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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 2056-2064
Isolation of a Highly Quiescent Subpopulation of Primitive Leukemic
Cells in Chronic Myeloid Leukemia
By
Tessa Holyoake,
Xiaoyan Jiang,
Connie Eaves, and
Allen Eaves
From the Terry Fox Laboratory, BC Cancer Agency, Vancouver, British
Columbia, Canada.
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ABSTRACT |
Chronic myeloid leukemia (CML) is characterized by an increased
proliferative activity of the leukemic progenitors that produce an
elevated number of mature granulocytes. Nevertheless, cell cycle-active
agents, even in very high doses, are alone unable to eradicate the
leukemic clone, suggesting the presence of a rare subset of quiescent
leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G0 and
G1/S/G2/M fractions of CD34+
cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and
cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic
(Ph+/BCR-ABL+) cells with in vitro
progenitor activity and capable of engrafting immunodeficient mice were
identified in the directly isolated G0 cells. Single-cell
reverse transcriptase-polymerase chain reaction (RT-PCR)
analysis showed that many leukemic CD34+ G0
cells also expressed BCR-ABL mRNA. CD34+ from 8 CML
patients were also labeled with carboxyfluorescein diacetate
succinimidyl diester (CFSE) before being cultured (with and without
added growth factors) to allow viable cells that had remained quiescent
(ie, CFSE+) after 4 days to be retrieved by FACS.
Leukemic progenitors were again detected in all quiescent populations
isolated by this second strategy, including those exposed to a
combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6,
and granulocyte colony-stimulating factor. These findings provide the
first direct and definitive evidence of a deeply but reversibly
quiescent subpopulation of leukemic cells in patients with CML with
both in vitro and in vivo stem cell properties.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOID leukemia (CML) is a
myeloproliferative disorder that appears after the deregulated clonal
expansion and differentiation of a totipotent hematopoietic stem
cell.1,2 The cytogenetic hallmark of the disease is the
Philadelphia (Ph) chromosome, which forms as a result of a reciprocal
translocation between the long arms of chromosomes 9 and
22.3 At the DNA level, the corresponding change is the
creation of a novel BCR-ABL fusion gene.4,5 Despite
overgrowth of the marrow in CML patients by the
Ph+/BCR-ABL+ clone, a residual population of
primitive normal (Ph /BCR-ABL)
hematopoietic stem cells typically persists (reviewed in Eaves et
al2).
In recent years, considerable interest has focused on the observation
that Ph+/BCR-ABL+ cells can show a reduced or
absent growth factor dependence for their survival.6,7 We
have demonstrated that highly purified populations of phenotypically
very primitive Ph+/BCR-ABL+ cells obtained from
CML patients will, in fact, proliferate in vitro for several weeks in
the absence of added growth factors8 and recently showed
that this involves an autocrine interleukin-3 (IL-3) and granulocyte
colony-stimulating factor (G-CSF) mechanism.9 These
observations could explain both the increased proliferative activity
exhibited by many types of Ph+/BCR-ABL+
progenitors in vivo2,10,11 and the decreased self-renewal exhibited by very primitive Ph+/BCR-ABL+ cells
identified as long-term culture-initiating cells
(LTC-IC),12,13 because exposure of primitive normal
hematopoietic cells to excess concentrations of IL-3 promotes their
differentiation as well as stimulating their
proliferation.14,15 Constitutive activation of the leukemic
progenitors in CML is also consistent with the ability of intensive
chemotherapy regimens to induce cytogenetic remissions in chronic-phase
patients.16,17 However, such remissions have generally been
short-lived and CML is considered incurable by conventional
chemotherapy alone. Therefore, it seemed likely that CML patients might
possess a rare subpopulation of quiescent Ph+/BCR-ABL+ stem cells that had not been
identified by previous strategies used to purify primitive populations
from CML samples. To try to isolate such cells, we pursued 2 approaches
for obtaining quiescent CD34+ cells. The first of these
exploited the dual staining procedure with Hoechst 33342 (Hst) and
Pyronin Y (Py) that allows G0 and G1/S/G2/M cells to be
separated.18-20 The second exploited the resolving power of
5- (and 6-) carboxyfluorescein diacetate succinimidyl diester (CFSE)
staining21 to distinguish undivided and divided cells in
short-term cultures of CD34+ cells.
