Stem Cells http://www.peprotech.com/
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Freedman, M. H.
Right arrow Articles by Squire, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Freedman, M. H.
Right arrow Articles by Squire, J.
Stem Cells, Vol. 14, No. 6, 690-701, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Juvenile Chronic Myelogenous Leukemia Multilineage CD34+ Cells: Aberrant Growth and Differentiation Properties

Melvin H. Freedman, Johann K. Hitzler, Nancy Bunin, Tom Grunberger, Jeremy Squire

Division of Hematology/Onocology, Department of Pediatrics, and Department of Pediatric Laboratory Medicine, Division of Pathology, Hospital for Sick Children, University of Toronto, Toronto, Canada; Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, USA

Key Words. Juvenile chronic myelogenous leukemia • CD34+ cells • Monocyte-macrophage colonies

Dr. Melvin H. Freedman, Division of Hematology/Oncology, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Juvenile chronic myelogenous leukemia (JCML) is a hematologic malignancy of monocyte-macrophage lineage in which leukemic progression is mediated in an autocrine manner by tumor necrosis factor (TNF-{alpha}), GM-CSF and possibly other growth factors. Cytogenetic data showing involvement of both erythroid and monocyte-macrophage lineages in the JCML leukemic clone, as well as an observed episode of B-lineage lymphoid blast crisis in JCML, has strengthened the thesis for a lymphohematopoietic pluripotent stem cell origin for the disorder. To study this further, JCML CD34+ cells from bone marrow (BM) or spleen from six newly diagnosed patients were isolated and cultured in clonogenic assays with combinations of recombinant cytokines. Compared to control CD34+ cells, JCML cells from all patients showed an aberrant growth pattern restricted almost exclusively to the monocyte-macrophage lineage. Most of the clonogenic activity was seen in a subsorted population of CD34+, HLA-Dr– cells. Additionally, an exaggerated growth response to minute doses of GM-CSF that had no effect on control cells was observed with JCML CD34+ cells. Recloning ("self-renewal") of JCML CD34+ cells was also strongly promoted by GM-CSF. JCML colonies also formed spontaneously in the absence of exogenous cytokines but were augmented by GM-CSF, interleukin 1 and TNF-{alpha}, the latter feature not seen with control CD34+ cells from normal BM. The abnormal spontaneous growth pattern of CD34+ JCML cells could be suppressed directly in vitro by anti-TNF-{alpha} antibodies and anti-GM-CSF antibodies alone or in combination, and by soluble TNF-{alpha} receptors (sTNF-R:Fc), consistent with the notion that JCML CD34+ cells are stimulated by both cytokines in an autocrine manner. In malignant CD34+ cells from one patient, the cytogenetic marker monosomy 7 proved leukemic involvement of monocyte-macrophage, erythroid and B-lymphoid lineages. We conclude that CD34+ JCML cells of multilineage potential exhibit excessive and aberrant monocyte-macrophage colony formation, a property that was previously observed in JCML progenitors found in light density cell fractions. Thus, within the CD34+ cellular compartment is a subpopulation of JCML "stem" cells that accounts for the abnormal leukemic proliferative activity in this disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In previous studies of patients with juvenile chronic myelogenous leukemia (JCML), we observed two reproducible abnormalities in vitro that have become hallmarks of the disease: impaired growth of normal bone marrow (BM) and peripheral blood (PB) hematopoietic progenitors, and excessive proliferation of malignant clonal monocyte-macrophage elements in the absence of an exogenous growth factor [1-8].

Further studies showed that JCML cells endogenously produce tumor necrosis factor (TNF-{alpha}) and GM-CSF that act as autocrine growth factors [9, 10]. Indeed, JCML cells show a selective hypersensitivity to the growth-promoting effect of GM-CSF compared to controls [11]. Data from Bagby et al. [12] also suggested that the abnormal proliferative activity of JCML cells was dependent, in part, on the endogenous production of interleukin 1 (IL-1) which, in a paracrine manner, stimulated the release of colony-stimulating activity by other cells.

All of these vitro studies were performed on heterogenous populations of BM or PB mononuclear cells, usually the light density elements obtained following a Percoll or Ficoll-Hypaque fractionation step. Within these mixed populations of cells, there must be a subpopulation of JCML progenitors that accounts for the abnormal proliferative properties [1-12] of the monocyte-macrophage progeny. Our goal is to identify and characterize these JCML progenitors in order to ask fundamental questions about the biology of this aggressive form of leukemia. Since it was proposed [13, 14] and recently confirmed [15] that JCML involves multiple hematopoietic lineages in a manner similar to Ph+ CML, we reasoned that the putative JCML "stem" cells should be within the pluripotent CD34+ cellular compartment.

In support of this hypothesis, this report describes the aberrant growth and differentiation characteristics of multilineage JCML CD34+ cells obtained from six patients with the disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Subjects
The six previously unpublished patients in this report fit the classical clinical and laboratory description of JCML [13, 14] and showed the characteristic in vitro hallmarks alluded to in the introduction. They were designated as patients 21-26 to correspond with our ongoing series. Details on the other patients in the series can be found as follows: patients 1-9, [1-7]; patient 10, [8]; patients 11-16, [9]; patient 17, [16]; and patients 18-20, [17].

