scholarly journals Expression of Ia antigens on myeloid progenitor cells in chronic myeloid leukemia: direct analysis using partially purified colony- forming cells

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 414-422 ◽  
Author(s):  
SA Cannistra ◽  
JF Daley ◽  
P Larcom ◽  
JD Griffin
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Antigen Expression ◽  
Colony Stimulating Factors ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Ia Antigens ◽  
Myeloid Progenitor Cells

Abstract The regulation of Ia (HLA-DR) antigen expression on myeloid progenitor cells may be closely related to the control of myelopoiesis in both normal individuals and chronic myeloid leukemia (CML) patients. In an effort to study directly the expression and behavior of Ia surface molecules on myeloid progenitor cells, we used an immunologic purification technique to enrich these cells approximately 100-fold from the peripheral blood of CML patients. The majority of cells in this blast population expressed HLA-DR antigens. Thirty percent to 40% of cells could form a granulocyte or monocyte colony in agar, and these cells tended to express the highest levels of HLA-DR. The number of HLA- DR molecules per cell increased about twofold as the cells tranversed the cell cycle from G0/G1 to G2/M. This was true for unstimulated cells or cells exposed to colony-stimulating factors. Some of this increase was related to a corresponding increase in cell size and is also seen with other cell surface antigens such as beta-2-microglobulin. Ia antigen expression was not modified by culture with colony-stimulating factors, fetal calf serum, or serum-free, prostaglandin-free medium for periods of up to 24 hours. These results demonstrate that Ia antigens are expressed on the myeloid progenitor cells of CML, are increased in the S and G2/M phases of the cell cycle, and are stable under most in vitro culture conditions for at least 24 hours of culture.

scholarly journals Expression of Ia antigens on myeloid progenitor cells in chronic myeloid leukemia: direct analysis using partially purified colony- forming cells

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 414-422
Author(s):  
SA Cannistra ◽  
JF Daley ◽  
P Larcom ◽  
JD Griffin
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Antigen Expression ◽  
Colony Stimulating Factors ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Ia Antigens ◽  
Myeloid Progenitor Cells

The regulation of Ia (HLA-DR) antigen expression on myeloid progenitor cells may be closely related to the control of myelopoiesis in both normal individuals and chronic myeloid leukemia (CML) patients. In an effort to study directly the expression and behavior of Ia surface molecules on myeloid progenitor cells, we used an immunologic purification technique to enrich these cells approximately 100-fold from the peripheral blood of CML patients. The majority of cells in this blast population expressed HLA-DR antigens. Thirty percent to 40% of cells could form a granulocyte or monocyte colony in agar, and these cells tended to express the highest levels of HLA-DR. The number of HLA- DR molecules per cell increased about twofold as the cells tranversed the cell cycle from G0/G1 to G2/M. This was true for unstimulated cells or cells exposed to colony-stimulating factors. Some of this increase was related to a corresponding increase in cell size and is also seen with other cell surface antigens such as beta-2-microglobulin. Ia antigen expression was not modified by culture with colony-stimulating factors, fetal calf serum, or serum-free, prostaglandin-free medium for periods of up to 24 hours. These results demonstrate that Ia antigens are expressed on the myeloid progenitor cells of CML, are increased in the S and G2/M phases of the cell cycle, and are stable under most in vitro culture conditions for at least 24 hours of culture.

scholarly journals The pattern of HLA-DR and HLA-DQ antigen expression on clonable subpopulations of human myeloid progenitor cells

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 379-384 ◽  
Author(s):  
RL Sparrow ◽  
N Williams
Keyword(s):  
Cell Membrane ◽  
Progenitor Cells ◽  
Marrow Cell ◽  
Myeloid Cell ◽  
Antigen Expression ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Hla Dq ◽  
Myeloid Progenitor Cells ◽  
Kinetics Of

