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Proliferating status of peripheral blood progenitor cells from patients with BCR/ABL-positive chronic myelogenous leukemia

A Krämer, H Löffler, J Bergmann, A Hochhaus & R Hehlmann

III Medizinische Klinik, Klinikum Mannheim, Universität Heidelberg, Mannheim, Germany

Correspondence to: A Krämer, Medizinische Klinik und Poliklinik V, Universität Heidelberg, Hospitalstr 3, 69115 Heidelberg, Germany; Fax: 49 6221 56 5722

To investigate the mechanisms behind the leukemic expansion of BCR/ABL-positive chronic myelogenous leukemia (CML), we examined the cell cycle status of hematopoietic progenitor cells from peripheral blood (PB) and bone marrow (BM) of 37 patients with newly diagnosed BCR/ABL-positive CML. We found a high proportion of 12.51 1.19% of CD34⁺peripheral blood progenitor cells (PBPC) in S/G₂M phase. Comparison of PB and BM from 19 cases revealed similar proliferation rates (10.74 1.41% vs 15.97 1.95%). Furthermore, even primitive CD34⁺/CD38^- PBPC displayed high proliferation rates (17.45 2.98%) in 10 cases examined. In contrast, PBPC from 11 patients with BCR/ABL-negative myeloproliferative disorders were almost noncycling (S/G₂M 1.46 0.47%). When matched pairs of PB and BM from six patients with BCR/ABL-negative myeloproliferative disorders were examined, only 0.89 0.41% of the CD34⁺ PBPC, but 8.29 3.13% CD34⁺ cells from BM were in S/G₂M phase. Consistently, as compared to 19 patients with newly diagnosed BCR/ABL-positive CML, a significantly lower PB/BM ratio of CD34⁺ cells in S/G₂M phase was found in these six patients with BCR/ABL-negative myeloprolifrative disorders. Administration of the tyrosine kinase inhibitor STI571 to 13 patients with CML in chronic phase, accelerated phase, or blast crisis lead to an inhibition of PBPC proliferation within a few days. Interestingly, CD34⁺ hematopoietic progenitor cells from BM remained proliferating in five cases examined, indicating that CML PBPC are more easily inhibited by STI571 as compared to CD34⁺ CML hematopoietic progenitor cells from BM. These data suggest that BCR/ABL leads to an enhanced cell cycle activation of CD34⁺ cells, which seems to be, at least in part, independent of additional factors provided by the bone marrow microenvironment. Leukemia (2001) 15, 62–68.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder that appears after the deregulated clonal expansion and differentiation of a totipotent hematopoietic stem cell.¹ It is associated in >95% of cases with a t(9;22) chromosomal translocation, the Philadelphia chromosome (Ph), which gives rise to the BCR/ABL fusion gene.^2,3,4 BCR/ABL mRNA encodes a 210 kDa BCR/ABL protein with increased and unregulated tyrosine kinase activity in comparison to the parent c-ABL protein.^3,5 Several reports suggest that this translocation may contribute to the apparent proliferative advantage of CML hematopoietic clones over normal clones during the course of the disease.^6,7,8 Others have found that abrogated apoptosis may lead to the increased cellularity in CML.^9,10 Studies using either unpurified or CD34⁺ selected hematopoietic progenitor cells (HPC) from bone marrow (BM) documented an increased proliferation of CML cells from this compartment.^9,11,12,13 In contrast, no data are available on the proliferating status of CML progenitors circulating in the peripheral blood (PB).

