidium iodide (PI) (R1 as shown in Figure 1A), granulocytes (R2 in Figure 1B) and lymphocytes (R3 in Figure 1B) were discriminated on the basis of size and granularity. Analysis was performed with and without fluorescein isothiocyanate (FITC)-labeled telomere-specific PNA probe (dark and light gray peaks in Figure 1C-D, respectively) to allow subtraction of autofluorescence of cells in the same light scatter window (Figure 1B) from telomere fluorescence (horizontal bars in Figure 1C-D). In 5% of patients, insufficient numbers of white blood cells were obtained. In around 10% of the samples analyzed, the distinction between granulocytes and lymphocytes was not possible. If fewer than 5% of the 5000 events acquired did not correspond to previously established gates for granulocytes or lymphocytes,32 the values were not used in the analysis. FITC-labeled fluorescent beads (QuantumTM-24 Premixed; Flow Cytometry Standards, San Juan, Puerto Rico) were used to correct for daily shifts in the linearity of the flow cytometer and fluctuations in the laser intensity and alignment.37 At the beginning of each experiment, the fluorescence signals from the 4 different populations of FITC-labeled microbeads suspended in PBS with 0.1% BSA were acquired. Voltage and amplification of FL1 parameter were set in such a way that blank, 5579, 15 842, and 36 990 molecular equivalents of soluble fluorochrome (MESF) units per bead corresponded to channel numbers 25, 162, 456, and 942, respectively, in the FL1 channel on a linear scale. The resulting calibration curve (y = 0.02604x) was then used to convert telomere fluorescence into molecular equivalents of MESF units, allowing comparison of results among experiments. To estimate the telomere length (in kilobases [kb]) from telomere fluorescence in MESF units, the slope of the calibration curve described previously31 was used (y = 0.019x) in the following equation: kb = MESF × 0.02604 × 0.019. In order to analyze the day-to-day variation in hybridization efficiency, we analyzed aliquots of the same frozen lymphoma cells in each experiment as reported previously.31
Statistics The following variables were used for statistical analysis of data: fluorescence values after gating on granulocyte and lymphocyte subpopulations of PBLs, peripheral blood counts at the time the sample was obtained, disease duration and duration after initiation of therapy in AA patients, and the patient's age. To avoid a mixture of cross-sectional and serial measurements, only the first sample analyzed was included in the analysis if sequential samples were available from one individual. Age-matched controls were from a cohort of 301 individuals analyzed previously.32 Individual patient measurements were age-corrected by subtraction from the linear regression line previously established for lymphocytes and granulocytes from normal individuals32; the resulting difference from the age-adjusted normal telomere length (deltaTEL) for a particular cell type (deltaTELgran or deltaTELlymph) was also expressed in MESF units. Statistical comparison of patient samples with controls (Figure 2) was performed after a maximum likelihood estimation of the parameters for the controls based on the assumption that telomere length values are normally distributed and that the means and the standard deviations depend linearly on age. The comparison between the patient groups among themselves and with the control was based on the age standardized values.
Telomere length analysis of granulocytes derived from subgroups of patients with AA and hemolytic PNH Telomere fluorescence in peripheral blood granulocytes from patients with AA was compared with measurements made previously in a population of 301 normal healthy controls.32 As shown in Figure 2, the age-adjusted granulocyte telomere fluorescence (deltaTELgran) in all patients with AA was significantly lower than expected ( 1500 ± 500 MESF,
P = .001). Average deltaTELgran in samples from patients with hemolytic PNH without cytopenia (mean + SE: 200 ± 2300 MESF, n = 6; data not shown) did not differ
significantly from age-adjusted normal controls32 (range:
7100 to 6300 MESF, data not shown).