All six patients had varying combinations of lymphadenopathy, skin manifestations, variable splenomegaly, pallor and hemorrhagic signs. The main hematological findings were anemia, thrombocytopenia, leukocytosis with a left shift and nucleated RBCs in the PB (Table 1Go). BM aspirates were cellular with granulocytic hyperplasia, active erythropoiesis and reduced to absent megakaryocytes. BM specimens from all six yielded high numbers of monocyte-macrophage colonies in a colony-forming unit (CFU)-GM assay in the absence of exogenous growth factor, typical of JCML. Routine cytogenetics studies of primary cellular specimens and colonies after 14 days in tissue culture were normal in five patients, but cells from patient 26 showed monosomy 7. For control studies, BM was obtained from hematologically normal donors for BM transplantation during the harvest under anesthesia, and from BM from adults with Ph+ CML in chronic phase. These specimens were obtained with informed consent and were approved by the Human Ethics Committee of our institution.


View this table:
[in this window]
[in a new window]
  		Table 1. Clinical and laboratory data on six patients with JCML at diagnosis
 
Preparation of CD34+ Cells
Patients 21, 23, 25, and 26 underwent splenectomy prior to BM transplantation. Splenic tissue was freshly obtained, minced and then filtered. Heparinized BM, PB or spleen cells were layered over Percoll (density 1.077 g/ml; Pharmacia Fine Chemicals; Piscataway, NJ) and centrifuged (400 g) for 10 min at 4°C to remove neutrophils and RBCs.

Ceprate LC Laboratory Cell Separation Systems (CellPro, Inc.; Bothwell, WA) were used to isolate CD34+cells in the following way: the post-Percoll mononuclear cells were incubated with biotinylated anti-CD34 monoclonal IgM and passed through a column of avidincoated beads [18-20]. The nonadsorbed cells were washed through with phosphate-buffered saline (PBS); the adsorbed cells were detached by squeezing the column several times followed by washing again with PBS. The enriched CD34+ cell population was analyzed by phenotype using cytofluorometry to assess the degree of purity.

Clonogenic Assays
To demonstrate the effect of specific cytokines on CD34+ cells, a modification of the CFU-granulocyte/erythroid/macrophage/megakaryocyte (GEMM) clonogenic assay was performed as previously described [2, 3]. A population of 3 x 103 CD34+ cells (BM, PB or spleen) was cultured in 0.8% methylcellulose with Iscove's modified Dulbeco's medium (Ontario Cancer Institute; Toronto, Canada), plasma obtained from a pool of five normal donors (for studies shown in Tables 2 and 3GoGo), and 5 x 10–5 M of 2-mercaptoethanol. For clonogenic assays other than those shown in Tables 2 and 3GoGo, 30% fetal calf serum was substituted for pooled human plasma. One ml of culture mixture was placed in 35 mm Petri dishes and incubated with 5% CO2 in air in a humidified atmosphere.


View this table:
[in this window]
[in a new window]
  		Table 2. Comparison of colony numbers from normal marrow before and after column fractionation for CD34+ cells
 

View this table:
[in this window]
[in a new window]
  		Table 3. Comparison of growth response of JCML and control CD34+ cells to IL-3, GM-CSF, Steel factor and erythropoietin
 
All cultures were evaluated after 14 days for the number of BFU-E colonies (defined as an aggregate of more than 500 hemoglobinized cells, or three or more erythroid subcolonies), CFU-megakaryocyte (MK) colonies (CFU-GEMM) containing all lineages and CFU-GM colonies defined as a cluster of more than 40 cells consisting of granulocytes, monocyte-macrophages, or both. For secondary and tertiary in vitro recloning experiments, colonies were counted after 14 days of incubation, resuspended and then replated in fresh semisolid medium. Cultures in the presence and absence of exogenous growth factor were processed in parallel.

Microscopically, pure granulocytic colonies could be easily distinguished from pure macrophage colonies because of smaller cells and looser clustering in the former. Individual colonies were plucked from the cultures with a micropipette and spread on glass slides, and the cellular composition was determined microscopically using Wright's stain and nonspecific (a-naphthyl butyrate) esterase staining with and without treatment with fluoride [21]. Staining with benzidine was used to confirm intracellular hemoglobin in colonies scored as BFU-E.

Fluorescence in Situ Hybridization (FISH)
After recording the coordinates, Wright-stained smears of individual BFU-E colonies were briefly destrained in ethanol prior to either fixation for FISH or for staining with benzidine. CD19+ lymphoid cell populations were prepared by fluorescence-activated cell sorting (FACStar, Becton Dickinson; San Jose, CA) by labeling CD34+ JCML spleen cells with fluorescein isothiocyanate (FITC)-conjugated anti-CD19 according to manufacturer's instructions (Coulter; Hialeah, FL). Resultant CD19+ cell populations (purity of 95% to 98%) were used to prepare cytospin slides for fixation and FISH.

To identify cells with monosomy 7, a biotinylated probe (D7ZI; Oncor, Inc; Gaithersburg, MD), hybridizing with the centromeric region of human chromosome 7 was used. Normal human lymphocytes were used as controls. FISH was based on published procedures [22]. After preparing the slides and hybridization mixture, hybridization was carried out for 16-20 h at 37°C. The slides were washed, soaked in 3% bovine serum albumin (BSA), and then incubated FITC (Oncor, Inc.). The slides were then washed and counter-stained with 0.6 µg/ml–1 propidium iodide in PBS). After washing in PBS for 5 min, the slides were mounted in 20 mM Tris-HC1, pH 8.0, 90% glycerol containing 2.3% of the DAPCO antifade, 1.4 diazabicyclo-(2,2,2) octane. Photomicrographs were obtained using a Nikon Microphot-FXA epifluorescence microscope equipped with dual band FITC/Texas red filters (Omega Optical Inc.; Montreal, Canada). Kodak color Ektachrome p800/1600 E-6p professional film was used with a camera setting of ASA 1600.

