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HEMATOPOIESIS
From the Terry Fox Laboratory, British Columbia Cancer
Agency, and Departments of Medicine, Pathology and Laboratory Medicine,
and Medical Genetics, University of British Columbia, Vancouver,
British Columbia, Canada.
Retroviral transduction of primary hematopoietic cells with human
oncogenes provides a powerful approach to investigating the molecular
mechanisms controlling the normal proliferation and differentiation of
these cells. Here we show that primitive human CD34+ cord
blood cells, including multipotent as well as granulopoietic- and
erythroid-restricted progenitors, can be efficiently transduced with a
MSCV-BCR-ABL-IRES-GFP retrovirus, resulting in the sustained expression
by their progeny of very high levels of tyrosine phosphorylated p210BCR-ABL. Interestingly, even in the presence of growth
factors that supported the exclusive production of granulopoietic cells
from green fluorescent protein (GFP)-transduced control cells,
BCR-ABL-transduced progenitor subpopulations generated large numbers
of erythropoietin-independent terminally differentiating erythroid
cells and reduced numbers of granulopoietic cells. Analyses of
individual clones generated by single transduced cells in both
semisolid and liquid cultures showed this BCR-ABL-induced erythroid
differentiation response to be elicited at a high frequency from all
types of transduced CD34+ cells independent of their
apparent prior lineage commitment status. Additional experiments showed
that this erythroid differentiation response was largely prevented when
the cells were transduced and maintained in the presence of the
BCR-ABL-specific tyrosine kinase inhibitor, STI-571. These findings
indicate that overexpression of BCR-ABL in primary human hematopoietic
cells can activate an erythroid differentiation program in apparently
granulopoietic-restricted cells through a BCR-ABL kinase-dependent
mechanism, thus providing a new molecular tool for elucidating
mechanisms underlying lineage fate determination in human hematopoietic
cells and infidelity in human leukemia.
(Blood. 2002;99:3197-3204) The hematopoietic system is composed of a hierarchy
of progenitors with the most primitive stem cells uniquely
characterized by their combined capacity for lymphomyeloid lineage
differentiation, extensive self-renewal, and, on transplantation,
lifelong maintenance of blood cell production. The mechanisms
regulating the initial differentiation steps these cells undergo are
still poorly understood, although considerable evidence now indicates
that this occurs in a consistently ordered fashion to give a relatively
simple picture of progressive restriction in differentiation ability to
specific lineages.1 Thus it has been possible to define a
series of phenotypic changes that distinguish progenitors with different lineage options2-4 and to identify a number of
transcription factors whose presence (or absence) is important to the
ability of early hematopoietic cells to undertake specific
differentiation steps.1,5 To date, dedifferentiation or
transdifferentiation during normal hematopoiesis has not been
described, although examples of such events have recently been reported
to occur in neural cells6,7 or in occasional examples of
transformed hematopoietic cells.8-13
The BCR-ABL oncogene is a fusion gene that characterizes the
neoplastic clone of patients with chronic myeloid leukemia
(CML).14,15 It encodes a 210-kd oncoprotein
(p210BCR-ABL)16 with constitutively active
tyrosine kinase activity17 and an abnormal cytoplasmic
location.18 Both of these features are important to its
transforming activity in model systems.19-24 The BCR-ABL
oncoprotein is also known to perturb many intracellular signaling
pathways, including the RAS, mitogen-activated protein (MAP) kinase,
phosphatidylinositol-3' kinase (PI3-K), janus-kinase/signal transducer
and activator of transcription (JAK/STAT), and MYC pathways (for a
review, see Deininger et al25). The development of
packaging cells that allow production of high titer preparations of
p210BCR-ABL complementary (cDNA)-containing retroviruses
has greatly facilitated analysis of the effects of this gene in primary
hematopoietic cells. These have shown that BCR-ABL-transduced mouse
bone marrow cells produce a rapidly fatal CML-like myeloid leukemia on
their transplantation into irradiated recipients.26-28
However, the speed with which the leukemia develops is clearly not
comparable to the approximately 7 year average duration of the latent
phase of human CML.29 This discrepancy in disease kinetics
might be caused by species-specific effects of p210BCR-ABL
or, alternatively, by differences in BCR-ABL message levels determined by differences in the promoters active in the 2 systems (the endogenous BCR promoter in human CML cells and the long terminal repeat
[LTR] in retrovirally transduced hematopoietic cells). To
investigate this question we began experiments to examine the effects
of BCR-ABL expression in primary human CD34+ cord blood
(CB) cells after transduction with a p210BCR-ABL
retrovirus. This resulted in the unexpected but consistent observation of a rapid outgrowth of an erythroid population. Additional experiments designed to investigate the basis of this response suggest that it
resulted from the very high level of p210BCR-ABL kinase
activity attained in the target cells independent of their apparent
prior "commitment" status.
