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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2461-2470
By
From the Leukemia Research Laboratory, Division of Haematology,
Hanson Centre for Cancer Research, IMVS, Adelaide, SA, Australia.
The interaction between p145c-KIT and
p210bcr-abl in transduced cell lines, and the selective
outgrowth of normal progenitors during long-term culture of chronic
myeloid leukemia (CML) cells on stroma deficient in stem-cell factor
(SCF) suggests that the response of CML cells to SCF may be abnormal.
We examined the proliferative effect of SCF(100 ng/mL), provided as the
sole stimulus, on individual CD34+ cells from five normal
donors and five chronic-phase CML patients. Forty-eight percent of
isolated single CML CD34+ cells proliferated after 6 days
of culture to a mean of 18 cells, whereas only 8% of normal
CD34+ cells proliferated (mean number of cells generated
was 4). SCF, as a single agent, supported the survival and expansion of
colony-forming unit-granulocyte-macrophage (CFU-GM) from CML
CD34+CD38+ cells and the more primitive CML
CD34+CD38
CHRONIC MYELOID leukemia (CML) is
characterized cytogenetically by the Philadelphia chromosome (Ph),
t(9;22)(q34;q1),1 and at the molecular level, by expression
of the bcr-abl fusion gene.2 The cell targeted by the Ph
translocation is usually a pluripotent stem cell,3 but
excessive proliferation is confined to the myeloid and megakaryocytic
lineages. Leukemic progenitors are capable of differentiation to
functional, mature hematopoietic cells.
In the early chronic phase of CML, the coexistence of normal
(Ph Normal hematopoiesis is a tightly regulated process involving a balance
between signals that stimulate and those that inhibit the proliferation
and differentiation of pluripotent progenitors. Many of these
regulatory signals are provided by interaction with the BM stromal
elements.11-19 The close association with stroma provides
hematopoietic cells with an array of cytokines that directly influence
cell survival, differentiation, and proliferation. Stromal cells
produce stem cell factor (SCF), which is an early acting cytokine of
248 amino acids that promotes the growth of all hematopoietic cell
lineages. SCF acts synergistically with other cytokines to promote
proliferation of normal progenitor cells in a variety of culture
systems. It does not increase the numbers of committed colony-forming
units (CFUs) but enhances granulocyte colony-stimulating factor-
(G-CSF), granulocyte-macrophage colony-stimulating factor- (GM-CSF),
interleukin-3- (IL-3), and IL-6-stimulated myelopoiesis in both
short- and long-term CD34+ cell cultures. SCF on its own
cannot recruit quiescent hematopoietic progenitor cells into cycle, but
it can prevent their apoptosis and is able to maintain the active
cell-cycle characteristics of isolated CD34+ cells in 2-day
cultures.20
SCF stimulates the growth of progenitor cells from most patients with
myelodysplastic syndromes21 and acute myeloid leukemia (AML),22,23 but reports on the effect of SCF on CML cell
proliferation have been conflicting. The addition of SCF to CML
cultures stimulated by G-CSF and/or GM-CSF has been reported to
induce little, or no, effect on proliferation.24
Conversely, erythroid CFU can be grown from CML cells in the absence of
erythropoietin, but only if SCF is present.25 Recently,
Hallek et al26 showed that the SCF receptors
p145c-KIT and p210bcr-abl form an intracellular
complex in bcr-abl positive, c-KIT positive cell lines. Fetal calf
serum (FCS)-supplemented colony-forming unit-granulocyte-macrophage
(CFU-GM) assays of CML bone marrow (BM) revealed a lack of response of
CML cells to SCF, and the authors concluded that activation of
p145c-KIT by p210bcr-abl usurped the role of
SCF and so rendered CML cells insensitive to this cytokine. Further
evidence of an abnormal response of CML cells to SCF is suggested by
the loss of CML cells, and a concomitant increase in normal
progenitors, during long-term culture on SCF-deficient
stroma.27
Cytokines also promote the attachment of normal hematopoietic
progenitors to stromal elements via activation of
The attachment of progenitors to FN in vitro has a negative effect on
proliferation that is independent of differentiation, necrosis, and
apoptosis.30,31 CML progenitors have an adhesive defect
that reduces their interaction with stroma in vitro.32 They
also show impaired attachment to FN.33 This reduced
attachment is brought about by a reduction in the steady-state affinity
of To investigate the effect of SCF on primary CML cells, we have examined
the proliferation of single leukemic CD34+ cells grown in
serum-deprived medium (SDM) with and without the addition of cytokines.