Recently, it has been demonstrated by several groups that CD34⁺ HPC mobilized into the PB of healthy donors by granulocyte colony-stimulating factor (G-CSF) and other cytokines are virtually noncycling, while premobilization BM CD34⁺ cells were actively proliferating.^{14,15,16,17,18,19} Whereas CD34⁺ HPC from steady-state PB have been reported to be predominantly in the G₀ phase of the cell cycle,²⁰ the noncycling status of normal PBPC harvested after mobilization with G-CSF alone and following G-CSF-supported chemotherapy seems to be increasingly due to a cell cycle arrest in late G₁ phase.^18,19 Furthermore, HPC from mice treated with cyclophosphamide and G-CSF proliferated only in the BM and spleen whereas HPC released into PB tended to be arrested in the G₀/G₁ phase over an observation period of 8 days.²¹ Interestingly, To and coworkers²² have described that this cell cycle arrest of normal human PBPC is not ascribable either to inhibitory elements in the blood or to reduced responsiveness to growth factors. Thus, PB obviously does not provide optimal growth conditions for normal CD34⁺ HPC.

To test for the hypothesis that expansion of the malignant clone in myeloproliferative disorders is not restricted to the BM, we compared the proliferation rates of HPC from BM and PB of patients with BCR/ABL-positive CML as well as BCR/ABL-negative chronic myeloproliferative disorders at diagnosis.

The tyrosine kinase inhibitor STI571, a 2-phenylaminopyrimidine derivative, selectively inhibits the ABL, platelet-derived growth factor receptor (PDGFR), and c-KIT tyrosine kinases.^23,24 In vitro as well as in vivo studies have demonstrated that STI571 specifically inhibits the growth of BCR/ABL-expressing CML progenitor cells without affecting the growth of normal cells or cells transformed by other tyrosine kinases.^25,26 To further elucidate the role of the p210^bcr/abl tyrosine kinase in the activation of HPC proliferation in vivo, we compared the proliferation rates of HPC from BM and PB of BCR/ABL-positive CML patients prior to and during the treatment with 400 mg STI571 per day.

Patients

For cell cycle analysis, PB and BM samples were obtained from 37 patients with newly diagnosed BCR/ABL-positive CML in early chronic phase (26 men and 11 women, 7 to 71 years old), three patients with chronic myelomonocytic leukemia (two men and one woman, 67 to 73 years old), three patients with polycythemia vera (two men and one woman, 42 to 71 years old), one patient with essential thrombocythemia (female, 58 years old), and four patients with BCR/ABL-negative CML (three men and one woman, 40 to 72 years old). All samples were obtained prior to any specific treatment. Five patients with BCR/ABL-positive CML blast crisis (four myeloid blast crises, one lymphoid blast crisis, three men and two women, 47 to 68 years old), one patient with accelerated phase of BCR/ABL-positive CML (female, 62 years old), and seven patients with chronic phase of BCR/ABL-positive CML (seven men, 45 to 66 years old) were treated with 400 mg STI571 per os daily according to the phase II open label study 0102 or 0110 protocol. As requested by the study protocols, all patients had received antineoplastic therapy prior to this treatment, including hydroxyurea in nine cases, interferon- in six cases, and other substances in five cases as the last antineoplastic pretreatment. PB and BM samples were taken immediately prior to as well as during the course of treatment with STI571. To determine the proportions of BCR/ABL-positive cells among CD34⁺ HPC, PB samples from 12 patients (six men and six women, 22 to 69 years old), and BM samples from seven patients (four men and three women, 22 to 54 years old) with newly diagnosed BCR/ABL-positive CML in early chronic phase were obtained prior to any specific treatment. This study has been approved by the Institutional Review Board. All samples were taken after informed consent according to the declaration of Helsinki.

Sample preparation and CD34⁺ cell purification

PB and BM samples were collected into heparinized syringes. Mononuclear cells were obtained by Ficoll gradient centrifugation (Ficoll, 1.077 g/ml, Pharmacia, Freiburg, Germany). Mononuclear cells were collected from the interphase and washed twice with phosphate-buffered saline (PBS). The MiniMacs CD34⁺ kit and MiniMacs columns (Miltenyi Biotec, Bergisch Gladbach, Germany) were used for CD34⁺ cell enrichment of the PB and BM mononuclear cell samples. For assessment of the selection efficiency, a CD34⁺ restaining was performed as described below.

