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Clinical Cancer Research Vol. 10, 7599-7606, November 15, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

CD34+ Cells from Acute Myeloid Leukemia, Myelodysplastic Syndromes, and Normal Bone Marrow Display Different Apoptosis and Drug Resistance–Associated Phenotypes

Lilia Suárez1,2, María-Belén Vidriales1,2, José García-Laraña4, Guillermo Sanz5, María-José Moreno6, Antonio López3, Susana Barrena2, Rafael Martínez7, Mar Tormo8, Luis Palomera9, Esperanza Lavilla10, Ma Consuelo López-Berges1, María de Santiago3, M. Encarnación Pérez de Equiza11, Jesús F. San Miguel1,2 and Alberto Orfao2,3 for the PETHEMA Cooperative Group

1 Department of Hematology, Hospital Universitario, 2 Centro de Investigación del Cáncer, and 3 Department of Cytometry, University of Salamanca, Salamanca, Spain; 4 Department of Hematology, Hospital Ramón y Cajal, and 5 Department of Hematology, Hospital La-Fe, Valencia; 6 Department of Hematology, Hospital Virgen de la Victoria, Málaga; 7 Department of Hematology, Hospital San Carlos Madrid; 8 Department of Hematology, Hospital Universitario Valencia; 9 Department of Hematology, Hospital Lozano Blesa, Zaragoza, 10 Department of Hematology, Hospital Xeral, Logo; and 11 Department of Hematology, Hospital de Navarra, Pamplona, Spain


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myelodysplastic syndromes and acute myeloid leukemia (AML) are heterogeneous disorders in which conflicting results in apoptosis and multidrug resistance (MDR) have been reported. We have evaluated by multiparameter flow cytometry the expression of apoptosis- (APO2.7, bcl-2, and bax) and MDR-related proteins [P-glycoprotein (P-gp), multidrug resistance protein (MRP), and lung resistance protein (LRP)] specifically on bone marrow (BM) CD34+ cells, and their major CD32–/dim and CD32+ subsets, in de novo AML (n = 90), high-risk myelodysplastic syndrome (n = 9), and low-risk myelodysplastic syndrome (n = 21) patients at diagnosis, and compared with normal BM CD34+ cells (n = 6). CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients displayed higher expression of bcl-2 (P < 0.0001) and lower reactivity for APO2.7 (P = 0.002) compared with low-risk myelodysplastic syndrome and normal controls. Similar results applied to the two predefined CD34+ myeloid cell subsets. No significant differences were found in the expression of P-gp, MRP, and LRP between low-risk myelodysplastic syndrome patients and normal BM, but decreased expression of MRP (P < 0.03) in AML and high-risk myelodysplastic syndromes and P-gp (P = 0.008) in high-risk myelodysplastic syndromes were detected. Hierarchical clustering analysis showed that low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, whereas high-risk myelodysplastic syndromes were clustered together and mixed with the de novo AML patients. In summary, increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would be explained more appropriately in terms of an increased antiapoptotic phenotype rather than a MDR phenotype. In low-risk myelodysplastic syndromes abnormally high apoptotic rates would be restricted to the CD34– cell compartments.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by peripheral cytopenias despite normal or hypercellular bone marrow (BM; ref. 1 ), which could evolve into an overt acute myeloid leukemia (AML). Underlying excessive apoptosis or an apoptosis-associated phenotype of BM cells, including clonal CD34+ precursors, has been identified as a potential explanation for the ineffective hematopoiesis, especially prominent in early myelodysplastic syndrome cases (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . AML also represents a heterogeneous group of malignant stem cell diseases in which CD34+ blast cells are frequently identified (13) .

Resistance of neoplastic cells to chemotherapy has been known to be multifactorial for some time. At present, it is well established that most chemotherapy drugs used in the treatment of hematologic malignancies produce cell death through apoptosis, and, as such, the increase of antiapoptotic mechanisms in tumor cells may represent a major factor responsible for resistance to chemotherapy (14, 15, 16) . Among other proteins, the members of the bcl-2 family either accelerate or inhibit apoptosis in response to a variety of stimuli, and the ratio between pro- and antiapoptotic proteins apparently determines the susceptibility of individual cells to death (15 , 16) . In addition, expression of proteins implicated in drug transport, and/or inactivation, has also been directly involved in modulating sensitivity and resistance to multiple drugs (14) . Among others, these include the P-glycoprotein (P-gp or MDR1), the multidrug resistance protein (MRP), and the lung resistance protein (LRP; ref. 14 ).

In AML, enhanced expression of antiapoptotic markers and MDR1 has been associated with CD34 expression, immature French-American-British (FAB) subtypes, and unfavorable karyotypes, as well as with low complete remission and/or survival rates (17, 18, 19, 20, 21, 22, 23) . In turn, in myelodysplastic syndromes, proapoptotic features are frequently observed, and a multidrug resistance (MDR) phenotype has been associated with progression (22, 23) and transformation into AML (24, 25, 26, 27) . Moreover, in both myelodysplastic syndromes and AML, MDR phenotypes have also been related to CD34 expression (23, 24, 25, 26) .

