Effect of recombinant GM-CSF and recombinant G-CSF on colony formation of blast progenitors in acute myeloblastic leukemia

The colony-promoting activities of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units [L-CFU]) from 21 patients with acute myeloblastic leukemia (AML) were examined using blast colony assay and compared to colony promotion stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Recombinant GM-CSF stimulated blast colonies in 13 out of 20 cases examined (1 case not done). The magnitude of stimulation by rGM-CSF varied significantly according to the type of AML, but in general was lower than that of PHA-LCM. Blast cells of type M1 did not form any colonies with rGM-CSF, although numerous colonies were produced with PHA-LCM. Type M4 blasts formed fairly large numbers of colonies, though slightly less than those stimulated by PHA-LCM. Blasts of type M2 and M5 formed colonies with the stimulation of rGM-CSF, but the numbers were considerably smaller than type M4 and those stimulated with PHA-LCM. Recombinant G-CSF stimulated blast colonies in only 5 out of 21 cases, 3 of them being type M2. The number of cases responding to rG-CSF was significantly smaller than that responding to rGM-CSF, and even in cases in which colonies were formed, the magnitude of stimulation was minimal. From these results it seems likely that blast cells of different types of AML require a different kind of CSF for their optimal growth; type M4 blasts responded to the stimulation of rGM-CSF well, but blasts of other types of AML responded poorly. Thus, except for type M4, CSF(s) other than rGM-CSF seems to be required for the sufficient growth of L-CFU. Recombinant G-CSF is not likely to play an essential role in the proliferation of leukemic blasts of most types. Previous exposure to rGM-CSF and rG-CSF did not alter the self-renewal capacity, cellular phenotype, and morphology of colony cells, indicating that the direction and degree of differentiation of L-CFU stimulated by rGM-CSF or rG-CSF were not different from those stimulated with PHA-LCM.     

Synergism between recombinant growth factors, GM-CSF and G-CSF, acting on the blast cells of acute myeloblastic leukemia

The genes for the hemopoietic growth factors, GM colony-stimulating factor (CSF) and G-CSF have been cloned, and recombinant material is available for both. We tested these recombinant factors for their effects on the blast cells of acute myeloblastic leukemia (AML). Culture methods are available that support both colony formation by AML blasts and the growth of blast stem cells in suspension. Recombinant GM-CSF is active in both culture systems, although to a varying degree. We found that recombinant G-CSF was also effective; however, the two recombinant factors showed striking synergism for the stimulation of blast growth of cells from five of eight AML patients. In these cases, the combination was equivalent to the stimulating activity of supernatants from the continuous cell line 5637. This conditioned medium (HTB9-CM) is considered the standard for blast growth. Blasts from one of the patients grew without added factor. In another instance, recombinant GM-CSF alone was almost as effective as HTB9-CM. In the third case, both recombinant factors were active, but synergism was not observed and their combined effect was not equivalent to that of HTB9-CM. Both GM-CSF and G-CSF were active on normal bone marrow granulopoietic progenitors, but synergism was not observed. We conclude that the marked heterogeneity observed when AML blasts are examined by other criteria is also observed when their response to growth factors is evaluated.     

Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukemia

The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) were compared to those of media conditioned by the continuous bladder carcinoma line, HTB9 (HTB9-CM), using three criteria. First, both GM-CSF and HTB9-CM stimulated blast colony formation in methylcellulose cultures, patient-to-patient variations were seen in the dose-response curves, and GM-CSF was effective, but less so that HTB9-CM. Second, GM-CSF also enhanced growth of blast progenitors in suspension culture, indicating its capacity to support self-renewal. GM-CSF was as effective as HTB9-CM in the production of adherent cells during the growth of blast cells in suspension, a finding that is interpreted to mean that GM-CSF also supports postdeterministic events in blast differentiation. Finally, colonies growing in the presence of GM-CSF were not phenotypically different than those stimulated by HTB9-CM.     

The effects of GM-CSF and G-CSF in promoting growth of clonogenic cells in acute myeloblastic leukemia

A small subset of leukemic cells from most patients with acute myeloblastic leukemia (AML) have properties of stem cells and can be assayed by colony formation in agar or methylcellulose. Colony formation generally requires the addition of exogenous growth factors, but the exact factors required are incompletely defined. The AML colony- promoting activities of two recombinant human colony-stimulating factors (GM-CSF and G-CSF) were investigated by using blasts from 48 patients with AML. In nine cases, no colonies formed with either CSF. In seven cases colonies formed only in response to G-CSF and in 11 cases only in response to GM-CSF. In 21 cases colonies formed in response to either GM-CSF or G-CSF, and in 12 of these cases there was an additive effect between the two CSFs in determining maximum colony size. For cases responding to both GM- and G-CSF, the total number of colonies formed in response to the combination of both CSFs was almost always less than additive compared with the number of colonies formed in response to the individual CSFs. Further, the AML-CFU responding to either GM-CSF or G-CSF could not be distinguished by surface markers or by the cytochemical staining pattern of the colonies. These results suggest that there is considerable overlap between the GM-CSF- and G- CSF-responsive AML-CFU subpopulations in most cases. For five of seven cases, the combination of GM-CSF and G-CSF could replace a leukocyte feeder layer in providing maximum growth stimulation. These results indicate that GM-CSF and G-CSF are active growth factors for AML cells and are frequently additive in promoting maximum colony size.     