Fluorescence activated cell sorting analysis

For surface staining of antigens, PB or BM cells were incubated for 15 min at room temperature with unconjugated, fluorescein-isothiocyanate (FITC)-, and phycoerythrin (PE)-conjugated monoclonal antibodies directed against the following epitopes: CD34 (unconjugated; Becton Dickinson, Heidelberg, Germany), CD34 (biotin, Immunotech, Marseille, France), CD38 (PE). Streptavidin-DTAF (Immunotech) was used for detection of the biotinylated anti-CD34 antibody. For detection of the unconjugated anti-CD34 antibody, goat anti-mouse IgG-FITC (Becton Dickinson) was used. Isotype-specific FITC and PE monoclonal antibodies (IgG₁-FITC, IgG₁-PE) served as controls. Staining of cellular DNA with propidium iodide (PI; Medac, Hamburg, Germany) was done using the QuickLysis lysing solution (Medac) according to the manufacturer's instructions. Cells were analyzed within 1 h after staining. For intracellular antigen staining and DNA analysis with 7-aminoactinomycin D (7-AAD, MoBiTec, Göttingen, Germany), a fixation and permeabilization protocol reported previously was followed.¹⁹ In brief, following surface antigen staining, CD34⁺ selected cells were washed once with ice-cold PBS and resuspended in 500 l PBS. While slowly vortexed, 500 l ice-cold PBS containing 2% paraformaldehyde (Merck, Darmstadt, Germany) and 160 g/ml L--lysolecithin (Sigma, Deisenhofen, Germany) were added dropwise and incubated for 5 min at 4°C. Lysolecithin activity was blocked by adding 2 ml PBS containing 1% bovine serum albumin (PBS/1% BSA w/v). Cells were washed twice with PBS/1% BSA w/v and resuspended in 100 l PBS/1% BSA w/v. After antigen staining, cell were washed twice in PBS/1% BSA w/v and resuspended in 100 l PBS/1% BSA w/v. For staining of cellular DNA with 7-AAD, cells were incubated for 30 min in a final concentration of 25 g/ml 7-AAD in PBS/1% BSA w/v at room temperature. Cells were analyzed within 1 h after staining. Acquisition and analysis were performed on a FACSCalibur flow cytometer (Becton Dickinson) mounted with an air-cooled 488 nm argon laser using Cell Quest software. Emissions from FITC and PI or FITC, PE, and 7-AAD were measured simultaneously using short pass 560 nm and 640 nm long pass filters, respectively. Between 60 000 and 100 000 CD34⁺ selected cells stained simultaneously for surface antigens and DNA were aquired at a flow rate of 10–70 events per s. Subsequent gates were set to exclude debris identified in the FSC vs SSC dot blot. A doublet discrimination module was used to discriminate between cells in the G₂/M phase and doublets. Only single cells were included in the analysis. All coexpression data were corrected for unspecific binding of isotype-matched control antibodies. Cell cycle phase distribution was calculated using ModFit LT software (Verity Software House, Topsham, ME, USA).

Dual-color fluorescence in situ hybridization (FISH)