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MATERIALS AND METHODS |
Cells.
Heparinized blood and bone marrow cells were obtained from 8 patients
with untreated BCR-ABL+ chronic-phase CML (see
Table 1 for details). Samples of normal adult marrow were either from harvests taken for allogeneic marrow transplants or were from cadaveric marrow samples (Northwest Tissue Center, Seattle, WA). Before use, all samples were enriched for CD34+ cells by negative immunomagnetic depletion of lineage
marker+ (lin+) cells using StemSep columns
(StemCell Technologies, Vancouver, British Columbia, Canada). For the
CML samples, antibodies to CD15 and IgE were added to the recommended
cocktail to enhance the removal of mature cells.22 All
human cell samples were obtained with informed consent according to
institutional guidelines.
Flow cytometry.
CD34+ cells were fractionated into G0 and
G1/S/G2/M fractions according to their staining
with Hst and Py.18-20 Briefly, cryopreserved lin+-depleted (lin) cells were thawed
and incubated overnight in a serum-free medium (SFM) consisting of
Iscove's medium (StemCell), a serum substitute (BIT; StemCell), 40 µg/mL low-density lipoproteins (Sigma Chemicals, St Louis, MO),
104 mol/L 2-mercaptoethanol, 300 ng/mL each of
recombinant human Steel factor (SF; Amgen, Thousand Oaks, CA) and
Flt3-ligand (FL; Immunex, Seattle, WA), and 60 ng/mL each of
recombinant human IL-3 (Novartis, Basel, Switzerland), IL-6 (Cangene,
Mississauga, Ontario, Canada), and G-CSF (StemCell) to allow
reactivation of RNA synthesis. The following day, the cells were washed
once in Hank's balanced salt solution supplemented with 2% fetal calf serum (HF/2) and then resuspended in HF/2 containing 10 µmol/L Hst
(Molecular Probes, Eugene, OR). After 45 minutes at 37°C, Py
(Sigma) was added to give a final concentration of 2.5 µg/mL and
cells were incubated for an additional 45 minutes.
Anti-CD34-fluorescein isothiocyanate (FITC) (8G12-FITC; Dr P. Lansdorp,
Terry Fox Laboratory, Vancouver, British Columbia, Canada) was then
added at 10 µg/mL for a final 20 minutes. Cells were washed once in
HF/2 containing Hst (10 µmol/L) and Py (2.5 µg/mL) and a second
time in the same medium containing 1 µg/mL propidium iodide (PI;
Sigma). Cells were finally resuspended in HF/2 without PI and kept on
ice in the dark until being sorted on a 3 laser FACStar Plus (Becton Dickinson [BD], San Jose, CA). Gates were set to collect
Hstlo Pylo (G0) and all remaining
(G1/S/G2/M) cells as separate fractions within
the PI CD34+ population.23
Relatively homogeneously CFSE (Molecular Probes)-labeled fractions of
CD34+ cells were also obtained and then cultured for 4 days
in SFM, with or without the following growth factors: 50 ng/mL
thrombopoietin (TPO; Genentech, San Francisco, CA), or FL, SF, IL-3,
IL-6, and G-CSF at the concentrations described above and with or
without 100 ng/mL colcemid (GIBCO BRL, Burlington, Ontario, Canada), as indicated. At the end of the 4 days, cells were harvested, washed once
in HF/2, and washed a second time in HF/2/PI. Cultures containing colcemid were used to establish the range of fluorescence expected for
all cells that had not divided during the 4-day interval.21 For both sorting strategies, single cells (and defined numbers of cells
where indicated) were collected using the automated cell deposition
unit of the fluorescence-activated cell sorter (FACS).
Detection of Ki-67 and D cyclins.