Autocrine secretion of GM-CSF in acute myeloblastic leukemia

Three cases of acute myeloblastic leukemia (AML) were identified in which clonogenic cells proliferated autonomously in vitro. Cells from two of these cases were found to secrete a colony-stimulating factor (CSF) that was immunologically and molecularly related to GM-CSF. Growth of AML-CFU could be blocked by the addition of a neutralizing antiserum to GM-CSF. Northern blot hybridization of leukemic cell mRNA with a cDNA probe for the GM-CSF gene revealed a 1-kb message identical in size to the normal GM-CSF message in stimulated T cells. No GM-CSF message was detected in the third case. These results indicate that constitutive expression of the GM-CSF gene, apparently by leukemic cells, can result in autonomous in vitro proliferation of AML-CFU in some cases of AML.     

Effects of recombinant interleukin 4 on the growth and differentiation of blast progenitors stimulated with G-CSF, GM-CSF and IL-3 from acute myeloblastic leukaemia patients

The effects of human recombinant interleukin 4 (rIL-4) on the growth of leukaemic blast progenitors were investigated. Cells obtained from 20 acute myeloblastic leukaemia (AML) patients were evaluated using the blast colony assay in methylcellulose and suspension cultures. While rIL-4 by itself did not show any colony stimulatory activity in the blast colony assay, it suppressed the blast colony formation in methylcellulose stimulated with G-CSF, GM-CSF or IL-3 in 14 patients. In another six patients, rIL-4 enhanced blast colony growth in four patients or did not show any significant effect with any CSF in two patients. In suspension cultures of 12 cases studied, the effects of rIL-4 on the clonogenic cell recoveries were essentially similar to the results of the blast colony assay in each case. In three patients, rIL-4 augmented the differentiation of the leukaemic cells to monocyte lineage. Further, the clinical outcome was significantly different between the patients whose blast progenitors were stimulated by rIL-4 and the patients whose blast progenitors were suppressed by rIL-4 (P less than 0.05); three out of four patients in the former group failed in achieving complete remission (CR), while 12 out of 14 patients in the latter group achieved CR. The results show that the effects of IL-4 on leukaemic blast progenitors were diverse and the responsiveness to IL-4 may be correlated with the prognoses of the patients.     

Self-renewal in culture of proliferative blast progenitor cells in acute myeloblastic leukemia

We have proposed that colonies of cells with blastlike morphology growing in culture are derived from a blast subpopulation with high proliferative potential. To test whether or not these blast progenitors have the capacity for self-renewal, blast colonies grown from the peripheral blood of the 21 patients with acute myeloblastic leukemia were replated; secondary colonies were observed in 17 instances, and these were similar to primary colonies in size, morphology, and culture requirements. Great patient-to-patient variation was observed in the frequency of secondary colonies, but low secondary plating efficiency was significantly correlated with successful remission induction. We conclude that the blast progenitors detected in the assay have at least limited self-renewal capacity and that this capacity may, along with other risk factors, contribute to clinical outcome.     

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

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.     

Effects of recombinant human GM-CSF on proliferation of clonogenic cells in acute myeloblastic leukemia

Proliferation of acute myeloblastic leukemia (AML) cells in vitro is limited in most cases to a small subset of blasts that have several properties of stem cells. These leukemic colony-forming cells (AML-CFU) generally require addition of exogenous growth factors for proliferation in agar or methylcellulose. These factors can be supplied by media conditioned by phytohemagglutinin-stimulated normal leukocytes or by CSF-secreting tumor cell lines. However, the exact factor or factors required for stimulation of AML-CFU growth have not been defined. We compared the AML-CFU stimulatory activity of a human recombinant GM-CSF with that of GCT-CM, Mo-CM, and the PHA-leukocyte feeder system in 15 cases of AML. In each of the 12 cases that required exogenous growth factors for maximum AML-CFU growth, recombinant GM-CSF could replace either GM-CSF or Mo-CM, and could partially replace the PHA-leukocyte feeder system. These results indicate that this GM-CSF is a growth promoter of AML-CFU in these culture systems.     

