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Stem Cells, Vol. 19, No. 4, 321-328, July 2001
© 2001 AlphaMed Press

Effect of the Hemoregulatory Peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1{alpha} is Reduced in Bone Marrow Cultures from Patients with Chronic Myeloid Leukemia (CML)

H. Karlica, N. Loudaa,c, M. Pfeilstöckera,b, F. Keilc, A. Lohningerd, E. Pittermanna,b, J. Paukovitse

a Ludwig Boltzmann Institute for Leukemia Research and Hematology, Hanusch Hospital, Vienna, Austria;
b 3rd Medical Department, Hanusch Hospital, Vienna, Austria;
c Department of Medicine I, Bone Marrow Transplantation Unit, University of Vienna, Austria;
d Institute of Medical Chemistry, University of Vienna, Austria;
e Institute for Cancer Research, University of Vienna, Austria

Key Words. Hemoregulatory peptide • MIP-1{alpha} • Long-term bone marrow cultures • Chronic myeloid leukemia

Heidrun Karlic, Ph.D., L. Boltzmann Institute for Leukemia Research, H.Collinstr. 30, A-1140 Vienna, Austria. Telephone: 43-1-91021-2347; Fax: 43-1-9143214; e-mail: heidrun.karlic{at}chello.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1{alpha}). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1{alpha}. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1{alpha} and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (G{alpha}16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients.

It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1{alpha}, thus inducing a downregulation of these factors in bone marrow from CML patients.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In hematopoiesis, synergistic effects are fairly common within the regulation of the cytokine network. It could be shown that the monomeric form of the granulocyte-derived hemoregulatory peptide (pEEDCK) [1] arrests hematopoietic cells at the G1/S transition [2]. There are indications that a suppressive synergism of the peptide with recombinant human (rHu)-tumor necrosis factor-{alpha} and interferon gamma [3] is responsible for this arrest.

pEEDCK has a close similarity to a five amino acid sequence in the effector domain of Gi{alpha} proteins, a group to which the hematopoiesis-specific G-protein {alpha} subunit G{alpha}16 belongs [4]. It is expressed in normal and malignant hematopoiesis [5]. Previous data also suggest a functional association of the hemoregulatory peptide with G-protein-mediated signal transduction and indicate that this type of G-protein {alpha}-subunit may cooperate with (pEEDCK)2 in triggering the cytokine response of immature hematopoietic cells [6].

The disulfide-bonded dimer (pEEDCK)2 is derived from pEEDCK which, like other thiols, is sensitive to oxidation which induces the formation of a disulfide-linked Cys-Cys motif. (pEEDCK)2 is known to be a stimulator of lineage-committed myeloid progenitors in vitro and in mice [7, 8] and also increases the production of M-CSF (macrophage colony-stimulating factor) from human marrow fibroblasts. It has been repeatedly shown that the activity of the dimeric form of the granulocyte-derived hemoregulatory peptide pEEDCK is closely associated with stroma-derived cytokines [9].

The macrophage inflammatory protein MIP-1{alpha} also harbors a Cys-Cys motif and has some functional similarities to the hemoregulatory peptide [10].

In this study, we tested the effect of the peptide dimer (pEEDCK)2 or MIP-1{alpha} and cytokines on stem cells in an assay system using LTBMCs (long-term bone marrow cultures) from patients with chronic myeloid leukemia (CML), healthy human donors, and mice. Expression of growth and differentiation-associated genes such as G{alpha}16, 5-lipoxygenase (5-LO), phospholipaseA2, c-kit, and CD34 and two isoforms of carnitine palmitoyltransferase (CPT), a key enzyme of oxidative metabolism, were analyzed in order to aid interpretation of different effects of (pEEDCK)2 or MIP-1{alpha} on LTBMCs from CML patients and healthy donors.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Hemoregulatory Peptide and Cytokines
The hemoregulatory peptide dimer was synthesized, purified, and stored as described [6]. In short, the synthesis procedure started with the monomer [11]. The dimer was generated by oxidation of S-acetamido-methyl-pEEDCK with iodine using the acetamidomethyl group for thiol protection during solid phase synthesis. The product was purified by anion exchange chromatography with the acetamidomethyl group still attached to the cysteine side chain. Excess iodine was removed by gel filtration through Sephadex G-10 (Pharmacia; Uppsala, Sweden; http://www.pnu.com). Cytokines were dissolved and diluted with a mixture containing nine parts Iscove's modified Dulbecco's medium (IMDM) (GIBCO BRL; Paisley, Great Britain) and one part fetal calf serum (FCS) (GIBCO BRL). The following stock solutions were used: stem cell factor (SCF) (Pepro Tech; Vienna, Austria; http://www.peprotech.com) at a concentration of 500 ng/ml and 1,000 ng/ml; GM-CSF (Novartis; Basel, Switzerland; http://www.novartis.com/) 10 µg/ml; interleukin 3 (IL-3) (Novartis) 10 µg/ml; macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) 1 µg/ml and leukemia inhibitory factor (LIF) (R&D Systems; Minneapolis, MN; http://www.rndsystems.com/) 2 x 104 U/ml; IL-6 10 µg/ml and anti-IL-1ß (Santa Cruz Biotechnology; Santa Cruz, CA) at a concentration of 10 µg/ml.