Virus production
Cells
Retroviral transduction Cells to be transduced were first cultured at 1 to 2 × 105/mL for 48 hours in complete serum-free medium (SFM) consisting of Iscoves medium supplemented with bovine serum albumin (BSA), insulin, and transferrin (BIT, StemCell), 40 µg/mL low-density lipoproteins, and 10 4 M 2-mercaptoethanol
(both from Sigma Chemicals, St Louis, MO) to which the following 5 purified recombinant human growth factors (GFs) were added: Flt-3
ligand (FL; 100 ng/mL, Immunex Corporation, Seattle, WA), Steel factor
(SF; 100 ng/mL, produced in the Terry Fox Laboratory), interleukin 3 (IL-3; 20 ng/mL, Novartis, Basel, Switzerland), interleukin-6 (IL-6; 20 ng/mL, Cangene, Mississauga, ON), and granulocyte colony-stimulating
factor (G-CSF; 20 ng/mL, StemCell). At the end of this 2-day
prestimulation period, the cells were resuspended in filtered VCM
supplemented with the same 5 GFs except for the studies shown in Table
3 where FL (100 ng/mL) plus granulocyte-macrophage CSF (GM-CSF, 20 ng/mL, Novartis) and G-CSF (20 ng/mL) were used as the GFs added during
the period of exposure to VCM. Protamine sulfate (5 µg/mL, Sigma) was
also added to the medium and the cells then placed in
fibronectin-coated Petri dishes previously "loaded" twice with VCM
(each time for 30 minutes). This infection procedure was repeated on
each of the next 2 consecutive days and another 24 hours later the
cells were transferred to fresh SFM plus GFs but no VCM and incubated for a further 24 hours before harvesting and further analysis. In 3 experiments, 0.25 µmol STI571 (Novartis) was added to the medium of
some of the transduction cultures.
Flow cytometry Viable (propidium iodide-negative [PI])
GFP+ and GFP CD34+ CB cells and
various subpopulations of CD34+ CB cells
(CD45RACD71;
CD45RA+CD71;
CD45RACD71+) were isolated at a purity of
more than 99% using a 3-laser FACStar+ (Becton Dickinson,
San Jose, CA) as described previously.2 Bulk cells were
collected in Eppendorf tubes containing complete SFM. Sorted
single cells were collected (using the single-cell deposition unit of
the FACS) into the individual round-bottom wells of 96-well plates that
had been prefilled with 100 µL of complete SFM. For the phenotype
analyses, cells were stained with anti-CD34 (8G12)-Cy5 and either
anti-CD15-phycoerythrin (PE) or anti-CD41a-PE (the latter both from
Pharmingen, Mississauga, ON) or anti-glycophorin-A-PE (10F7) or
anti-CD71-PE (OKT-9) or anti-CD13-PE or anti-CD33-PE or
anti-c-kit-PE or anti-CD36-PE (the last 4, all from Becton
Dickinson). They were then analyzed on a FACScalibur using Cell Quest
software (Becton Dickinson) with gates set to exclude more than 99.9%
of cells stained with irrelevant antibodies labeled with the
corresponding fluorochromes.
Cultures Transduced cells were cultured either in bulk cultures in complete SFM with FL, SF, IL-3, IL-6, and G-CSF, or as single cells in 96-well plates in the same medium with or without erythropoietin (epo, 3 U/mL, StemCell) as indicated. In some experiments 0.25 µmol STI 571 was also added. Colony-forming cell (CFC) assays were performed as described32 in SFM-containing methylcellulose cultures (StemCell) with human SF (50 ng/mL), 20 ng/mL each of IL-3, IL-6, G-CSF, and GM-CSF with or without epo (3 U/mL), or with FL (100 ng/mL), and 20 ng/mL G-CSF and GM-CSF, as indicated.Reverse transcriptase-polymerase chain reaction analyses Total RNA was extracted from aliquots of 104 cells as described previously.33 The reverse transcriptase (RT) reaction was performed in 20 µL with superscript II RNase H RT (Gibco/BRL, Rockville, MD) using random hexamer
oligonucleotides as primers (Pharmacia). Then, 5 µL of the RT
reaction was used for polymerase chain reaction (PCR) amplification in
50 µL volumes of 1 times PCR buffer (Gibco/BRL) containing 20 mmol
Tris-HCl (pH 8.4), 50 mmol KCL, 2 mM MgCl2, 200 µmol of
each dNTP (Amersham Pharmacia), 1 unit of Taq
polymerase, and 10 pmol of specific primers for BCR-ABL
5'-CAGGGTGCACAGCCGCAACGGCAA-3' and
5'-GTCCAGCGAGAAGGTTTTCCTTGGA-3'), actin 5'-GTGCGTGACATTAAGGAGAA-3' and
5'-GGAGGGGCCGGACTCGTCA-3') to give DNA fragments of 374 base pairs
(bp; BCR-ABL) and 471 bp (actin). Thirty-five cycles of amplification
(94°C for 30 seconds, 62°C for 1 minute, 72°C for 1 minute) were
then performed.