Purified CD34⁺ HPC were dropped on to poly-L-lysine-coated slides, incubated for 1 h in a wet chamber, air-dried, fixed in methanol/ acetic acid (3:1), and stored at -20°C. Dual-color FISH was performed using commercially available DNA probes for BCR covering approximately 300 kb immediately 5' of the major breakpoint cluster region and for ABL covering approximately 200 kb 3' of exon 4, readily labeled with Spectrum Green (BCR) and Spectrum Orange (ABL) (Vysis, Stuttgart, Germany), according to the manufacturer's recommendations. Briefly, slides were digested (13 min, 37°C) in 0.01 M HCl containing 3–5 mg/100 ml pepsin (Serva, Heidelberg, Germany), washed twice in PBS, fixed with PBS containing 1% paraformaldehyde (Sigma) and 5 mM MgCl₂ for 5 min, and again washed twice in PBS. Dehydration in 70%, 90%, and 100% ethanol was followed by denaturation (2 min, 70°C) with 70% formamide (Merck) in 2 SSC, incubation in ice-cold CaCl₂ for 20 min, and dehydration in 70%, 90%, and 100% ethanol. After addition of 10 l BCR/ABL probe mixture at 48°C, the covered and sealed slides were incubated for 12 to 16 h at 37°C in a wet chamber. Finally, the slides were uncovered, washed in 0.4% SSC (2 min, 72°C) and in 2% SSC containing 0.1% NP40 (Sigma) (2 min, room temperature), counterstained with 4,6-diamidino-2-phenylindol (DAPI), mounted (Vectashield; Vector Laboratories, Burlingame, CA, USA), and covered again. All FISH analyses were performed by a single investigator using dual-color fluorescence microscopy (Zeiss, Oberkochen, Germany). Evaluation of 200 interphase nuclei was attempted in each sample, and samples with at least 100 evaluable nuclei were included in this study. Following the manufacturer's guidelines, nuclei displaying a yellow BCR/ABL fusion signal or touching green BCR and red ABL signals were scored BCR/ABL positive, while nuclei displaying no touching or fusion signal and at least two BCR and two ABL signals were scored BCR/ABL negative. Nuclei not matching these criteria were considered not evaluable. Using this method, the mean proportion of false-positive cells as determined by the same investigator in PB mononuclear cells of 12 healthy volunteers was 2.38 1.09% (mean 1 standard deviation), leading to a cut-off value of 6% (mean + 3 standard deviations).

Statistics

If not stated otherwise, data are given as mean values 1 standard error of mean (s.e.m.). Significance levels for BM to PB ratios of patients with BCR/ABL-positive CML vs patients with BCR/ABL-negative myeloproliferative disorders were determined by Mann–Whitney test analysis. Significance levels for BM vs PB proliferation rates or proliferation rates prior to and after STI571 treatment were calculated using the Wilcoxon test for matched pair analysis.

Cell cycle analysis of CD34⁺ PBPC from patients with newly diagnosed BCR/ABL-positive CML in early chronic phase

PB samples from 37 patients with BCR/ABL-positive CML at diagnosis were examined. After immunomagnetic CD34⁺ cell selection, a total of 60 000 to 100 000 CD34⁺ selected cells stained simultaneously for CD34 and DNA at a flow rate of 10–70 events per s were acquired. DNA histograms were displayed from primitive hematopoietic cells fitting into a lymphoblastoid region in a forward vs sideward scatter dot plot, in a CD34⁺ gate, and in a singlet gate defined by a fluorescence-area against fluorescence-width display (Figure 1). 12.51 1.19% of the CD34⁺ CML PBPC were found to be in S/G₂M phase of the cell cycle. The coefficient of variation (CV) values for the G₀/G₁ peak of the DNA histograms following propidium iodide staining were 3.62 1.10% (mean 1 standard deviation, n = 114), which was in line with previous reports on the use of this dye.

Comparison of the cell cycle status of CD34⁺ HPC from PB and BM of patients with newly diagnosed BCR/ABL-positive CML in early chronic phase

Matched pairs of CD34⁺ HPC from PB and BM of 19 patients with BCR/ABL-positive CML at diagnosis were analyzed. Following immunomagnetic CD34⁺ cell selection, 60 000 to 100 000 CD34⁺ selected cells were acquired from each sample. The proportions of CD34⁺ cells in S/G₂M phase from PB (10.74 1.41%) and BM (15.97 1.95%) were similar (Figure 2).