Interestingly, in AML and myelodysplastic syndromes, the number of CD34+ blast cells is higher in the advanced stages of the disease and at relapse (1 , 28) . This suggests that CD34+ blast cells are particularly resistant to chemotherapy and less susceptible to apoptosis (29 , 30) as compared with their normal counterpart (2 , 15 , 22 , 31, 32, 33) . However, despite the increasing number of studies in which apoptosis and/or drug resistance phenotypes are analyzed in myelodysplastic syndromes and/or AML, conflicting results have frequently been reported (34, 35, 36, 37) , and few studies have been published in which multiparameter analyses of these phenotypes are specifically done on neoplastic CD34+ cells, in comparison to normal CD34+ BM progenitors.

In the present study, we have comparatively analyzed the expression of several apoptosis (APO2.7, bcl-2, and bax) and MDR- associated proteins (P-gp, MRP, and LRP) specifically in BM CD34+ cells and their major CD32–/dim and CD32+ subsets, in de novo AML and myelodysplastic syndromes patients at diagnosis, and compared them to normal CD34+ BM cells. Our results indicate that increased resistance to chemotherapy of CD34+ cells from both AML and high-risk myelodysplastic syndromes would most probably be related to increased resistance to apoptosis rather than to the mechanisms involved in MDR. In turn, in low-risk myelodysplastic syndromes, CD34+ myeloid cells display a high degree of similarity to normal precursors regarding the expression of apoptosis- and MDR-associated proteins, and high apoptotic rates would be restricted to the CD34– cell compartments.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Samples.
BM samples were obtained from 120 patients with myeloid malignancies (90 de novo acute non-promyelocytic leukemia and 30 myelodysplastic syndromes) and from 6 healthy donors. In all of the cases, BM samples were collected in EDTA anticoagulant before the initiation of therapy and processed immediately or within the first 24 hours after collection, at the latest. In the de novo AML patient group, only cases in which a CD34+ blast cell subset was identified were included in this study.

The distribution of the 90 de novo AML cases according to the FAB classification (38) was as follows: M0, 15%; M1, 20%; M2, 29%; M4, 15%; M5, 15%; M6, 5%; and M7, 1%. From the 30 myelodysplastic syndrome patients (39) , 35% were classified as refractory anemia (RA), 19% as RA with ringed sideroblasts, 35% as RA with an excess of blasts, and 11% as RA with an excess of blasts in transformation. Myelodysplastic syndrome patients were grouped according to both the International Prognostic Scoring System (40) and the Spanish Prognostic Scoring System (41) into low-risk (International Prognostic Scoring System < 1.5; Spanish Prognostic Scoring System < 3)–21 cases (70%)–and high-risk myelodysplastic syndromes (International Prognostic Scoring System > 1.5; Spanish Prognostic Scoring System > 3)–9 cases (30%).

Immunophenotypic Studies.
Erythrocyte-lysed, freshly obtained BM samples were analyzed with a four-color immunofluorescence technique for the simultaneous staining of surface markers alone or surface markers together with cytoplasmic (cyt) antigens, according to well-established methods, which have been previously described in detail (15 , 42) . The following combinations of monoclonal antibodies conjugated to FITC/phycoerythrin (PE)/PE-cyanine 5 (Cy5)/allophycocyanine (APC) were used: cytbcl-2/CD32/CD34/CD45, cytbax/CD32/CD34/CD45, CD32/cytAPO2.7/CD34/CD45, CD19/P-gp/CD34/CD33, cytMRP/CD34/CD19/CD33, and cytLRP/CD34/CD19/CD33. Each of these 4-color combinations allowed the specific identification of the whole CD34+ cell population present in the BM samples analyzed (Fig. 1A)Citation and the discrimination within all of the CD34+ cells between B-lymphoid and myeloid CD34+ cells (Fig. 1B)Citation , providing identical results as regards the identification and enumeration of these cell subsets. Additionally, two subpopulations of CD34+ non-B cells could also be identified with the first three combinations, one corresponding to the more immature (CD34+/CD32-/dim) and the other to the more mature myeloid cells (CD34+/CD32high; Fig. 1CCitation ). The specific monoclonal antibodies clones used and their source was as follows: anti-APO2.7–PE (clone 2.7A6A3), anti-bax (clone 4F11), CD19-PECy5 (clone J4.119), and CD34-PECy5 (clone 581) were purchased from Beckman-Coulter (Miami, FL); anti-Bcl-2–FITC (clone 124) was obtained from DakoCytomation (Glostrup, Denmark); anti-MRP (clone MRPm6) and anti-LRP (clone LRP-56) were from Chemicon (Temecula, CA); CD19-FITC (clone 4G7), CD34-PE (clone 8G12), CD33-APC (clone p67.6) and anti-P–gp (clone 15D3) were from Becton Dickinson Biosciences (San José, CA); CD32-FITC (clone AT 10) and CD32-PE (clone AT 10) were obtained from Serotec (Oxford, United Kingdom); and CD45-APC (clone HI30) was purchased from Caltag Laboratories (San Francisco, CA). Appropriate isotype-matched negative controls were stained in parallel.



