Klin. Pädiatr. 202 (1990)
Control of blast cell proliferation and differentiation in acute myelogenous leukemia by soluble polypeptide growth factors F. Herrmann, W. Oster, R. Mertelsmann Abt. Hämatologie und Onkologie, Medizinische Klinik und Poliklinik, Klinikum der Albert-Ludwigs-Universität, Freiburg
Summary
Proliferation of acute myelogenous leukemia (AML) derived blast cells requires the presence in culture of one or more growth factors. In the majority of cases Interleukin-3 (lL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate c1onogenicity of AML blasts, which can be synergised by Interleukin-6 (lL-6), Interleukin-I (lL-I) and granulocyte colony-stimulating factor (G-CSF). In contrast, macrophage colony-stimulating factor (M-CSF) favors deterministic divisions. A substantial part of AML sampIes have c1onogenic cells which, however, proliferate autonomously in vitro. The production by leukemic cells of a variety of growth or synergizing factors including GMCSF, G-CSF, IL-I, IL-6, and Tumor Necrosis Factor (TNF) has been demonstrated and a fraction of cases will use these molecules to support c1onogenic growth in an autocrine or paracrine fashion. However, unlike the situation with retrovirus-induced murine or avian leukemias, the role of production of CSFs and other cytokines by human leukemic cells in the transformational process remains uncertain.
Introduction
Normal hematopoiesis is known to be controlled by a broad spectrum of polypeptide factors. The question of how malignant hematopoietic cells escape physiological growth restrictions has, however, remained largely obscur. Rapid and unrestricted expansion of transformed hematopoietic cells can be observed most impressively in acute myelogenous leukemia (AML), suggesting, that fundamental regulatory principles of hematopoiesis are inoperative in this disease. Several investigators including our own group have shown, that a group of hormoneIike glycoproteins, named colony-stimulating factors (CSF), which are essential for in vitro proliferation and differentiation of normal hematopoietic cells (see 7 for Klin. Pädiatr. 202 (1990) 212-217 @ 1990 F. Enke Verlag Stuttgart
Kontrolle der Proliferation mit Differenzierung von Blasten bei der akuten myeloischen Leukämie durch lösliche Polypeptid-Wachstumsfaktoren
Die Proliferation von Blasten akuter myeloischer Leukämien (AML) erfordert in vitro die Anwesenheit eines oder mehrerer hämatopoetischer Wachstumsfaktoren. Interleukin-3- und Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) stimulieren in der überwiegenden Mehrzahl leukämischer Zellproben die Klonogenität von AML-Blasten. lnterleukin-6, Interleukin-I und Granulozyten-stimulierender Faktor können synergistisch die durch IL-3 und GM-CSF induzierte B1astenproliferation steigern. Im Gegensatz hierzu steuert der Makrophagen-Kolonie-stimulierende Faktor (M-CSF) überwiegend deterministische Teilungen. Ein erheblicher Anteil von AML-Proben weist jedoch klonogene Zellen auf, die in vitro autonom, d. h. in Abwesenheit exogener Wachstumsfaktoren, proliferieren. Wir zeigen, daß die Produktion von hämatopoetischen Wachstumsfaktoren und synergisierenden Faktoren GM-CSF, G-CSF, li-I, IL-6 und Tumor-Nekrose-Faktor (TNF) durch Leukämieblasten möglich ist und daß in einer Reihe von Fällen diese Moleküle von Leukämiezellen autorkin oder parakrin genutzt werden, um das eigene Wachstum zu steuern. Der Grund für die konstitutive Wachstumsfaktorexpression in AML-Blasten ist noch unbekannt.
review), mayaIso be involved in the process of malignant transformation of hematopoietic cells. This article summarizes effects of CSFs and related molecules on proliferation and differentiation of AML blasts, by focussing on autocrine and paracrine mechanisms used by AML blasts to provide CSF supply and points out possible implications of CSF-involvement in the pathophysiology of this disease.
Regulation 01 normal hematopoiesis by colony-stimulating lactors The process of blood cell formation by which a small number of self-renewing stern cells undergoes commitment and spurs irreversibly committed progenitor cells to proliferate and 10 differentiate along a restricted lineage, involves interactions of cell-derived biomolecules with their target cells (2, 3). Much of what we
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212
Control of blast ce// proliferation and differentiation in acute myelogenous leukemia Cytokines involved in hemalopoiesis
MOLECULE
SYNONYM
CHROMOS. LOCATION
mRNA (kb)
MW (kD)
SECRETION IKb)
multi-CSF GM-CSF G-CSF M-CSF EPO
IL-3 CSF-alpha CSF-beta CSF-1
5q 5q21-32 17qll-22 5q33 7qll-22
1 1 1.6 1.5-4.5 1.6
14-28 14-35 18-22 36-90 34-39
e;p' e; e;p e;a e
IL-1-alpha IL-1-beta IL-2 IL-3 IL-4 IL-5 IL-6
Hemopoitein-1
2.2 16
TCGF
2q14 2q14 4p
31; 17 31; 17 15
e;p;a e;p;a e;p;a
BSF-1 BCGF-II, TRF IFN-beta2, BSF2
5q 5q 7q
0.9 1.7 13
15-20 12-18 24
e;p;a e;p P
Cachectin TNF-beta type-li IFN type-I IFN type-I IFN
6p23 6p23 12 9p13-21 9p22
1.6 1.4 1.7 1.0 0.9
17 25 15-45 15-24 20
e;p;a P P P e;p
TNF-alpha Lymphotoxin IFN-gamma IFN-alpha IFN-beta 1 • e; endocrine
p; paracrine
a; autocri ne
currently know about these interactions has been derived form in vitro cell culture techniques, enabeling us to identify progenitor cells by their ability to form colonies of various Iineages in semisolid medium in response to specific growth factors stimuli (39). Recently rapid progress has occurred in characterizing the molecular nature of these biologically active factors by purification and cloning of these molecules. Five human growth factor species have been identified so far, i. e. CSF for granulocytes (G-CSF) (30, 46), for macrophages (M-CSF or CSF-l) (18), for mixed granulocyte-macrophage colonies (GM-CSF) (6), for multilineage colonies (lL-3) (36,52), and erythropoietin (EPO) (23). Other factors that not strictly belong to the terminological entity of CSFs or hematopoietic growth factors, have also been shown to provide synergistic growth regulatory effects on hematopoiesis, or to induce release of hematopoietic growth factors by producer cells (Table 1). All CSFs are encoded by single copy genes and some are c1ustered on chromosome 5: The M-CSF gene has been mapped to 5q 33.1 (38), the GM-CSF gene to 5 q 21-q32 (17), the IL-3 gene to the same band of chromosome 5 as GM-CSF (22). Additionally, chromosome 5 carries multiple genes coding for growth related proteins and protein receptars, including the M-CSF receptor gene, closely related and possibly identical to the proto-oncogene c-fms (44), the receptor for platelet derived growth factor (5 q 31.3-32) (53), the beta-2 adrenergic receptor (5 q 31-32) (20), and genes coding for endothelial cell growth factor (5 q 31.3-32). Chromosome 5 is frequently involved in cytogenetic aberrations in AML, e. g. partialloss of the long arm of the chromosome (deI 5 q), most often occurring secondary to cytotoxic therapy but also arrising in patients with de novo AML (43). It has been shown that the genes far GM-CSF, M-CSF, and IL-3 are directly affected by structural changes of chromosome 5 (17, 22). G-CSF has been mapped to the chromosomal region 17 q 11.2-21 proximal to the breakpoint involved in the translocation (15; 17) (45), which is the hallmark karyotypic aberration of acute promyeIocytic leukemia (FAß M3) (43).