Autocrine growth mechanisms of the progenitors of blast cells in acute myeloblastic leukemia

Autocrine growth mechanisms of leukemic blast progenitors in acute myeloblastic leukemia (AML) were investigated. Colony formation of leukemic blast progenitors was observed in 14 of 14 patients tested when purified blast cell fraction depleted of both T cells and monocytes was plated in methylcellulose without any colony-stimulating factor (CSF). However, there existed a minimal cell density required to initiate blast progenitor growth with marked patient-to-patient variation. To clarify the role of cell density on the spontaneous growth of blast progenitors, we tested whether leukemic cells produced and secreted some stimulatory humoral factor(s). Production of colony- stimulating activity (CSA) by blast cells was observed in 17 of 18 patients tested. Following further depletion of monocytes, the CSA levels decreased markedly in 14 patients, indicating that blast cells with monocytoid differentiation were responsible for CSA production. We also confirmed granulocyte colony-stimulating factor (G-CSF) and/or granulocyte macrophage-colony-stimulating factor (GM-CSF) production by leukemic blasts using specific immunologic assays. When leukemic cells were divided into nonadherent nonphagocytic cell fraction and adherent cell fraction, only nonadherent nonphagocytic cells showed clonogenecity and adherent blast cells lacked the colony-forming capacity. The results indicate that there are at least two blast cell subpopulations in AML: one is proliferating subpopulation with self- renewal capacity and the other is supporting subpopulation with functions such as CSF production. The quite intimate relationship between these two blast cell subpopulations in AML may play an important role on the growth of leukemic blast progenitors in vitro.     

Complete remission in three patients with acute myeloblastic leukemia by administration of G-CSF without antileukemic agents

We describe 3 patients with acute myeloblastic leukemia (AML), who received rhG-CSF for infections such as pneumonia or for prophylaxis of infection, and who achieved complete remission. They had not received any antileukemic therapy before or during the administration of rhG-CSF. These findings suggest the possibility that complete remission can be brought about by G-CSF itself in some patients with AML. Am. J. Hematol. 56:42–44, 1997. © 1997 Wiley-Liss, Inc.     

Separation of blast cell and T-lymphocyte progenitors in the blood of patients with acute myeloblastic leukemia.

The peripheral blood of acute myeloblastic leukemia (AML) patients often contains large numbers of two distinct cell populations, both capable of forming colonies in culture under similar conditions. The first population consists of the precursors of blast cells and has specificity for AML; the second population consists of T-lymphocyte precursors, also found in normal blood. The two progenitor populations can be separated by exploiting the capacity of T-lymphocyte (but not blasts) progenitors to form rosettes with sheep erythrocytes (E rosettes). After E-rosette formation, T-lymphocyte precursors can be removed by centrifugation on Ficoll-Hypaque. Such separation has a number of consequences: (1) Blast progenitors can be detected where unseparated mononuclear preparations have yielded either no colonies or only T-lymphocyte colonies (20 of 21 patients). (2) The stimulator requirements of the blast progenitors change, indicating that cell-cell interactions may take place between blast and T-lymphocyte progenitors. (3) It is feasible to characterize blast and T-lymphocyte precursors independently, even though they may coexist in peripheral blood. This may be important if progenitor properties are attributes contributing to the variance in outcome in AML.     

The proliferation in suspension of the progenitors of the blast cells in acute myeloblastic leukemia

A minority of blast cells in acute myeloblastic leukemia (AML) form colonies in culture in methylcellulose when stimulated by media conditioned by normal leukocytes in the presence of phytohemagglutinin (PHA-LCM). Blast colonies can be replated successfully, either as pooled cells or suspensions from single colonies. However, the plating efficiency declines with repeated passages, and more than four subcultures have not been achieved. In this study, blast populations were cultured in suspension, with fetal calf serum, alpha-minimal essential medium and PHA-LCM. In cells from 17 of 18 patients, exponential growth of clonogenic blast cells was maintained for six to seven days without reculturing. Colonies obtained from progenitors taken from liquid culture and replated in methylcellulose were replated to obtain the secondary plating efficiency (PE2). In 14 cases, this value was maintained or increased. In three instances, PE2 fell following culture in methylcellulose. When cells in suspension were recultured, exponential growth continued. In nine instances, exponential growth was maintained for from seven to 70 days. During this time, PE2 was maintained. Results from experiments using velocity sedimentation separation and analysis of single colonies were consistent with the view that the increase in clonogenic cells in suspension was a manifestation of their self-renewal capacity. The observations also support a model of blast progenitor growth that contains the postulate that these are capable not only of self-renewal but also of determination-like events leading to loss of proliferative capacity.     

Effect of platelet-derived growth factor on the proliferation of blast progenitors from acute myeloblastic leukemia patients

The effect of platelet-derived growth factor (PDGF) on self-renewal and terminal divisions of blast progenitors of acute myeloblastic leukemia was studied. The terminal divisions of blast progenitors were determined as the primary blast colony formation in methylcellulose culture; self-renewal was assessed by secondary colony formation by replating in methylcellulose and the recovery of clonogenic cells in suspension culture. PDGF neither enhanced nor suppressed primary or secondary blast colony formation in methylcellulose culture nor recovery of clonogenic cells in suspension. The results show that PDGF does not affect the self-renewal or terminal divisions of leukemic blast progenitors.     

Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia

Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface-bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM-CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5-36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.