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  		Fig. 1. Representative SSC/CD45/CD34 three-dimensional dot plot corresponding to the whole cellularity of a BM sample from a patient with low-risk myelodysplastic syndromes, illustrating in A how total CD34+ cells (black dots) were identified based on their low/intermediate SSC and strong reactivity for CD34; B shows the distribution of the B-lymphoid (black dots) and myeloid (gray dots) precursor cells within the total (gated) CD34+ BM cells, based on their FSC/SSC characteristics. A representative bivariate dot plot of CD32 versus CD34 gated CD34+ cells in which CD34+/CD32– B-lymphoid precursors (dark gray dots; 13%) as well as both the CD34+/CD32–/dim (gray dots; 77%) and CD34+/CD32+ (black dots; 10%) subsets of myeloid precursors are displayed is shown in C.

 
Data acquisition was done in a FACScalibur flow cytometer (Becton Dickinson Biosciences) with the CellQuest software program (Becton Dickinson Biosciences). In all of the cases analyzed, a minimum of 2 x 104 BM events corresponding to the whole BM cellularity were acquired for each four-color staining. In addition, a second acquisition step was done with an electronic "live gate," in which only those cells displaying low/intermediate SSC and strong reactivity for CD34 were selected from a total of 3 x 105 events, to obtain information on high numbers of CD34+ cells for each combination of monoclonal antibodies. For data analysis, the Paint-a-Gate software program (Becton Dickinson Biosciences) was used.

The percentage of CD34+ ranged from 5 to 95% (median 42%) of all of the BM nucleated cells in de novo AML and between 0.3 and 22% (median 3.6%) of all of the nucleated cells in myelodysplastic syndromes, significantly (P < 0.0001) higher numbers of CD34+ cells were being found in high-risk as compared with low-risk myelodysplastic syndromes–median of 8.5% (range, 1.5 to 22%) versus 1.5% (range, 0.3 to 4.4%), respectively. In normal BM, the median number of CD34+ cells was of 0.5% (range, 0.22 to 0.7%). In both de novo AML and high-risk myelodysplastic syndrome patients, all of the BM CD34+ cells corresponded to myeloid cells, whereas CD34+ B-lymphoid precursors were present in all of the normal BM samples and in 8 of 21 (38%) low-risk myelodysplastic syndromes patients, where they represented 0.06 ± 0.03% and 0.1 ± 0.4% of the overall cellularity, respectively (Table 1)Citation .


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  		Table 1 Distribution of CD34+ cells and their major B-lymphoid and myeloid subsets in BM samples from patients with AML, high-risk MDS, and low-risk MDS as compared to normal volunteers

 
Among CD34+ non-B lymphoid progenitors, a clear predominance of the more immature CD34+/CD32–/dim precursors, as compared with the CD34+/CD32+ cells, was observed in all groups of individuals (high-risk myelodysplastic syndromes, 8.2 ± 7.7 versus 0.7 ± 0.8; P = 0.002; low-risk myelodysplastic syndromes, 1.1 ± 0.9 versus 0.3 ± 0.3; P < 0.0001; and normal BM, 0.32 ± 0.1 versus 0.09 ± 0.03; P = 0.004), except in the de novo AML cases who had similar proportions of both subpopulations (42 ± 22% versus 41 ± 24%, respectively; P > 0.05; Table 1Citation ).

The mean fluorescence intensity obtained for each individual apoptosis- and MDR-associated marker analyzed and the corresponding isotype-matched negative control were specifically recorded for each cell subpopulation. For each individual marker, results were reported as relative fluorescence intensity units (RFI) calculated as the ratio between the mean fluorescence intensity of the cells under analysis specifically stained for a given apoptosis- or MDR-associated protein and the mean fluorescence intensity obtained for the same cell subset in the corresponding isotype-matched negative control. In all of the cases, the consensus recommendations for staining and analysis of multidrug resistance-associated proteins were strictly followed (34, 35, 36) .

Statistical Methods.
For all of the phenotypic variables under study, median and mean values as well as their SD and range were calculated with the SPSS software program (SPSS 10.0. Inc., Chicago, IL). The Kruskal-Wallis and the Mann-Whitney U tests were used to estimate the statistical significance of the differences observed between groups. P < 0.05 was considered statistically significance.

For hierarchical clustering analyses, log-transformed RFI values of each apoptotic and MDR-related protein measured on BM CD34+ cells for each individual sample were used. Before the analysis, data were normalized dividing the RFI value of each apoptosis- and MDR-associated protein obtained for each individual sample by the mean RFI value observed for the same protein in normal BM samples; a logarithmic transformation was applied to the values of the ratio for individual data sets.