To detect intracellular Ki-67, the staining procedure described by
Jordan et al24 was followed. Cells were analyzed for expression of cyclins D1, D2, and D3 after fixation and staining with
an FITC-conjugated antibody (Ab) (Pharmingen, Mississauga, Ontario,
Canada) according to the manufacturer's directions.
Analysis of BCR-ABL mRNA expression.
Cells were centrifuged and lyzed in guanidinium isothiocyanate solution
(5 mol/L GIT, 20 mmol/L 1,4-diothioerythritol [DTT], 25 mmol/L sodium citrate, pH 7.0, 0.5% Sarcosyl) before the application of a 2-step (nested) reverse transcriptase-polymerase chain reaction (RT-PCR) procedure using an initial oligo (dT)-based
primer and poly (A) tailing strategy.25,26 After
electrophoresis of amplified products, BCR-ABL-specific and
actin-specific fragments were detected by Southern blotting and
hybridization with BCR-ABL and actin probes.25,26 Blots
were exposed for 6 to 16 hours.
In vitro progenitor assays.
Assays for different types of colony-forming cells (CFC) capable of
generating pure or mixed colonies of erythroid cells, granulocytes, and
macrophages in fetal calf serum (FCS)-containing methylcellulose medium
(H4330; StemCell) supplemented with 50 ng/mL SF and 20 ng/mL each of
IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF;
Novartis), and G-CSF, and 3 U/mL of erythropoietin (StemCell) were
performed as previously described.27 Cells were assayed for
LTC-IC by measurement of the CFC content of 6-week cocultures
containing pre-established, irradiated murine fibroblasts genetically
engineered to produce human IL-3, G-CSF, and SF.27,28
Transplantation and detection of human cells in immunodeficient
mice.
Breeding pairs of NOD/LtSz-scid/scid mice with homozygous
disruption of their  2 microglobulin genes
(NOD/SCID-2M/
mice29; kindly provided by Dr L. Schultz, Jackson
Laboratory, Bar Harbor, ME) were expanded and maintained in the animal
facility of the British Columbia Cancer Research Centre (Vancouver,
British Columbia, Canada) in microisolators under defined sterile
conditions. Six- to 8-week-old mice were irradiated with 300 cGy of
137Cs  -rays not more than 24 hours before being injected
intravenously with human cells. In each case, 106
irradiated (1,500 cGy) normal human bone marrow cells (as carriers) were coinjected into each mouse. After 6 weeks, the cells from both
femurs and tibias from each mouse were labeled with various antimouse
and antihuman antibodies and depleted immunomagnetically of all murine
cells using a StemSep column,22 and human
CD34CD45RA+ and/or CD71+
(total human) and CD34+ (primitive human) cells were
specifically identified and sorted using the FACS.26
Aliquots of the sorted human CD34 and
CD34+ cells were analyzed by RT-PCR for BCR-ABL mRNA as
described above.
Statistics.
Error estimates shown on mean values from greater than 2 samples in a
group are the SEM.
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RESULTS |
Direct isolation of G0 leukemic cells from chronic-phase
CML patients.
In a first series of experiments, CD34+ cells from normal
marrow (n = 3) and CML samples (no. 1 through 6, Table 1) were
separated into 2 fractions: 1 containing
HstloPylo (G0) cells and 1 containing all other (HstloPy+ and all
Hst+) (G1/S/G2/M) cells
(Fig 1). In all 9 samples, a relatively
discrete population of HstloPylo cells was
identified. On average, this population was largest in the 3 normal
samples (39% ± 18%), intermediate in the 2 CML samples with
predominantly normal (Ph) LTC-IC (21% and 31%),
and smallest in the 4 CML samples with predominantly leukemic LTC-IC
(4% ± 2%).
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| Fig 1.
Representative examples of Hst and Py staining of viable
CD34+ cells from normal marrow (left) and from CML
patients with predominantly Ph (middle, no. 2) or
Ph+ (right, no. 3) LTC-IC.