Recombinant Human Cytokines and Their Respective Antibodies
Human TNF-{alpha} (lot #N9030AX; Genentech; San Francisco, CA) with the specific activity of 5.6 x 107 U/mg was used in a concentration of 1 x 103 U/ml. Monoclonal anti-TNF-{alpha} neutralizing antibodies (mAb-Hu TNF-{alpha}, lot #5890-90; Genentech) were purified from ascites fluid to a concentration of 2 mg/ml, and had a neutralizing titre of more than 5 x 105 neutralizing U/ml. The same lot number of mAb-Hu TNF-{alpha} was used in all of the studies. For our assays, anti-TNF-{alpha} antibodies were added directly to cell cultures, in which an exogenous source of growth factor or cytokine was added or omitted. In quality control studies, 2,000 U/ml for anti-TNF-{alpha} antibodies neutralized 1,000 U of TNF-{alpha}, and hence 2,000 U/ml were used in all of our assays. The efficiency of neutralization of TNF-{alpha} by anti-TNF-{alpha} antibodies was documented in a TNF-{alpha} cytoxicity assay using L929 mouse fibrosarcoma cells [23]. Soluble TNF receptor (p80), linked to the Fc portion of human IgG1 (rHu TNF-R:FC, lot #3353- 090; Immunex; Seattle, WA) was used in a concentration of 5 mg/ml to neutralize the in vitro effect on TNF-{alpha} [24].

GM-CSF (Genetics Institute; Cambridge, MA) with a specific activity in a CML clonogenic assay of 10.6 x 106 U/mg was used in a concentration of 5 U/ml. Anti-GM-CSF neutralizing antibodies (Genetics Institute) were used in a concentration of 10 mg/ml. In quality control bioassay neutralization testing, 1 mg/ml blocked the activity of 5 U/ml of GM-CSF. In our clonogenic assays, the anti-GM-CSF antibodies were added directly to the cell cultures.

IL-1{alpha} (Hoffmann-LaRoche; Nutley, NJ) with specific activity of 3 x 108 U/mg was used in concentration of 100 U/ml. Rabbit polyclonal antihuman IL-1 neutralizing antibodies (product LP-712; Genzyme; Boston, MA) were used directly in cultures in a concentration of 10 U/ml, which was effective in reducing proliferation of hematopoietic colonies from Ph+ CML patients. Erythropoietin (Eprex; Ortho Biological Inc.; Raritan, NJ) with activity of 1 x 104 U/ml was used in a concentration of 2 U/ml. Il-3 (lot #15168-138 B, Genetics Institute) with a specific activity of 1.58 x 106 U/ml was used in a concentration of 40 U/ml. MGF (Steel factor, c-kit ligand, lot #3913-035; Immunex) with a specific activity of 1 x 105 ng/ml was used in a concentration of 50 ng/ml.

Statistical Analysis
The probability of a significant difference between colony numbers was determined by Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Enrichment of Marrow and Spleen Specimens for CD34+ Cells
The Ceprate LC Laboratory Cell Separation columns were highly effective in fractionating CD34+ cells from monouclear elements in post-Percoll mononuclear preparations. With normal BM specimens (n = 7), the average yield of CD34+ cells was 1.6% of the total (range 0.5%-2.5%); the purity of the fractionation by flow cytometry varied from 85% to 99%. With the JCML marrow or spleen specimens (n = 6), the average yield of CD34+ cells was 2.7% (range 1.5%-5%) with the same degree of purity. With Ph+ CML marrow (n = 2), the average yield was 0.36% (range 0.3%-0.4%).

To test the magnitude of enrichment of clonogenic CD34+ cells by the column fractionation technique, normal marrow was cultured in a CFU-GEMM assay before and after the fractionation step. With the postcolumn CD34+ cell preparation, 3 x 103 cells plated yielded comparable colony numbers 2 x 105 precolumn, post-Precoll mononuclear cells (Table 2Go). Thus, there was approximately a two-log enrichment of clonogenic cells using the postcolumn CD34+ cell preparation.

Abnormal Colony Formation by JCML CD34+ Cells
Table 3Go shows the colony growth pattern of JCML CD34+ cells in response to a cytokine "cocktail" of IL-3, GM-CSF, Steel factor and erythropoietin. Compared to CD34+ cells fractionated from the BM of healthy controls and a newly diagnosed patient with Ph+ CML in chronic phase, the JCML colony numbers and cellular composition of individual colonies were markedly and consistently different for all six patients. JCML BFU-E-derived colonies were reduced in number and size, mixed colonies were absent, and CFU-GM colony numbers were consistently higher compared to the control study and the Ph+ CML results. Individual JCML CFU-"GM" colonies at harvest showed all stages of the monocytic lineage including blast forms, promonocytes, monocytes and histiocytic macrophages, whereas in the control and Ph+ CML cultures, at least 50% of the colonies were granulocytic. These findings were confirmed by morphological and cyto-chemical analysis of individual colonies plucked from the cultures and by examination of the cellular composition of entire harvested culture plates. It is noteworthy that the CD34+ cells from patients 21, 25 and 26 in these experiments were fractionated from the spleen after its surgical removal; the abnormal growth pattern of the cells was identical to that seen with the marrow specimens from the other three patients.