Cell cycle analysis of CD34⁺ PBPC from patients with newly diagnosed BCR/ABL-negative myeloproliferative disorders

To examine whether proliferation in PB is specific to HPC from patients with BCR/ABL-positive CML, the S/G₂M phase fractions of PBPC from 11 patients with BCR/ABL-negative myeloproliferative disorders at diagnosis were determined. Specifically, four patients with BCR/ABL-negative CML, three patients with polycythemia vera, one patient with essential thrombocythemia, and three patients with chronic myelomonocytic leukemia were examined. After immunomagnetic CD34⁺ cell selection, 60 000 to 100 000 CD34⁺ selected cells were aquired from each sample. In contrast to PBPC from patients with BCR/ABL-positive CML, PBPC from these patients were almost noncycling. Only 1.46 0.47% of the CD34⁺ PBPC of these patients were found to be in S/G₂M phase of the cell cycle (Figure 2). When matched pairs of PB and BM from six patients with BCR/ABL-negative myeloproliferative disorders were examined, only 0.89 0.41% of the CD34⁺ PBPC but 8.29 3.13% CD34⁺ cells from BM were in S/G₂M phase. Consistently, as compared to 19 patients with newly diagnosed BCR/ABL-positive CML, a significantly lower PB/BM ratio of CD34⁺ cells in S/G₂M phase was found in these six patients with BCR/ABL-negative myeloproliferative disorders (P < 0.05; Figure 2).

Relationship between cell cycle status and CD38 expression of CD34⁺ PBPC from patients with newly diagnosed BCR/ABL-positive CML in early chronic phase

To examine whether proliferation in PB of CD34⁺ CML HPC is related to their maturation status, the S/G₂M phase fractions of CD38⁺ and CD38^- subpopulations of CD34⁺ PBPC from 10 patients with BCR/ABL-positive CML at diagnosis were determined. Cells were stained simultaneously for the surface expression of CD34 and CD38, as well as for DNA content analysis with 7-AAD. Following CD34⁺ cell selection, 60 000 to 100 000 CD34⁺ selected cells were aquired from each sample. Subpopulation analysis demonstrated that primitive CD34⁺/38^- CML HPC have similar proliferation rates as compared to more mature CD34⁺/38⁺ CML PBPC from the same patients (17.45 2.98% for CD34⁺/CD38^low vs 17.48 2.86% for CD34⁺/CD38^dim vs 13.28 2.12% for CD34⁺/CD38^high; Figure 3). The CV values for the G₀/G₁ peak of the DNA histograms following 7-AAD staining were 6.61% 1.78% (mean 1 standard deviation), which was in line with previous reports on the use of this dye.²⁷

Comparison of the cell cycle status of CD34⁺ PBPC from PB and BM of CML patients prior to and during treatment with the tyrosine kinase inhibitor STI571

PB and BM samples from six patients with blast crisis or accelerated phase, and from seven patients with chronic phase of BCR/ABL-positive CML were taken prior to and during the course of a treatment with 400 mg STI571 per os daily. Between 60 000 and 100 000 CD34⁺ selected cells were acquired from each sample. As shown in Figure 4, administration of STI571 to blast crisis or accelerated phase CML patients lead to an inhibition of PBPC proliferation within a few days. Whereas prior to STI571 treatment, 4.53 1.32% of the CD34⁺ PBPC were proliferating, only 0.93 0.24% of the CD34⁺ PBPC were in S/G₂M phase 1 week after the initiation of STI571 therapy (P < 0.05). In contrast, the S/G₂M phase fractions of the HPC from BM of five of these patients remained high even after 25 to 30 days of treatment with STI571 (6.96 1.72%), and matched pair analysis of BM samples taken from these patients prior to and 4 weeks after the initiation of STI571 treatment did not reveal a significant difference between the proportions of CD34⁺ cells in S/G₂M phase of the cell cycle. In the patients with chronic phase CML (Figure 5), PBPC proliferation was significantly inhibited as well within a few days of STI571 treatment from 4.59 0.96% to 2.25 0.50% (P < 0.05). The relatively low initial proliferation rates in most cases are explained by the antineoplastic pretreatment as specified in Materials and methods.

Dual-color interphase FISH of CD34⁺ HPC

To exclude the possibility that our results might be confounded by varying amounts of BCR/ABL-negative cells present among isolated CD34⁺ HPC, we determined the proportions of BCR/ABL-positive cells among CD34⁺ HPC isolated from 12 PB and seven BM samples of patients with newly diagnosed BCR/ABL-positive CML in early chronic phase using dual-color interphase FISH. We found that 96.74 0.67% (range: 91.18%–99.50%) of PBPC and 97.53 1.00% (range: 92.50% – 100%) of BM HPC were BCR/ABL-positive.