Hierarchical clustering analysis was done with the J-Express Pro V2.1 software (MolMine AS, Bergen, Norway). The average-linkage method, based on the analysis of Euclidean distance matrices, was used for the clustering of individual cases from each diagnostic group and normal controls. In this part of the study, the following variables were considered: relative number of CD34+ cells present in the BM and the bcl-2, APO2.7, bax, P-gp, MRP, LRP, and RFI ratios specifically obtained for BM CD34+ cells in each individual sample.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Concerning the expression of apoptosis-associated proteins (Fig. 2)Citation , BM CD34+ cells in AML and high-risk myelodysplastic syndrome cases showed a significantly (P < 0.0001) higher bcl-2 expression than low-risk myelodysplastic syndrome patients and control subjects (mean RFI ± SD of 13.1 ± 7.2 and 12.4 ± 7.0 versus 7.0 ± 3.0 and 7.3 ± 1.5, respectively), no statistically significant differences were found between AML and high-risk myelodysplastic syndromes nor between low-risk myelodysplastic syndromes and control BM. As shown in Fig. 2Citation , these differences similarly applied to the two predefined CD34+/CD32–/dim (P < 0.0001) and CD34+/CD32high (P < 0.0001) myeloid subsets. On comparing these two cell subpopulations, it was observed that in AML patients the expression of bcl-2 was significantly higher in the more immature subset (CD34+/CD32–/dim) as compared with the more mature group [CD34+/CD32high; mean RFI ± SD of 14.8 ± 7.9 versus 10.8 ± 5.2 (P = 0.01)]. By contrast, in myelodysplastic syndromes or normal individuals, no significant differences were observed between these two subpopulations according to bcl-2 expression. In line with bcl-2 results, CD34+ cells from de novo AML and high-risk myelodysplastic syndrome patients exhibited lower reactivity for APO2.7 (mean RFI ± SD of 11.9 ± 16.7 and 6.8 ± 4.6, respectively) than BM CD34+ cells from low-risk myelodysplastic syndromes and normal individuals (mean RFI ± SD of 18.9 ± 10.3 and 18.7 ± 6.7, respectively; P < 0.005). Concerning the two CD34+ myeloid cell subsets defined according to their reactivity for CD32, similar results were observed. Accordingly, the levels of APO2.7 were significantly lower in both cell subsets in de novo AML patients and high-risk myelodysplastic syndrome cases as compared with low-risk myelodysplastic syndromes and normal controls [mean RFI ± SD for the CD34+/CD32–/dim (P < 0.0001) and CD34+/CD32+ (P < 0.0001) cell subsets of 11.3 ± 12.9 and 12.7 ± 21.1 in de novo AML and 6.3 ± 4.0 and 9.3 ± 6.8 in high-risk myelodysplastic syndromes versus 17.0 ± 10.2 and 24.4 ± 13.8 in low-risk myelodysplastic syndromes and 16.3 ± 5.3 and 25.5 ± 9.6 in normal controls, respectively]. No statistically significant differences were found in the expression of APO2.7 in the CD34+/CD32–/dim population, as compared with CD34+/CD32+ cells, in any of the groups of individuals included in this study. The pattern of expression of bax was similar in de novo AML, myelodysplastic syndromes, and normal BM in all of the populations of the CD34+ BM cells analyzed (Fig. 2)Citation .



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  		Fig. 2. RFI reflecting cyt expression of the bcl-2, APO2.7, and bax apoptosis-related intracellular proteins on CD34+ cells and their CD32–/dim and CD32+ myeloid cell subsets from de novo AML (n = 90), high-risk (hr) myelodysplastic syndromes (n = 9), low-risk (lr) myelodysplastic syndromes (n = 21), and normal BM (NBM; n = 6), as analyzed by multiparameter flow cytometry. Boxes extend from the 25th to the 75th percentiles; the line in the middle and vertical lines represent median values and the 95% confidence intervals, respectively. The following statistically significant differences were observed: *, P ≤ 0.0001 on comparing cytbcl-2 RFI of CD34+ cells from AML and hr-myelodysplastic syndrome cases with lr-myelodysplastic syndrome cases and normal BM; **, P ≤ 0.005 and ***, P ≤ 0.01 on comparing the cytAPO2.7 RFI of CD34+ cells from de novo AML patients with low-myelodysplastic syndrome patients and normal BM; and •, P ≤ 0.005 on comparing the cytAPO2.7 RFI of CD34+ cells from hr-myelodysplastic syndrome cases with that of lr-myelodysplastic syndrome patients. Bars, ±SD.

