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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-05-1324.
NEOPLASIA
From the Terry Fox Laboratory, British Columbia Cancer
Agency, and the Departments of Medicine, Pathology, and Laboratory
Medicine, and Medical Genetics, University of British Columbia,
Vancouver, BC, Canada.
Primitive chronic myeloid leukemia cells display a unique autocrine
interleukin 3 (IL-3)/granulocyte-colony-stimluating factor (G-CSF)
mechanism that may explain their abnormal proliferation and
differentiation control. Here we show that BCR-ABL transduction of
primitive Sca-1+ lin Autocrine mechanisms have been well documented in many
types of hematologic neoplasms, but whether or how they contribute to
the initial development or subsequent maintenance of the malignant stem
cell compartment is not known. Expression of BCR-ABL,
the oncogene responsible for human chronic myeloid leukemia
(CML; reviewed in Sawyers1 and Holyoake2)
confers autonomous growth properties on a variety of growth
factor-dependent hematopoietic cell lines.3-6 In several
cases, acquisition of this growth factor independence has been shown to
be accompanied by, and at least partially dependent on, activation of
interleukin 3 (IL-3) production3,5,6 and activation of the
Janus kinase/signal transducer and activator of transcription
(JAK/STAT) pathway (reviewed in Sattler and Salgia7). Recently we showed that an autocrine
IL-3/granulocyte-colony-stimulating factor (G-CSF) mechanism is
activated in the primitive (CD34+) neoplastic cells from
patients with CML and the associated activation of STAT5 in these cells
is specifically dependent on their autocrine IL-3
response.8 An unusual feature of this autocrine mechanism is that it declines when the leukemic cells reach the terminal stages
of differentiation. This explains the well-known growth factor
dependence that human CML progenitors display when plated in semisolid
assays to detect their ability to form colonies of mature
cells,9 which contrasts with the extensive
factor-independent proliferative activity seen when primitive CML cells
are incubated in liquid suspension cultures,10 because in
liquid suspension cultures, the growth factor requirement at the later
stages of differentiation can be met by paracrine
mechanisms.8
IL-3 is also produced by the rapidly fatal leukemic cell populations
generated in mice injected with BCR-ABL-transduced mouse bone marrow
(BM) cells11,12 and by transformed hematopoietic cell lines
derived from BCR-ABL-transduced embryonic stem cells.13 Interestingly, it was recently shown that BCR-ABL-transduced
IL-3 To address this issue, we have re-examined the role of IL-3 in the
BCR-ABL-mediated transformation of very primitive hematopoietic cells
in a model in which the generation of disease in vivo is more
attenuated. The results of these studies show that BCR-ABL rapidly
activates a stable but initially redundant autocrine IL-3 mechanism in primitive hematopoietic cells. However, after
several weeks in culture, both autonomous growth in vitro and
leukemogenic activity in vivo are lost by
BCR-ABL+-transformed IL-3 Retroviral vectors and virus production
Plasmid DNA was isolated by Qiagen plasmid purification (Qiagen,
Hilden, Germany) and ecotropic Phoenix packaging
cells15 then transfected with 10 to 30 µg retroviral
plasmid DNA using calcium phosphate.16 The medium was
changed 24 hours later and virus-containing supernatants (VCS)
harvested after another 24 hours. Supernatants were passed through a
0.45 µ filter, aliquoted, and frozen at Infection of mouse BM cells
Cell sorting and flow cytometry
Cell culture Sorted cells were cultured at 1-5 × 105 cells/mL in DMEM plus 10% FCS with or without 100 ng/mL SF and 10 ng/mL IL-6 at 37°C. Viable cell numbers were determined from hematocytometer counts of trypan blue-excluding cells. Clone formation (presence of 2 refractile cells) and clone size (total number
of refractile cells) in wells seeded with single cells (using the FACS
cloning device) were assessed by direct visual inspection using an
inverted microscope.
Progenitor assays Colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were assayed using culture media from StemCell Technologies. No IL-3 was added to the CFC methylcellulose assay medium (which contained FCS plus 10 ng/mL human IL-6, 50 ng/mL murine SF and 3 U/mL human erythropoietin [EPO; StemCell Technologies] unless otherwise indicated) to ensure equal stimulation of IL-3 / and +/+ CFCs. Colony counts were performed after
12 to 14 days of incubation at 37°C using standard scoring
criteria.20 LTCs were set up using pre-established
irradiated mouse marrow LTC feeder layers and weekly half medium
changes of LTC medium containing fresh hydrocortisone as previously
described.20 In some CFC assays the following antibodies
were added alone or in combination: an anti-IL-3 antibody (SH15 purAB,
from H. Ziltener, Biomedical Research Centre, University of British
Columbia, Vancouver, BC, Canada), an anti-GM-CSF antibody (RAB 143, also from H. Ziltener), an anti-G-CSF antibody (R&D Systems,
Minneapolis, MN), or control sheep or rabbit IgG (from H. Ziltener).