In a separate experiment, CD34+ cells from patients 24 were subsorted by flow cytometry into HLA-Dr+ and HLA-Dr– populations, each of 99% purity. Each population was cultured (3 x 103 cells plated) with IL-3, GM-CSF, Steel factor and erythropoietin. The HLA-Dr+ population yielded six BFU-E colonies, zero mixed colonies and 175 monocyte-macrophage colonies; the HLA-Dr– population yielded 18 BFU-E colonies, zero mixed colonies, and > 1,000 monocyte-macrophage colonies.

Removal of Adherent Cell Does Not Impair JCML CD34+ Plating Efficiency
PB cells from patient 26 were fractionated into CD34+ cells then divided into two samples. The first sample was not processed further; the other sample (106 cells/ml) was incubated in 100 mm plastic Petri dishes with culture medium and 10% fetal calf serum for 6 h. The nonadherent cells were then harvested and subjected to a second 12 h incubation in plastic Petri dishes in order to achieve a thorough depletion of adherent cells. The resultant cellular preparation (reduced in number by 60%) was analyzed by flow cytometry using monocyte-associated surface markers; no monocytic elements were detected.

The two preparations of JCML CD34+ cells were then compared for clonogenic activity in the presence or absence of added GM-CSF (Table 4Go). Using the sample prior to adherent cell removal, spontaneous monocyte-macrophage colony growth was observed in the absence of added GM-CSF; colony number was dependent on the cell number plated. Added GM-CSF markedly enhanced the clonogenic activity and induced higher monocyte-macrophage colony numbers in all the studies compared to cultures lacking GM-CSF.


View this table:
[in this window]
[in a new window]
  		Table 4. Comparison of colony growth of PB JCML CD34+ cell (patient 26) before and after removal of adherent cells
 
Using the sample with adherent cells removed, spontaneous growth was still observed without added GM-CSF, and the numbers were higher compared to the sample prior to adherent cell removal. Added GM-CSF augmented colony numbers in all of the studies compared to cultures lacking GM-CSF. The colony numbers were also higher when contrasted to those seen from the sample prior to adherent cell removal.

A similar study was performed on spleen derived CD34+ cells after adherent cell depletion from patient 25 and spontaneous monocyte-macrophage colony growth in the absence of added growth factors was again observed (data not shown).

TNF-{alpha}, IL-1 and GM-CSF Augment JCML CD34+ Colony Numbers
Table 5Go shows the growth response of JCML CD34+ cells from four patients to TNF-{alpha}, IL-1 and to GM-CSF when studied in CFU-GM clonogenic assays. Compared to CD34+ cells from a normal control marrow and from a Ph+ CML marrow specimen, the growth properties of the JCML cells were very different. Remarkable, without added cytokines, all four JCML specimens showed "autonomous" colony formation, a feature not seen with the controls. Once again, these "autonomous" CFU-"GM" colonies were restricted exclusively to the monocyte-macrophage lineage. Daily observation of the culture plates showed very early "autonomous" colony growth after only 48 h of culture which, by day 7, became excessive numerically and very large and cellular. The monocytic macrophage colony growth was further augmented by adding IL-1, TNF-{alpha} and GM-CSF individually, and TNF-{alpha} and GM-CSF together. (Colony increments induced by all three cytokines reached significant p values). The JCML CD34+ cell fractions for these experiments were obtained from spleen specimens from patients 21 and 23, and from marrow from patients 22 and 24; yet the unusual growth pattern and cytokine response were identical for all four patients. In the controls, CFU-GM colony formation was only observed in cultures containing added GM-CSF, and the colonies in these plates were about 50% granulocytic in composition.


View this table:
[in this window]
[in a new window]
  		Table 5. Response of JCML CD34+ cells to IL-1{alpha}, TNF-{alpha} and GM-CSF
 
Hyper-Responsiveness of JCML CD34+ Cells to GM-CSF
Table 6Go shows the colony growth of JCML BM CD34+ cells from patient 21 in response to graded doses of GM-CSF. Without added GM-CSF, exuberant "autonomous" colony growth was again observed, and the colonies were exclusively monocyte-macrophage in composition. With increasing concentrations of added GM-CSF, there was a dose-responsive increase in monocyte-macrophage colonies, a characteristic not seen in the Ph+ CML or the normal control arms of the study in which no growth of any sort was seen. Even with minute concentrations of GM-CSF, for example with 0.3 and 0.6 U/ml, enhanced monocyte-macrophage JCML colony growth was observed. Some CFU-GM granulocytic colony formation was noted in the Ph+ CML cultures, but only at the highest concentration of GM-CSF that was used (5.0 U/ml). The number of JCML colonies in response to 5.0 U/ml of GM-CSF was 8.7 fold higher than the Ph+ CML CFU-GM colony numbers, however (p < 0.001).


View this table:
[in this window]
[in a new window]
  		Table 6. GM-CSF dose response of JCML BM CD34+ cells (patient 21)
 
The exaggerated response of JCML CD34+ cells to GM-CSF was confirmed in a similar but modified study of BM from patient 24. Without added cytokine, a mean of 136 "autonomous" colonies/culture were seen; in response to two doses of GM-CSF, 0.6 U/ml and to 5.0 U/ml, monocyte-macrophage colony growth increased to 179 (p < 0.1) and 240 (p < 0.01) colonies/culture, respectively.