Bone Marrow Samples and Separation Procedure
Aliquots of bone marrow samples from CML Philadelphia chromosome-positive patients were obtained by posterior iliac crest aspiration after informed consent. Mononuclear cells were isolated on Ficoll-Hypaque gradients (density 1.077) (SeroMed, Biochrom KG; Berlin, Germany; http://www.Biochrom.de/), centrifuged and washed twice with RPMI 1640 medium (Serva-Novex; San Diego, CA; http://www.serva.de/).

LTBMC
For LTBMCs, bone marrow cells were grown at a concentration of 2 x 106 cells/ml in a total volume of 4 ml per dish in IMDM (GIBCO BRL) containing 12.5% FCS (GIBCO BRL), 12.5% horse serum (GIBCO BRL) and 1 µM hydrocortisone (Sigma Chemical Co.; St. Louis, Mo; http://www.sigma-aldrich.com). Recombinant growth factors were added in different combinations using the concentrations given in Table 1Go. Peptides were added to the cultures every second day.


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  		Table 1. Combinations of cytokines
 
Colony Assays
For colony assays, aliquots of nonadherent cells from LTBMCs (and on the last day also adherent cells) were resuspended in medium composed of IMDM + Glutamax I (GIBCO BRL), supplemented with 20% FCS and methylcellulose (0.8%, 4,000 cps, Colorcon; Königstein, Germany; http://www.colorcon.com) initially at a concentration of 0.5 x 105, from the first to fifth week at 1 x 105 and at the end of week 5 the adherent cells at 2 x 105. Colony formation was induced by rHuIL-3 and rHu-GM-CSF (each 10 ng/ml). Duplicates or triplicates from each well were incubated at 37°C in a humidified atmosphere of 7.5% CO2 in air. After 14 days, aggregates of 50 or more cells were considered to be colonies.

mRNA Isolation, cDNA Preparation and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
mRNA isolation and cDNA preparation were performed using commercially available systems (Amersham Pharmacia Biotech Europe; Freiburg, Germany; http://www.apbiotech.com).

PCR conditions were the same for all assays (1 minute 95°C/1 minute 55°C/1 minute 72°C, 35 cycles). For studies of G{alpha}16, 5-LO, c-kit, CD34, phospholipase A2 and expression of bcr/abl, c-abl and ß-actin were used as positive controls and assays were set up on a block cycler (Perkin-Elmer; Norwalk, CT).

RT-PCR was carried out using a LightCyclerTM System (Roche Molecular Biochemicals; Mannheim, Germany) which allows amplification and detection (by fluorescence) in the same tube, using a kinetic approach. LightCycler-PCR reactions were set up in microcapillary tubes using 1 µl (10-50 ng) cDNA with 19 µl of a SYBR Green I [12] master mix containing upstream and downstream PCR primers, MgCl2, and SYBR Green. The final concentrations of the reaction components were 1.0 µM each primer, 4 µM MgCl2 and 1 x SYBR Green master mix. Primers and PCR conditions are shown in Table 2Go.