Expanded populations of BCR-ABL-transduced +/+ and
IL-3 Reverse transcriptase-polymerase chain reaction analyses A semiquantitative procedure to assess cytokine mRNA expression was performed as previously described.8,22,23 Briefly, total RNA was extracted from aliquots of 1-2 × 104 cells and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using an oligo (dT)-based primer and poly (A) tailing procedure. For specific RT-PCR amplification, the RT reaction was performed in 20 µL with superscript II reverse transcriptase (Gibco/BRL, Rockville, MD) using random hexamer oligonucleotides in 50 µL 1X PCR buffer (Amersham Pharmacia) containing 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 2mM MgCl2, 200 µM of each dNTP (Gibco/BRL), 1 U Taq polymerase, and 10 pM of specific primers for BCR-ABL (5'-CAGGGTGCACACCGCAACGGCAA-3' and 5'-GTCCAGCGAGAAGGTTTTCCTTGGA-3'), mouse IL-3 (5'-CTCCAAGCTTCAATCAGTGGCC-3' and 5'-CGGTTCCACGGTTAGGAGAGAC-3'), mouse GM-CSF (5'-CTGCAGAATTTACTTTTCCTGGGC-3' and 5'-GCATTCAAAGGGGATATCAGTCAG-3'), mouse G-CSF (5'-CAAGTGAGGAAGATCCAGGCCA-3' and 5'-AGCCCTGCAGGTACGAAATGGC-3'), glyceraldehyde phosphate dehydrogenase (GAPDH) (5'-GTCTTCACCACCATGGAGAAGG-3' and 5'-GCCTGCTTCACCACCTTCTTGA-3') to give DNA fragments of 374 base pair (bp) (BCR-ABL), 455 bp (IL-3), 309 bp (GM-CSF), 409 bp (G-CSF), and 493 bp (GAPDH). We then performed 35 cycles of amplification (94°C for 30 seconds, 62°C for 60 seconds, 72°C for 60 seconds). Aliquots (10 µL) of the total amplified cDNA or specific amplified PCR products were then electrophoresed in a 1% agarose gel stained with ethidium bromide for quantification using a Kodak Digital Science ID scanner and image analysis software (Eastman Kodak Company, Rochester, NY). The total amplified cDNA was then transferred onto a nylon membrane (Zeta-probe; BioRad) and hybridized. cDNA probes for murine IL-3, murine GM-CSF, and murine G-CSF were provided by R. Kay and K. Humphries. Blots were exposed to Kodak XAR film (Eastman-Kodak) at 70°C for periods of 3 to 24 hours.
Southern blotting High-molecular-weight DNA was isolated using DNAZol (Gibco/BRL). DNA (15 µg) was digested with EcoR1, electrophoresed, and transferred to a nylon membrane.8 The blot was then hybridized with a GFP probe derived from the retroviral vector, and a BCR-ABL cDNA used to confirm the presence of an intact BCR-ABL gene.Western blotting Cells (105) were lysed in phosphorylation solubilization buffer (PSB; 50 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] buffer, 100 mM NaF, 10 nM sodium pyrophosphate, 2 mM Na3VO4, 4 mM ethylenediaminetetraacetic acid [EDTA]) containing 0.5% Nonidet P-4010 and Western analyses performed using a monoclonal anti-ABL antibody (8E9; Pharmingen, Mississauga, ON, Canada), an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Lake Placid, NY) and rabbit polyclonal anti-phospho-STAT5A/B and anti-STAT5A antibodies (Upstate Biotechnology).Animals C57BL/6 mice with a homozygous null mutation in the IL-3 gene24 (IL-3/
mice, provided by G. Dranoff, Dana Farber Cancer Institute), C57BL/6+/+ mice, nonobese diabetic-scid
(NOD/SCID) mice25 and NOD/SCID- 2 microglobulin (2m)/ mice26 were all bred
and maintained in the animal facility of the British Columbia Cancer
Research Centre (Vancouver, BC, Canada) in microisolator cages and were
provided with autoclaved food and water. NOD/SCID and
NOD/SCID-2m/ mice were irradiated at 8 to 12 weeks
of age with 350 cGy 137Cs  -rays and thereafter
received acidified water containing 100 mg/mL Ciprofloxacin (Bayer AG,
Leverkusen, Germany). Cells were injected into mice intravenously a few
hours after they had been irradiated. Mice were monitored daily for
signs of weight loss or lethargy. Peripheral blood was collected every
1 to 2 weeks and leukocytes analyzed by FACS as described above after
lysis of the red cells with ammonium chloride.