Interplay of TNF-{alpha}, GM-CSF and Their Blockers on JCML CD34+ Cell Growth
Regulation of JCML CD34+ cell growth by TNF-{alpha}, GM-CSF and their respective antagonists was studied using fractionated marrow cells from patient 24 (Figs. 1 and 2GoGo). Data in Figure 1Go confirm that in the absence of added cytokines, "autonomous" JCML colony formation ensued, and that GM-CSF and TNF-{alpha} individually augmented the colony formation. When the respective antibodies were added to cultures containing GM-CSF or TNF-{alpha}, the growth augmentation was reversed and JCML CD34+ cell growth was inhibited. The suppressive effect by the respective antibodies could be reversed by adding excess doses of cytokine (data not shown). Remarkably, anti-TNF-{alpha} antibodies reversed the growth-promoting effect of GM-CSF, and anti-GM-CSF antibodies reversed the growth-promoting effect of TNF-{alpha}.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. Interplay of GM-CSF, TNF-{alpha} and their respective antibodies on JCML CD34+ colony formation (patient 24). Cultures were initiated with 3 x 103 CD34+ cells. Values shown are the mean of duplicate cultures. The concentrations of added products were: GM-CSF, 5 U/ml; TNF-{alpha}, 103 U/ml; anti-GM-CSF antibodies, 10 mg/ml; anti-TNF-{alpha} antibodies, 2,000 U/ml.

 


View larger version (11K):
[in this window]
[in a new window]
  		Figure 2. Direct suppression of JCML CD34+ colony formation (patient 24) by anti- PT TNF-{alpha} antibodies, anti-GM-CSF antibodies and by soluble TNF-{alpha} receptors (sTNF-{alpha}-R:Fc). Values shown are the mean of duplicate cultures. The concentrations of the added products were: anti-TNF-{alpha} antibodies 2,000 U/ml: anit-GM-CSF antibodies, 10 mg/ml; soluble TNF-{alpha} receptors, 5 mg/ml.

 
In Figure 2Go, a direct suppression of JCML CD34+ colony formation was observed when either anti-TNF-{alpha} antibodies or anti-GM-CSF antibodies were added directly to the cultures that lacked an exogenous source of cytokines. Both antibodies added together inhibited "autonomous" JCML colony growth in a synergistic manner and produced a 75% decline in numbers (p < 0.02). Soluble TNF-{alpha} receptors had a similar inhibitory effect on JCML CD34+ colony formation that was comparable to combined effect of the two anticytokine antibodies.

Recloning of CD34+ JCML Spleen Cells
Figure 3Go shows a representative recloning ("self-renewal") experiment of CD34+ JCML spleen cells in the presence and absence of GM-CSF. After 10-14 days in a standard assay, colonies were counted, resuspended and replated in fresh semisolid medium with or without GM-CSF. Cultures exposed to GM-CSF during the primary culture could be "self-renewed" for three passages whether or not GM-CSF was added to further subcultures. JCML colonies, that were initially cultured in the absence of GM-CSF, however, had markedly restricted recloning capability.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Recloning of JCML CD34+ spleen cells in the presence or absence of GM-CSF (patient 26). CD34+ cells (3x 103) were incubated with and without 5 U/ml GM-CSF and serially recloned. Values shown are the mean of duplicate cultures of a representative experiment.

 
Multilineage Potential of JCML CD34+ Cells
The cytogenetic marker monosomy 7 in malignant spleen cells from patient 26 allowed us to investigate leukemic involvement in lineages other than monocyte-macrophage from CD34+ cells and their progeny. Although the number of BFU-E colonies was very low compared to controls (Table 3Go), individual colonies could be isolated for study. Monosomy 7 was clearly demonstrated in benzidine-positive erythroid cells generated from CD34+ spleen cells (Fig. 4Go). Similarly, CD19-enriched lymphoid populations from CD34+ spleen cells from the same patient showed monosomy 7, as well as normal disomy 7 B-lineage cells (Fig. 5Go).



View larger version (75K):
[in this window]
[in a new window]
  		Figure 4. Erythroid colonies generated from CD34+ JCML spleen cells showing monosomy 7 (patient 26). Middle: Wright stain of cells prepared from single BFU-E colony. Right: FISH analysis with chromosome 7-specific probe (same specimen as in middle). Left: benzidine staining of colony cells showing intracellular hemoglobin.

 


View larger version (60K):
[in this window]
[in a new window]
  		Figure 5. Representative CD19+ cells (patient 26) sorted from CD34+ JCML spleen cells show two populations of cells, monosomy 7 clonal cells (long arrows) and normal disomy 7 B-lineage lymphocytes (arrowheads). Cytospin preparations of these CD19+ cells were analysed by FISH using a chromosome 7-specific centromere probe, as described.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Although the hematologic phenotype is similar for all JCML patients, about 20% show a clonal cytogenetic change in myeloid cells, such as trisomy 8 and monosomy 7. The role of these cytogenetic abnormalities in the pathogenesis of JCML is unclear, but they serve as useful markers for tracking the cellular origin of the disease.