Physiological sources of CSFs are monocyte/macrophage containing tissues, T-Iymphocytes, granulocytes, endothelial cells, and fibroblasts (3, 12,24, 29, 32, 33, 35). Normal immature hematopoietic progenitor cells, however, are not known to have the capacity to produce CSFs, thus suggesting lack of autocrine growth regulation in the normal bone marrow.
Effects of CSFs on myeloid leukemia ce//s Formation of blast colonies in agar cultures originating from a small proportion of fresh leukemic cell populations has also been shown (9, 28). These leukemic colony forming cells (LCFC) have several features in common with normal hematopoietic stern cells: For colony growth they require in most cases supply of exogenously provided growth factors, which are commonly available in media conditioned by lectin-activated leukocytes (9) or media from various tumor cell Iines, including the histiocytic GCT-line, the T-Iymphoblastoid Mo-line, and the bladder carcinoma line 5637; furthermore, L-CFC possess self renewal capacity , a high thymidine suicide index, and the potential to undergo at least limited, although abnormal differentiation to nonproliferating cells (4). Several investigators have assessed the ability of recombinant purified CSFs to substitute far standard conditioned media using the L-CFC assay by demonstrating that IL-3, GM-CSF, and G-CSF are promoters of L-CFC growth from many patients with AML (8, 10, 19, 49) (see Table 2). However, it became obvious that AML is not a homogenous disease by demonstrating that L-CFC exhibit a broad spectrum of reponses to CSFs, with marked variation from patient to patient. CSFs have been shown to affect both self renewal and deterministic divisions of L-CFC. Miauchy et al (26, 27) have shown, that
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Tab. 1
Klin. Pädiatr. 202 (1990) 213
214 Klin. Pädiatr. 202 (1990) Tab.2
F. Herrmann et al.
Proliferative response of L-CFC to soluble polypeptide growth factors' No. positive/No. investigated (%)
5637-CM (15%v/v)
GM-CSF (50 ng/ml)
M-CSF (1000 U/ml)
IL-3 150 ng/ml)
IL-l beta 120 U/ml)
38/49 (77)
31/49 (63)
25/49 (51)
24/38 167)
0/20 (01
TNFalpha 125 ng/mll 0/19101
• colony lormation 01 L-CFC at day 1001 culture in a double layer agar assay
30
o
20
medium IL-3 II1II GM-CSF [] G-CSF 11 M-CSF ~
10
o ..L-
"--~===
Fig. 1 Effect 01 various CSFs on the development 01 adherent celis in AML cultures. The va lues represent means 01 lour dllferent experiments.
200
l i 8 1 ..!! :0
• 11 l1li ~
0
100
~
• ~
EI ~
0
'?
•
o:!
'Cl
....
medium IL-3 GM-CSF G-CSF M-CSF IL-3+G-CSF IL-3+GM-CSF IL-3+M-CSF GM-CSF+G-CSG GM-CSF+M-CSF G-CSF+M-CSF
o..L-_ _---'_ Fig. 2 Synergistlcs or additive effect of various CSFs on colony lormation 01 L-CFC. The values represent means of two different experiments and are expressed as colony numbers compared to numbers 01 colonies that grew in response to IL-3 alone.