BCR-ABL rapidly activates an autocrine mechanism mediated by IL-3, GM-CSF, and G-CSF in primitive adult mouse BM cells To obtain a highly stem cell-enriched population of BCR-ABL (or control) virus-transduced cells, BM cells from 5-FU-treated +/+ mice were first exposed to a BCR-ABL or control MIG virus (Figure 1). The transduced (GFP+) fraction of Sca-1+ lin cells (11% ± 4% of the total cells present
immediately after transduction, n = 6) was then isolated by FACS
(Figure 2A). Already by this time, that
is, 4 days after the first exposure of the cells to BCR-ABL virus
(labeled as day 0 in the representative experiment shown in Figure
3), BCR-ABL transcripts were at maximum levels, as shown by semiquantitative gene expression analysis. Continued culture of both BCR-ABL-transduced and control
(MIG-transduced) cells in the presence of growth factors supported
their expansion at an indistinguishable rate (data not shown) and
sequential gene expression analyses of the BCR-ABL-transduced cells
demonstrated that IL-3 and GM-CSF transcripts consistently appeared in
them 3 days later (Figure 3). G-CSF transcripts were also detected in
them after another 3 days. No transcripts for any of these growth
factors were detected at any time in MIG-transduced cells. The
activated expression of IL-3, GM-CSF, and G-CSF in the
BCR-ABL-transduced cells initiated a multifactorial autocrine growth
mechanism. This was demonstrated by the inhibitory effects of adding
neutralizing IL-3, GM-CSF, or G-CSF antibodies to CFC assays of the
initially BCR-ABL-transduced (GFP+) Sca-1+
lin cells. As shown in Table
1, addition of antibody to a single growth factor gave a 15% to 35% reduction in colony formation and,
when antibodies to all 3 growth factors were combined, up to 50%
inhibition of factor-independent colony formation was seen.
To assess effects on a more primitive, functionally defined subset of
the initially transduced cells, LTC-IC assays were also performed on
freshly isolated GFP+ Sca-1+ lin
Reduced growth autonomy of BCR-ABL-transduced
IL-3 / mice. As expected, the
Sca-1+ lin BCR-ABL-transduced
IL-3/ cells produced no IL-3 transcripts. However, both
GM-CSF and G-CSF transcripts appeared with the same rapid kinetics as
seen in the +/+ cells (Figure 3). Consistent with this finding was the
observation of similar growth rates of the BCR-ABL-transduced IL-3/ and +/+ cells during the first 2 weeks both in
liquid suspension culture and in semisolid medium either with or
without added growth factors (data not shown). However, as shown in
Figure 4, the output of all types of CFCs generated in 4-week LTC-IC
assays of BCR-ABL-transduced IL-3/ cells was
consistently lower than in the corresponding +/+ assays (~2-fold
less, P < .002, although still increased above
MIG-transduced control values, P < .05). In addition, the
BCR-ABL-transduced IL-3/ LTC-ICs generated very few
factor-independent CFCs. To determine whether a unique role of
BCR-ABL-activated autocrine IL-3 production might be demonstrable
sooner, we assessed the growth potential of single BCR-ABL-transduced
Sca-1+ lin GFP+ +/+ and
IL-3/ cells in serum-free liquid medium without added
growth factors. Under these very stringent conditions, which preclude
any initial paracrine effects, individual BCR-ABL-transduced
IL-3/ cells showed a significantly reduced ability to
proliferate (P < .05, Figure
5).
To further examine the role of IL-3 in contributing to this reduced
growth factor autonomy, we evaluated the behavior of primitive BCR-ABL-transduced IL-3
Similar results were obtained when the effects of forced expression of
IL-3 were examined in single cell cultures of Sca-1+
lin Characterization of clonal isolates generated from primitive
BCR-ABL-transduced +/+ and IL-3 / cells for several weeks in medium
containing growth factors yielded rapidly growing populations of cells,
more than 95% of which showed a Sca-1+ c-kit+
lin IgE receptor+ (IgER+)
phenotype (Figure 6A) and mast cell
morphology (Figure 6B). Southern blot analysis and hybridization with a
GFP probe of EcoR1-digested extracts of DNA from these
expanded clones showed that most contained a single copy of the
retroviral cDNA with occasional examples of 2 integration sites (Figure
7). Rehybridization with a BCR-ABL cDNA
probe confirmed the presence of the full-length 7.3-kb
BCR-ABL gene in each case.