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To further characterize the cycling status of these 2 subpopulations of
CD34+ cells, particularly in the CML samples with no
detectable normal elements, their expression of intracellular Ki-67 and
D cyclins was investigated. Ki-67 is a nuclear antigen found
exclusively in proliferating cells,24,30 and the D cyclins
are induced upon mitogenic stimulation of quiescent
cells.31 Less than 2% of the
HstloPylo cells from all 3 CML samples examined
(no. 4 through 6, Table 1) were found to contain detectable levels of
Ki-67 or any of the D cyclins, whereas a high level of expression of
both Ki-67 and at least 1 of the D cyclins was consistently evident in
the majority of the remaining CD34+ cells
(Fig 2).
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| Fig 2.
Representative examples of G0 and
G1/S/G2/M fractions of CD34+
cells after staining with anti-Ki-67-FITC/7AAD and anti-cyclins D1, D2,
and D3-FITC/PI (CML no. 4).
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In vitro assays for progenitor activity showed that most of the CML CFC
and many of the LTC-IC were cycling (Hst+ and/or
Hstlo Py+). However, as summarized in
Table 2, both CML progenitor types could
also be detected in the quiescent (HstloPylo)
fraction of the CD34+ cells, although proportionately less
so than was the case for normal marrow. Cytogenetic
analyses32 of the colonies generated in the assays of the
cells isolated from the CML samples showed that leukemic
(Ph+) progenitors were present in 4 of 5 cases in which
analyzable colony metaphases were obtained (Table 2). To determine
whether these quiescent cells also produce BCR-ABL transcripts, RT-PCR analyses were performed directly on the freshly isolated cells from 3 patients (all of whom had predominantly Ph+ LTC-IC).
BCR-ABL mRNA was detected in aliquots of 10, 100, and 1,000 CD34+ cells from all 3 patients. Analyses of single
HstloPylo cells showed 8 of 12 (67%), 6 of 12 (50%), and 6 of 12 (50%) of these (from each of the 3 patients,
respectively) to be BCR-ABL+
(Fig 3).
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Table 2.
Proportion and Genotype of Progenitors in the
G0 (HstloPylo) Subpopulation of
CD34+ Cells in CML Samples by Comparison to Normal
Marrow
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| Fig 3.
BCR-ABL expression in G0 and
G1/S/G2/M CD34+ cells isolated
from CML patient samples no. 3 through 5. Controls included 1,000 G1/S/G2/M cells without RT (CON 1) and
H2O blank (CON 2).
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To determine whether the quiescent cells isolated from CML patients
could also generate leukemic populations in vivo, 30 sublethally irradiated immunodeficient
NOD/SCID-2M/ mice were
injected either with HstloPylo
CD34+ cells (1 to 2 × 105/mouse) or with
the remaining CD34+ cells (1 to 20 × 105/mouse) isolated from 3 CML samples (no. 4 through 6, Table 1). When assessed 6 weeks later, all of the mice showed evidence
of engraftment with human cells, including CD34+ as well as
CD34 phenotypes (see example shown in
Fig 4). Both of these subpopulations were
then isolated by FACS and examined for the presence of leukemic (BCR-ABL+) cells by RT-PCR. Most mice transplanted with
either G0 cells (6/6) or G1/S/G2/M
cells (14/15) from 2 of the CML samples were found to contain both
CD34 and CD34+ leukemic cells (see
example in Fig 5). In the third case (CML no. 5, Table 1), no leukemic cells were detected in the
CD34 population of human cells present in 1 of the 2 mice transplanted with G0 cells or in any of the 7 mice
transplanted with G1/S/G2/M cells (despite the
consistent presence of detectable human actin transcripts). Thus, some
normal transplantable human hematopoietic cells must have been present
in both the quiescent and cycling populations isolated from this CML
sample (no. 5). The known ability of transplantable stem cells from
normal human marrow and cord blood to generate predominantly
CD34 B-lineage progeny in NOD/SCID
mice33-35 may explain the prevalence of normal human
CD34 cells in mice containing
BCR-ABL+CD34+ cells.