Abstract Three subpopulations of human myeloid progenitor cells (CFU-GM) can be distinguished by differences in their kinetics of development; the liquid phase pre-CFU-GM, the day 14 CFU-GM, and the day 7 CFU-GM. The relative cell membrane densities of the HLA-DR and HLA-DQ antigens expressed by the three subpopulations was investigated by comparing the amount of antibody required to deplete bone marrow cell preparations of each cell type. Three separate approaches were used--complement (C') cytotoxicity, antiglobulin/C'-cytotoxicity and immune rosette depletion. Similar results were obtained for all three procedures, although the latter two gave a tenfold greater sensitivity over the standard C'-cytotoxicity method. At saturating anti-HLA-DR antibody concentrations, 85% to 95% of cells within the three myeloid subpopulations were found to express HLA-DR antigens. However, the relative amount of HLA-DR expressed by these subpopulations increased from the pre-CFU-GM to the day 7 CFU-GM. The expression of HLA-DQ antigens was considerably lower and could only be detected by using the more sensitive procedures. Only 50% of day 7 and 14 CFU-GM progenitor cells expressed detectable HLA-DQ antigens, whereas a greater proportion (80%) of the pre-CFU-GM were HLA-DQ positive. The pattern of HLA-DQ expression on these clonable precursors was quite distinct and opposite to the cell membrane density of the HLA-DR antigens. Because these three progenitor cell populations are thought to be linked in differentiation sequence, these results provide indirect support for the hypothesis that HLA class II antigens are implicated in regulatory mechanisms during normal myeloid cell differentiation.

Interlaboratory Validation of a 3-Tube 10-Color Antibody Flow Cytometry Panel for Diagnosis of Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4941-4941
Author(s):  
Wolfgang Kern ◽  
Marie-Christine Béné ◽  
Anna Porwit
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Research Funding ◽  
Expression Patterns ◽  
Antigen Expression ◽  
Erythroid Cells ◽  
Cell Compartments ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Myeloid Progenitor Cells

Abstract Abstract 4941 Background: Multiparameter flow cytometry (MFC) is increasingly used in the diagnostic work-up of patients with suspected myelodysplastic syndromes (MDS). Modern MFC allows the simultaneous detection of 10 fluorochrome-conjugated antibodies recognizing different antigens and thus provides the basis for a refined detection of MDS-related aberrant antigen expression. While efforts are made to harmonize MFC approaches to diagnose MDS, especially on the markers useful to investigate (Westers et al., Leukemia 2012), there has been no interlaboratory evaluation yet of uniform procedures including a common antibody panel and thus reproducibility remains to be proven. Aims: Prove the interlaboratory comparability of diagnostic read-out using a uniform MFC approach to diagnose MDS. Methods: A 10-color/3-tube panel was designed using the fluorochromes FITC, PE, ECD, PC5. 5, PC7, APC, APCA700, APCA750, Pacific Blue, and Krome Orange conjugated to the following antibodies: tube 1) CD14, CD13, CD38, CD123, CD117, CD11b, CD34, CD33, CD16, CD45; tube 2) CD71, CD4, CD64, CD56, CD117, CD36, CD34, CD33, HLA-DR, CD45; tube 3) CD7, CD10, CD8, CD5, CD2, CD3, CD34, CD19, CD15, CD45. Tube 1 was designed for the assessment of myeloid progenitor cells and granulocytes, tube 2 for monocytes and erythroid cells, tube 3 for lymphoid cells and granulocytes. Data acquisition was performed using Navios flow cytometers (Beckman Coulter, Miami, FL) in each of three laboratories from Germany, France, and Canada. Instrument settings were adjusted at all sites to match pre-defined target channels using beads as controls. During the pilot phase, data of a total of 10 MDS cases acquired at the different sites were analyzed by all of the 3 sites following systematic procedures agreed upon. The parameters evaluated included the percentages of respective cell compartments, expression patterns of CD13/CD16/CD11b in granulocytes, aberrant expression of myeloid markers, coexpression of lymphoid markers in granulocytes, monocytes, myeloid progenitor cells and erythroid cells. Coefficients of variation (CV) were calculated for each analyzed parameter for each patient. Results: The quantification of cell compartments was homogeneously performed by the different sites with median CVs amounting to 5. 7% for granulocytes, 10. 5% for monocytes, 7. 1% for lymphocytes, and 5. 0% for CD34 positive progenitor cells. The mean side scatter signal for granulocytes was consistently determined with a median CV of only 3. 4%. Mean fluorescent intensities (MFIs) for distinct markers in general were homogeneously determined between the three sites with the following median CVs: CD33 1. 8% in granulocytes; CD33 2. 6%, CD14 15. 6%, CD13 4. 8%, CD11b 3. 6%, CD56 23. 8%, HLA-DR 5. 1%, CD2 16. 7% in monocytes; CD71 6. 9%, CD36 2. 9% in erythroid cells; CD34 4. 8% in progenitor cells. This provides an important basis for the harmonization of rating aberrantly expressed antigens in MDS which often are identified by only relatively small changes in the respective MFIs. Importantly, also the percentages of positivity for cross-lineage expression of lymphatic antigens in myeloid progenitor cells, which have previously been shown to carry prognostic significance, were determined homogeneously between the three sites with median CVs of 22. 0%, 13. 4% and 33. 9% for the very small subsets of CD2, CD7 and CD5 positive cells. Another aim of the present pilot study has been the objective read-out of parameters which in general have been judged on subjectively. In this regard, the expression patterns of CD11b and CD16 as well as of CD13 and CD16 in granulocytes were subjectively judged aberrant or not aberrant with complete agreement between the three sites. Importantly, the separation of the giraffe-like pattern of CD11b/CD16 expression in granulocytes into 4 subpopulations was performed homogeneously (median CVs 4. 8%, 22. 1%, 18. 0%, 13. 6%) and the resulting MFIs for CD11b (median CVs 7. 2%, 12. 8%, 10. 8%, 11. 5%) in these 4 subpopulations also were consistently quantified between the three sites. Conclusions: This data indicates that harmonization of procedures across different sites in the flow cytometric evaluation of patients with MDS is feasible. While agreement for judgement on antigen expression patterns is possible by experienced scientists it may be complemented or even substituted by standardized data analysis procedures following testing rounds. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership; Beckman Coulter, Miami, Florida: Research Funding. Béné:Beckman Coulter, Miami, Florida: Research Funding. Porwit:Beckman Coulter, Miami, Florida: Research Funding.