To further analyze telomere shortening in patients with AA, we
subdivided the patients on the basis of the severity of the disease and
their response to clinical intervention. There were significant
differences in deltaTELgran, dependent on the clinical stage of the disease (Figure 2). Patients with untreated severe or
moderate AA (sAA/mAA) as well as patients who had failed treatment and
manifested continued severe pancytopenia (sAANR) showed shorter telomere length as compared with healthy individuals ( Telomere length dynamics with age in different subgroups of patients with AA In normal individuals, both the average length of telomeres in nucleated blood cells and the rate of telomere shortening have been found to vary with age.32,38 We thus used linear regression analysis to examine the telomere fluorescence as a function of age in samples obtained from patients with recAA (Figure 3) and in samples from patients who belonged to either the sAANR or the untreated sAA/mAA group (Figure 4). Comparisons were performed separately for gated granulocytes (Figure 3A-B; Figure 4A-B) and lymphocytes (Figure 3C-D; Figure 4C-D) in view of the substantial differences in the age-related telomere length dynamics in those populations.32 When telomere fluorescence values (TEL, Figure 3A,C; Figure 4A,C) were plotted separately for each group, a significant decline in telomere length with age was found in granulocytes ( 45 base pairs (bp)/y, Figure 3A) and lymphocytes
( 64 bp/y, Figure 3C) from patients with recAA. The rate of telomere
shortening in this group was not different from the control population,
as shown by the distribution of the age-adjusted deltaTEL values (Figure 3B,D). In contrast, in untreated or nonresponding AA patients, no significant decline in calculated telomere length with age was found
in the granulocyte gate (Figure 4A). This effect translated into a
significantly positive slope of the linear regression line (+ 40 bp/y)
when deltaTELgran was plotted versus age (Figure 4B). Thus,
although as a group patients with sAANR or untreated AA show mostly
negative deltaTELgran values throughout the entire age
range, this relative telomere length deficit appeared to be most
pronounced in young patients and to decline with age. In the same
patients, the rate of telomere loss per year in lymphocytes was
slightly lower than in normal controls ( 45 bp/y, Figure 4C).
Correlation of peripheral blood counts with telomere length in patients with AA and PNH We next addressed the question of whether age-adjusted telomere length correlated with peripheral blood counts, which are used as an indicator of disease severity in patients with AA and PNH. As shown in Table 1, corresponding peripheral blood parameters were obtained for samples of all subgroups analyzed. Average peripheral blood counts for all samples were as follows: WBCs, 3.2 (0.2 × 103/µL; absolute neutrophil count (Gran), 1.5 (0.1 × 103/µL; absolute lymphocyte count (Lymph) 1.3 (0.1 × 103/µL; absolute monocyte count (Mono) 0.3, 0.2 × 103/µL; platelet count (Plts) 70.7, 7.7 × 103/µL; and hemoglobin (Hb) 10.3 (0.5 g/dL). Using linear regression analysis, we found significant positive correlations with deltaTELgran for WBC (P = .0003), Gran (P = .006), Plts (P = .002), Mono (P = .02), and Hb (P = .04) (Figure 5).
In this study, we measured the telomere length in nucleated blood cells from a cohort of patients with bone marrow failure syndromes using a newly introduced technique combining FISH and flow cytometry (flow-FISH).31,32 Measurements for different cell types were expressed as absolute telomere fluorescence values or as the difference in telomere fluorescence relative to age-adjusted controls (deltaTEL). We found significantly decreased deltaTEL values in granulocytes derived from patients with AA. In addition, we found that in patients who responded to immunosuppressive therapy, the estimated telomere length was not significantly different from controls, and a relatively normal rate of telomere shortening with age was observed in this group of patients. The current study was based on the predicate that, when hereditary differences in telomere length are taken into account, the telomere length in mature circulating leukocytes directly reflects the divisional history of the hematopoietic stem cells from which the leukocytes are derived. One important underlying assumption is that the number of cell divisions required for differentiation from a stem cell to a mature blood cell is both independent of patient age and also relatively constant for a particular differentiation lineage even in patients with marrow failure syndromes. Although these propositions are difficult to test directly, they are in agreement with telomere length measurement obtained with purified normal bone marrow cells,27 blood cells from bone marrow transplant recipients,28,29 and granulocytes and T-cell populations from normal individuals31,32 and patients with chronic myeloid leukemia.34 We furthermore hypothesized that patients with marrow failure syndromes would show variable telomere shortening in circulating cells relative to normal controls, depending on the extent and the duration of the damage to the stem cell compartment and the