Thus, Inoue et al. [25] used cytogenetic data of patients with JCML to demonstrate the presence of the same karyotypic abnormality (monosomy 7) in both initial marrow specimen and pooled erythroid colonies, suggesting erythroid involvement in the malignant JCML cell clone in addition to the monocyte-macrophage lineage. Lau et al. [16] described a case of JCML terminating in B-lymphoid blast crisis. FISH demonstrated that lymphoblasts isolated after blast transformation, as well as myelomonocytic marrow cells isolated during the "chronic" phase of the disease, were positive for monosomy 7. Thus, the leukemic "event" in JCML clearly occurs at an early stage of hematopoiesis judging from these data showing multilineage involvement. This thesis is in complete agreement with data by Busque et al. who demonstrated in one female JCML patient, using a G6PD CT polymorphism clonality assay, that the leukemic clone involved multiple hematopoietic lineages [15].

To examine this directly, CD34+ spleen cells from patient 26 were used to analyze lineage potential of JCML cells. The CD34 antigen identifies cell populations that are enriched for pluripotent and lineage-restricted hematopoietic progenitor cells in vitro [26-36], some of which are capable of marrow reconstitution in vivo [31-33]. The phenotype of the most primitive human hematopoietic cells isolated so far shows expression of this membrane protein [34-36]. Thus, we reasoned that CD34+ JCML cells were a suitably defined subset of cells for an appropriate analysis of their multilineage potential.

The hemoglobin content of BFU-E colonies derived from the CD34+ cells of patient 26 was confirmed by benzidine staining of cell smears. In addition, cells of B-lymphoid phenotype were prepared from CD34+ JCML cells by fluorescence-activated cell sorting using antibody to CD19. Both cell populations, erythroid and B-lymphoid cells, were shown by FISH to be positive for monosomy 7, the marker present in the diagnostic patient marrow and in monocyte-macrophage colonies derived from marrow and spleen cells. The malignant transformation in JCML therefore occurs in a multipotential cell which can generate monocyte-macrophage, erythroid and B-lymphoid lineages. This in vitro finding is in keeping with reported data [15] and suggests a "stem" cell origin of JCML.

Excessive formation of monocyte-macrophage colonies with impaired normal residual hematopoiesis is a characteristic pattern of JCML cell proliferation in vitro [2, 3, 6]. Endogenous production of cytokines such as TNF-{alpha}, GM-CSF and IL-1 [9-12] by JCML cultures has been proposed as an explanation for this unique proliferative behavior. Constitutive secretion of TNF-{alpha} by JCML cells inhibits normal hematopoietic progenitors in vitro [9] and, at the same time with GM-CSF, acts as an autocrine stimulator of excessive malignant monocyte-macrophage colony formation [9, 10]. Emmanuel et al. [11] demonstrated the hypersensitivity of mononuclear JCML cells to exogenous GM-CSF as measured by monocyte-macrophage colony formation. IL-1 was found to stimulate the release of JCML growth-promoting cytokines by other BM or PB cells [12] and was implicated as a paracrine growth factor of JCML cells. Importantly, in vitro proliferation of JCML cells could be decreased by antibodies to TNF-{alpha} and GM-CSF [9], by IL-1 receptor antagonist [37], soluble IL-1 receptors, IL-4 (which inhibits IL-1 and TNF-{alpha} production), and pentoxiphylline which downregulates TNF-{alpha} mRNA [17, 26].

The fractionation of CD34+ JCML cells allowed experiments with a more clearly defined JCML cell population which did not appear "contaminated" by a growth factor secreting adherent cells, as evidenced by enhanced clonogenic efficiency after exhaustive adherent cell removal (see below). The results were clear-cut, consistent in every patient studied and provided compelling evidence that JCML is a hematopoetic malignancy with in vitro growth characteristics entirely reproducible by CD34+ JCML cells.

First, the colony growth pattern, in response to a combination of IL-3, GM-CSF, Steel factor and erythropoietin, was strikingly different from normal BM controls as well as from Ph+ CML cells. The JCML cultures always showed reduced to absent erythroid colony growth and increased monocyte-macrophage colony formation.

Second in contrast to normal BM cells, CD34+ JCML cells grew spontaneously in the absence of exogenous growth factors. The addition of GM-CSF, TNF-{alpha} and IL-1 resulted in further augmentation of the already excessive monocyte-macrophage growth. CD34+ JCML cells demonstrated the hypersensitivity of colony formation in response to GM-CSF at minute concentrations that have no proliferative effect on normal marrow cells. This feature was previously described in mixed, nonadherent, T cell depleted populations of light density JCML cells [11].

The spontaneous colony formation by JCML CD34+ cells in the absence of added cytokines prompts the question of whether growth factor-secreting "contaminating" monocytes are present in the samples. Although this is unlikely because of the high degree of purity of the CD34+ preparations proven by flow cytometry, detailed experiments were performed to address this question directly. We observed that total removal of adherent cells, proven by flow cytometry by an exhaustive double-depletion technique over 18 h of incubation with plastic, did not impair or decrease the plating efficiency of JCML CD34+ cells in the absence of added growth factors. Indeed, spontaneous formation of JCML monocyte-macrophage colonies were augmented, possibly due to enrichment because of removal of nonclonogenic CD34+ cells with surface adherence properties. Thus, the results suggest that a highly purified population of JCML CD34+ can "self-start" and form colonies in keeping with an autocrine growth pattern. Our data differ from previous studies by others that showed impaired spontaneous JCML colony formation after adherent cell depletion [12, 38]. Their studies were performed on light density cells and ours on highly purified CD34+ cells. Nonetheless, the explanation for the contradictory results is not readily evident. Taking an overview of their data and ours, we would have to conclude that both autocrine and paracrine growth loops are operative in JCML cell proliferation and contribute to the cytokine-driven leukemic process.