not only the degree of responsiveness to one single CSF but also the spectrum of its effects varies considerably among individual AML sampies. IL-3 was most effective by contributing mitogenic effects to AML growth, whereas GCSF promoted both growth and differentiation to a varying degree of intensity. GM-CSF was also found to be a potent growth inducing factor for human myeloid leukemia cells (37), whereas M-CSF was shown to inhibit self renewal of leukemic stern cells in culture (27), and thus favouring differentiation (Figure I). In some AML cases, CSFs have been described to co-operate synergistically to induce in vitro blast colony growth, e. g. G- and GM-CSF or IL-3
The growth factor requirements of L-CFC in short-term and long-term proliferation assays have also been compared. In short term cultures IL-3 exhibited a greater biological activity than G-CSF and an equivalent activity to GM-CSF to induce L-CFC growth. Responsiveness of AML to CSFs could, however, not be correlated to the immunophenotype of the entire leukemic populations, to the immunophenotype of L-CFC, or to morphology according to FAß criteria (I). In long term cultures the combinations of IL-3 with G-CSF and GM-CSF resulted in higher plating efficiency compared to the effects seen with individual CSFs. Interestingly, the tendency of AML cells to differentiate in this assay was not reported to accelerate in response to exogenously added CSFs (50), as compared to the maturation process appearing spontaneously. In rare cases of AML (FAß M7) IL-I-beta has been identified as an essential growth promoter but also as inducing molecule for Interleukin-2-receptor expression (CD 25 antigen) (42). Although not a topic of this article, there is also evidence for suppressive effects on growth of AML, exerted by direct action of various growth factor related cytokines, like TNF-alpha, synergizing with interferon-gamma (13), and of transforming growth factor-beta, causing a reversible delay of cell cycle progression into Sand G2IM phase (48). CSF and cytokine production by AML blasts
Several recent reports have described production of growth factors by myeloid leukemia cell lines. One example is the murine myelomonocytic leukemia line WEHI-3ß, which constitutively synthesizes IL-3, due to the insertion of an endogenous retrovirus-like element c10se to the 5' end of the gene (54). In fresh human myeloid leukemia sampIes autonomous cluster and/or colony formation has also been demonstrated in some cases of AML (13, 14). With data of CSF responsiveness of AML celllines and fresh AML sampIes and of CSF production by leukemic celliines available, it was rat her conclusive to search for autocrine production of CSF in cases of AML, in which c1onogenic cells proliferated autonomously in vitro. Cells from some of these cases were found to transcribe and secrete GM-CSF constitutively (10, 14,55). Growth
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(Figure 2), whereas in other cases only additive effects were seen (19, 50). IL-6 has been identified as a co-stimulator to augment CSF induced c1onogenicity of AML blasts (34).
Control of blast cell proliferation and differentiation in acute myelogenous leukemia
In recent studies it became obvious, that a high percentage of autonomously growing AML sampies synthesized mRNA for various CSF species (32, 41, 56). However, no signifieant correlation of autonomous colony growth to a particular pattern of mRNA or protein production of CSFs by AMLs or to morphological subtypes of AML had been detected. Several investigators have screened series of AML populations for their ability to accumulate CSF mRNA including our own group (Table 3). GCSF mRNA was detected in 25-30070, GM-CSF in 23-25%, and M-CSF in 12-50% ofthe AML sampies investigated (32, 41,55,56). Whereas in the majority of cases, G-CSF and GM-CSF mRNA expression was accompanied by secretion of a biologically active protein, M-CSF activity could not be detected in culture supernatants conditioned by AML blasts (Table 3). In most cases, M-CSF was, however, detected in an membrane anchored form (41). Failure to secrete biologically active CSF proteins despite the presence of CSF gene transeripts may be explained by the association of several eukaryotic genes to alternative promoters. Thus consecutively appearing alternative modes of splicing could result in generation of other mRNAs coding for polypeptide products, which potentially differ in biologieal activity or exhibit different secretion patterns. Different splicing of G-CSF precursor mRNA by the alternative use of the 5'splice donor sites in intron 2 has been shown in human squamous carcinoma line (CHU-2), which produces high quantities of G-CSF constitutively (30). Other reports describe utilization of alternative promoters to transcribe the murine GM-CSF gene, thereby altering the hydrophobie NH2-terminalleader sequence, and generating a nonsecreted form of the protein (47). The presence of a hydrophobie transmembrane domain and two forms of protein encoded by two differently spliced mRNAs has been shown for M-CSF (21). Uninduced serum-free cell cultures of the pancreatic carcinoma line Mia
Tab. 3
PaCa showed the largest, most abundant 4,5 kb M-CSF mRNA species in the cytoplasm without detectable protein levels in the culture medium, suggesting the existence of a secretory and a membrane anchored form (40). Recent findings demonstrate that some leukemie cell populations might express CSF proteins at their cell membranes without releasing detectable amounts of CSFs, like M-CSF as mentioned above (41), but still might provide growth stimulatory effects to leukemic blast cells, e. g. by cell-cell contact (31). Furthermore, some leukemie cells might suffer defects on the translationallevel resulting in failure of CSF protein synthesis or production of a biologically less active or inactive protein. On the other hand, if CSF expression by myeloid cells in early differentiation stages is uncommon, these primitive cells might not be sufficientIy equipped to translate high amounts of proteins to secrete them properly. Other studies have focused on the expression of other cytokines with growth regulatory effects by AML blasts. It was found, that a high proportion of AML populations secrete IL-6, IL-I-beta and TNF-alpha (11, 34) (Table 3). Whereas IL-6 was shown to promote AML growth in vitro directly in a synergistic mode (34), IL-I-beta and TNF-alpha exert their positive growth regulating function by recruiting accessory cells, such as endothelial cells to provide CSF supply (G-, GM-, and M-CSF) (11,34) (Figure 3). However, IL-I-beta has also been reported as an essential growth promoter in AML of the FAB M7 type by exerting its action directly. These AML sampies experienced growth arrest when IL-I-beta was antagonized by specific monoclonal antibodies. A large number of AML sampies also express M-CSF receptors. No significant relationship was, however, found between growth of AML in vitro and expression of the fms oncogene, which is reported to be idential to be M-CSF receptor (44). It has been shown, that AML sampies with monocytic phenotypes, (FAB M4 and M5) can be induced to express surface binding sites for IL-2 by interferon-gamma (15). As already mentioned above, the CD 25 antigen could also be upregulated by IL-I-beta in AML M7 subtypes (42).
CSF-mRNA and -Protein Synthesis by AML blasts
G-CSF GM-CSF M-CSF IL-3 IL-1 beta IL-2 IL-5 IL-6 TNFalpha
mRNA
Protein
17/53' 13/53 6/49
15/53 11/53 0/49 nd" 15/49 nd nd 314 12/49
0170 15/49 0/40 0/15 20/49 17/49
• No. positive/No. investigated
"not determined
Fig. 3 Role of hematopoietlc polypeptides in growth control of blasts in acute myelogenous leukemia. Autocrine secretion of GM-, G-, and M-CSF by AML blasts, comodulated by autocrinously produced IL-6. Paracrinously secreted IL-1 beta and TNF-alpha induce CSF release by marrow stroma I cells .