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  		Table 2. Primers and PCR conditions
 
Statistical Analysis
Results were presented as mean ± standard deviation from a series of experiments. The p-value was calculated using the Students t-test or the Wilcoxon Sign Rank test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Cytokines Combined with (pEEDCK)2 or MIP-1{alpha} Do Not Affect Growth of LTBMCs from CML Patients but Stimulate Stem Cells of Healthy Donors
The induction of colony-forming units-culture (CFU-C) and CFU-granulocyte/erythroid/macrophage/megakarycyte (GEMM) from adherent cells by (pEEDCK)2 or MIP-1{alpha} in combination with other cytokines was higher in cells derived from normal bone marrow than from CML bone marrow (Figs. 1A and 1CGo). In normal bone marrow, the stimulatory effect of MIP-1{alpha} on adherent cell-derived CFU-GEMM was significantly higher (fourfold with MIP-1{alpha} alone or sixfold in combination with cytokines) than with (pEEDCK)2. By contrast, Figures 1B and 1DGo show that MIP-1{alpha} had no stimulatory effect on CFU-C and CFU-GEMM derived from nonadherent cells of either CML or healthy subjects, but a significant stimulation on CFU-GEMM was induced by (pEEDCK)2 in combination with cytokines in cultures from healthy donors.






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  		Figure 1. The fold increase of progenitors (CFU-C) and committed progenitors (CFU-GEMM) from adherent cells (Figs. 1A and 1CGo) or nonadherent cells (Figs. 1B and 1DGo) from LTBMC of CML patients and healthy donors, harvested after 5 weeks. The methylcellulose assays were initiated at a concentration of 1 x 105 cells/ml and a stimulation with GM-CSF, IL-3, SCF (30 ng/ml), and erythropoietin for cells from the adherent layer. After 2 weeks the colonies were scored as CFU-GEMM. Open and closed bars show the fold increase (mean values obtained from each group). The number above each bar indicates the number of parallel samples, the dots their median value.

 
Expression of Cell Type-Associated Genes G{alpha}16, c-kit, CD34, 5-LO, and Phospholipase A2 and Genes from Key Enzymes of Oxidative Metabolism (CPT1A and CPT1B)
To investigate whether the differences in response to growth factors are reflected in the activity of genes are typical for certain types of blood cells and progenitors (Table 3Go), RT-PCR and immunofluorescence techniques were used.


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  		Table 3. Cell type specific expression of genes analyzed in this study
 
Expression of G{alpha}16 correlated with growth of CFU-GEMM in bone marrow of healthy donors (range of CFU-GEMM 10-1,200), in 4/4 cases. However, in bone marrow from CML patients (range of CFU-GEMM 1-400), all samples were positive for G{alpha}16 expression but less than 50% formed CFU-GEMM after one or more weeks of culture.

As shown in Figure 2Go, the expression of other cell type-associated genes, such as c-kit and CD34, which are also expressed in immature blood cells, phospholipase A2, which is present in mono- and megakaryocytes, and 5-LO, which is typical for maturing granulocytes, was similar in both normal and leukemic bone marrow. Evaluation by both RT-PCR and fluorescence in situ hybridization demonstrated that no reduction in the number of BCR/ABL+ cells took place. Thus no selective stimulation of normal cells from CML patients could be achieved.



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  		Figure 2. Relative gene expression in HLTBMCs from CML patients and healthy donors. The bars represent mean values of expression rates relative to standard genes (ß-actin or G6PDH) from four samples/ group calculated after semiquantitative densitometric evaluation of PCR products of G{alpha}16, 5-LO, phospholipase A2, c-kit, and CD34 on agarose gels. Quantification of expression rates from carnitine palmitoyltransferase isoforms CPT1A and CPT1B was performed using real time PCR and LightCyclerSYBR Green technology.

 
However, quantitative analysis of the expression rate of genes of two isoforms of carnitine palmitoyltransferase 1 (CPT1A and CPT1B), which are key enzymes of oxidative metabolism, showed that the expression rate of these genes in cells from CML patients was downregulated to 3% of that of healthy donors.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The hemoregulatory peptide (pEEDCK)2 has the potential to replace the activity of growth factor cocktails in in vitro systems of murine hematopoiesis [13, 14] and to enhance the effect of cytokines such as IL-3 and SCF on colony outgrowth of human CD34+ cells (stem cells [6]). Considering its activity on hematopoietic cells, the hemoregulatory peptide pEEDCK has some functional similarity to MIP-1{alpha} that is reflected in a structural analogy involving the presence of Cys-Cys motifs.

This prompted us to investigate differences regarding the response to such stimuli in cultured bone marrow cells from patients with CML compared to healthy donors.

In this study, we demonstrated that the synergistic activity of (pEEDCK)2 or MIP-1{alpha} and cytokines can be found in LTBMC from healthy donors but cannot be detected in cultures from CML patients.