scholarly journals The pattern of HLA-DR and HLA-DQ antigen expression on clonable subpopulations of human myeloid progenitor cells

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 379-384
Author(s):  
RL Sparrow ◽  
N Williams
Keyword(s):  
Cell Membrane ◽  
Progenitor Cells ◽  
Marrow Cell ◽  
Myeloid Cell ◽  
Antigen Expression ◽  
Myeloid Progenitor ◽  
Hla Dr ◽  
Hla Dq ◽  
Myeloid Progenitor Cells ◽  
Kinetics Of

Three subpopulations of human myeloid progenitor cells (CFU-GM) can be distinguished by differences in their kinetics of development; the liquid phase pre-CFU-GM, the day 14 CFU-GM, and the day 7 CFU-GM. The relative cell membrane densities of the HLA-DR and HLA-DQ antigens expressed by the three subpopulations was investigated by comparing the amount of antibody required to deplete bone marrow cell preparations of each cell type. Three separate approaches were used--complement (C') cytotoxicity, antiglobulin/C'-cytotoxicity and immune rosette depletion. Similar results were obtained for all three procedures, although the latter two gave a tenfold greater sensitivity over the standard C'-cytotoxicity method. At saturating anti-HLA-DR antibody concentrations, 85% to 95% of cells within the three myeloid subpopulations were found to express HLA-DR antigens. However, the relative amount of HLA-DR expressed by these subpopulations increased from the pre-CFU-GM to the day 7 CFU-GM. The expression of HLA-DQ antigens was considerably lower and could only be detected by using the more sensitive procedures. Only 50% of day 7 and 14 CFU-GM progenitor cells expressed detectable HLA-DQ antigens, whereas a greater proportion (80%) of the pre-CFU-GM were HLA-DQ positive. The pattern of HLA-DQ expression on these clonable precursors was quite distinct and opposite to the cell membrane density of the HLA-DR antigens. Because these three progenitor cell populations are thought to be linked in differentiation sequence, these results provide indirect support for the hypothesis that HLA class II antigens are implicated in regulatory mechanisms during normal myeloid cell differentiation.

scholarly journals Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2229-2236 ◽  
Author(s):  
MJ Robertson ◽  
RJ Soiffer ◽  
AS Freedman ◽  
SL Rabinowe ◽  
KC Anderson ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Red Blood Cell Transfusion ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Acute Myeloid