number of compensatory stem cell divisions. On the basis of these considerations, cytopenia per se does not have to be associated with a decrease in telomere length (eg, upon acute toxicity to stem cells or in situations of increased demand for mature blood cells but suppressed stem cell turnover) whereas normal blood counts can be associated with a decrease in telomere length (increased demand on production of blood cells matched by increased stem cell turnover, or normal demand, matched by a reduced number of stem cells with a higher number of accumulated cell divisions). Of course, other factors than stem cell turnover also determine the telomere length in peripheral blood cells and the results of flow-FISH analysis. First, there are marked hereditary differences in telomere length among normal individuals of the same age.32,33 However, method-related variability (estimated to be up to 15% of telomere fluorescence values) and, possibly, telomerase and/or other regulators of telomere length in human cells, as has been suggested by others,38 may also have contributed to variation in measurements that is unrelated to actual differences in the mitotic history of nucleated blood cells and their precursors. Although recovered patients showed some improvement in the LTC-IC numbers, a profound deficit in the number of these functional progenitor cells persists in these patients even in the presence of a normal blood count.42 In view of these findings, the observation that the telomere length in granulocytes and the rate of telomere shortening with age in this group of patients did not differ significantly from controls is of interest. One would perhaps have expected that extensive damage to the stem cell pool would have required increased mitotic activity in residual stem cells accompanied by a measurable decline in telomere length. However, to date, it is not clear how many hematopoietic stem cell clones are actually contributing to steady-state normal (physiologic) or stressed (pathophysiologic) hematopoiesis at any given point in time. Therefore, one possible explanation for the normal telomere length in patients with recAA is that the damage to the stem cell pool was insufficient to require detectable compensatory cell divisions in remaining stem cells. A possible reduction in stem cell numbers and functional reserve in patients with recAA might become apparent upon increased hematopoietic stress, eg, cytostatic chemotherapy or infection. Alternatively, the primary targets of immune destruction in these patients may not have been stem cells but progenitor cells. In this case, the number of additional, compensatory cell divisions from stem cells may also have been too small to result in a measurable change in granulocyte telomere length. Importantly, in patients with moderate depression of blood counts but a long history of the disease, a significant decrease in granulocyte telomere length was found. Most likely, this finding reflects a gradual and more general depletion of stem cell numbers with progressively fewer stem cell clones being forced to divide more frequently than normal to sustain hematopoiesis. A similar postulate may apply to patients with severe refractory AA in whom the telomere length was most significantly depressed. In a previous study on total peripheral blood leukocytes using conventional Southern blot analysis, no difference in telomere length was found in a group of patients with active AA compared with a group of patients with recovered AA.30 Both subgroups showed a similar average amount of telomere shortening (approximately 0.8 kb), which is comparable to our findings for patients with severe refractory AA (estimated to be approximately 1.2 kb). This discrepancy might be explained by the fact that the differences in telomere lengths seen in the granulocyte compartment in our study were hidden because Ball et al30 used unfractionated peripheral blood cells as a source for the telomere length analysis. Since considerable differences in telomere length in lymphocytes and granulocytes had been reported previously,32 differences solely in the cellular composition within the leukocyte fraction in the 2 groups could prevent these differences from being detectable. Interestingly, Ball et al found deltaTEL to progressively decrease with the duration of the disease at an additional rate of 216 bp/y in the "active AA group" whereas no such correlation was found in patients with fully recovered blood counts.30 Early therapeutic intervention may have prevented substantial losses of stem cells, allowing a full reconstitution of hematopoiesis in our recovered AA patient population. The telomere length in granulocytes of patients with untreated or refractory AA did not seem to decline significantly with age as observed in normal individuals. As a result, an increase in deltaTELgran with age was observed in this subgroup (Figure 4). A potential explanation for this observation is that the stem cell compartment in younger patients is capable of compensating losses of stem cells for a longer period of time by increasing the turnover of remaining stem cells. However, at the time of clinical presentation, this capacity may have been exhausted. The lack