This assay system removes the potentially confounding influence of FCS
and possible paracrine growth factor production by accessory cells and
reveals the true response of leukemic hematopoietic progenitors to
individual cytokines or cytokine combinations.
We have further investigated the role of SCF in serum-deprived
conditions in augmentation of attachment of CML cells to immobilized FN
because proliferation and adhesion are correlated events in normal
CD34+ cells.34
Though p210bcr-abl may indeed activate
p145c-KIT in CML cells, 26 our findings
indicate that this activation is not complete because SCF, through
p145c-KIT, can provide additional adhesive and
proliferative signals. However, stimulation of CML cells with SCF alone
results in a potent proliferative signal that is not observed in cells
lacking bcr-abl gene expression.
BM Cells
Antibody Labeling and Cell Sorting
Antibody Labeling for Immunofluorescence Analysis Cells were resuspended in blocking buffer (HBSS/5% FCS, 1% BSA, 5% heat-inactivated normal human serum) and incubated on ice for up to 1 hour to block Fc-mediated antibody binding. For single-layer staining, cells were then incubated with fluorochrome-conjugated antibodies recognizing CD34, CD14, CD15, CD61, glycophorin A, CD19, or CD3 on ice for 45 minutes and then washed twice in ice cold immunofluorescence (IF) buffer (HBSS/5% FCS, 0.2 g/500 mL sodium azide) and fixed in 1% formamide in PBS. For two-layer staining, the cells were incubated with the anti-p145c-KIT antibody YB5.8 on ice for 45 minutes. After two washes in IF buffer, a secondary antibody was added (PE-conjugated sheep F(ab)2 fragments directed against mouse IgG) and incubated on ice for 30 minutes. The cells were washed twice in IF buffer and fixed in 1% formamide in PBS. Antibodies used in this study are shown in Table 1.
Cytokines and Chemicals Recombinant human SCF, IL-3, IL-6, G-CSF, and GM-CSF were generously provided by Amgen Inc (Thousand Oaks, CA). FN was purchased from Boehringer Mannheim (Mannheim, Germany). Culture media were purchased from Life Technologies.Pre-CFU Culture This is a stroma-free, cytokine-dependent, suspension culture system based on the method of Iscove et al.35 It measures the de novo generation of CFU-GM colonies as an index of preprogenitors. One thousand sorted CD34+ cells were resuspended in 1 mL of SDM with appropriate growth factors in 24-well plates. Cells were incubated at 37°C in a humidified atmosphere and, at 7-day intervals, were given fresh medium and growth factors. Before medium changes, an aliquot of cells was removed and plated in the CFU-GM assay.CFU-GM Assay Cells were plated in triplicate in 1-mL cultures containing 0.9% methylcellulose in IMDM supplemented with 30% FCS, 3 mmol/l L-glutamine, IL-3 (10 ng/mL), GM-CSF (10 ng/mL), IL-6 (20 ng/mL), G-CSF (100 ng/mL), and SCF (100 ng/mL). After 14 days incubation at 37°C in a humidified atmosphere with 5% CO2, CFU-GM colonies were scored as aggregates of greater than 50 cells.Reverse-Transcription Polymerase Chain Reaction (RT-PCR) of CFU-GM Colonies RNA extraction. Individual colonies were plucked with 5 µL of semisolid culture medium and vigorously mixed in 100 µL RNAZOL B (Biotecx Laboratories, Houston, TX). RNA extraction was continued according to the manufacturer's recommendations with the addition of 20 µg Glycogen (Boehringer Mannheim). cDNA synthesis. RNA was dissolved in 8 µL of diethyl pyrocarbonate-treated water and heated at 65°C for 10 minutes. After cooling the RNA rapidly on ice, 12.5 µL of RT mix was added and cDNA synthesis was performed at 37°C for 2 hours. RT mix consists of 80 mmol/L Tris pH 8.3; 120 mmol/L KCl; 4.8 mmol/L MgCl2; 16 mmol/L DTT; 0.1 mmol/L each of dATP, dCTP, dGTP, and dTTP; 0.01 µg/µL pdN6 (Pharmacia, Victoria, Australia); 1.4 U/µL RNAguard (Pharmacia); and 16 U/µL M-MLV RT (GIBCO-BRL, Gaithersburg, MD). RT was inactivated by heating at 65°C for 10 minutes. Nested PCR. The