Serial recloning experiments showed that exposure of CD34+ JCML cells to GM-CSF during early culture passage was associated with successful "self-renewal" for up to three passages. Absence of GM-CSF during this phase of proliferation could not be compensated by stimulation with GM-CSF during subsequent culture generations. In addition to hypersensitivity of the overall JCML population to GM-CSF, a transient growth factor-sensitive period during early in vitro proliferation was demonstrated using the CD34+ JCML cells.

Finally, the abnormal growth of CD34+ JCML cells could be suppressed by antagonists to TNF-{alpha} and GM-CSF. Working with light density cells, Emmanuel et al. also found that anti-GM-CSF, antibodies suppressed spontaneous JCML colony growth but anti-TNF antibodies had no significant effect [38]. In our hands, however, mAbs to TNF-{alpha} and GM-CSF, alone or in combination, resulted in growth inhibition comparable to the effect of soluble TNF-{alpha} receptors. These data confirm our previous work [9] and suggest interference by these agents on the effect of autocrine growth stimulation that was previously identified in the light density population of cells [9]. Reversal of the effects of GM-CSF by anti-TNF-{alpha} and TNF-{alpha} by anti-GM-CSF illustrates the complex interplay of cytokines involved in JCML growth stimulation.

Thus, JCML is a disease that originates in pluripotent cells and can be identified in the CD34+ population. All of the in vitro features previously ascribed to JCML "progenitors" in the mixed light density cell population can be reproduced by CD34+ JCML cells. In addition, CD34+ JCML cells can engraft in severe combined immunodeficiency mice and cause rapid infiltration of murine tissues, cachexia and hematopoetic failure similar to the human disease [39]. Therefore, JCML CD34+ cells represent a close approximation of the cell of origin of the disease and provide an entry point for fundamental investigations of this disorder.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We are grateful to Wilma Vanek for her cell culture expertise, and to Zong Mei Zheung and Teresa Scheidl for excellent technical assistance with the FISH studies.

Supported by the National Cancer Institute of Canada, the Histiocytosis Association of America, and the Medical Research Council of Canada.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Estrov Z. Zimmerman B, Grunberger T et al. Characterization of malignant peripheral blood cells of juvenile chronic myelogenous leukemia. Cancer Res 1986;46:6456-6461.[Medline]

  2. Estrov Z, Grunberger T, Chan HSL et al. Juvenile chronic myelogenous leukemia: characterization of the disease using cell cultures. Blood 1986;67:1382-1387.[Abstract/Free Full Text]

  3. Estrov Z, Dube IA, Chan HSL et al. Residual juvenile chronic myelogenous leukemia cells detected in peripheral blood during clinical remission. Blood 1987;70:1466-1469.[Abstract/Free Full Text]

  4. Estrov Z, Lau AS, Williams BRG et al. Recombinant human interferon alpha-2 and juvenile chronic myelogenous leukemia: cell receptor binding, enzymatic induction, and growth suppression in vitro. Exp Hematol 1987;15:127-132.[Medline]

  5. Freedman MH, Estrov Z. Juvenile chronic myelogenous leukemia and histiocytosis syndromes in children. Lancet 1987;1:754.[Medline]

  6. Freedman MH, Estrov Z, Chan HSL. Juvenile chronic myelogenous leukemia. Am J Pediatr Hematol Oncol 1988;10:261-267.[Medline]

  7. Chan HSL, Estrov Z, Weitzman SS et al. The value of intensive combination chemotherapy for juvenile chronic myelogenous leukemia. J Clin Oncol 1987;5:1960-1967.[Abstract/Free Full Text]

  8. Kirby MA, Weitzman S, Freedman MH. Juvenile chronic myelogenous leukemia: differentiation from infantile cytomegalovirus infection. Am J Pediatr Hematol Oncol 1990;12:292-296.[Medline]

  9. Freedman MH, Cohen A, Grunberger T et al. Central role of tumour necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia. Br J Haematol 1992;80:40-48.[Medline]

  10. Gualtieri RJ, Emanuel PD, Zuckerman KS et al. Granulocyte-macrophage colony-stimulating factor is an endogenous regulator of cell proliferation in juvenile chronic myelogenous leukemia. Blood 1989;74:2360-2367.[Abstract/Free Full Text]

  11. Emanuel PD, Bates LJ, Castleberry RP et al. Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood 1991;77:925-929.[Abstract/Free Full Text]

  12. Bagby GC, Dinarello CA, Neerhout RC et al. Interleukin-1-dependent paracrine granulopoiesis in chronic granuloycytic leukemia of the juvenile type. J Clin Invest 1988;82:1430-1436.

  13. Altman AJ. Schwartz AD. Malignant Diseases of Infancy, Childhood and Adolescence. Philadelphia: W.B. Saunders, 1983:228-229.