of a decline in telomere length with age in the granulocytes from this patient group supports this hypothesis and argues in favor of a critical telomere length threshold that needs to be reached before patients fail to regenerate hematopoiesis. This threshold may be reached earlier in older patients who have shorter telomeres to start with than in younger patients. Analysis of telomere length in lymphocyte populations did not reveal significant differences in telomere length dynamics between both subgroups of AA patients and controls. A more detailed analysis of lymphocyte subpopulations is required to discriminate between possible underlying immune or autoimmune phenomena and to study potential shifts in subpopulations of naive/memory/CD4/CD8 cells in AA. In contrast, the positive correlation between age-adjusted telomere length in granulocytes with blood cell counts in all leukocyte subpopulations (except lymphocytes) appears to reflect the severity of the disease. No consistent shortening of telomere length was reported in patients with PNH in an earlier study.30 On the basis of these findings, the authors concluded that oligoclonality may not be required for the evolution of PNH. In our study, the telomere length in the granulocytes from a limited number of PNH patients was also highly variable, which supports the previous conclusion and perhaps reflects heterogeneity of the associated bone marrow disease. A normal or even increased telomere length was observed in cells from patients in whom the majority of leukocytes displayed the PNH phenotype. As GPI-deficient cells arise from a mutation in a single hematopoietic stem cell, lack of significant telomere shortening in the cells derived from this clone is unexpected and difficult to explain. On the basis of the observation that purified candidate stem cells show asymmetric cell divisions in vitro, we previously postulated the existence of an extensive hierarchy within the stem cell compartment on the basis of differences in replicative history between individual stem cells.39,40 Perhaps GPI-deficient clones are sometimes derived from a stem cell that has divided fewer times than the majority of the stem cells contributing to hematopoiesis. Alternatively, the molecular defect in PNH may result in altered regulation of telomere length in affected cells. We prefer the replicative hierarchy model because telomere-mediated replicative senescence could explain the apparent exhaustion of PNH clones in (rare) patients with spontaneous remission of the disease.41 Future studies performed with sequential samples will help to circumvent some of the limitations of cross-sectional studies that are associated with the pronounced interindividual variation in telomere length. These studies are warranted to validate the use of telomere length in granulocytes as a measure of the mitotic history of stem cells.
Submitted February 8, 2000; accepted September 15, 2000.
Supported by National Institutes of Health grant AI29524 and by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run as well as a grant from the Deutsche Forschungsgemeinschaft (T.H.B.).
T.H.B. and J.P.M. contributed equally to this article.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Peter M. Lansdorp, Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada; e-mail: plansdor{at}bccancer.bc.ca.
1.
Marsh JC, Chang J, Testa NG, Hows JM, Dexter TM.
The hematopoietic defect in aplastic anemia assessed by long-term marrow culture.
Blood.
1990;76:1748-1757 2. Maciejewski JP, Anderson S, Katevas P, Young NS. Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure. Br J Haematol. 1994;87:227-234[Medline] [Order article via Infotrieve]. 3. Scopes J, Bagnara M, Gordon-Smith EC, Ball SE, Gibson FM. Haemopoietic progenitor cells are reduced in aplastic anaemia. Br J Haematol. 1994;86:427-430[Medline] [Order article via Infotrieve].
4.
Maciejewski JP, Selleri C, Sato T, Anderson S, Young NS.
A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia.
Blood.
1996;88:1983-1991
5.
Schrezenmeier H, Jenal M, Herrmann F, Heimpel H, Raghavachar A.
Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay.
Blood.
1996;88:4474-4480
6.
Podesta M, Piaggio G, Frassoni F, et al.
The assessment of the hematopoietic reservoir after immunosuppressive therapy or bone marrow transplantation in severe aplastic anemia.
Blood.
1998;91:1959-1965 7. Podesta M, Piaggio G, Frassoni F, et al. Deficient reconstitution of early progenitors after allogeneic bone marrow transplantation. Bone Marrow Transplant. 1997;19:1011-1017[CrossRef][Medline] [Order article via Infotrieve]. 8. Bacigalupo A, Figari O, Tong J, et al. Long-term marrow culture in patients with aplastic anemia compared with marrow transplant recipients and normal controls. Exp Hematol. 1992;20:425-430[Medline] [Order article via Infotrieve]. 9. Selleri C, Maciejewski JP, De Rosa G, et al. Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant. Bone Marrow Transplant. 1999;23:1029-1037[CrossRef][Medline] [Order article via Infotrieve]. 10. Blackburn EH. Structure and function of telomeres. Nature. 1991;350:569-572[CrossRef][Medline] [Order article via Infotrieve].
11.
Zakian VA.
Telomeres: beginning to understand the end.
Science.