presence of abl gene transcripts (cDNA control) and bcr-abl transcripts was determined by two rounds of PCR using nested primers as previously described.36 Fluorescence in Situ Hybridization (FISH) Analysis FISH using bcr-abl probes (Vysis pty ltd, Downers Grove, IL) was performed as previously described.37 Cells were placed on a poly-L-lysine-coated microscope slide (Polysine; Biolab Scientific, Auckland, New Zealand) and swollen in situ in hypotonic (0.075 mol/L) KCl for 20 minutes. The cytoplasm was removed by three changes of ice cold 3:1 methanol:acetic acid, and then the slides were allowed to air dry. Cells thus treated were stored at 4°C for a maximum of 2 weeks before hybridization. Cells were treated with RNaseA (0.1 mg/mL 37°C) for 1 hour and were then heat denatured in 70% formamide/2× SSC. The denatured probe mixture was added to the slides, which had been dehydrated through graded ethanol and air dried. Hybridization continued overnight at 37°C. Posthybridization washes in 50% formamide/2× SSC were performed, and slides were mounted in antifade solution before examination by fluorescence microscope. On examination of interphase nuclei, the normal abl gene is seen as a red dot, the normal bcr gene is seen as a green dot, and the bcr-abl fusion gene appears either as a yellow dot or as adjacent red and green signals.Functional Assays Of Adhesion Estimation of the percent of the total cell population that is adherent. Ninety-six-well tissue culture-treated plates (Nunc, Roskilde, Denmark) were incubated overnight at 4°C with 50 µg/mL FN in PBS. This solution was removed by aspiration, and nonspecific binding to the wells was minimized by incubation at 37°C for 2 hours in 100 µL 2% BSA in RPMI 1640. Plates were washed three times with 0.2% BSA in RPMI 1640 (adhesion medium), transferred onto ice, and used immediately. Cytokines were added to protein-coated wells as specified. Cells, which had been deprived of growth factors overnight, were washed twice and resuspended in approximately 500 µL of RPMI 1640 containing 5 µmol/L Calcein-AM (Molecular Probes, Eugene, OR). The cells were incubated at 37°C for one hour, then washed three times in IMDM with 0.2% BSA, resuspended at 2 to 4 × 105 cells/mL in adhesion medium, and incubated on ice alone or with appropriate antibodies for 30 minutes. One hundred microliters of the cell suspension was then added, in triplicate, to FN- or BSA-coated microtitre plates on ice. The plates were centrifuged at 1,000 rpm for 5 minutes at 4°C to ensure direct and even contact with treated surfaces. After warming the plates to 37°C for 2 minutes on an incublock, they were transferred to a humidified 37°C incubator for 30 minutes. Nonadherent cells were removed by aspiration, and the wells were washed three times (or until the BSA-coated wells were clear) by the addition of 150 µL of adhesion medium. One hundred and fifty microliters of 1% sodium dodecyl sulfate (SDS), 1% NaOH solution was added to lyse the cells, and the fluorescence was measured using a Fluorimager (Molecular Dynamics, Sunnyvale, CA). Analysis of data was performed with ImageQuant software (Molecular Dynamics). The percentage of adherent cells was determined by dividing the fluorescence of the adherent fraction lysate by the fluorescence of the initial cell loading. Estimation of the number of CFU-GM that are adherent. The assays were performed as above in 6-well clusters (Nunc), except that cells were not prestained with calcein-AM. After removal of nonadherent cells, methylcellulose mixture and cytokines (as described in the CFU-GM assay) were added to the adherent cells and incubated at 37°C for 14 days. CFU-GM assays were also established for nonadherent cells. The number of colonies grown at day 14 was expressed as a percent of the total number of colonies that were obtained from 1,000 CD34+ cells that were plated directly in the CFU-GM assay and that had not undergone the adhesion assay.