Southern blot analysis High-molecular-weight DNA from p210- or MIG-transduced cells was isolated using DNAzol (Gibco/BRL) and 15 µg DNA was digested with XbaI (which cuts once in the LTRs of the provirus) to release a full-length proviral fragment. The digested DNA was then electrophoresed on 0.8% agarose gel and processed as previously described34 using a 32P-dCTP-labeled (Amersham Pharmacia) GFP probe derived from the MIG retroviral vector.Western analyses Cells (105 cells per treatment group) were lysed in phosphorylation solubilization buffer (PSB; 50 mmol HEPES buffer, 100 mmol NaF, 10 mmol sodium pyrophosphate, 2 mmol Na3VO4, 4 mmol EDTA) containing 0.5% Nonidet P-40 and then Western analyses performed as previously described35 using a mouse monoclonal anti-Abl antibody (8E9, Pharmingen) and antiphosphotyrosine antibody (4G10, Upstate Biotechnology, Lake Placid, NY).Statistical analysis Results are shown as the mean ± SEM of values obtained in independent experiments. Differences between groups were assessed using the 2-tailed Student t test.
BCR-ABL-transduced CD34+ human CB cells generate predominantly erythroid cells in suspension cultures containing FL, SF, IL-3, IL-6, and G-CSF but no epo LinCD34+ CB cells were transduced with a
p210-GFP or MIG (control GFP) MSCV-IRES vector as described in
"Materials and methods" using a 6-day culture procedure in which
the cells were maintained in FL, SF, IL-3, IL-6, and G-CSF and exposed
on the third, fourth, and fifth days to fresh virus. FACS analysis of
GFP expression 2 days after the last exposure to fresh virus showed
that the overall efficiency of transduction by the p210 and MIG viruses was similar at 17% ± 6% versus 25% ± 8%, respectively
(n = 5). Corresponding proportions of GFP+ cells among
the CD34+ present at the end of the transduction procedure
were also not significantly different (10% ± 2% and 21% ± 8%,
respectively). Because the yield of CD34+ cells at the end
of the transduction procedure was similar between the 2 groups, the
absolute yields of GFP+ CD34+ cells were also
similar (1.8 ± 1.0 and 5.7 ± 5.1, respectively, per 100 initial
CD34+ cells, P = .3).
FACS-sorted GFP+ CD34+ cells isolated from both
transduced populations were then cultured further in the presence of
the same growth factors (FL, SF, IL-3, IL-6, and G-CSF). As shown in
Figure 1, both transduced populations
expanded rapidly under these conditions but the growth of the
p210-transduced cells was much faster (~105- versus
~103-fold over the first 14 days, P < .05,
n = 3). Southern blot analysis confirmed the presence of the expected
full-length BCR-ABL proviral sequence in DNA extracted from the
expanded p210-transduced cells (Figure
2A, lane 1) and RT-PCR analyses
demonstrated that they were expressing BCR-ABL messenger RNA (mRNA;
Figure 2B). Interestingly, as shown in Figure 2C, the p210-transduced
CB cells produced a higher level of p210BCR-ABL than was
seen in K562 cells (an erythroleukemic cell line isolated from a
patient with CML in blast crisis36) and the
p210BCR-ABL in the transduced CB cells also appeared to be
more highly tyrosine-phosphorylated.
Surprisingly, both morphologic (Figure 3)
and phenotype analyses (Figure 4)
revealed that, within 1 week in culture (after the transduction
procedure was completed), most of the cells generated by the
p210-transduced CD34+ cells were glycophorin A+
erythroblasts even though no epo had been added to the medium. By 2 weeks after transduction, the number of erythroid cells present in
these cultures had increased another 100-fold (Figure
5A,B) and many showed evidence of
terminal differentiation as indicated both by the presence of large
numbers of orthochromatic erythroblasts in Wright-Giemsa-stained
cytospin preparations and the red pellets obtained when the cells were
centrifuged. In contrast, the MIG-transduced cells continued to expand
less rapidly and did not produce recognizable erythroid cells.
Interestingly, the massive production of erythroblasts in the cultures
of p210-transduced cells was accompanied by a marked absolute decrease
in the output of terminally differentiating myeloid cells (Figure
5C,D). Follow-up studies of the cultures of p210-transduced cells after
more prolonged times showed that terminally differentiating erythroid
cells continued to be produced and were maintained as the predominant
cell type for many weeks (data not shown), in contrast to the
MIG-transduced cells that remained predominantly granulopoietic.