1995;270:1601-1607 12. de Lange T. Telomere dynamics and genome instability in human cancer. In: Blackburn EH,Greider CW, eds. Telomeres. Plainview, NY: Cold Spring Harbor Laboratory Press; 1995:265-293. 13. Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature. 1990;345:458-460[CrossRef][Medline] [Order article via Infotrieve]. 14. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC. Telomere reduction in human colorectal carcinoma and with ageing. Nature. 1990;346:866-868[CrossRef][Medline] [Order article via Infotrieve]. 15. Olovnikov AM. A theory of marginotomy. J Theor Biol. 1973;41:181-190[CrossRef][Medline] [Order article via Infotrieve]. 16. Watson JD. Origin of concatameric T4 DNA. Nat New Biol. 1972;239:197-201[CrossRef][Medline] [Order article via Infotrieve]. 17. Greider CW. Telomere length regulation. Annu Rev Biochem. 1996;65:337-365[CrossRef][Medline] [Order article via Infotrieve].
18.
Kim NW, Piatyszek MA, Prowse KR, et al.
Specific association of human telomerase activity with immortal cells and cancer.
Science.
1994;266:2011-2015 19. Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet. 1996;18:173-179[CrossRef][Medline] [Order article via Infotrieve].
20.
Harley CB, Kim NW, Prowse KR, et al.
Telomerase, cell immortality, and cancer.
Cold Spring Harb Symp Quant Biol.
1994;59:307-315 21. Blasco MA, Lee H-W, Hande MP, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997;91:25-34[CrossRef][Medline] [Order article via Infotrieve].
22.
Hande MP, Samper E, Lansdorp P, Blasco MA.
Telomere length dynamics and chromosomal instability in cells derived from telomerase null mice.
J Cell Biol.
1999;144:589-601 23. Rudolph KL, Chang S, Lee H-W, et al. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell. 1999;96:701-712[CrossRef][Medline] [Order article via Infotrieve].
24.
Bodnar AG, Ouellette M, Frolkis M, et al.
Extension of life-span by introduction of telomerase into normal human cells.
Science.
1998;279:349-353 25. Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol. 1998;8:279-282[CrossRef][Medline] [Order article via Infotrieve].
26.
Lansdorp PM, Dragowska W, Mayani H.
Ontogeny-related changes in proliferative potential of human hematopoietic cells.
J Exp Med.
1993;178:787-791
27.
Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM.
Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age.
Proc Natl Acad Sci U S A.
1994;91:9857-9860 28. Wynn RF, Cross MA, Hatton C, et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet. 1998;351:178-181[CrossRef][Medline] [Order article via Infotrieve].
29.
Notaro R, Cimmino A, Tabarini D, Rotoli B, Luzzatto L.
In vivo telomere dynamics of human hematopoietic stem cells.
Proc Natl Acad Sci U S A.
1997;94:13782-13785
30.
Ball SE, Gibson FM, Rizzo S, Tooze JA, Marsh JCW, Gordon-Smith EC.
Progressive telomere shortening in aplastic anemia.
Blood.
1998;91:3582-3592 31. Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol. 1998;16:743-747[CrossRef][Medline] [Order article via Infotrieve].
32.
Rufer N, Brummendorf TH, Kolvraa S, et al.
Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood.
J Exp Med.
1999;190:157-167 33. Slagboom PE, Droog S, Boomsma DI. Genetic determination of telomere size in humans: a twin study of three age groups. Am J Hum Genet. 1994;55:876-882[Medline] [Order article via Infotrieve].
34.
Brummendorf TH, Holyoake TL, Rufer N, et al.
Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry.
Blood.
2000;95:1883-1890
35.
Camitta BM, Thomas ED, Nathan DG, et al.
Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality.
Blood.
1976;48:63-70
36.
Dunn DE, Tanawattanacharoen P, Boccuni P, et al.
Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes.
Ann Intern Med.
1999;131:401-408 37. Henderson LO, Marti GE, Gaigalas A, Hannon WH, Vogt RF Jr. Terminology and nomenclature for standardization in quantitative fluorescence cytometry. Cytometry. 1998;33:97-105[CrossRef][Medline] [Order article via Infotrieve].
38.
Frenck RW Jr, Blackburn EH, Shannon KM.
The rate of telomere sequence loss in human leukocytes varies with age.
Proc Natl Acad Sci U S A.
1998;95:5607-5610
39.
Brummendorf TH, Dragowska W, Zijlmans JMJM, Thornbury G, Lansdorp PM.