Even in the presence of SF and IL-6, the cells in all of the clones
derived from BCR-ABL-transduced IL-3
Cultures expanded from single BCR-ABL-transduced
IL-3 / cells, RT-PCR analyses were
undertaken to examine their growth factor gene expression profile. All
8 of the expanded clones of BCR-ABL-transduced +/+ cells still
contained IL-3 transcripts and 2 of these also still contained GM-CSF
transcripts (see Figure 9A versus Figure
3). However, none contained detectable levels of G-CSF mRNA (data not
shown). Interestingly, no transcripts for any of these 3 growth factors
were detected in any of the 6 expanded clones of BCR-ABL-transduced
IL-3/ BM cells, although their expression of BCR-ABL
appeared undiminished (Figure 9A).
Western analyses were also performed on extracts of the
BCR-ABL-transduced +/+ and IL-3 Expanded clonal populations of primitive, BCR-ABL-transduced +/+
cells produce a delayed but multilineage and ultimately fatal
myeloproliferative disease in vivo whereas IL-3 / BM cells confirmed the previously reported
ability of these cells to generate rapidly lethal leukemias (within 20 days) in irradiated (900 cGy) syngeneic recipients injected with large
numbers of unselected cells (3 to 5 × 105
cells/mouse) immediately after transduction.14 Even when
limiting numbers of transplantable transduced cells were injected by
decreasing the transplant 30- to 50-fold, death from the transduced +/+
cells occurred within 30 days and from the IL-3/ cells
within 45 days. Because the growth factor dependence of the in
vitro-expanded clones of BCR-ABL-transduced IL-3/ BM
cells appeared more profoundly compromised than the initial cells from
which they were derived, we also tested their in vivo leukemogenic
potential. In these experiments, we used sublethally irradiated (350 cGy) NOD/SCID and NOD/SCID-2m/ mice as hosts. This
was based on the need for strategies to avoid the lethal consequences
of a myeloablative preparative regimen and at the same time minimize
immune reactivity in a nonmyeloablated host to potential neoantigens
acquired by the cultured BCR-ABL-transduced cells. Cells from 3 different expanded clones of BCR-ABL-transduced +/+ BM cells (2 producing only IL-3 and one producing IL-3 and GM-CSF) and from 3 independently isolated clones of IL-3/ cells were
injected into both types of mice after a total period in vitro of 4 to
6 weeks. As shown in Figure 10A, no
mice injected with clonally expanded populations of BCR-ABL-transduced
+/+ cells died during the first 3 weeks and the first deaths in the
NOD/SCID mice did not occur until another 5 weeks later. Nevertheless, all mice injected with BCR-ABL-transduced +/+ cells eventually developed a GFP+ leukemia (Figure 10B) and were dead within
100 days, with the more immunodeficient NOD/SCID-2m/
mice dying approximately 2 weeks faster than the NOD/SCID hosts. However, there was no significant difference between the rate of death
in mice injected with BCR-ABL-transduced +/+ cells producing IL-3 only
and those producing GM-CSF as well as IL-3 (data not shown).