BCR-ABL transduction of CD34+ human CB cells alters the types of CFCs detectable immediately after transduction To determine which and how many progenitors were responsible for the altered growth and differentiation seen in bulk cultures of p210-transduced CD34+ CB cells, aliquots of the same transduced CD34+ GFP+ cells isolated immediately after transduction were plated in standard semisolid methylcellulose assays (both with and without epo). When epo was present, the p210-transduced cells made significantly fewer pure granulopoietic colonies than did the same number of MIG (control)-transduced cells (11 ± 3/100 versus 50 ± 7/100 initial CD34+ cells, P < .05, n = 5; see the footnote to Table 1 for a more detailed description of this normalization.). Under the same (plus epo) conditions, the p210-transduced cells also usually made fewer colonies containing both erythroid and granulopoietic cells. However, the numbers of these were more variable between experiments and overall the difference with the control cells did not reach significance (69 ± 17/100 versus 106 ± 18/100 initial CD34+ CB cells, respectively, P = .08). Conversely, an approximately 3-fold higher number of erythroid colonies was produced by the p210-transduced cells in the same assays, although the variability in these values again made the difference not significant (300 ± 150/100 versus 110 ± 52/100 initial CD34+ CB cells, P = .13). When epo was not added to the methylcellulose medium, the yield of granulopoietic colonies was the same as in the assays to which epo was added. However, in the absence of epo, the p210-transduced cells also produced many obviously hemoglobinized (red) colonies. As expected, these were not seen in parallel assays of the MIG-transduced cells (data not shown). Taken together, these findings suggest that overexpression of p210BCR-ABL in CD34+ CB cells can suppress normal granulopoietic differentiation and promote erythroid differentiation both in pluripotent and in apparently committed granulopoietic progenitors (rather than simply stimulating the amplified outgrowth of a subset of progenitors already restricted to differentiation along the erythroid pathway).
Evidence for altered programming of committed human CB progenitors by p210BCR-ABL To examine more rigorously the types of progenitors that could be stimulated to generate erythroid progeny following transduction with BCR-ABL, the following experiment was performed. LinCD34+ CB cells were first physically
separated by FACS into 3 biologically distinct subsets according to
their levels of expression of CD45RA and CD7137
before being transduced. As confirmed in Table 1, which
shows the CFC content of the 3 fractions of CD34+ cells
isolated (as assessed immediately after their isolation), this sort
strategy reproducibly yields subpopulations of CD34+ cells
in which more than 90% of the CFCs are either granulopoietic (in the
CD34+CD45RA+CD71 fraction) or
erythroid (in the
CD34+CD45RACD71+ fraction), or
contain the majority of the pluripotent CFCs (in the
CD34+CD45RACD71 fraction). The
remainder of the cells isolated in each of these CD34+
subsets were then transduced with either the p210 or the MIG virus
using the same 6-day transduction protocol as used before for the
unseparated CD34+ CB cells.
Analysis of the proportion of GFP+ cells in each fraction
at the end of the transduction procedure showed that the efficiency of
transduction of the
CD34+CD45RA The immediate effect of expressing p210BCR-ABL in the
progenitors present in the various subpopulations of transduced
CD34+ cells was first assessed by isolating the
GFP+ cells from each of the 6 transduced populations by
FACS (directly at the end of the transduction procedure) and then
plating these cells in equal numbers either in methylcellulose colony
assays (plus epo, Table 1) or in single-cell liquid suspension cultures (both with and without epo, Table 2).
Because all types of CFCs are amplified during the 6 days of GF
stimulation used in the transduction procedure, all values have been
normalized to 100 initial cells in each of the 3 CD34+
subsets. The magnitude of these increases can thus be seen by comparison of the "pretransduction" values and the corresponding "post-(MIG) transduction" values. Interestingly, when the results of the assays of the p210 and MIG-transduced cells are compared, there
is no significant difference in the total numbers of colonies produced
from any of the 3 subsets of CD34+ cells transduced.
However, p210 transduction of the granulopoietic (CD34+CD45RA+CD71
Because less than 40% (6%-32%) of the transduced cells from any of
the CD34+ subsets were able to produce a colony in
methylcellulose, the CFC data in Table 1 do not exclude the possibility
that the increased numbers of erythroid CFCs and decreased numbers of
mixed and granulopoietic CFCs detected could result from separate
effects of p210BCR-ABL on different cell types. To address
this, we also analyzed the differentiation of clones of cells produced
in single-cell liquid cultures containing FL, SF, IL-3, IL-6, and G-CSF
with and without epo, to maximize and minimize, respectively, the
detection of cells with "normal" erythroid potential. As expected,
under these conditions, the frequency of clone formation
( In 2 further experiments, the granulopoietic progenitor-enriched
(CD45RA+CD71
Low doses of STI571 normalize the type of progeny obtained from p210-transduced CD34+ CB cells Because of the very high levels of p210BCR-ABL found in the transduced cells, we hypothesized that the excessive kinase activity of this oncoprotein was responsible for their increased growth rate and deregulated production of erythroid progeny. To test this hypothesis, we took advantage of the specificity of STI571 as an inhibitor of the p210BCR-ABL kinase.23 As shown in Figure 1, when CB cells were transduced and subsequently cultured in the presence of 0.25 µmol STI571, the growth rate of the p210-transduced cells became similar to that of the MIG-transduced cells. Moreover, the STI571-treated p210-transduced cells also showed a more "normal" differentiation behavior with a predominance of granulopoietic cells among those produced in the first 2 weeks (at which time the cultures were terminated for analysis, Figure 5). As expected, STI571 had no influence on the growth or differentiation of the MIG-transduced cells. Western analyses confirmed that the dose of STI 571 used caused a decrease in the level of tyrosine phosphorylated p210BCR-ABL and other proteins present in the progeny of the p210-transduced cells (data not shown).