CML CD34+ Cells Proliferate With SCF as the Sole Stimulus BM CD34+ cells from five CML patients and from five normal donors were cultured at a concentration of 103/mL in SDM + SCF (100 ng/mL), SDM alone, or SDM + 4 hematopoietic growth factor (HGF; IL-3 10 ng/mL, IL-6 20 ng/mL, G-CSF 100 ng/mL, and SCF 100 ng/mL). Growth of normal and leukemic CD34+ cells was below the limits of detection in the cytokine-free cultures. Normal CD34+ cells and CML CD34+ cells showed a 3,000-fold expansion in the presence of 4HGF after 21 days in culture. In contrast, only the CML cells proliferated in response to SCF alone (2,400-fold expansion ), whereas normal CD34 cells did not (Fig 1A).
CML CD34+ Cells Proliferate in a Single-Cell Assay With SCF as the Sole Stimulus Because SCF induces marked proliferation of normal CD34+ cells only when it acts in synergy with other cytokines, we postulated that the survival and subsequent growth of CML cells in SCF alone may have resulted from paracrine production of additional cytokines. To investigate whether individual CD34+ cells will survive in the absence of other cells, and to examine the number of cells that respond to SCF and the extent of their proliferation, single CD34+ cells were deposited by the ACDU of the FACStarplus into SDM alone, SDM supplemented with 100 ng/mL SCF, or SDM + 4HGF. CD34+ cells from normal donors failed to proliferate to a significant extent in SDM without cytokines and showed only minimal expansion in SCF (100 ng/mL) alone. At day 6 in SCF, 8% of normal CD34+ cells had proliferated to a mean of 4 cells/well (range 3 to 8, n = 48). Expansion in response to 4HGF was seen in 52% of normal CD34+ cells at day 6, and the mean number of cells grown from a single cell at this time point was 32 (range 3 to 65, n = 123). CML CD34+ cells showed only 8% proliferation in SDM without cytokines (mean number of cells generated was 5, range 3 to 9, n = 67). CML CD34+ cells showed significantly greater proliferation in SCF compared with normal cells (P < .001); 48% of CML cells showed proliferation in the presence of SCF alone. The mean number of cells grown from a single cell at this time point was 18, (range 3 to 72, n = 403). In 4HGF, a higher percentage (88%) of CML CD34+ cells proliferated compared with normal cells (P < .001), and the mean number of cells grown from a single cell was 49 (range 3 to 71, n = 152; Fig 2, Table 2)
CML CD34+ Cells Show Dose-Dependent Proliferation in SCF Whereas Cells Derived From Normal Donors Do Not Total CD34+ cells from three CML patients and three normal donors were cultured for 7 days in SDM with SCF in doses ranging from 0 ng/mL to 1,000 ng/mL in approximately half log increments. Cells were also cultured in 4HGF to assess viability. The CML cells showed a dose-dependent increase in proliferation to SCF, whereas the normal cells were not observed to proliferate at any concentration of SCF. All CML samples and normal samples proliferated in 4HGF (data not shown). In two of the three normal cell cultures, enumeration of cells at day 7 was not possible because the cell numbers were below the level of detection. The third normal cell culture was seeded at a concentration of 13.5 × 103 cells/mL and yielded enumerable cells after 7 days in SDM + SCF. A representative CML dose response curve to SCF and the single assay of normal cells for which results were obtained are shown in Fig 3.
Cells That Proliferate in Response to SCF Alone Are bcr-abl Positive Single CML CD34+ cells from two CML patients were cultured for 14 days in SCF alone or in 4HGF. RNA was extracted from expanded clones, and RT-PCR for the bcr-abl fusion gene was performed to assess the origin of the proliferating cells. All 18 clones expanded in SCF alone were positive for abl and bcr-abl gene transcripts by RT-PCR. This finding indicates that only the leukemic cells were SCF responsive. Furthermore, fusion of bcr and abl was shown in an additional five SCF responsive clones from one patient by FISH. Twenty-four clones expanded in 4HGF for 14 days were subjected to RT-PCR. Only 16 were bcr-abl positive, but all 24 were positive for expression of the normal abl allele (Table 3).