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| Fig 4.
Example of human CD34 cells (expressing
either CD45RA or CD71) and human CD34+ cells obtained
from the marrow of a NOD/SCID-2M/
mouse transplanted 6 weeks previously with 105
G0 cells from CML no. 5. Cells were first depleted of
coexisting mouse cells by immunodepletion as described in Materials and
Methods.
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| Fig 5.
Examples of BCR-ABL expression in CD34 and
CD34+ cells isolated from the marrow of immunodeficient
mice transplanted 6 weeks earlier with G0 or
G1/S/G2/M fractions of CD34+ CML
cells (CML no. 6 in Table 1). Controls included 1,000 K562 cells
(K562+), 1,000 K562 cells without RT
(K562), and H2O blank.
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Ability of some leukemic progenitors from CML patients to remain
quiescent in vitro for at least 4 days independent of exposure to
exogenous growth factors.
In a second series of experiments, the ability of quiescent leukemic
CD34+ cells to survive and resist mitogenic activation for
at least 4 days under different conditions of growth factor stimulation was compared with that characteristic of normal CD34+
cells. Cells were labeled with CFSE, incubated overnight, sorted to
obtain a homogeneously CFSE-labeled CD34+ population, and
then cultured for another 4 days in SFM containing either no other
factors, TPO only, or a combination of FL, SF, IL-3, IL-6, and G-CSF.
The cultured cells were then harvested and resorted into 2 fractions
according to their proliferative histories, as indicated by their
individual levels of persisting CFSE fluorescence
(Fig 6). Cultures containing no growth
factors were included to favor the selection of quiescent leukemic
cells. TPO alone has been previously shown to maintain the viability of
primitive normal cells with minimal stimulation of their
proliferation36 and was thus expected to maximize the
detection of quiescent cells of either genotype. The 5 growth factor
combination was included to identify quiescent cells that would be
maximally resistant to activation, because these conditions have been
found to mitotically stimulate most primitive adult normal
hematopoietic cells within this time frame.21,23
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| Fig 6.
Distribution of cells according to their CFSE
fluorescence after being cultured for 4 days in the presence or absence
of growth factors as described in the text. The unshaded areas indicate
the fluorescence distribution of (still viable) cells that had been
prevented from dividing by the addition of colcemid to the cultures.
The black peaks indicate the cells that had undergone 0 to 7 divisions
during the 4-day culture period.
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The various populations of cells obtained from these cultures were then
assayed for the presence of CFC and LTC-IC. As shown in
Table 3, some persisting quiescent cells
could be identified in all 8 CML samples studied, regardless of the
growth factors to which the cells were exposed during the period they
were cultured before analysis. Moreover, the CD34+ cells
that remained quiescent for 4 days also always included a subset with
CFC activity. As expected, the relative proportion of the persisting
quiescent cells retrieved from the cultures (initiated with either
normal or CML cells) decreased with increasing growth factor
supplementation of the medium. This proportion was consistently lower
in the cultures initiated with cells from the CML samples.
Interestingly, the quiescent cells also showed lower forward light
scattering characteristics indicative of a reduced cell size,
regardless of their genotype (data not shown).
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Table 3.
A Proportion of CD34+ Normal Bone Marrow
(NBM) and CML Cells Can Remain Viable and Quiescent for at Least 4 Days
When Cultured in the Presence or Absence of Growth Factors
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DISCUSSION |
In designing the present study, we first sought a method that would
allow the direct isolation from a CD34+ cell population of
a viable G0 fraction of cells distinct from those in
G1 as well as S/G2/M. For this, we used a
nontoxic double staining procedure with Hoechst 33342 and Pyronin Y and
multiparameter flow cytometry. This allowed the viable
(PI) CD34+ cells to be divided into 2 fractions: 1 containing cells with a 2n DNA content and a low RNA
content (Hstlo/Pylo; ie, the G0
cells) and 1 containing all of the remaining
(G1/S/G2/M) cells.18-20 Analysis of
quiescent (G0) cells in other systems has shown that they
typically are smaller in size and have reduced numbers of mitochondria,
a decreased protein content, and a higher degree of chromatin
condensation.37 In addition, expression of certain genes
(eg, growth-arrest-specific, gas, genes,38 and
statin39) is turned on and expression of others (eg,
proliferating cell nuclear antigen,40
Ki-67,24,30 and the D cyclins31) may be
silenced. Validation of the G0 status of the cells assigned a Hstlo/Pylo phenotype here was provided by
demonstrating the specific absence of 2 of the above-noted markers
(Ki-67 and the D cyclins) in these cells in addition to noting their
reduced size.