The human myeloid leukemias, like many malignancies, are clonal disorders believed to arise through a series of mutational events that first occur in the stem cell compartment of the tissue. Although the first event is assumed to confer a proliferative advantage on the affected cell, evolution to a "malignant" leukemia is characterized by a failure of the cells to mature normally. As a result, large numbers of cells that appear "undifferentiated" accumulate. In some cases, leukemic cells that display an abnormal, mixed lineage phenotype are also produced.38 These latter effects on differentiation are believed to be caused by the acquisition of additional mutations. Nevertheless, the nature of these mutations and how they may perturb normal mechanisms of hematopoietic progenitor commitment and differentiation remain poorly understood. Here we provide evidence that p210BCR-ABL, when expressed at sufficiently high levels in primary human hematopoietic CB cells, can alter their normal pattern of differentiation. This was first suggested by the increased output of differentiating erythroid cells and decreased output of differentiating granulopoietic cells seen in bulk suspension cultures of p210-transduced CB cells. Subsequent experiments showed this reflected a reprogramming of the majority of all progenitors in the p210-transduced CD34+ CB population, independent of their original lineage restriction, even under growth factor conditions that normally selectively promote granulopoietic differentiation. To our knowledge this is the first report of experimentally induced altered programming of a primary human hematopoietic cell, although several examples of oncogene or growth factor-induced lineage switching in previously immortalized or transformed murine hematopoietic cell lines have been described in the past.8-11,13 Of particular relevance here is the single report of spontaneous lineage switching from a mast cell to an erythroid or myeloid phenotype in certain established lines of BCR-ABL-transformed murine cells.12 More recently p185BCR-ABL transduction of human HL60 cells (a myeloid human leukemic cell line39) was found to activate an erythroid program in these cells.40 Taken together, these findings suggest that lineage commitment mechanisms in the hematopoietic system are more flexible and susceptible to perturbation at much later stages of differentiation than previously appreciated. Such a concept has recently been provided with a mechanistic basis through emerging evidence that suppression of alternative lineage-specific transcription factors and the downstream genes they regulate may be as important to the process of lineage determination as lineage-specific gene activation.41-44 Accordingly, it will be of interest in future work to examine the sequence of gene expression and biochemical changes that occur as a result of the highly up-regulated expression of p210BCR-ABL that is achieved in the novel model described here. The ability of STI571 to partially reverse the p210BCR-ABL- induced suppression of granulopoietic differentiation and concomitant up-regulation of the generation of erythroid cells shows that these effects require the kinase activity of p210BCR-ABL. Preliminary experiments indicate that these effects are also obtained in transduced CD34+ cells from normal adult human bone marrow. Thus, the mechanisms involved do not appear to be exclusive to ontogenically early target cells although this may influence the likelihood of obtaining the reprogramming response observed here. In contrast to the essential role of the kinase domain of p210BCR-ABL, experiments using a mutant BCR-ABL vector indicate an intact SH2 domain is not required (Y.C. et al, manuscript in preparation). It should be noted that in CML patients' CD34+ cells, a level of p210BCR-ABL equivalent to that obtained here using an MSCV LTR promoter may never be achieved in the chronic phase of the disease and perhaps only rarely in blast crisis. Interestingly in blast crisis, leukemic cells expressing markers of erythroid differentiation are sometimes seen.45,46 Even in the chronic phase, both erythroid and myeloid progenitors are proportionately increased and, although only the output of mature granulocytes is usually elevated,47 occasional examples of Philadelphia chromosome-positive (Ph+) patients presenting with an erythrocytosis have been reported.48 Thus, in retrospect, the present findings may be less at odds with the pathologic features of human CML than would appear at first glance. Differences in the level of BCR-ABL expression may also explain the differences between the results described here and those of Zhao et al49 who reported an increase in granulopoietic but not erythroid progenitors in BCR-ABL-transduced CD34+ CB cells, although in their study, objective documentation of the amplified granulopoiesis described was not provided. Our findings also provide the first reported example of epo-independent erythropoiesis induced by the introduction of an oncogene in primary human hematopoietic cells. Epo independence is a well-established, albeit variably detectable, feature of the neoplastic erythroid progenitors from patients with CML.50 The present findings confirm this to be a consequence of BCR-ABL expression in human erythroid cells, consistent with a number of previous studies with murine targets showing a similar effect of v-abl51 and more recently of BCR-ABL even in progenitor cells lacking erythropoietin receptors.52 However, further analyses will be required to determine what are the downstream targets in p210BCR-ABL-expressing human erythroid cells. In summary, we have found that expression of high levels of the p210BCR-ABL oncoprotein in primitive human CB cells suppresses granulopoietic differentiation and simultaneously activates entry and progression down the erythroid pathway. This occurs even in apparently committed granulopoietic progenitors and is accompanied by a partial abrogation of the normal requirement of terminally differentiating erythroid cells for stimulation by exogenous epo. Further investigation of this novel transdifferentiation response may offer insights into the normal mechanisms of lineage restriction in primitive human hematopoietic cells as well as the mechanisms by which they can be perturbed in advanced stages of human myeloid leukemia.