Asymmetric cell divisions sustain long-term hematopoiesis from single-sorted human fetal liver cells.
J Exp Med.
1998;188:1117-1124 40. Lansdorp PM. Self-renewal of stem cells. Biol Blood Marrow Transplant. 1997;3:171-178[Medline] [Order article via Infotrieve].
41.
Hillmen P, Lewis SM, Bessler M, Luzzatto L, Dacie JV.
Natural history of paroxysmal nocturnal hemoglobinuria.
N Engl J Med.
1995;333:1253-1258 42. Maciejewski JP, Kim S, Sloand E, Selleri C, Young NS. Sustained long-term hematologic recovery despite a marked quantitative defect in the stem cell compartment of patients with aplastic anemia after immunosuppressive therapy. Am J Hematol. 2000;65:123-131[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  J. T. Cronkhite, C. Xing, G. Raghu, K. M. Chin, F. Torres, R. L. Rosenblatt, and C. K. Garcia Telomere Shortening in Familial and Sporadic Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., October 1, 2008; 178(7): 729 - 737. [Abstract] [Full Text] [PDF]
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 I. Spyridopoulos, Y. Erben, T. H. Brummendorf, J. Haendeler, K. Dietz, F. Seeger, C. K. Kissel, H. Martin, J. Hoffmann, B. Assmus, et al. Telomere Gap Between Granulocytes and Lymphocytes Is a Determinant for Hematopoetic Progenitor Cell Impairment in Patients With Previous Myocardial Infarction Arterioscler. Thromb. Vasc. Biol., May 1, 2008; 28(5): 968 - 974. [Abstract] [Full Text] [PDF]
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 R. T. Calado and N. S. Young Telomere maintenance and human bone marrow failure Blood, May 1, 2008; 111(9): 4446 - 4455. [Abstract] [Full Text] [PDF]
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F. D. Goldman, G. Aubert, A. J. Klingelhutz, M. Hills, S. R. Cooper, W. S. Hamilton, A. J. Schlueter, K. Lambie, C. J. Eaves, and P. M. Lansdorp Characterization of primitive hematopoietic cells from patients with dyskeratosis congenita Blood, May 1, 2008; 111(9): 4523 - 4531. [Abstract] [Full Text] [PDF]
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 G. Aubert and P. M. Lansdorp Telomeres and Aging Physiol Rev, April 1, 2008; 88(2): 557 - 579. [Abstract] [Full Text] [PDF]
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A. Marrone, A. Walne, H. Tamary, Y. Masunari, M. Kirwan, R. Beswick, T. Vulliamy, and I. Dokal Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome Blood, December 15, 2007; 110(13): 4198 - 4205. [Abstract] [Full Text] [PDF]
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 C. K. Garcia, W. E. Wright, and J. W. Shay Human diseases of telomerase dysfunction: insights into tissue aging Nucleic Acids Res., December 3, 2007; 35(22): 7406 - 7416. [Abstract] [Full Text] [PDF]
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 Y. Wang, H. Yagasaki, A. Hama, N. Nishio, Y. Takahashi, and S. Kojima Mutation of SBDS and SH2D1A is not associated with aplastic anemia in Japanese children Haematologica, November 1, 2007; 92(11): 1573 - 1573. [Abstract] [Full Text] [PDF]
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B. P. Alter, G. M. Baerlocher, S. A. Savage, S. J. Chanock, B. B. Weksler, J. P. Willner, J. A. Peters, N. Giri, and P. M. Lansdorp Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita Blood, September 1, 2007; 110(5): 1439 - 1447. [Abstract] [Full Text] [PDF]
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R. T. Calado, S. A. Graf, K. L. Wilkerson, S. Kajigaya, P. J. Ancliff, Y. Dror, S. J. Chanock, P. M. Lansdorp, and N. S. Young Mutations in the SBDS gene in acquired aplastic anemia Blood, August 15, 2007; 110(4): 1141 - 1146. [Abstract] [Full Text] [PDF]
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A. Marrone, P. Sokhal, A. Walne, R. Beswick, M. Kirwan, S. Killick, M. Williams, J. Marsh, T. Vulliamy, and I. Dokal Functional characterization of novel telomerase RNA (TERC) mutations in patients with diverse clinical and pathological presentations Haematologica, August 1, 2007; 92(8): 1013 - 1020. [Abstract] [Full Text] [PDF]