 
As regards the expression of MDR-associated proteins, BM CD34+ myeloid cells from de novo AML showed lower (P = 0.03) MRP expression than normal controls (mean RFI ± SD of 2.4 ± 1.8 versus 3.1 ± 0.8, respectively), but no differences were observed for P-pg nor for LRP between either groups of individuals. Moreover, in the expression of these three MDR-proteins, we did not find any statistically significant differences between de novo AML and either low-risk or high-risk myelodysplastic syndrome patients. Regarding myelodysplastic syndromes patients, BM CD34+ cells from high-risk myelodysplastic syndromes cases showed a lower expression of both P-gp (P = 0.008) and MRP (P = 0.009), but not LRP, as compared with normal controls (mean RFI ± SD for P-gp of 1.9 ± 0.9 versus 4.0 ± 2.0 and for MRP of 1.5 ± 0.5 versus 3.1 ± 0.8, respectively). No statistically significant differences were found between low-risk myelodysplastic syndromes, high-risk myelodysplastic syndromes, and normal controls in terms of the expression of P-gp, MRP, and LRP on BM CD34+ cells (Fig. 3)Citation .



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  		Fig. 3. RFI reflecting membrane reactivity for P-gp and intracellular (cyt) expression of the MRP and LRP multidrug resistance-associated proteins on CD34+CD33+ BM precursor cells from de novo AML (n = 90), high-risk (hr) myelodysplastic syndromes (n = 9), low-risk (lr) myelodysplastic syndromes (n = 21), and normal BM (NBM; n = 6), as analyzed by multiparameter flow cytometry. Boxes extend from the 25th to the 75th percentiles; the line in the middle and vertical lines represent median values and the 95% confidence intervals, respectively. *, P = 0.03 on comparing the MRP RFI of AML cases with that of normal BM and •, P < 0.005 on comparing P-gp and MRP RFI of hr myelodysplastic syndrome cases with that of normal BM. Bars, ±SD.

 
Hierarchical clustering analysis based on the relative number of CD34+ cells present in the BM and the amount expression of the different apoptosis- (bcl-2, APO2.7, and bax) and MDR-associated proteins (P-gp, MRP, and LRP) allowed the classification of all of the individual samples into two major groups (Fig. 4)Citation . In the first group, low-risk myelodysplastic syndrome patients were clustered next to normal BM samples, and in the second group, high-risk myelodysplastic syndromes were clustered together and mixed with de novo AML patients.



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  		Fig. 4. Hierarchical clustering analysis of de novo AML (n = 90), high-risk myelodysplastic syndromes (n = 9), low-risk myelodysplastic syndromes (n = 21), and normal BM samples (n = 6) based on the number and phenotype of CD34+ BM cells for the apoptosis (bcl-2, APO2.7, and bax) and MDR-associated proteins (P-gp, MRP, and LRP) analyzed. Rows represent the different cases studied, labeled in a gray scale according to diagnosis, and columns represent each individual variable included in the analysis. The relative level of expression of each marker is represented by a color: red = expression > mean; green = expression < mean; color intensity represents the magnitude of the deviation from the mean. As indicated, the scale extends from –7.630 to +7.630 (in log2units).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The CD34+ compartment of BM cells in both AML and myelodysplastic syndromes is believed to play a crucial role in the behavior of the disease because it potentially contains the neoplastic precursors with clonogenic capability. Until now, limited information based on conflicting results has been available concerning the expression of apoptosis- and MDR-related proteins on BM CD34+ cells from myelodysplastic syndrome and AML patients (3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 23, 24, 25, 26, 27) . Such controversial results could be attributed, at least in part, to differences in the study design (34, 35, 36, 37 , 43) . To avoid methodological pitfalls, in the present study we have restricted the analysis of apoptosis and MDR phenotypes to the study of the expression of several well-characterized proteins by multiparametric flow cytometry in fresh whole BM samples, obtained from untreated AML and myelodysplastic syndrome patients that contained a well-defined population of CD34+ cells, and compared them to normal BM CD34+ cells.

At present, it is well established that entry into the execution phase of apoptosis greatly depends on the balance between the cytoplasmic levels of several pro- and antiapoptotic proteins from which the members of the bcl-2 protein play an essential role (15 , 16 , 37) . In the present study, we found high levels of the bcl-2 antiapoptotic protein and low amounts of the APO2.7 proapoptotic protein on BM CD34+ myeloid cells from AML and high-risk myelodysplastic syndrome patients. This concurs with previous studies (3 , 5 , 7, 8, 9) . Of particular interest is the observation that high-risk myelodysplastic syndrome cases displayed greater amounts of bcl-2 and reduced expression of APO2.7 as compared with low-risk myelodysplastic syndrome patients, resembling the phenotype of AML cases. In line with these findings, several authors (3, 4, 5, 6, 7 , 9) have reported that in high-risk myelodysplastic syndromes, CD34+ cells are more resistant to apoptosis than they are in low-risk myelodysplastic syndromes, and that an increased ratio between proapoptotic and antiapoptotic proteins from the bcl-2 family is associated with disease evolution into aggressive forms of the disease (4 , 7 , 9) . In contrast, Huh et al. (10) did not find any differences between RA with an excess of blasts in transformation and other subtypes of myelodysplastic syndromes, although when compared with AML patients, CD34+ cells from RA with an excess of blasts in transformation cases were more prone to apoptosis. Although we have not explored the expression of either bad or bcl-X in the present study, no statistically significant differences were observed in bax levels in any of the groups analyzed, supporting the notion (4 , 9) that some proapoptotic proteins like bax do not play a crucial role in these myeloid malignancies.