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of L-CFC could be blocked by the addition of neutralizing antibodies to GM-CSF, suggesting growth promoting effects of autocrinuously secreted GM-CSF, whereas other autonomously growing AML specimens failed to express GM-CSF and their growth could not be abrogated by GMCSF antiserum. This observation has to be ranked as highIy abnormal, since stable GM-CSF mRNA transeripts have not been detected in resting or stimulated normal myeloid progenitor cells, and purified normal myeloid progenitor cells do not proliferate autonomously in vitro (11, 32, 55, 56).
Klin. Pädiatr. 202 (1990) 215
216 Klin. Pädiatr. 202 (1990)
The coordinated expression and close linkage of IL-3 and GM-CSF genes in human activated T cells (36), led us to examine AML cells for IL-3 expression; however, mRNA or protein activity for IL-3 was not detected in any of the cases studied (n = 70, unpublished). Several investigators including oUf own group have shown that GM-CSF induces M-CSF, IL-l and TNF-alpha in the monoblast cellline U937 (5,25). With other data available on normal hematopoietic cells, showing cascade induction of various cytokines (16, 24, 34, 51), it must be considered that growth factors mayaiso recruit each other in an autocrine or paracrine fashion. Further studies on the inducible expression of CSFs by AML blasts are therefore needed and are CUfrently under way to show, wheter a distinct differentiation state may affect the pattern of CSF expression by AML blasts.
Acknowledgment Drs. L. Souza (Amgen), D. Krumwieh (Behringwerke), P. Ralpha (Cetus), M. Schreier and C. Nissen (Sandoz), D. Blohm (BASF) are acknowledged far their gifts of cDNA probes and recombinant cytokines. This work was supported by the Deutsche Forschungsgemeinschaft He 138012-1.
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10
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In our own series, however, IL2 mRNA was not detectable in a large number of AML sampies of various subtypes [(n = 60), thus suggesting that IL-2 does not playa role in autocrine growth control of AML.
F. Herrmann et al.
Nagata S., M. Tsuchia, S. Asano, Y. Osami, Y. Hirata, N. Kubata, M. Oheda, H. Nomura, T. Yamazak: Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor. EMBO J. 5 (1986) 575-581 II OSler, W., A. Lindemann, S. Horn, R. Mertelsmann, F. Herrmann: Tumor necrosis factor (TNF)-alpha but not TNF-beta induces secretion of colony stimulating factor for macrophages by human monocytes. Blood 70 (1987) 170ü-1703 12 Oster, W., A. Lindemann, R. Mertelsmann, F. Herrmann: Regulation of gene expression of M-, G-, GM-, and Multi-CSF in normal and malignant hematopoietic cells. Blood Cells 14, in press (1988) ]J OSler, W., A. Lindemann, R. Marlelsmann, F. Herrmann: Cellular sources of CSF in the peripheral blood. J. Immunol, in press (1988) .14 Oster, W, N. A: Cicco, H. Klein, B. Fleischer; T. Hirano, T. Kishimo10, A. Lindemann, R. Merlelsmann, F. Herrmann: Participation of the monokines IL-6, TNF-alpha, and IL-I-beta secreted by acute myelogenous leukemia blasts in autocrine ans paracrine leukemia growth control. Submitted (1988) 35 Oster, W., A. Lindemann, R. Mertelsmann, F. Herrmann: GM-CSF and Multi-CSF regulate monocyte secretion of G-CSF. Blood, in press (1988) 36 Olsuka, T., A. Miyajima, N. Brown, K. Otsu, J. Abrams. S. Sealand, C. Caux, R. D. W Malefijl, J. De Vries, P. Meyerson, K. Yokota, L. Gemmel, D. Rennick, F. Lee, N. Arai, K.-I. Arai, T. Yokota: Isolation and characterization of an expressible cDNA encoding human IL-3. J. Immunol. 140 (1988) 2288-2295 37 Pebusque. M.-J., M. Lopez, H. Torres, A. Carroti, L. Guilben. P. Mannoni: Growth response of human myeloid leukemia cells to colonystimulating factors. Exp. Hematol. 16 (1988) 360-366 .1' Peltenati, M. J., M. M. Le Beau, R. S. Lemons, E. A. Shima, E: S. Kawasaki, R. A. Larson, C. J. Sherr, M. O. Diaz, J. D. Rowley: Assignment of CSF-] to 5q33.1: Evidence for clustering of genes regulating hematopoiesis and for their involvement in the deletion of the long arm of chromosome 5 in myeloid disorders. Proc. Natl. Acad. Sci. USA 83 (1987) 2970-2974 .19 Pike, B. L., W. A. Rohinsan: Human bone marrow colony growth in agar gel. J. Cell Physiol. 76 (1970) 77-83 40 Ralph, P., M. K. Warren, M. T. Lee, J. Csejtey, J. F. Weaver, H. E. Broxmeyer, D. E. Williams, E. A. Slanley, E. S. Kawasaki: Inducible production of human macrophage growth factor, CSF-1. Blood 68 (1986) 633-639 4' Rambaldi, A., N. Wahamiya, E. Vellenga, J. Heriguchi, K. Warren, D. Kufe, J. D. Griffin: Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells. J. Clin. Invesl. 8] (1988) 10330-1035 42 Sakai, K., T. Haltori, M. Matsuoka, N. Asou, S. Yamamolo, K. Sagawa, K. Takatsuki: Autocrine stimulation of interleukin-1-beta in acute myelogenous leukemia cells. J. Exp. Med. 166 (1987) 1597-1602 4.1 Sandherg, A. A.: The chromosomes in human leukemia. Sem. Hematol. 23 (1986) 201-217 44 Sherr, C. J., C. W. Rettenmier, R. SaLTa, M. F. Roussel, A. T. Look, E. R. Stanley: The c-fms proto-oncogene product is related to the receptor fort he mononuclear phagocyte growth factor, CSF-l. Cell41 (1985) 665-676 30