We thank the members of the Stem Cell Assay Service of the Terry Fox Laboratory for initial cell processing; Gayle Thornbury, Giovanna Cameron, and Rick Zapf for operating the FACStar+; J. Griffin (Dana Farber Cancer Institute, Boston MA) for the BCR-ABL plasmid; K. Humphries and P. Lansdorp (both from the Terry Fox Laboratory) for various cDNA probes and antibodies; and StemCell, Novartis, Cangene, and Immunex for gifts of recombinant growth factors and other reagents.
Submitted August 13, 2001; accepted December 26, 2001.
Supported by grants from the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society and the Terry Fox Run. Y.C. was supported by a Terwindt Foundation Fellowship, and C.E. was a Terry Fox Cancer Research Scientist of the NCIC.
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: Connie J. Eaves, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC, V5Z 1L3, Canada; e-mail: ceaves{at}bccancer.bc.ca.
1. Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat Genet. 2000;1:57-64[CrossRef][Medline] [Order article via Infotrieve].
2.
Lansdorp PM, Dragowska W.
Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow.
J Exp Med.
1992;175:1501-1509 3. Kondo M, Weissman IL, Akashi K. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell. 1997;91:661-672[CrossRef][Medline] [Order article via Infotrieve]. 4. Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature. 2000;404:193-197[CrossRef][Medline] [Order article via Infotrieve]. 5. Lawrence HJ, Sauvageau G, Largman C, Humphries RK. Homeobox gene networks and the regulation of hematopoiesis. In: Zon G, ed. Hematopoiesis A Developmental Approach. New York, NY: Oxford University Press; 2001:402-414.
6.
Laywell ED, Rakic P, Kukekov VG, Holland EC, Steindler DA.
Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain.
Proc Natl Acad Sci U S A.
2000;97:13883-13888
7.
Kondo T, Raff M.
Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells.
Science.
2000;289:1754-1757 8. Klinken SP, Alexander WS, Adams JM. Hemopoietic lineage switch: v-raf oncogene converts Em-myc transgenic B cells into macrophages. Cell. 1988;53:857-867[CrossRef][Medline] [Order article via Infotrieve].
9.
Borzillo GV, Ashmun RA, Sherr CJ.
Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.
Mol Cell Biol.
1990;10:2703-2714
10.
Chiba T, Nagata Y, Kishi A, et al.
Induction of erythroid-specific gene expression in lymphoid cells.
Proc Natl Acad Sci U S A.
1993;90:11593-11597 11. Martin M, Strasser A, Baumgarth N, et al. A novel cellular model (SPGM 1) of switching between the pre-B cell and myelomonocytic lineages. J Immunol. 1993;150:4395-4406[Abstract].
12.
Elefanty AG, Cory S.
bcr-abl-Induced cell lines can switch from mast cell to erythroid or myeloid differentiation in vitro.
Blood.
1992;79:1271-1281 13. Kondo M, Scherer DC, Miyamoto T, et al. Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature. 2000;407:383-386[CrossRef][Medline] [Order article via Infotrieve]. 14. Groffen J, Stephenson JR, Heisterkamp N, De Klein A, Bartram CR, Grosveld G. Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984;36:93-99[CrossRef][Medline] [Order article via Infotrieve]. 15. Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature. 1985;315:550-554[CrossRef][Medline] [Order article via Infotrieve].
16.
Ben-Neriah Y, Daley GQ, Mes-Masson AM, Witte ON, Baltimore D.
The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene.
Science.
1986;233:212-214
17.
Konopka JB, Witte ON.
Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl gene products.
Mol Cell Biol.
1985;5:3116-3123 18. Van Etten RA, Jackson P, Baltimore D. The mouse type IV c-abl gene product is a nuclear protein, and activation of transforming ability is associated with cytoplasmic localization. Cell. 1989;58:669-678[CrossRef][Medline] [Order article via Infotrieve].
19.
Daley GQ, Van Etten RA, Baltimore D.
Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.
Science.
1990;247:824-830 20. Heisterkamp N, Jenster G, ten Hoeve J, Zovich D, Pattengale PK, Groffen J. Acute leukemia in bcr/abl transgenic mice. Nature. 1990;344:251-253[CrossRef][Medline] [Order article via Infotrieve].
21.
Lugo TG, Pendergast AM, Muller AJ, Witte ON.
Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.
Science.