Interestingly, we found that expression of the APO2.7, bcl-2, and bax proteins on BM CD34+ cells from low-risk myelodysplastic syndromes were within the normal range. These results are in line with those reported by Davis et al. (5) and Pecci et al. (12) who found no differences in the percentage of bcl-2 and the bcl-2 index or the degree of apoptosis between early myelodysplastic syndromes and normal BM. These findings are of particular interest in light of our previous observations (44) from a similar series of myelodysplastic syndrome patients, in which we showed that particularly in low-risk myelodysplastic syndromes, CD34– nucleated red cells and myelomonocytic cells display a proapoptotic phenotype, probably reflecting the greater susceptibility of the cell to apoptosis. Such differences in the apoptotic phenotype of the CD34+ and CD34– compartments of BM cells in myelodysplastic syndromes concur with the clinical behavior of the disease, and they suggest that the more mature CD34– cells, but not the CD34+ cell compartment, could be responsible for the increased apoptosis observed in myelodysplastic syndromes (12 , 44) . On the basis of these findings, we aimed to investigate whether or not within the different maturational compartments of CD34+ myeloid precursors, there could also exist differences in the apoptotic-related phenotype. Our results show no significant differences in the expression of bcl-2, bax, and APO2.7 between the more immature CD34+/CD32–/dim and the more differentiated CD34+/CD32+ myeloid cells from myelodysplastic syndromes patients nor from normal BM. In contrast, in AML, the CD34+/CD32–/+dim subpopulation displayed greater expression of bcl-2 than the more mature CD34+/CD32+ cells, which is consistent with previous results (17 , 18 , 31) showing that expression of bcl-2 decreases with maturation of blast cells.

In contrast to AML (14 , 20 , 23) , until now few studies have been reported (24, 25, 26, 27 , 45) in which the MDR phenotype of CD34+ cells is analyzed in myelodysplastic syndrome patients. In the present study, with a quantitative and objective approach, we found no statistically significant differences in the expression of P-gp, MRP, and LRP between CD34+ BM cells from low-risk myelodysplastic syndrome patients and normal BM. In contrast, high-risk myelodysplastic syndrome and AML patients showed a lower MRP expression but normal LRP levels as compared with normal BM. Regarding P-gp expression, decreased levels were found in high-risk myelodysplastic syndromes but not in AML. Previous studies (24, 25, 26, 27) suggested that P-gp and MRP are less frequently expressed in low-risk myelodysplastic syndromes compared with high-risk myelodysplastic syndromes. In another study (45) , LRP expression in myelodysplastic syndromes did not show any correlation with the FAB subtype, and it was suggested that Pgp and LRP expression may be more frequent in myelodysplastic syndromes than in de novo AML. These discrepancies with respect to our findings may be attributed to differences in methodological approaches that were used for the definition of the MDR phenotype. However, in line with others (46) , overall our data supports the notion that the pattern of expression of MDR-associated proteins on CD34+ cells is conserved during leukemic transformation. Similarly, it has been recently reported that other independent MDR-related proteins involved in drug efflux mechanisms, such as the breast cancer resistant protein (BCRP/ABCG2), are expressed at the mRNA level in both normal CD34+ cells and AML cells at similar quantities (47 , 48) ; despite numerous reports of BCRP/ABCG2 expression in AML, little evidence supports a role for this drug-efflux protein in patient outcome (49) . This could be because its expression did not prove to confer resistance to idarubicin (47) , a drug frequently used in AML polychemotherapy protocols.

Until now, no reports have been published concerning hierarchical clustering analysis based on the number of CD34+ cells present in the BM and/or the expression of apoptosis and MDR-associated proteins. Interestingly, in our hands, hierarchical clustering analysis grouped low-risk myelodysplastic syndrome patients with normal BM and high-risk myelodysplastic syndromes together with de novo AML patients, thereby supporting our results.

These results would suggest that resistance to chemotherapy in both AML and high-risk myelodysplastic syndromes could most probably be related to the increased resistance in CD34+ leukemic cells to apoptosis than to the mechanisms evolved in intracellular or extracellular drug transport and extrusion. In addition, our results show that in low-risk myelodysplastic syndromes, CD34+ myeloid cells display a high degree of similarity to normal precursors regarding the expression of apoptosis- and MDR-associated proteins.