Klin. Pädiatr. 202 (1990) 217
., Simmers, R. N., L. M. Webber, M. F. Shannon, O. M. Garsan, G. Wong, M. A. Vadas, G. R. Sutherland: Localisation of the G-CSF gene on chromosome 17 proximal to the breakpoint in t (15, 17) in acute promyelocytic leukemia. Blood 70 (1987) 330-332 46 Souza, L. M., T. C. Raone, J. L. Gabrilove, P. H. Lai, K. M. Zsabo, D. C. Murdock, V. R. Chazin, J. Bruszewski, H. Lu, K. K. Chen, J. Barendt, E. Platzer, M. A. S. Moore, K. Weite: Recombinant human granulocyte colony-stimulating factor: Effects on normal and leukemic myeloid cells. Science 232 (1986) 61-65 47 Stanley, E., D. Metcalf, P. Sobieszcuk, N. M. Gough. A. R. Dunn: The structure and expression of the murine gene encoding granulocyte-macrophage colony-stimulating factor: Evidence for utilisation of alternative promoters. EMBO J. 4 (1985) 2569-2573 4' Tessier, N., T. Hoang: Transforming growth factor-beta inhibits the proliferation of blast cells of acute myeloblastic leukemia. Blood 72 (1988) 159-164 49 Vellenga, E., D. C. Young, K. Wagner, D. Wiper, D. Ostaporicz, J. D. Griffin: The effects of GM-CSF and G-CSF in promoting growth of clonogenic cells in acute myeloblastic leukemia. Blood 69 (1987) 1771-1776 '0 Vellenga, E., D. Ostapovicz, B. O'Rourke, J. D. Griffin: Effects of recombinant IL-3, GM-CSF, and G-CSF on proliferation of leukemic clonogenic cells in short-term and long-term cultures. Leukemia I (1987) 584-589 " Vellenga, E., A. Rambaldi, T. J. Ernst, D. Ostapovicz, J. D. Griffin: Independent regulation of M-CSF and G-CAF gene expression in human monocytes. Blood 71 (/988) 1529-2532 U Yang. Y. C., A. B. Ciarletta, P. A. Temple, M. P. Chung, S. Kovacic, J. S. Witek-Giannotti, A. C. Leary, R. Kriz, R. E. Donahue, G. G. Wong, S. C. Clark: Human IL-3 (multi-CSF): Identification by expression cloning of a novel hematopoietic growth factor related ta murine IL-3. Cell47 (1986) 3-10 5J Yarden, Y., i. A. Escobedo, W.-i. Kuang, T. L. Yang-Feng, R. N. Harkins, U. Franke, V. A. Fried, A. Ullrich, L. T. Williams: Structure of the receptor for plateletderived growth factor helps define a familiy of closely related growth factar receptor. Nature 323 (/986) 226-232 5. Ymer, S., Q. T. Tucker, C. J. Sanderson, A. J. Hapel, H. D. Campbell, I. G. Young: Constitutive synthesis of interleukin-3 by leukemia cdlline WEHI-3B is due to retroviral insertion near the gene. Nature 317 (/985) 255-258 " Young, D. c., J. D. Griffin: Autocrine Secretion of GM-CSF in acute myeloblastic leukemia. Blood 68 (1986) 1178-1181 56 Young, D. C., G. D. Demelri, T. i. ErnsI. S. A. Cannislra, J. D. Griffin: In vitro expression of colony-stimulating factor genes by human acute myeloblastic leukemia. Exp. Hematol. 16 (1988) 378-382
Dr. F. Herrmann Abt. Hämatologie 1II. Medizinische Klinik und Poliklinik Klinikum der Johannes Gutenberg-Universität Langenbeckstr. I D-65Oü Mainz
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Control of blast cell proliferation and differentiation in acute myelogenous leukemia
Control of blast cell proliferation and differentiation in acute myelogenous leukemia by soluble polypeptide growth factors F. Herrmann, W. Oster, R. Mertelsmann Abt. Hämatologie und Onkologie, Medizinische Klinik und Poliklinik, Klinikum der Albert-Ludwigs-Universität, Freiburg
Summary
Proliferation of acute myelogenous leukemia (AML) derived blast cells requires the presence in culture of one or more growth factors. In the majority of cases Interleukin-3 (lL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate c1onogenicity of AML blasts, which can be synergised by Interleukin-6 (lL-6), Interleukin-I (lL-I) and granulocyte colony-stimulating factor (G-CSF). In contrast, macrophage colony-stimulating factor (M-CSF) favors deterministic divisions. A substantial part of AML sampIes have c1onogenic cells which, however, proliferate autonomously in vitro. The production by leukemic cells of a variety of growth or synergizing factors including GMCSF, G-CSF, IL-I, IL-6, and Tumor Necrosis Factor (TNF) has been demonstrated and a fraction of cases will use these molecules to support c1onogenic growth in an autocrine or paracrine fashion. However, unlike the situation with retrovirus-induced murine or avian leukemias, the role of production of CSFs and other cytokines by human leukemic cells in the transformational process remains uncertain.