The Progeny of Cells Stimulated With SCF Alone Is Morphologically and Immunophenotypically Distinct From That Cultured in 4HGF The morphology of Jenner Giemsa-stained preparations of cultured normal and CML BM cells was examined. Six days after culture in SCF alone, the expanded CML cells showed an increase in size and resembled large blast cells morphologically. They had a high nuclear to cytoplasmic ratio and densely stained chromatin. Following an additional 7 days in SCF, some of these cells retained their primitive appearance, whereas others assumed a monocytoid appearance. Normal and CML cells grown in 4HGF differentiated into granulocytes and monocytoid cells by day 14. Typical cell morphology is shown in Fig 4. After 24 days of culture, the difference in morphology between CML cells grown in SCF and those grown in 4HGF was even more pronounced. SCF-derived cells appeared smaller and more homogeneous in size and shape than the 4HGF-derived cells, which varied in size, granularity, and nuclear shape (Fig 5).
Culture in SCF Alone Supports Survival of Leukemic Preprogenitor
Cells
Expression of P145c-kit Is Not Elevated in CML
CD34+ Cells
Autocrine Secretion of HGF by CML CD34+ Cells Is Not Sufficient to Stimulate Proliferation of Normal Cells in the Presence of SCF Alone Synergy between exogenous SCF and cytokines secreted by CML cells may explain why these cells respond to SCF in the absence of other exogenous cytokines. We explored this possibility by culturing mixtures of 1,000 male CML CD34+ cells and 1,000 female normal CD34+ cells in SCF or in 4HGF. The ratio of male (CML) cells to female (normal) cells was determined by FISH at day 14 using X and Y chromosome-specific probes. Two separate experiments were performed and contained cells from two male CML patients admixed with cells from two individual normal donors. The expansion of female cells in the SCF-stimulated cultures would suggest that their proliferation was enhanced by coculture with CML cells. Results of the FISH analyses are shown in Table 5. Proliferation of CML cells in 4HGF was quantitatively similar to proliferation of cells from a normal donor in 4HGF, but CML cells stimulated with only SCF had a marked growth advantage over normal donor cells grown in these conditions. Inability to detect female cells in 100 cells examined by FISH after 14 days in culture with SCF alone shows that these cells comprised fewer than 3% of the total cell number.38 These results indicate that autocrine cytokine secretion by CML cells is not sufficient to support the proliferation of other cytokine-dependent cells.
Enhancement of Adhesion to FN in Response to SCF Is Reduced in CML CD34+ Cells and Is Not Coupled to the Proliferative Response In normal CD34+ cells, increased proliferation in response to SCF stimulation is coupled with enhanced adhesion to FN.34 In accord with previously published data,29 a 30-minute stimulation with SCF resulted in a 2.4-fold increase (P < .01) in the number of normal CD34+ cells adhering to immobilized FN (n = 4), and the augmentation of adhesion of CML CD34+ cells in response to SCF stimulation was also significant (P < .05), but it was limited to 1.4-fold (n = 4). Maximal response was seen at 10 ng/mL for CML and normal CD34+ cells. A representative experiment (performed in triplicate) is shown in Fig 8.
In the present study, we have examined the role of SCF in the
proliferation and adhesion of CML Ph+ progenitors in vitro.
Our results show that SCF alone is sufficient to provide a strong
proliferative signal for CML CD34+ cells. This is in
contrast to the response of normal CD34+ cells, which show
substantial proliferation in SCF only when other growth factors are
present. In CML both the CD34+CD38+ cells and
the more primitive CD34+CD38 Submitted February 11, 1998;
accepted June 1, 1998.
The authors thank Dr J. Marty of Amgen Inc for gifts of recombinant
growth factors, Mario Nicola and Jeff Suttle for assistance with
diagnostic cytogenetic and RT-PCR analysis of CML samples, and Alan
Bishop and Sandy McIntyre for their expertise in flow cytometry.
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Abstract
Introduction
cells. These CFU-GM colonies
were all bcr-abl positive, showing the specificity of SCF stimulation
for the leukemic cell population. Coculture of CML and normal
CD34+ cells showed exclusive growth of Ph+
cells, suggesting that growth in SCF alone is not dependent on secretion of cytokines by CML cells. SCF augmentation of
1-integrin-mediated adhesion of CML CD34+
cells to fibronectin was not increased when compared with the effect on
normal CD34+ cells, suggesting that the proliferative and
adhesive responses resulting from SCF stimulation are uncoupled. The
increased proliferation may contribute to the accumulation of leukemic
progenitors, which is a feature of CML.
Abstract
.
, CML in SCF; 
, CML in 4HGF; 
, normal in SCF;
, normal in 4HGF. (B) 