In the present study, we have analyzed the proliferation rates of CD34⁺ HPC from PB and BM of patients with BCR/ABL-positive CML. We have observed that (1) a significant proportion of CD34⁺ CML PBPC is in S/G₂M phase; (2) there is no major difference between the proliferation rates of CML HPC from PB and BM; (3) even primitive CD34⁺/38^- PBPC from patients with CML are proliferating; and (4) treatment of CML patients with the tyrosine kinase inhibitor STI571 leads to a complete inhibition of PBPC, but not BM HPC proliferation. In contrast to our findings with CML PBPC, we have found that PBPC from patients with BCR/ABL-negative myeloproliferative disorders are almost noncycling.

To exclude therapy effects as confounders, only patients with newly diagnosed disease prior to any specific treatment were included in the first part of our study. For patients with newly diagnosed BCR/ABL-positive CML, we showed that CD34⁺ HPC isolated from PB or BM are predominantly BCR/ABL-positive, which is in accordance with results reported by others.²⁸ We conclude that in the CML cases, the significantly increased proliferation is caused by BCR/ABL-positive HPC. As an additional confounder, one might argue that BCR/ABL-negative PBPC could be enriched among CD34⁺/CD38^low cells. However, the abnormally high proliferation rates in this subpopulation are not readily explained by this assumption. Moreover, CD38 expression has been described to be not a useful marker for discriminating between BCR/ABL-positive and negative PBPC.²⁹

In the second part of our study, we sought to confirm our results by assessing the effect of the tyrosine kinase inhibitor STI571 on the cell cycle status of CML CD34⁺ HPC. It should be noted that all patients treated with STI571 had received antineoplastic therapies prior to inclusion in this study, as requested by the study protocols, including hydroxyurea, which is known to cause cell cycle arrest in early S phase,³⁰ in nine of 13 cases. Thus, the lower proliferation rates of CD34⁺ HPC from these patients as compared to the patients with newly diagnosed CML are explained by this pretreatment. Nevertheless, STI571 treatment lead to a further, significant decrease in PBPC proliferation rates.

The mechanisms underlying the growth advantage of CML over normal hematopoietic cells are unknown. Whereas enhanced proliferation of CML HPC in the BM environment has been well documented,^11,12,13 only little is known on the proliferative behavior of these cells in PB. Elevated numbers of predominantly Ph-positive long-term culture-initiating cells (LTC-IC) have been found in the PB of CML patients and interpreted as reflecting a selective mobilization of Ph-positive LTC-IC in association with an expansion of the neoplastic clone.^12,31 In murine and human hematopoietic cell lines, BCR/ABL transfection replaces some of the growth factor,^32,33,34,35 as well as adhesion requirements.^36,37,38 Also, previous studies have shown that adhesive interactions between CML progenitor cells and BM stroma are defective, leading to abnormal trafficking of malignant progenitors into the PB in BCR/ABL-positive CML.^39,40 Since space in BM is limited, proliferation of CML HPC in PB might therefore contribute to the proliferative advantage of BCR/ABL-positive HPC over their normal counterparts.

In contrast to BCR/ABL-positive CML, no data on the proliferative behavior of CD34⁺ HPC from patients with BCR/ABL-negative myeloproliferative disorders are available. Our finding that PBPC from patients with several different BCR/ABL-negative myeloproliferative disorders are almost noncycling argues in favor of a BCR/ABL-specific effect on cell cycle activation of CD34⁺ cells, which seems to be, at least in part, independent from additional factors provided by the BM microenvironment.