Abstract The CD33 antigen, identified by murine monoclonal antibody anti-MY9, is expressed by clonogenic leukemic cells from almost all patients with acute myeloid leukemia; it is also expressed by normal myeloid progenitor cells. Twelve consecutive patients with de novo acute myeloid leukemia received myeloablative therapy followed by infusion of autologous marrow previously treated in vitro with anti-MY9 and complement. Anti-MY9 and complement treatment eliminated virtually all committed myeloid progenitors (colony-forming unit granulocyte- macrophage) from the autografts. Nevertheless, in the absence of early relapse of leukemia, all patients showed durable trilineage engraftment. The median interval post bone marrow transplantation (BMT) required to achieve an absolute neutrophil count greater than 500/microL was 43 days (range, 16 to 75), to achieve a platelet count greater than 20,000/microL without transfusion was 92 days (range, 35 to 679), and to achieve red blood cell transfusion independence was 105 days (range, 37 to 670). At the time of BM harvest, 10 patients were in second remission, one patient was in first remission, and one patient was in third remission. Eight patients relapsed 3 to 18 months after BMT. Four patients transplanted in second remission remain disease-free 34+, 37+, 52+, and 57+ months after BMT. There was no treatment-related mortality. Early engraftment was significantly delayed in patients receiving CD33-purged autografts compared with concurrently treated patients receiving CD9/CD10-purged autografts for acute lymphoblastic leukemia or patients receiving CD6-purged allografts from HLA- compatible sibling donors. In contrast, both groups of autograft patients required a significantly longer time to achieve neutrophil counts greater than 500/microL and greater than 1,000/microL than did patients receiving normal allogeneic marrow. CD33(+)-committed myeloid progenitor cells thus appear to play an important role in the early phase of hematopoietic reconstitution after BMT. However, our results also show that human marrow depleted of CD33+ cells can sustain durable engraftment after myeloablative therapy, and provide further evidence that the CD33 antigen is absent from the human pluripotent hematopoietic stem cell.

scholarly journals Induction of differentiation in blast cells and leukemia colony-forming cells from patients with acute myeloid leukemia

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 721-729 ◽  
Author(s):  
AL Howell ◽  
TA Stukel ◽  
CD Bloomfield ◽  
FR Davey ◽  
ED Ball
Keyword(s):  
Myeloid Leukemia ◽  
Antigen Expression ◽  
Myeloid Differentiation ◽  
Short Term ◽  
Differentiation Antigens ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Blast Cells ◽  
Short Term Culture

Abstract The characteristic lesion in acute myeloid leukemia (AML) is the failure of myeloid cells to differentiate normally, leading to the accumulation of immature blast cells (BC) in the bone marrow. We determined whether BC and leukemia colony-forming cells (L-CFC) from AML patients could differentiate in vitro after short-term culture with interferon-gamma (IFN gamma), 1,25 dihydroxyvitamin D3 (D3), retinoic acid (RA), tumor necrosis factor-alpha (TNF alpha), and granulocyte- monocyte colony-stimulating factor (GM-CSF). Expression of myeloid differentiation antigens CD15, CD14, CD33, and p124 was determined on the BC by immunofluorescence and on the L-CFC by monoclonal antibody (MoAb) and complement (C')-mediated cytotoxicity followed by cloning in methylcellulose. We found that 26 of 39 (67%) cases demonstrated changes in the expression of myeloid differentiation antigens on the BC, and 6 of 7 (86%) cases showed an altered L-CFC myeloid antigen phenotype after short-term culture with differentiating agents. Alterations in myeloid antigen expression in the L-CFC population correlated with a reduction in L-CFC cloning potential. In the BC, alterations of myeloid differentiation antigens occurred in a manner consistent with those observed during normal myelopoiesis. For example, CD14 antigen expression (a late-stage monocyte antigen) increased on BC from 12 of 39 (31%) cases, and p124 (an antigen expressed both by myeloid progenitor cells and by a subset of monocytes) increased on 15 of 39 (38%) cases. Changes in the expression of CD33 antigens (expressed normally by myeloid progenitor cells and by mature monocytes) on the BC were variable, with 7 of 29 cases (24%) showing a decrease and 7 of 29 cases (24%) showing an increase. When comparisons were made between pairs of differentiation agents that caused the altered expression of an antigen on either the BC or L-CFC of a patient, the majority of changes were in the same direction (either both “increased” or both “decreased”). This suggests that the direction of antigen change is characteristic of the leukemia cell subpopulation for each patient and not of the stimulatory agent. This study demonstrates that cells from more than two thirds of AML cases examined responded to various differentiation agents in vitro as measured by changes in the expression of myeloid cell-associated surface antigens and by alterations in cloning potential of the L-CFC, a finding of potential clinical significance.