We chose growth conditions free of reducing agents (such as mercaptoethanol). This should favor the stability of MIP-1{alpha} and lead to an immediate oxidation of residual endogenous pEEDCK to (pEEDCK)2, thus avoiding the inhibitory activity of the monomer. However, the results of this study clearly show that the activities of both (pEEDCK)2 and MIP-1{alpha} could be detected in LTBMCs from healthy donors but not in CML patients. However, since morphological analysis as well as expression patterns of a series of genes such as G{alpha}16, c-kit, CD34 phospholipase A2, and 5-LO were similar in LTBMC from healthy subjects and CML patients, this difference was not due to an effect of culture conditions.

Data exist showing that the gene regulating expression of G{alpha}16 harbors a sequence homologous to pEEDCK in normal hematopoietic cells as well as in myeloid malignancies [2, 5]. There is also a correlation between G{alpha}16 mRNA and expression of the CD34 protein which is only transcribed in quiescent cells and downregulated earlier during differentiation than is known for c-kit, explaining the lower transcription rate of CD34 compared to G{alpha}16. Additionally, G{alpha}16 is also expressed in monocytes that are negative for CD34. The reason for the observation that the transcription rate of phospholipase A2 was only 25% compared to G{alpha}16 or 5-LO is probably associated with the fact that, in contrast to the latter two, it is expressed only in monocytes but not in early (CD34+) progenitors or in mature granulocytes (Table 3Go). The fact that the level of 5-LO was the same as G{alpha}16 may be attributed to their expression by the two prominent cell types which are present within LTBMC (Table 3Go).

In CML bone marrow, residual primitive normal hematopoietic cells coexist with their leukemic counterparts [15]. The Philadelphia translocation t(9;22) and the hybrid BCR-ABL gene are well-established markers for discriminating normal and leukemic cell populations. Allogeneic stem cell transplantation has the potential to cure this disease [16]. However, age restriction and lack of suitable donors limit this therapeutic approach. An alternative therapeutic option is myeloablative chemotherapy with infusion of autologous stem cells. However, the potential of this is impeded by a high risk of relapse due to reinfusion of BCR-ABL+ cells. Recent approaches showed promising results regarding in vitro enrichment of BCR-ABL– cells [17]. However, an optimal combination of cytokines for 100% enrichment of BCR-ABL– progenitor cells from cultured CML bone marrow has not yet been found and could not be demonstrated in this study.

Considering the fact that BCR-ABL exerts its antiapoptotic effect at the mitochondrial level [18], we decided to investigate the expression of mitochondrial isoforms of carnitine palmitoyl-transferase (CPT1A and CPT1B). Besides their participation in apoptotic regulation [19, 20], CPTs are known to be key enzymes of oxidative metabolism. As they are regulated at the transcriptional level [21], quantitative RT-PCR using the SYBR GreenTM technique was applied for investigating their transcription rate. We found that both CPT1A and CPT1B were downregulated in LTBMCs from CML patients to a level of 3% of that of healthy donors. As LTBMCs were cultivated in the presence of horse serum, this effect could not be assigned to a carnitine deficiency in the culture medium that has recently been associated with a downregulation of CPT [22]. It is known that CML cells like other leukemic cells are similar to cancer cells in that they are known to rely on anaerobic glycolysis [23-26] and not oxidative metabolism as do their normal counterparts [27, 28].

This may be associated with an elevated intracellular redox status in cultured CML cells which is known to upregulate thioredoxin in cancer cells [29]. Thioredoxin in turn reduces oxidized cysteine groups on proteins which might lead to a reduction of (pEEDCK)2 to pEEDCK and downregulation of MIP-1{alpha} which also harbors a Cys-Cys motif (a direct measurement of this was not possible within the scope of this study). With respect to a possible formation of pEEDCK, this may rely on the structural relationship of specific sequence motifs from IL-11, IL-6-R, or c-kit that interact hydropathically with the monomeric form of this peptide pEEDCK [30]. As the bone marrow stroma of CML patients is known for its potential for autologous growth factor production [31], this regulatory network may be disturbed in the microenvironment of their bone marrow.

In conclusion, our data indicate that there are remarkable differences in growth characteristics of HLTBMCs between bone marrow from healthy donors and CML patients, especially regarding the response to cytokines and growth factors. These differences appear to be based on a disturbance of redox control in CML bone marrow cells. Our data provide hints for a model that may explain regulation of the cytokine network within the bone marrow microenvironment by a new mechanism acting on a multitude of cytokine subsets.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by the Jubiläumsfonds der Österreichischen Nationalbank (Project No. 7451) and the Austrian Federal Chancellery.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received February 27, 2001; accepted for publication April 2, 2001.




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