Introduction
Normal hematopoiesis is known to be controlled by a broad spectrum of polypeptide factors. The question of how malignant hematopoietic cells escape physiological growth restrictions has, however, remained largely obscur. Rapid and unrestricted expansion of transformed hematopoietic cells can be observed most impressively in acute myelogenous leukemia (AML), suggesting, that fundamental regulatory principles of hematopoiesis are inoperative in this disease. Several investigators including our own group have shown, that a group of hormoneIike glycoproteins, named colony-stimulating factors (CSF), which are essential for in vitro proliferation and differentiation of normal hematopoietic cells (see 7 for Klin. Pädiatr. 202 (1990) 212-217 @ 1990 F. Enke Verlag Stuttgart
Kontrolle der Proliferation mit Differenzierung von Blasten bei der akuten myeloischen Leukämie durch lösliche Polypeptid-Wachstumsfaktoren
Die Proliferation von Blasten akuter myeloischer Leukämien (AML) erfordert in vitro die Anwesenheit eines oder mehrerer hämatopoetischer Wachstumsfaktoren. Interleukin-3- und Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) stimulieren in der überwiegenden Mehrzahl leukämischer Zellproben die Klonogenität von AML-Blasten. lnterleukin-6, Interleukin-I und Granulozyten-stimulierender Faktor können synergistisch die durch IL-3 und GM-CSF induzierte B1astenproliferation steigern. Im Gegensatz hierzu steuert der Makrophagen-Kolonie-stimulierende Faktor (M-CSF) überwiegend deterministische Teilungen. Ein erheblicher Anteil von AML-Proben weist jedoch klonogene Zellen auf, die in vitro autonom, d. h. in Abwesenheit exogener Wachstumsfaktoren, proliferieren. Wir zeigen, daß die Produktion von hämatopoetischen Wachstumsfaktoren und synergisierenden Faktoren GM-CSF, G-CSF, li-I, IL-6 und Tumor-Nekrose-Faktor (TNF) durch Leukämieblasten möglich ist und daß in einer Reihe von Fällen diese Moleküle von Leukämiezellen autorkin oder parakrin genutzt werden, um das eigene Wachstum zu steuern. Der Grund für die konstitutive Wachstumsfaktorexpression in AML-Blasten ist noch unbekannt.
review), mayaIso be involved in the process of malignant transformation of hematopoietic cells. This article summarizes effects of CSFs and related molecules on proliferation and differentiation of AML blasts, by focussing on autocrine and paracrine mechanisms used by AML blasts to provide CSF supply and points out possible implications of CSF-involvement in the pathophysiology of this disease.
Regulation 01 normal hematopoiesis by colony-stimulating lactors The process of blood cell formation by which a small number of self-renewing stern cells undergoes commitment and spurs irreversibly committed progenitor cells to proliferate and 10 differentiate along a restricted lineage, involves interactions of cell-derived biomolecules with their target cells (2, 3). Much of what we
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212
Control of blast ce// proliferation and differentiation in acute myelogenous leukemia Cytokines involved in hemalopoiesis
MOLECULE
SYNONYM
CHROMOS. LOCATION
mRNA (kb)
MW (kD)
SECRETION IKb)
multi-CSF GM-CSF G-CSF M-CSF EPO
IL-3 CSF-alpha CSF-beta CSF-1
5q 5q21-32 17qll-22 5q33 7qll-22
1 1 1.6 1.5-4.5 1.6
14-28 14-35 18-22 36-90 34-39
e;p' e; e;p e;a e
IL-1-alpha IL-1-beta IL-2 IL-3 IL-4 IL-5 IL-6
Hemopoitein-1
2.2 16
TCGF
2q14 2q14 4p
31; 17 31; 17 15
e;p;a e;p;a e;p;a
BSF-1 BCGF-II, TRF IFN-beta2, BSF2
5q 5q 7q
0.9 1.7 13
15-20 12-18 24
e;p;a e;p P
Cachectin TNF-beta type-li IFN type-I IFN type-I IFN
6p23 6p23 12 9p13-21 9p22
1.6 1.4 1.7 1.0 0.9
17 25 15-45 15-24 20
e;p;a P P P e;p
TNF-alpha Lymphotoxin IFN-gamma IFN-alpha IFN-beta 1 • e; endocrine
p; paracrine
a; autocri ne
currently know about these interactions has been derived form in vitro cell culture techniques, enabeling us to identify progenitor cells by their ability to form colonies of various Iineages in semisolid medium in response to specific growth factors stimuli (39). Recently rapid progress has occurred in characterizing the molecular nature of these biologically active factors by purification and cloning of these molecules. Five human growth factor species have been identified so far, i. e. CSF for granulocytes (G-CSF) (30, 46), for macrophages (M-CSF or CSF-l) (18), for mixed granulocyte-macrophage colonies (GM-CSF) (6), for multilineage colonies (lL-3) (36,52), and erythropoietin (EPO) (23). Other factors that not strictly belong to the terminological entity of CSFs or hematopoietic growth factors, have also been shown to provide synergistic growth regulatory effects on hematopoiesis, or to induce release of hematopoietic growth factors by producer cells (Table 1). All CSFs are encoded by single copy genes and some are c1ustered on chromosome 5: The M-CSF gene has been mapped to 5q 33.1 (38), the GM-CSF gene to 5 q 21-q32 (17), the IL-3 gene to the same band of chromosome 5 as GM-CSF (22). Additionally, chromosome 5 carries multiple genes coding for growth related proteins and protein receptars, including the M-CSF receptor gene, closely related and possibly identical to the proto-oncogene c-fms (44), the receptor for platelet derived growth factor (5 q 31.3-32) (53), the beta-2 adrenergic receptor (5 q 31-32) (20), and genes coding for endothelial cell growth factor (5 q 31.3-32). Chromosome 5 is frequently involved in cytogenetic aberrations in AML, e. g. partialloss of the long arm of the chromosome (deI 5 q), most often occurring secondary to cytotoxic therapy but also arrising in patients with de novo AML (43). It has been shown that the genes far GM-CSF, M-CSF, and IL-3 are directly affected by structural changes of chromosome 5 (17, 22). G-CSF has been mapped to the chromosomal region 17 q 11.2-21 proximal to the breakpoint involved in the translocation (15; 17) (45), which is the hallmark karyotypic aberration of acute promyeIocytic leukemia (FAß M3) (43).
Physiological sources of CSFs are monocyte/macrophage containing tissues, T-Iymphocytes, granulocytes, endothelial cells, and fibroblasts (3, 12,24, 29, 32, 33, 35). Normal immature hematopoietic progenitor cells, however, are not known to have the capacity to produce CSFs, thus suggesting lack of autocrine growth regulation in the normal bone marrow.