Functional characterization of these directly isolated G0
cells confirmed that a significant proportion of the CFC and LTC-IC in
normal bone marrow are quiescent,10,20,41 indicative of multilevel regulatory mechanisms normally constraining the expansion of
primitive hematopoietic cells. Parallel studies of CML cells showed
most of the leukemic CFC and LTC-IC to be cycling, as
expected.2,10 However, quiescent leukemic CFC and LTC-IC
could also be consistently isolated. Both quiescent and cycling
populations of leukemic cells were capable of generating leukemic
progeny in irradiated immunodeficient mice. Thus, the quiescent status
of Ph+ progenitors, like that of their normal counterparts,
is reversible both in vitro and in vivo.
Recent studies suggest that growth factor activation may modulate the
engraftment capacity of transplantable hematopoietic cells.42 Gothot et al43 have proposed that this
may result from early growth factor-induced changes in the homing
function(s) of these cells rather than in their differentiated state.
If the majority of the leukemic stem cells in patients with CML are
proliferating more rapidly than is typical of their normal
counterparts, as suggested by LTC-IC analyses,2 then it
might be anticipated that the ability of CML stem cells to be
transplanted might also be compromised. This would be consistent with
the widely encountered difficulty in rapidly obtaining high levels of
leukemic (Ph+/BCR-ABL+) cells in NOD/SCID
mice.44-46 In the present studies, we found that both
G0 and, to a lesser extent,
G1/S/G2/M cells were able to engraft mice with
leukemic cells. However, a more immunodeficient strain of NOD/SCID mice
(also lacking 2-microglobulin29) was used,
and limiting dilution analyses of the frequency of engrafting cells
were not feasible. Thus, the intriguing question of whether an
engraftment endpoint may underestimate the leukemic stem cell population in CML by a bias in favor of those that are quiescent must
await resolution from further studies. Similarly, the possibility that
some cycling leukemic stem cells contaminating the G0
fraction may have contributed to the engraftment seen here cannot be
completely ruled out.
The presence in CML patients of a quiescent population of leukemic
cells with progenitor activity was also demonstrated using a completely
independent approach. In this second strategy, we used CFSE labeling to
allow the specific isolation of cells that would remain viable but not
divide when cultured under different conditions. These experiments
confirmed that primitive CML cells can proliferate in the absence of
added growth factors,6,8,9 although a small number will
also remain quiescent for at least 4 days, even in the presence of a
strong mitogenic stimulus. The fact that such cells subsequently formed
colonies in vitro again indicates the transient nature of their arrest
in G0.
The mechanisms underlying the control of normal human hematopoietic
progenitor cell cycling are known to be complex and to involve the
integration of signals from the environment that either promote or
block cell cycle progression. Defects in a subset of these mechanisms
have been shown to exist in CML progenitors.47-49 However,
it is now clear that very primitive normal hematopoietic cells undergo
early changes in the types of external stimuli they respond to as well
as in the types of responses elicited by a given
stimulus.15,50,51 It is thus interesting to note that the
chemokines able to inhibit the proliferation of primitive normal but
not CML clonogenic cells are not effective in inhibiting the
proliferation of more primitive normal progenitors detected as
LTC-IC.52 Thus, the increased turnover exhibited by the
leukemic LTC-IC isolated from CML patients2 is likely to be
explained by another mechanism. One possibility would be the autocrine
production of IL-3 that we have recently described to occur in
primitive CML cells.9
However, these findings are not readily reconciled with the existence
of a subpopulation of primitive quiescent leukemic cells in patients
with CML. As a first approach to investigating an underlying mechanism,
we used single-cell RT-PCR to determine whether these cells might not
express BCR-ABL. However, in all 3 patients studied,  50% of the
freshly isolated G0 cells contained BCR-ABL mRNA. Thus,
BCR-ABL expression alone is not sufficient to force the rapid transit
through G1 of primitive hematopoietic cells. Interestingly,
preliminary studies indicate that such G0 BCR-ABL+ cells may not contain IL-3 or G-CSF mRNA. At least
some of these leukemic G0 cells can, nevertheless, survive
and subsequently proliferate even in the absence of further growth
factor stimulation (Holyoake et al, manuscript submitted).