   FOOTNOTES
 
Grant support: Grant 63022 from the Agencia Española de Cooperación Internacional from Madrid, Spain, and Grant SB 200-0089 Secretaría de Estado de Educación y Universidades from Spain (L. Suárez), included in the program Estancia de Profesores, Investigadores, Doctores y Tecnólogos Extranjeros en España and a National Grant from the Spanish Dirección General de Ciencia y Technología (SAF 2001/1687).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: María-Belén Vidriales, Department of Hematology, Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain. Phone: 34-93-29-13-84; Fax: 34-93-29-46-24; E-mail: mbvidri{at}usal.es

Received 3/27/04; revised 6/18/04; accepted 7/12/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. San Miguel JF, Sanz GF, Vallespí T, del Cañizo MC, Sanz MA. Myelodysplastic syndromes. Crit Rev Oncol-Hematol 1996;23:57-93.[Medline]
  2. Domenico D, Aiello A, Soligo D, et al Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells. Blood 1992;5:1291-8.
  3. Rajapaksa R, Ginzton N, Rott LS, Greenberg PL. Altered oncoprotein expression and apoptosis in myelodysplastic syndrome marrow cells. Blood 1996;88:4275-87.[Abstract/Free Full Text]
  4. Parker JE, Fishlock KL, Mijovic A, et al Low-risk myelodysplastic syndrome is associated with excessive apoptosis and an increased ratio of pro- versus anti-apoptotic bcl-2-related proteins. Br J Haematol 1998;103:1075-82.[CrossRef][Medline]
  5. Davis RE, Greenberg PL. Bcl-2 expression by myeloid precursors in myelodysplastic syndromes: relation to disease progression. Leuk Res 1998;22:767-77.[CrossRef][Medline]
  6. Tsoplou P, Kouraklis-Symeonidis A, Thanopoulou E, et al Apoptosis in patients with myelodysplastic syndromes: differential involvement of marrow cells in ‘good’ versus ‘poor’ prognosis patients and correlation with apoptosis-related genes. Leukemia (Baltimore) 1999;13:1554-63.
  7. Parker JE, Mufti GJ, Rasool F, et al The role of apoptosis, proliferation, and the Bcl-2-related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood 2000;96:3932-8.[Abstract/Free Full Text]
  8. Berger G, Hunault-Berger M, Rachieru P, et al Increased apoptosis in mononucleated cells but not in CD34 (+) cells in blastic forms of myelodysplastic syndromes. Hematol J 2001;2:87-96.[CrossRef][Medline]
  9. Boudard D, Vasselon C, Bertheas MF, et al Expression and prognostic significance of Bcl-2 family proteins in myelodysplastic syndromes. Am J Hematol 2002;70:115-25.[CrossRef][Medline]
  10. Huh YO, Jilani I, Estey E, et al More cell death in refractory anemia with excess blasts in transformation than in acute myeloid leukemia. Leukemia (Baltimore) 2002;16:2249-52.
  11. Lin CW, Manshouri T, Jilani I, et al Proliferation and apoptosis in acute and chronic leukemias and myelodysplastic syndrome. Leuk Res 2002;26:551-9.[CrossRef][Medline]
  12. Pecci A, Travaglino E, Klersy C, Invernizzi R. Apoptosis in relation to CD34 antigen expression in normal and myelodysplastic bone marrow. Acta Haematol (Basel) 2003;109:29-34.
  13. Macedo A, Orfao A, González M, et al Immunological detection of blast cell subpopulations in acute myeloblastic leukemia at diagnosis: implications for minimal residual disease studies. Leukemia (Baltimore) 1995;9:993-8.
  14. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature Rev Cancer 2002;2:48-58.[CrossRef][Medline]
  15. Igney FH, Krammer PH. Death and anti-death: tumor resistance to apoptosis. Nature Rev Cancer 2002;2:277-88.[CrossRef][Medline]
  16. Kaufmann SH, Vaux DL. Alterations in the apoptotic machinery and their potential role in anticancer drug resistance. Oncogene 2003;22:7414-30.[CrossRef][Medline]
  17. Bradbury DA, Russell NH. Comparative quantitative expression of bcl-2 by normal and leukaemic myeloid cells. Br J Haematol 1995;91:374-9.[Medline]
  18. Suárez L, Vidriales B, García-Laraña J, et al Multiparametric analysis of apoptotic and multi-drug resistance phenotypes according to the blast cell maturation stage in elderly patients with acute myeloid leukemia. Haematologica 2001;86:1287-95.[Medline]
  19. Tothova E, Fricova M, Stecova N, Kafkova A, Elbertova A. High expression of Bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Neoplasma (Bratisl) 2002;49:141-4.
  20. Wuchter C, Karawajew L, Ruppert V, et al Clinical significance of CD95, bcl-2 and bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics. Leukemia (Baltimore) 1999;13:1943-53.
  21. Del Poeta G, Venditti A, Del Principe MI, et al Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003;101:2125-31.[Abstract/Free Full Text]
  22. Takeshita A, Shinjo K, Ohnishi K, Ohno R. Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells. Br J Haematol 1996;93:18-21.[CrossRef][Medline]
  23. List AF. The role of multidrug resistance and its pharmacological modulation in acute myeloid leukemia. Leukemia (Baltimore) 1996;10:937-42.