Effects of CSFs on myeloid leukemia ce//s Formation of blast colonies in agar cultures originating from a small proportion of fresh leukemic cell populations has also been shown (9, 28). These leukemic colony forming cells (LCFC) have several features in common with normal hematopoietic stern cells: For colony growth they require in most cases supply of exogenously provided growth factors, which are commonly available in media conditioned by lectin-activated leukocytes (9) or media from various tumor cell Iines, including the histiocytic GCT-line, the T-Iymphoblastoid Mo-line, and the bladder carcinoma line 5637; furthermore, L-CFC possess self renewal capacity , a high thymidine suicide index, and the potential to undergo at least limited, although abnormal differentiation to nonproliferating cells (4). Several investigators have assessed the ability of recombinant purified CSFs to substitute far standard conditioned media using the L-CFC assay by demonstrating that IL-3, GM-CSF, and G-CSF are promoters of L-CFC growth from many patients with AML (8, 10, 19, 49) (see Table 2). However, it became obvious that AML is not a homogenous disease by demonstrating that L-CFC exhibit a broad spectrum of reponses to CSFs, with marked variation from patient to patient. CSFs have been shown to affect both self renewal and deterministic divisions of L-CFC. Miauchy et al (26, 27) have shown, that
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Tab. 1
Klin. Pädiatr. 202 (1990) 213
214 Klin. Pädiatr. 202 (1990) Tab.2
F. Herrmann et al.
Proliferative response of L-CFC to soluble polypeptide growth factors' No. positive/No. investigated (%)
5637-CM (15%v/v)
GM-CSF (50 ng/ml)
M-CSF (1000 U/ml)
IL-3 150 ng/ml)
IL-l beta 120 U/ml)
38/49 (77)
31/49 (63)
25/49 (51)
24/38 167)
0/20 (01
TNFalpha 125 ng/mll 0/19101
• colony lormation 01 L-CFC at day 1001 culture in a double layer agar assay
30
o
20
medium IL-3 II1II GM-CSF [] G-CSF 11 M-CSF ~
10
o ..L-
"--~===
Fig. 1 Effect 01 various CSFs on the development 01 adherent celis in AML cultures. The va lues represent means 01 lour dllferent experiments.
200
l i 8 1 ..!! :0
• 11 l1li ~
0
100
~
• ~
EI ~
0
'?
•
o:!
'Cl
....
medium IL-3 GM-CSF G-CSF M-CSF IL-3+G-CSF IL-3+GM-CSF IL-3+M-CSF GM-CSF+G-CSG GM-CSF+M-CSF G-CSF+M-CSF
o..L-_ _---'_ Fig. 2 Synergistlcs or additive effect of various CSFs on colony lormation 01 L-CFC. The values represent means of two different experiments and are expressed as colony numbers compared to numbers 01 colonies that grew in response to IL-3 alone.
not only the degree of responsiveness to one single CSF but also the spectrum of its effects varies considerably among individual AML sampies. IL-3 was most effective by contributing mitogenic effects to AML growth, whereas GCSF promoted both growth and differentiation to a varying degree of intensity. GM-CSF was also found to be a potent growth inducing factor for human myeloid leukemia cells (37), whereas M-CSF was shown to inhibit self renewal of leukemic stern cells in culture (27), and thus favouring differentiation (Figure I). In some AML cases, CSFs have been described to co-operate synergistically to induce in vitro blast colony growth, e. g. G- and GM-CSF or IL-3
The growth factor requirements of L-CFC in short-term and long-term proliferation assays have also been compared. In short term cultures IL-3 exhibited a greater biological activity than G-CSF and an equivalent activity to GM-CSF to induce L-CFC growth. Responsiveness of AML to CSFs could, however, not be correlated to the immunophenotype of the entire leukemic populations, to the immunophenotype of L-CFC, or to morphology according to FAß criteria (I). In long term cultures the combinations of IL-3 with G-CSF and GM-CSF resulted in higher plating efficiency compared to the effects seen with individual CSFs. Interestingly, the tendency of AML cells to differentiate in this assay was not reported to accelerate in response to exogenously added CSFs (50), as compared to the maturation process appearing spontaneously. In rare cases of AML (FAß M7) IL-I-beta has been identified as an essential growth promoter but also as inducing molecule for Interleukin-2-receptor expression (CD 25 antigen) (42). Although not a topic of this article, there is also evidence for suppressive effects on growth of AML, exerted by direct action of various growth factor related cytokines, like TNF-alpha, synergizing with interferon-gamma (13), and of transforming growth factor-beta, causing a reversible delay of cell cycle progression into Sand G2IM phase (48). CSF and cytokine production by AML blasts
Several recent reports have described production of growth factors by myeloid leukemia cell lines. One example is the murine myelomonocytic leukemia line WEHI-3ß, which constitutively synthesizes IL-3, due to the insertion of an endogenous retrovirus-like element c10se to the 5' end of the gene (54). In fresh human myeloid leukemia sampIes autonomous cluster and/or colony formation has also been demonstrated in some cases of AML (13, 14). With data of CSF responsiveness of AML celllines and fresh AML sampIes and of CSF production by leukemic celliines available, it was rat her conclusive to search for autocrine production of CSF in cases of AML, in which c1onogenic cells proliferated autonomously in vitro. Cells from some of these cases were found to transcribe and secrete GM-CSF constitutively (10, 14,55). Growth
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(Figure 2), whereas in other cases only additive effects were seen (19, 50). IL-6 has been identified as a co-stimulator to augment CSF induced c1onogenicity of AML blasts (34).