Peripheral Blood Cytogenetic Studies in Hematological Neoplasms: Predictors of Obtaining Metaphases for Analysis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3504-3504
Author(s):  
Kebede Hussein ◽  
Rhett P. Ketterling ◽  
Gordon W. Dewald ◽  
Rachael L. Hulshizer ◽  
Daniel G. Kuffel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Clinical Diagnosis ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Cytogenetic Study ◽  
Lymphocytic Leukemia ◽  
Myeloid Progenitor ◽  
Cytogenetic Studies ◽  
Myeloid Progenitor Cells

Abstract Background: Peripheral blood (PB) is sometimes used in place of bone marrow (BM) for cytogenetic studies during the evaluation of hematologic malignancies. We looked for clinical or laboratory features that predict success in obtaining analyzable metaphases during PB chromosome studies. Methods: The Mayo Clinic cytogenetics database was queried to identify adult cases (age > 18 years) with suspected or established hematologic neoplasm in whom PB cytogenetic studies were performed. Success defined as the acquisition of at least two metaphases, was correlated with clinical and laboratory information corresponding to the time of the PB cytogenetic study. Results: A total of 242 PB cytogenetic studies were performed: clinical diagnosis was a myeloid neoplasm in 169 patients (70%), lymphoid neoplasm in 50 (21%), and unexplained cytopenia or leukocytosis in 23 (9%). The 169 myeloid cases included 59 patients with either primary (n=39) or post-polycythemia vera/essential thrombocythemia (post-PV/ET MF) myelofibrosis (n=20), 42 with acute myeloid leukemia (AML), 15 with chronic myeloid leukemia, 9 with myelodysplastic syndrome (MDS), 8 with ET, 6 with PV, and 30 with other MPDs. The 50 lymphoid cases included 19 with chronic lymphocytic leukemia, 12 with lymphoma, 11 with acute lymphocytic leukemia (ALL), and 8 with plasma cell proliferative disorders. PB cytogenetic studies resulted in at least two analyzable metaphases (median 20, range 2–31) in 142 of the 242 study cases (59%); in univariate analysis, this was predicted by the specific clinical diagnosis (p<0.0001), presence and degree of circulating myeloid progenitor cells (p<0.0001), higher leukocyte count (p<0.001), lower platelet count (p=0.003), lower hemoglobin level (p=0.002), and presence of palpable splenomegaly (p=0.002). In multivariable analysis, only the presence of circulating myeloid progenitor cells sustained its significance and this was consistent with the high yield rates seen in PMF (80%), post-PV/ET MF (85%), AML (76%), and ALL (80%) as opposed to the low rates seen in ET (0%) and PV (2%). In 104 cases, BM cytogenetic studies were performed within one month of the PB cytogenetic studies; an abnormal BM cytogenetic finding was another independent predictor of a successful PB study (p=0.002). Conclusion: PB cytogenetic studies are most appropriate in diseases characterized by presence of circulating myeloid progenitors or blasts (e.g. PMF, AML, ALL); the yield otherwise is too small to be cost-effective. The current study also suggests a higher likelihood of a successful PB cytogenetic study in the presence of an abnormal bone marrow karyotype.