The CD34⁺/38^low immunophenotype defines a highly primitive subpopulation of HPC which are highly enriched for blast colony-forming cells while CD34⁺/38^high cells are associated with lineage commitment and lack the ability to generate clonogenic precursors.⁴¹ Whereas CD34⁺/38^high cells from normal human BM are highly proliferative, CD34⁺/38^low cells have been shown to be noncycling even after a 24 h in vitro exposure to recombinant growth factors.^18,42,43 In contrast to these observations, we have found that CD34⁺ PBPC derived from patients with newly diagnosed BCR/ABL-positive CML are highly proliferative regardless of their CD38 expression status. Since CML originates within the pluripotent stem cell compartment, this finding further supports the view that continuation of cycling under suboptimal growth conditions as present in PB is a pathophysiological mechanism in BCR/ABL-positive CML in vivo independent of the maturation status of the transformed cell.

To further elucidate our finding of CML HPC proliferation outside the BM compartment, we have analyzed the proliferation rates of CD34⁺ HPC from CML patients prior to, as well as during the treatment with the tyrosine kinase inhibitor STI571. Druker et al²⁴ demonstrated that proliferation of lineage-committed CML cells was specifically inhibited by STI571 with no effects on normal cells. In addition, it has been reported that STI571 also inhibits the proliferation of primitive CML progenitor cells from PB that give rise to long-term hematopoiesis.⁴⁴ As mechanisms of action, in vitro assays revealed that STI571 inhibits thymidine uptake, stops DNA synthesis, and induces apoptosis in BCR/ABL-positive cells.⁴⁵ When apoptosis is suppressed, STI571, in turn, induces a cell cycle arrest in G₁ phase.⁴⁶ Accordingly, growth factor-independent G₁/S phase progression of both immature and differentiated primary CML cells was completely corrected by STI571.³⁵ Consistent with these observations, we have found that oral administration of 400 mg STI571 daily lead to a rapid decrease in the proportion of CD34⁺ PBPC in S/G₂M phase in all patients examined. In contrast, the proportions of BM HPC in S/G₂M phase from these patients remained high after 4 weeks of treatment, indicating that CML PBPC are more easily inhibited by STI571 as compared to CD34⁺ CML HPC from BM.

Taken together, our results indicate that p210^bcr/abl stimulates cell cycle entry of both primitive and committed CD34⁺ HPC and substitutes for growth promoting factors provided by the BM environment, a finding that might, at least in part, explain the proliferative advantage of BCR/ABL-positive HPC over their normal counterparts.

Acknowledgements

We thank Ms Susanne Brendel for excellent technical assistance. This work was supported by the Deutsche Krebshilfe (Grant No. 10-1179-Krl) and the Forschungsfonds, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Germany.

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Figure 2  Abnormal cell cycle status of CD34⁺ PBPC in BCR/ABL-positive CML. Proportions of CD34⁺ hematopoietic progenitor cells in S/G₂M phase isolated from peripheral blood (PB) and bone marrow (BM) of 37 patients with BCR/ABL-positive chronic myelogenous leukemia (CML) and 11 patients with BCR/ABL-negative chronic myeloproliferative disorders (MPD).

Figure 3  Abnormal cell cycle status of CD34⁺/CD38^low CML PBPC. Proportions of CD34⁺ hematopoietic progenitor cells in S/G₂M phase isolated from peripheral blood (PB) of 10 CML patients. Correlation of cell cycle state to CD38 expression.

Figure 4  Effect of the tyrosine kinase inhibitor STI571 on the cell cycle status of peripheral blood progenitor cells (PBPC) from six patients with BCR/ABL-positive CML in accelerated phase or blast crisis. Shown are the proportions of CD34⁺ hematopoietic progenitor cells in S/G₂M phase isolated from peripheral blood (PB) prior to (day 0) and during oral administration of 400 mg STI571 daily.

Figure 5  Effect of the tyrosine kinase inhibitor STI571 on the cell cycle status of peripheral blood progenitor cells (PBPC) from seven patients with BCR/ABL-positive CML in chronic phase. Shown are the proportions of CD34⁺ hematopoietic progenitor cells in S/G₂M phase isolated from peripheral blood (PB) prior to (day 0) and during oral administration of 400 mg STI571 daily.

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