1990;247:1079-1082
22.
Evans CA, Owen-Lynch J, Whetton AD, Dive C.
Activation of the Abelson tyrosine kinase activity is associated with suppression of apoptosis in hemopoietic cells.
Cancer Res.
1993;53:1735-1738 23. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566[CrossRef][Medline] [Order article via Infotrieve]. 24. Vigneri P, Wang JYJ. Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase. Nat Med. 2001;7:228-234[CrossRef][Medline] [Order article via Infotrieve]. 25. Deininger MWN, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia. Blood. 2000;3343-3356.
26.
Pear WS, Miller JP, Xu L, et al.
Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.
Blood.
1998;92:3780-3792
27.
Li S, Ilaria RL, Million RP, Daley GQ, Van Etten RA.
The p190, p210, and p230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.
J Exp Med.
1999;189:1399-1412
28.
Zhang X, Ren R.
BCR-ABL efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia.
Blood.
1998;92:3829-3840 29. Ichimaru M, Ishimaru T, Mikami M, Yamada Y, Ohkita T. Incidence of leukemia in a fixed cohort of atomic bomb survivors and controls, Hiroshima and Nagasaki October 1950-December 1978. Technical Report RERF TR 13-81. Hiroshima: Radiation Effects Research Foundation; 1981. 30. Kinsella TM, Nolan GP. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum Gene Ther. 1996;7:1405-1413[Medline] [Order article via Infotrieve].
31.
Pear WS, Nolan GP, Scott ML, Baltimore D.
Production of high-titer helper-free retroviruses by transient transfection.
Proc Natl Acad Sci U S A.
1993;90:8392-8396
32.
Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ.
Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor.
Blood.
1996;88:3765-3773
33.
Maguer-Satta V, Petzer AL, Eaves AC, Eaves CJ.
BCR-ABL expression in different subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic myeloid leukemia.
Blood.
1996;88:1796-1804
34.
Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C.
Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia.
Proc Natl Acad Sci U S A.
1999;96:12804-12809 35. Maguer-Satta V, Burl S, Liu L, et al. BCR-ABL accelerates C2-ceramide-induced apoptosis. Oncogene. 1998;16:237-248[CrossRef][Medline] [Order article via Infotrieve]. 36. Andersson LC, Jokinen M, Gahmberg CG. Induction of erythroid differentiation in the human leukaemia cell line K562. Nature. 1979;278:364-365[CrossRef][Medline] [Order article via Infotrieve].
37.
Mayani H, Dragowska W, Lansdorp PM.
Characterization of functionally distinct subpopulations of CD34+ cord blood cells in serum-free long-term cultures supplemented with hematopoietic cytokines.
Blood.
1993;82:2664-2672
38.
McCulloch EA.
Stem cells in normal and leukemic hemopoiesis.
Blood.
1983;62:1-13 39. Collins SJ, Gallo RC, Gallagher RE. Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature. 1977;270:347-349[CrossRef][Medline] [Order article via Infotrieve].
40.
Fang G, Kim CN, Perkins CL, et al.
CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs.
Blood.
2000;96:2246-2253 41. Nutt SL, Heavey B, Rolink AG, Busslinger M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature. 1999;401:556-562[CrossRef][Medline] [Order article via Infotrieve].
42.
Rekhtman N, Radparvar F, Evans T, Skoultchi AI.
Direct interaction of hematopoietic transcription factors PU.1 and GATA-1: functional antagonism in erythroid cells.
Genes Dev.
1999;13:1398-1411
43.
Nerlov C, Querfurth E, Kulessa H, Graf T.
GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription.
Blood.
2000;95:2543-2551
44.
Zhang P, Behre G, Pan J, et al.
Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1.
Proc Natl Acad Sci U S A.
1999;96:8705-8710
45.
Ekblom M, Borgstrom G, Von Willebrand E, Gahmberg CG, Vuopio P, Andersson LC.
Erythroid blast crisis in chronic myelogenous leukemia.
Blood.
1983;62:591-596
46.
Saikia T, Advani S, Dasgupta A, et al.
Characterization of blast cells during blastic phase of chronic myeloid leukaemia by immunophenotyping 47. Eaves C, Cashman J, Eaves A. Defective regulation of leukemic hematopoiesis in chronic myeloid leukemia. Leuk Res. 1998;22:1085-1096[CrossRef][Medline] [Order article via Infotrieve]. 48. Hoppin EC, Lewis JP. Polycythemia rubra vera progressing to Ph1-positive chronic myelogenous leukemia. Ann Intern Med. 1975;83:820-823.
49.
Zhao RC, Jiang Y, Verfaillie CM.
A model of human p210bcr/ABL mediated CML by transducing primary normal human CD34+ cells with a BCR/ABL containing retroviral vector.
Blood.