This raises the possibility that quiescence and a non-growth factor
producing phenotype may be linked properties. Investigation of this
possibility is the subject of ongoing studies and could provide
important clues to potential therapeutic targets.
| |
ACKNOWLEDGMENT |
The authors acknowledge the help of Dr Michael Barnett and the staff in
the Leukemia and Bone Marrow Transplant Program as well as the Stem
Cell Assay Service for the provision, storage, and preparation of
normal bone marrow and CML samples. We also thank Pamela Austin, Chris
Laird, and Margaret Hale for technical assistance; Gloria Shaw for the
cytogenetic data; Maya Sinclaire for assistance with the animal
procedures; Gayle Thornbury and Giovanna Cameron for help with the flow
cytometry; Dr Peter Lansdorp (Terry Fox Laboratory); Amgen, Cangene,
Genentech, Immunex, Novartis, and StemCell for generous gifts of
reagents; and Bernadine Fox for typing the manuscript.
| |
FOOTNOTES |
Submitted February 19, 1999; accepted April 22, 1999.
Supported by grants from the National Cancer Institute of Canada (NCIC)
with funds from the Canadian Cancer Society and the Terry Fox Run and a
grant from Novartis Canada. T.H. holds a United Kingdom Leukaemia
Research Fund Senior Lectureship and C.E. is a Terry Fox Cancer
Research Scientist of the NCIC.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Allen Eaves, MD, PhD, Terry
Fox Laboratory, 601 W 10th Ave, Vancouver, British Columbia, Canada V5Z
1L3; e-mail: allen{at}terryfox.ubc.ca.
| |
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A. Bagg
Chronic Myeloid Leukemia: A Minimalistic View of Post-Therapeutic Monitoring
J. Mol. Diagn.,
February 1, 2002;
4(1):
1 - 10.
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H. G. Jorgensen, M. A. Elliott, E. K. Allan, C. E. Carr, T. L. Holyoake, and K. D. Smith
alpha 1-Acid glycoprotein expressed in the plasma of chronic myeloid leukemia patients does not mediate significant in vitro resistance to STI571
Blood,
January 15, 2002;
99(2):
713 - 715.
[Abstract]
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S. M. Graham, H. G. Jorgensen, E. Allan, C. Pearson, M. J. Alcorn, L. Richmond, and T. L. Holyoake
Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro
Blood,
January 1, 2002;
99(1):
319 - 325.
[Abstract]
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V. J. Weston, C. M. McConville, J. R. Mann, P. J. Darbyshire, S. Lawson, J. Gordon, P. A. H. Moss, A. Malcolm, R. Taylor, and T. Stankovic
Molecular Analysis of Single Colonies Reveals a Diverse Origin of Initial Clonal Proliferation in B-Precursor Acute Lymphoblastic Leukemia that Can Precede the t(12;21) Translocation
Cancer Res.,
December 1, 2001;
61(23):
8547 - 8553.
[Abstract]
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M. L. Guzman, S. J. Neering, D. Upchurch, B. Grimes, D. S. Howard, D. A. Rizzieri, S. M. Luger, and C. T. Jordan
Nuclear factor-{kappa}B is constitutively activated in primitive human acute myelogenous leukemia cells
Blood,
October 15, 2001;
98(8):
2301 - 2307.