Control of blast cell proliferation and differentiation in acute myelogenous leukemia
In recent studies it became obvious, that a high percentage of autonomously growing AML sampies synthesized mRNA for various CSF species (32, 41, 56). However, no signifieant correlation of autonomous colony growth to a particular pattern of mRNA or protein production of CSFs by AMLs or to morphological subtypes of AML had been detected. Several investigators have screened series of AML populations for their ability to accumulate CSF mRNA including our own group (Table 3). GCSF mRNA was detected in 25-30070, GM-CSF in 23-25%, and M-CSF in 12-50% ofthe AML sampies investigated (32, 41,55,56). Whereas in the majority of cases, G-CSF and GM-CSF mRNA expression was accompanied by secretion of a biologically active protein, M-CSF activity could not be detected in culture supernatants conditioned by AML blasts (Table 3). In most cases, M-CSF was, however, detected in an membrane anchored form (41). Failure to secrete biologically active CSF proteins despite the presence of CSF gene transeripts may be explained by the association of several eukaryotic genes to alternative promoters. Thus consecutively appearing alternative modes of splicing could result in generation of other mRNAs coding for polypeptide products, which potentially differ in biologieal activity or exhibit different secretion patterns. Different splicing of G-CSF precursor mRNA by the alternative use of the 5'splice donor sites in intron 2 has been shown in human squamous carcinoma line (CHU-2), which produces high quantities of G-CSF constitutively (30). Other reports describe utilization of alternative promoters to transcribe the murine GM-CSF gene, thereby altering the hydrophobie NH2-terminalleader sequence, and generating a nonsecreted form of the protein (47). The presence of a hydrophobie transmembrane domain and two forms of protein encoded by two differently spliced mRNAs has been shown for M-CSF (21). Uninduced serum-free cell cultures of the pancreatic carcinoma line Mia
Tab. 3
PaCa showed the largest, most abundant 4,5 kb M-CSF mRNA species in the cytoplasm without detectable protein levels in the culture medium, suggesting the existence of a secretory and a membrane anchored form (40). Recent findings demonstrate that some leukemie cell populations might express CSF proteins at their cell membranes without releasing detectable amounts of CSFs, like M-CSF as mentioned above (41), but still might provide growth stimulatory effects to leukemic blast cells, e. g. by cell-cell contact (31). Furthermore, some leukemie cells might suffer defects on the translationallevel resulting in failure of CSF protein synthesis or production of a biologically less active or inactive protein. On the other hand, if CSF expression by myeloid cells in early differentiation stages is uncommon, these primitive cells might not be sufficientIy equipped to translate high amounts of proteins to secrete them properly. Other studies have focused on the expression of other cytokines with growth regulatory effects by AML blasts. It was found, that a high proportion of AML populations secrete IL-6, IL-I-beta and TNF-alpha (11, 34) (Table 3). Whereas IL-6 was shown to promote AML growth in vitro directly in a synergistic mode (34), IL-I-beta and TNF-alpha exert their positive growth regulating function by recruiting accessory cells, such as endothelial cells to provide CSF supply (G-, GM-, and M-CSF) (11,34) (Figure 3). However, IL-I-beta has also been reported as an essential growth promoter in AML of the FAB M7 type by exerting its action directly. These AML sampies experienced growth arrest when IL-I-beta was antagonized by specific monoclonal antibodies. A large number of AML sampies also express M-CSF receptors. No significant relationship was, however, found between growth of AML in vitro and expression of the fms oncogene, which is reported to be idential to be M-CSF receptor (44). It has been shown, that AML sampies with monocytic phenotypes, (FAB M4 and M5) can be induced to express surface binding sites for IL-2 by interferon-gamma (15). As already mentioned above, the CD 25 antigen could also be upregulated by IL-I-beta in AML M7 subtypes (42).
CSF-mRNA and -Protein Synthesis by AML blasts
G-CSF GM-CSF M-CSF IL-3 IL-1 beta IL-2 IL-5 IL-6 TNFalpha
mRNA
Protein
17/53' 13/53 6/49
15/53 11/53 0/49 nd" 15/49 nd nd 314 12/49
0170 15/49 0/40 0/15 20/49 17/49
• No. positive/No. investigated
"not determined
Fig. 3 Role of hematopoietlc polypeptides in growth control of blasts in acute myelogenous leukemia. Autocrine secretion of GM-, G-, and M-CSF by AML blasts, comodulated by autocrinously produced IL-6. Paracrinously secreted IL-1 beta and TNF-alpha induce CSF release by marrow stroma I cells .
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of L-CFC could be blocked by the addition of neutralizing antibodies to GM-CSF, suggesting growth promoting effects of autocrinuously secreted GM-CSF, whereas other autonomously growing AML specimens failed to express GM-CSF and their growth could not be abrogated by GMCSF antiserum. This observation has to be ranked as highIy abnormal, since stable GM-CSF mRNA transeripts have not been detected in resting or stimulated normal myeloid progenitor cells, and purified normal myeloid progenitor cells do not proliferate autonomously in vitro (11, 32, 55, 56).
Klin. Pädiatr. 202 (1990) 215
216 Klin. Pädiatr. 202 (1990)
The coordinated expression and close linkage of IL-3 and GM-CSF genes in human activated T cells (36), led us to examine AML cells for IL-3 expression; however, mRNA or protein activity for IL-3 was not detected in any of the cases studied (n = 70, unpublished). Several investigators including oUf own group have shown that GM-CSF induces M-CSF, IL-l and TNF-alpha in the monoblast cellline U937 (5,25). With other data available on normal hematopoietic cells, showing cascade induction of various cytokines (16, 24, 34, 51), it must be considered that growth factors mayaiso recruit each other in an autocrine or paracrine fashion. Further studies on the inducible expression of CSFs by AML blasts are therefore needed and are CUfrently under way to show, wheter a distinct differentiation state may affect the pattern of CSF expression by AML blasts.
Acknowledgment Drs. L. Souza (Amgen), D. Krumwieh (Behringwerke), P. Ralpha (Cetus), M. Schreier and C. Nissen (Sandoz), D. Blohm (BASF) are acknowledged far their gifts of cDNA probes and recombinant cytokines. This work was supported by the Deutsche Forschungsgemeinschaft He 138012-1.
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In our own series, however, IL2 mRNA was not detectable in a large number of AML sampies of various subtypes [(n = 60), thus suggesting that IL-2 does not playa role in autocrine growth control of AML.
F. Herrmann et al.
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Dr. F. Herrmann Abt. Hämatologie 1II. Medizinische Klinik und Poliklinik Klinikum der Johannes Gutenberg-Universität Langenbeckstr. I D-65Oü Mainz
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Control of blast cell proliferation and differentiation in acute myelogenous leukemia