  24. List AF, Spier CM, Cline A, et al Expression of the multidrug resistance gene product (P-glycoprotein) in myelodysplasia is associated with a stem cell phenotype. Br J Haematol 1991;78:28-34.[Medline]
  25. Sonneveld P, van Dongen JJ, Hagemeijer A, et al High expression of the multidrug resistance P-glycoprotein in high-risk myelodysplasia is associated with immature phenotype. Leukemia (Baltimore) 1993;7:963-9.
  26. Lepelly P, Soenen V, Preudhomme C, et al Expression of the multidrug resistance p-glycoprotein and its relationship to hematological characteristic and response to treatment in myelodysplastic syndromes. Leukemia (Baltimore) 1994;8:998-1004.
  27. Poulain S, Leppelly P, Preudhomme C, et al Expression of multidrug resistance-associated protein in myelodysplastic syndromes. Br J Haematol 2000;110:591-8.[CrossRef][Medline]
  28. Macedo A, San Miguel JF, Vidriales MB, et al Phenotypic changes in acute myeloid leukemia: implications in the detection of minimal residual disease. J Clin Pathol 1996;49:15-8.[Abstract/Free Full Text]
  29. Raspadori D, Lauria F, Ventura MA, et al Incidence and prognostic relevance of CD34 expression in acute myeloblastic leukemia: analysis of 141 cases. Leuk Res 1997;21:603-7.[CrossRef][Medline]
  30. Konopleva M, Zhao S, Hu W, et al The anti-apoptotic genes Bcl-X(L) and Bcl-2 are over-expressed and contribute to chemoresistance of non-proliferating leukaemic CD34+ cells. Br J Haematol 2002;118:521-34.[CrossRef][Medline]
  31. Porwit-MacDonald A, Ivory K, Wilkinson S, et al Bcl-2 protein expression in normal human bone marrow precursors and in acute myelogenous leukemia. Leukemia (Baltimore) 1995;9:1191-8.
  32. Drach D, Zhao S, Drach J, et al Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood 1992;80:2729-34.[Abstract/Free Full Text]
  33. Legrand O, Perrot JY, Tang R, et al Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal pheripheral blood and bone marrow haemopoietic cells. Br J Haematol 1996;94:23-33.[CrossRef][Medline]
  34. Beck WT, Grogan TM. Methods to detect P-glycoprotein and implications for other drug resistance-associated proteins. Leukemia (Baltimore) 1997;11:1107-9.
  35. Legrand O, Simonin G, Zittoun R, Marie JP. Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia: a critical evaluation by three techniques. Leukemia (Baltimore) 1998;12:1367-74.
  36. Marie JP, Legrand O, Perrot JY, et al Measuring multidrug resistance expression in human malignancies: elaboration of consensus recommendations. Semin Hematol 1997;4(Supp 5):63-71.
  37. Parker JE, Mufti GJ. The role of apoptosis in the pathogenesis of the myelodysplastic syndromes. Int J Hematol 2001;73:416-28.[Medline]
  38. Bennett JM, Catovsky D, Daniel MT, et al Proposed revised criteria for the classification of the acute myeloid leukaemia. Ann Intern Med 1985;103:620-9.
  39. Bennett JM, Catovsky D, Daniel MT, et al Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982;51:189-99.[Medline]
  40. Greenberg P, Cox C, Lebeau MM, et al International scoring system for evaluation prognosis in myelodysplastic syndromes. Blood 1997;89:2079-88.[Abstract/Free Full Text]
  41. Sanz GF, Sanz MA, Vallespí T, et al Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients. Blood 1989;74:395-408.[Abstract/Free Full Text]
  42. Kappelmayer J, Gratama JW, Karaszi E, et al Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols. J Immunol Methods 2000;242:53-65.[CrossRef][Medline]
  43. Shetty V, Hussaini S, Broady-Robinson L, et al Intramedullary apoptosis of hematopoietic cells in myelodysplastic syndrome patients can be massive: apoptotic cells recovered from high-density fraction of bone marrow aspirates. Blood 2000;96:1388-92.[Abstract/Free Full Text]
  44. Suárez L, Vidriales MB, Sanz G, et al Expression of APO2.7, bcl-2 and bax apoptosis-associated proteins in CD34-ve bone marrow cell compartments from patients with myelodysplastic syndromes (MDS). Leukemia (Baltimore). 2004;18:1311-13.
  45. Leppelly P, Poulain S, Grardel N, et al Expression of lung resistance protein and correlation with other drug resistance proteins and outcome in myelodysplastic syndromes. Leuk Lymphoma 1998;29:547-51.[Medline]
  46. Sonneveld P. Multidrug resistance in haematological malignancies. J Intern Med 2000;247:521-34.[Medline]
  47. Abbott BL, Colapietro AM, Barnes Y, et al Low levels of ABCG2 expression in adult AML blast samples. Blood 2002;100:4594-601.[Abstract/Free Full Text]
  48. Nakanishi T, Karp JE, Tan M, et al Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients. Clin Cancer Res 2003;9:3320-8.[Abstract/Free Full Text]
  49. Abbott BL. ABCG2 (BCRP) expression in normal and malignant hematopoietic cells. Hematol Oncol 2003;21:115-30.[CrossRef][Medline]



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