Remarkably, in spite of the apparently homogeneous mast cell phenotype
of the cells transplanted (Figure 6), the GFP+ leukemia
generated from the +/+ cells in vivo had multilineage features that
included the production of GFP+ B220+
(B-lineage) cells, GFP+ CD4+ or
CD8+ (T-lineage) cells, and GFP+
Ter119+ (erythroid) cells as well as large numbers of
GFP+ Gr-1+ or Mac-1+ (myeloid)
cells (Figure 10C). Although double-staining studies were not
performed, the fact that more than 80% of all the circulating leukocytes appeared as morphologically recognizeable neutrophils and
monocytes means that many of these must have also phenotypically abnormal in their expression of Sca-1 and/or c-kit. Conversely, only a
minority of the cells produced in vivo (< 2%) exhibited a mast
cell/basophil morphology (Table 3). The
leukemic mice also developed splenomegaly (Figure 10D), hepatomegaly,
and multilineage GFP+ hematopoiesis in all hematopoietic
tissues (Figure 10C) and BM and spleen cells from primary diseased mice
produced GFP+ leukemia with the same prolonged latent
period when transplanted into secondary recipients (n = 4, data
not shown). In marked contrast, none of the clonally expanded
populations of BCR-ABL-transduced IL-3
In the generation of human CML, expression of the BCR-ABL oncogene is initiated in a hematopoietic stem cell and this leads to the generation of an amplified clone of multiple lineages of differentiating myeloid and lymphoid progeny and a deregulated overproduction of granulocytes. These biologic features of the chronic phase of the human disease are explained by multiple abnormalities in the complex biologic mechanisms that control normal hematopoiesis. However, a clear understanding of molecular changes important at particular stages in the expansion of the chronic phase clone has remained elusive. Recently, we demonstrated the consistent activation in primitive CML cells of a novel IL-3- and G-CSF-mediated autocrine growth-stimulatory mechanism that appears to explain many of the altered biologic properties of the earliest neoplastic elements.8 In the present study we provide new evidence that IL-3-mediated
growth factor autonomy is important for the maintenance of leukemogenic
activity in BCR-ABL-transduced cells able to display hematopoietic
stem cell activity in vivo. First, phenotypically enriched
(Sca-1+ lin Previous investigations have described more modest effects on the
induction of leukemia by BCR-ABL-transduced murine
IL-3 The ability of BCR-ABL expression in primitive murine +/+ and
IL-3 An important and unexpected outcome of the present study was
the observation that apparently homogeneous populations of
proliferating mast cells generated in vitro from single primitive
BCR-ABL-transduced +/+ mouse BM cells consistently produced an
eventually fatal clone of leukemic cells in vivo that showed features
of multilineage differentiation. This was shown by the appearance
several months after the injection of the cultured cells of large
numbers of GFP+ cells in many hematopoietic tissues
including the blood, marrow, spleen, and liver that expressed antigens
characteristic of mature myeloid, erythroid, and lymphoid cells.
Although no attempt was made to explain how these phenotypes might have
been generated in vivo, BCR-ABL-induced lineage switching in
hematopoietic cells is an emerging theme.32,33 Thus, one
possibility would be that certain cells within the
proliferating clones of BCR-ABL+ mast cells generated in
this study were able to dedifferentiate or transdifferentiate in vivo
to produce a spectrum of myeloid and lymphoid cell
types.34,35 However, evidence that BCR-ABL can transform
primary adult mouse BM cells with multilineage potential is also well
established,11 and recently, clonal analysis of differentiating murine embryonic stem cells expressing high levels of
BCR-ABL revealed some that could produce primitive erythroid as well as
adult lymphoid and myeloid progeny.36 A second possibility would therefore be that the clonally expanded cells transplanted in
this study contained a rare subpopulation of BCR-ABL-transduced stem
cells with multilineage potential. Although the majority of cells in
the cultured BCR-ABL+ populations studied had a
lin In summary, we describe here an experimental protocol for obtaining BCR-ABL-transduced adult mouse BM cells that give rise to multilineage leukemias after a prolonged latency in nonmyeloablated immunodeficient hosts. We also show that a rapidly activated and persistent autocrine IL-3 mechanism is essential to the sustained leukemogenicity of these cells in this model. These findings reinforce the likely importance of autocrine IL-3 in the early stages of development of human CML and provide a new approach for further investigations of BCR-ABL-induced myeloid leukemia.
The authors thank Gayle Thornbury, Giovanna Cameron, and Rick Zapf
for FACS operation; Dianne Reid, Maya Sinclair, Margaret Hale, and
Karen Lambie for technical assistance; and Amy Ahamed for assistance in
preparing the manuscript. We also thank G. Dranoff (Dana Farber Cancer
Institute, Boston, MA) for providing the IL-3 C.J.E. was a Terry Fox Cancer Research Scientist of the NCIC.
Submitted May 8, 2002; accepted June 21, 2002.
Prepublished online as Blood First Edition Paper, July 5, 2002; DOI 10.1182/blood-2002-05-1324.
Supported by grants from the Leukemia Research Fund of Canada and the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society and from the Terry Fox Run.
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: C. J. Eaves, Terry Fox Laboratory, 601 West 10th Ave, Vancouver, BC, Canada V5Z 1L3; e-mail: ceaves{at}bccancer.bc.ca.
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X. Jiang, M. Stuible, Y. Chalandon, A. Li, W. Y. Chan, W. Eisterer, G. Krystal, A. Eaves, and C. Eaves Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia Blood, October 15, 2003; 102(8): 2976 - 2984. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or without any added growth factors (
) are shown
for the various combinations of target cells and viruses indicated.
Values shown are the means ± SEM from 3 experiments each with
MIG-, BCR-ABL- and BCR-ABL plus IL-3-transduced +/+ and
IL-3