  14. Nathan DG, Oski FA, Hematology of Infancy and Childhood, 4th Edition. Philadelphia: W.B. Saunders, 1993:1293-1295.

  15. Busque L, Gilliland DG, Pryhcal JT et al. Clonality in juvenile chronic myelogenous leukemia. Blood 1995;85:21-30.[Abstract/Free Full Text]

  16. Lau RC, Squire J. Brisson L et al. Lymphoid blast crisis of B-lineage phenotype with monosomy 7 in a patient with juvenile chronic myelogenous leukemia (JCML). Leukemia 1994;8:903-908.[Medline]

  17. Estrov Z, O'Brien PM, Horvath A et al. Interruption of autocrine/paracrine growth factor stimulatory mechanisms: a potential approach for juvenile chronic myelogenous leukemia therapy. Int J Pediatr Hematol Oncol 1994;1:253-258.

  18. Berenson RJ, Bensinger WI, Kalamasz D. Positive selection of viable cell populations using avidin-biotin immunoadsorption. J Immunol Methods 1986;91:11-19.[Medline]

  19. Berenson RJ, Bensinger WI, Kalamasz D et al. Elimination of Daudi lymphoblasts from human bone marrow using avidin-biotin immunoabsorption. Blood 1986;67:509-515.[Abstract/Free Full Text]

  20. Berenson RJ, Bensinger WI, Kalamasz D et al. Engraftment of dogs with Ia-positive marrow cells isolated by avidin-biotin immunoadsorption. Blood 1987;69:1363-1367.[Abstract/Free Full Text]

  21. Tucker SB, Pierre RV, Jordan RE. Rapid identification of monocytes in mixed mononuclear cell preparations. J Immunol Methods 1977;14:267-269.[Medline]

  22. Lichter P, Tang CJ, Call K et al. High resolution mapping of human chromosome II by in situ hybridization with cosmid clones. Science 1990;247:64-69.[Abstract/Free Full Text]

  23. Lau AS, Livesey JF. Endotoxin induction of tumor necrosis factor is enhanced by acid-labile interferon-alpha in acquired immunodeficiency syndrome. J Clin Invest 1989;84:738-743.

  24. Mohler KM, Torrance DS, Smith CA et al. Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. J Immunol 1993;151:1548-1561.[Abstract]

  25. Inoue S, Shibata T, Ravindranath Y et al. Clonal origin of erythroid cells in juvenile chronic myelogenous leukemia. Blood 1987;69:975-976.[Free Full Text]

  26. Andrews RG, Singer JW, Bernstein ID. Monoclonal antibody 12-8 recognizes a 115-kd molecule present on both unipotent and multipotent hematopoietic colony-forming cells and their precursors. Blood 1986;67:842-845.[Abstract/Free Full Text]

  27. Brandt JE, Baird N, Lu L et al. Characterization of a human hematopoietic progenitor cell capable of forming blast cells containing colonies in vitro. J Clin Invest 1988;82:1017-1027.

  28. Andrews RG, Singer JW, Bernstein ID. Precursors of colony-forming cells in humans can be distinguished from colony forming cells by expression of the CD33 and CD34 antigen and light scatter properties. J Exp Med 1989;169:1721-1731.[Abstract/Free Full Text]

  29. Sutherland HJ, Eaves CJ, Eaves AC et al. Characterization and partial purification of human marrow cells capable of initiation long-term hematopoiesis in vitro. Blood 1989;74:1563-1570.[Abstract/Free Full Text]

  30. Sutherland HJ, Lansdorp PM, Henkelman DH et al. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive stromal layers. Proc Natl Acad Sci USA 1990;87:3584-3588.[Abstract/Free Full Text]

  31. Berenson RJ, Andrews RG, Bensinger WI et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 1988;81:951-955.

  32. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991;77:1717-1722.[Abstract/Free Full Text]

  33. Peault B, Weissman IL, Baum C et al. Lymphoid reconstitution of the human fetal thymus in SCID mice with CD34+ precursor cells. J Exp Med 1991;174:1283-1286.[Abstract/Free Full Text]

  34. Baum CM, Weissman IL, Tsukamoto AS et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 1992;89:2804-2808.[Abstract/Free Full Text]

  35. Craig W, Kay R, Cutler RL et al. Expression of Thy-1 on human hematopoietic progenitor cells. J Exp Med 1993;177:1331-1342.[Abstract/Free Full Text]

  36. Huang S, Terstappen WMM. Lymphoid and myeloid differentiation of single human CD34+, HLA-Dr+, CD38– hematopoietic stem cells. Blood 1994;83:1515-1526.[Abstract/Free Full Text]

  37. Schiro R, Longoni D, Rossi V et al. Suppression of juvenile myelogenous leukemia colony growth by interleukin-1 receptor antagonist. Blood 1994;83:460-465.[Abstract/Free Full Text]

  38. Emanuel PD, Bates LJ, Zhu S-W et al. The role of monocyte-derived hematopoietic growth factors in the regulation of myeloproliferation in juvenile chronic myelogenous leukemia. Exp Hematol 1991;19:1017-1024.[Medline]

  39. Lapidot T, Grunberger T, Vermoor J et al. Identification of immature juvenile chronic myelogenous leukemia cells capable of initiating disease in primary and secondary SCID mice. Blood 1996;88:2655-2664.[Abstract/Free Full Text]

Received March 28, 1996; accepted for publication July 30, 1996.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Freedman, M. H.
Right arrow Articles by Squire, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Freedman, M. H.
Right arrow Articles by Squire, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
Email Content Delivery