Elevated EGR2 Expression Provides a Potential Link Between Cell Cycle Arrest and Induced Differentiation in Myeloid Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2733-2733
Author(s):  
Joshua B. Bland ◽  
Jose R. Peralta ◽  
William T. Tse
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Progenitor Cells ◽  
Myeloid Differentiation ◽  
Phenotypic Effect ◽  
Induced Differentiation ◽  
Hl60 Cells ◽  
Cycle Arrest ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Abstract Similar to many immature cell types, myeloid progenitor cells need to exit cell cycle to undergo terminal differentiation, but the mechanism linking the two is still unclear. Elucidating this mechanism could lead to the development of new differentiation therapies to treat myeloid leukemia. Recent studies have suggested that the processes regulating myeloid differentiation and cell cycle progression together constitute a positive feedback loop where each process reciprocally affects the other. To study the relationship between these processes, we examined early cellular and molecular events associated with induced differentiation of the HL60 human promyelocytic leukemia cells. We treated HL60 cells with 3 classical inducers of differentiation (vitamin D3 analog EB1089 (EB), all-trans retinoic acid (ATRA), and dimethyl sulfoxide (DMSO)), along with PD0332991 (PD), a selective cyclin D-dependent kinase 4/6 inhibitor that caused G1-phase-specific cell-cycle arrest. We evaluated differentiation of the treated cells by flow cytometric analysis of CD11b (integrin αM) and CD71 (transferrin receptor) expression. In untreated HL60 cells, a baseline subset of 3-5% of cells exhibits a differentiated, CD11b+CD71- phenotype. Exposure to the various inducers revealed a progressive increase in the percentage of CD11b+CD71- cells with time, such that by day 4 of treatment, it has increased to 50-90% in the treated samples, indicating that all 4 agents tested were effective in inducing myeloid differentiation. To understand how differentiation induced by each agent affects cell cycle progression, the cell cycle status of the induced cells were evaluated by a BrdU-incorporation assay after a 30-minute pulse of BrdU labeling. Uninduced cells exhibited a baseline cell cycle phase distribution of 64%-28%-8% (G1-S-G2/M phases). After 1 day of induction, EB-treated sample showed no changes in the distribution (58%-33%-9%), but ATRA, DMSO and PD-treated samples showed significant changes, with an increase of cell numbers in G1 phase and decrease in S phase (74%-18%-8%, 79%-13%-8%, and 93%-4%-3%, respectively). These results reveal that an early induction of G1 arrest was caused by treatment with ATRA, DMSO and PD, but not EB, and that the cell cycle arrest occurred before major changes in the myeloid phenotype were observed. To determine how the cell cycle perturbation relates to changes in the underlying genetic regulatory network, we examined by quantitative RT-PCR analysis the expression of several transcription factors associated with myeloid differentiation. PU.1 and CEBPA were found to be expressed at high levels but these levels did not change upon treatment with the inducing agents. Similarly, the expression levels of GFI1 and EGR1 did not change significantly with induction. In contrast, the expression level of EGR2 (Early Growth Response 2) was found to be low initially but became elevated upon treatment with 3 of the 4 inducers. EGR2 is a zinc finger transcription factor implicated in the control of a switch between pro- and anti-proliferation pathways. EGR2 has been shown to regulate the transition between differentiation states of Schwann cells, induction of anergic and regulatory T cells, growth and survival of osteoclasts, and proliferation and apoptosis of acute myeloid leukemia blasts. We found that EGR2 expression, after 1 day of treatment with ATRA, DMSO or PD, was increased by 5.2 ± 0.9, 7.6 ± 1.9, 5.8 ± 0.9 folds, respectively, whereas treatment with EB led to no significant change (1.5 ± 0.2 fold). We evaluated whether simultaneous treatment of the cells with 2 inducers would result in an additive effect. Treatment of HL60 cells with a combination of ATRA/DMSO, ATRA/PD, or DMSO/PD increased the percentage of CD11b+CD71- cells to 55%, 70% and 25% after just 1 day of treatment. In line with the enhanced phenotypic effect, the expression level of EGR2 was further elevated to 7.7 ± 1.4, 15.4 ± 3.5, and 11.3 ± 3.4 folds, respectively, when the cells were treated with the above inducer combinations, indicating a tight association between EGR2 expression and the phenotypic effect. In summary, our data suggest that elevated expression of EGR2 is an early event in the induction of myeloid differentiation in HL60 cells. Because of its known role in cell cycle regulation, EGR2 could function as a mechanistic link between cell cycle arrest and induced differentiation in myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document