2001;97:2406-2412 50. Eaves AC, Henkelman DH, Eaves CJ. Abnormal erythropoiesis in the myeloproliferative disorders: an analysis of underlying cellular and humoral mechanisms. Exp Hematol. 1980;8(Suppl 8):235-245. 51. Waneck GL, Keyes L, Rosenberg N. Abelson virus drives the differentiation of Harvey virus-infected erythroid cells. Cell. 1986;44:337-344[CrossRef][Medline] [Order article via Infotrieve].
52.
Ghaffari S, Wu H, Gerlach M, Han Y, Lodish HF, Daley GQ.
BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells.
Proc Natl Acad Sci U S A.
1999;96:13186-13190
© 2002 by The American Society of Hematology.
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  L. L. Zhou, Y. Zhao, A. Ringrose, D. DeGeer, E. Kennah, A. E.-J. Lin, G. Sheng, X.-J. Li, A. Turhan, and X. Jiang AHI-1 interacts with BCR-ABL and modulates BCR-ABL transforming activity and imatinib response of CML stem/progenitor cells J. Exp. Med., October 27, 2008; 205(11): 2657 - 2671. [Abstract] [Full Text] [PDF]
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 S. Chu, L. Li, H. Singh, and R. Bhatia BCR-Tyrosine 177 Plays an Essential Role in Ras and Akt Activation and in Human Hematopoietic Progenitor Transformation in Chronic Myelogenous Leukemia Cancer Res., July 15, 2007; 67(14): 7045 - 7053. [Abstract] [Full Text] [PDF]
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 J. A. Kennedy, F. Barabe, B. J. Patterson, J. Bayani, J. A. Squire, D. L. Barber, and J. E. Dick Expression of TEL-JAK2 in primary human hematopoietic cells drives erythropoietin-independent erythropoiesis and induces myelofibrosis in vivo PNAS, November 7, 2006; 103(45): 16930 - 16935. [Abstract] [Full Text] [PDF]
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A. Takeda, C. Goolsby, and N. R. Yaseen NUP98-HOXA9 Induces Long-term Proliferation and Blocks Differentiation of Primary Human CD34+ Hematopoietic Cells. Cancer Res., July 1, 2006; 66(13): 6628 - 6637. [Abstract] [Full Text] [PDF]
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 S. M. Vercauteren and H. J. Sutherland Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo Blood, October 15, 2004; 104(8): 2315 - 2322. [Abstract] [Full Text] [PDF]
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P. Ramaraj, H. Singh, N. Niu, S. Chu, M. Holtz, J. K. Yee, and R. Bhatia Effect of Mutational Inactivation of Tyrosine Kinase Activity on BCR/ABL-Induced Abnormalities in Cell Growth and Adhesion in Human Hematopoietic Progenitors Cancer Res., August 1, 2004; 64(15): 5322 - 5331. [Abstract] [Full Text] [PDF]
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C. S. Huettner, S. Koschmieder, H. Iwasaki, J. Iwasaki-Arai, H. S. Radomska, K. Akashi, and D. G. Tenen Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome Blood, November 1, 2003; 102(9): 3363 - 3370. [Abstract] [Full Text] [PDF]
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 C. Wang, N. Pattabiraman, J. N. Zhou, M. Fu, T. Sakamaki, C. Albanese, Z. Li, K. Wu, J. Hulit, P. Neumeister, et al. Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor {gamma} Expression and Transactivation Mol. Cell. Biol., September 1, 2003; 23(17): 6159 - 6173. [Abstract] [Full Text] [PDF]
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 L. Haughn, R. G. Hawley, D. K. Morrison, H. von Boehmer, and D. M. Hockenbery BCL-2 and BCL-XL Restrict Lineage Choice during Hematopoietic Differentiation J. Biol. Chem., June 27, 2003; 278(27): 25158 - 25165. [Abstract] [Full Text] [PDF]
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S. Wong, J. McLaughlin, D. Cheng, and O. N. Witte Cell context-specific effects of the BCR-ABL oncogene monitored in hematopoietic progenitors Blood, May 15, 2003; 101(10): 4088 - 4097. [Abstract] [Full Text] [PDF]
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R. L. Darley, L. Pearn, N. Omidvar, M. Sweeney, J. Fisher, S. Phillips, T. Hoy, and A. K. Burnett Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells Blood, December 1, 2002; 100(12): 4185 - 4192. [Abstract] [Full Text] [PDF]
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X. Jiang, E. Ng, C. Yip, W. Eisterer, Y. Chalandon, M. Stuible, A. Eaves, and C. J. Eaves Primitive interleukin 3 null hematopoietic cells transduced with BCR-ABL show accelerated loss after culture of factor-independence in vitro and leukemogenic activity in vivo Blood, November 15, 2002; 100(10): 3731 - 3740. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
190°C. Titers of the p210 and MIG virus were 2 to 5 and 5 to
9 × 105/mL, respectively, as assessed by green
fluorescence protein (GFP) expression in NIH 3T3 cells.
32 cells/well) when epo was present was generally
higher than 50% (Table 
experience in 60 patients.
Leuk Res.
1988;12:499-506