[Abstract]
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S. Fukuda and L. M. Pelus
Regulation of the inhibitor-of-apoptosis family member survivin in normal cord blood and bone marrow CD34+ cells by hematopoietic growth factors: implication of survivin expression in normal hematopoiesis
Blood,
October 1, 2001;
98(7):
2091 - 2100.
[Abstract]
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D. P. Dialynas, M.-J. Lee, D. P. Gold, L.-e. Shao, A. L. Yu, M. J. Borowitz, and J. Yu
Preconditioning with fetal cord blood facilitates engraftment of primary childhood T-cell acute lymphoblastic leukemia in immunodeficient mice
Blood,
May 15, 2001;
97(10):
3218 - 3225.
[Abstract]
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I. Thornley, D. R. Sutherland, R. Nayar, L. Sung, M. H. Freedman, and H. A. Messner
Replicative stress after allogeneic bone marrow transplantation: changes in cycling of CD34+CD90+ and CD34+CD90{-} hematopoietic progenitors
Blood,
March 15, 2001;
97(6):
1876 - 1878.
[Abstract]
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S. Li, S. Gillessen, M. H. Tomasson, G. Dranoff, D. G. Gilliland, and R. A. Van Etten
Interleukin 3 and granulocyte-macrophage colony-stimulating factor are not required for induction of chronic myeloid leukemia-like myeloproliferative disease in mice by BCR/ABL
Blood,
March 1, 2001;
97(5):
1442 - 1450.
[Abstract]
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T. L. Holyoake, X. Jiang, H. G. Jorgensen, S. Graham, M. J. Alcorn, C. Laird, A. C. Eaves, and C. J. Eaves
Primitive quiescent leukemic cells from patients with chronic myeloid leukemia spontaneously initiate factor-independent growth in vitro in association with up-regulation of expression of interleukin-3
Blood,
February 1, 2001;
97(3):
720 - 728.
[Abstract]
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A. Bennaceur-Griscelli, C. Pondarre, V. Schiavon, W. Vainchenker, and L. Coulombel
Stromal cells retard the differentiation of CD34+CD38low/neg human primitive progenitors exposed to cytokines independent of their mitotic history
Blood,
January 15, 2001;
97(2):
435 - 441.
[Abstract]
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I.-H. Oh, A. Lau, and C. J. Eaves
During ontogeny primitive (CD34+CD38-) hematopoietic cells show altered expression of a subset of genes associated with early cytokine and differentiation responses of their adult counterparts
Blood,
December 15, 2000;
96(13):
4160 - 4168.
[Abstract]
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H. Glimm, I.-H. Oh, and C. J. Eaves
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0
Blood,
December 15, 2000;
96(13):
4185 - 4193.
[Abstract]
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B. Hennemann, I.-H. Oh, J. Y. Chuo, C. P. Kalberer, P. D. Schley, S. Rose-John, R. K. Humphries, and C. J. Eaves
Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin
Blood,
October 1, 2000;
96(7):
2432 - 2439.
[Abstract]
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T. H. Brummendorf, T. L. Holyoake, N. Rufer, M. J. Barnett, M. Schulzer, C. J. Eaves, A. C. Eaves, and P. M. Lansdorp
Prognostic implications of differences in telomere length between normal and malignant cells from patients with chronic myeloid leukemia measured by flow cytometry
Blood,
March 15, 2000;
95(6):
1883 - 1890.
[Abstract]
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T. Cheng, N. Rodrigues, H. Shen, Y. Yang, D. Dombkowski, M. Sykes, and D. T. Scadden
Hematopoietic Stem Cell Quiescence Maintained by p21cip1/waf1
Science,
March 10, 2000;
287(5459):
1804 - 1808.
[Abstract]
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X. Jiang, A. Lopez, T. Holyoake, A. Eaves, and C. Eaves
Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia
PNAS,
October 26, 1999;
96(22):
12804 - 12809.
[Abstract]
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TOP
ABSTRACT
INTRODUCTION