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 M. W. Drummond, S. Balabanov, T. L. Holyoake, and T. H. Brummendorf Concise Review: Telomere Biology in Normal and Leukemic Hematopoietic Stem Cells Stem Cells, August 1, 2007; 25(8): 1853 - 1861. [Abstract] [Full Text] [PDF]
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 B. P. Alter Diagnosis, Genetics, and Management of Inherited Bone Marrow Failure Syndromes Hematology, January 1, 2007; 2007(1): 29 - 39. [Abstract] [Full Text] [PDF]
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N. S. Young, R. T. Calado, and P. Scheinberg Current concepts in the pathophysiology and treatment of aplastic anemia Blood, October 15, 2006; 108(8): 2509 - 2519. [Abstract] [Full Text] [PDF]
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T. J. Vulliamy, A. Marrone, S. W. Knight, A. Walne, P. J. Mason, and I. Dokal Mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation Blood, April 1, 2006; 107(7): 2680 - 2685. [Abstract] [Full Text] [PDF]
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K. M. Austin, R. J. Leary, and A. Shimamura The Shwachman-Diamond SBDS protein localizes to the nucleolus Blood, August 15, 2005; 106(4): 1253 - 1258. [Abstract] [Full Text] [PDF]
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F. Beier, S. Balabanov, T. Buckley, K. Dietz, U. Hartmann, M. Rojewski, L. Kanz, H. Schrezenmeier, and T. H. Brummendorf Accelerated telomere shortening in glycosylphosphatidylinositol (GPI)-negative compared with GPI-positive granulocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) detected by proaerolysin flow-FISH Blood, July 15, 2005; 106(2): 531 - 533. [Abstract] [Full Text] [PDF]
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 H. Yamaguchi, R. T. Calado, H. Ly, S. Kajigaya, G. M. Baerlocher, S. J. Chanock, P. M. Lansdorp, and N. S. Young Mutations in TERT, the Gene for Telomerase Reverse Transcriptase, in Aplastic Anemia N. Engl. J. Med., April 7, 2005; 352(14): 1413 - 1424. [Abstract] [Full Text] [PDF]
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W. E. Fibbe Telomerase Mutations in Aplastic Anemia N. Engl. J. Med., April 7, 2005; 352(14): 1481 - 1483. [Full Text] [PDF]
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S. Zimmermann, S. Glaser, R. Ketteler, C. F. Waller, U. Klingmuller, and U. M. Martens Effects of Telomerase Modulation in Human Hematopoietic Progenitor Cells Stem Cells, September 1, 2004; 22(5): 741 - 749. [Abstract] [Full Text] [PDF]
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J. A.G. Van Ziffle, G. M. Baerlocher, and P. M. Lansdorp Telomere Length in Subpopulations of Human Hematopoietic Cells Stem Cells, November 1, 2003; 21(6): 654 - 660. [Abstract] [Full Text] [PDF]
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H. Yamaguchi, G. M. Baerlocher, P. M. Lansdorp, S. J. Chanock, O. Nunez, E. Sloand, and N. S. Young Mutations of the human telomerase RNA gene (TERC) in aplastic anemia and myelodysplastic syndrome Blood, August 1, 2003; 102(3): 916 - 918. [Abstract] [Full Text] [PDF]
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A. Karadimitris, D. J. Araten, L. Luzzatto, and R. Notaro Severe telomere shortening in patients with paroxysmal nocturnal hemoglobinuria affects both GPI- and GPI+ hematopoiesis Blood, July 15, 2003; 102(2): 514 - 516. [Abstract] [Full Text] [PDF]
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 M. Bogliolo, O. Cabre, E. Callen, V. Castillo, A. Creus, R. Marcos, and J. Surralles The Fanconi anaemia genome stability and tumour suppressor network Mutagenesis, November 1, 2002; 17(6): 529 - 538. [Abstract] [Full Text] [PDF]
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 E. Callen, E. Samper, M. J. Ramirez, A. Creus, R. Marcos, J. J. Ortega, T. Olive, I. Badell, M. A. Blasco, and J. Surralles Breaks at telomeres and TRF2-independent end fusions in Fanconi anemia Hum. Mol. Genet., February 1, 2002; 11(4): 439 - 444. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
