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Stem Cells, Vol. 14, No. 2, 215-224, March 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Inhibition of Enriched Stem Cells in Vivo and in Vitro by the Hemoregulatory Peptide SK&F108636

O.P. Veibya, S. LoCastrob, P. Bhatnagarb, W.M. Olsena

a Nycomed Pharma AS, Diagnostica and Exploratory Therapy R&D, Oslo, Norway;
b SmithKline Beecham Pharmaceuticals, Medicinal Chemistry Dept., King of Prussia, Pennsylvania, USA

Key Words. Stem cells • Hematopoiesis • Hemoregulatory peptide • SK&F108636 • Inhibition

Dr. W.M. Olsen, Nycomed Pharma, Gaustadalléen 21, N-0371 Oslo, Norway.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Replacement of the labile sulfhydryl group (-SH) of the hemoregulatory peptide monomer pyroGluGluAspCysLys (HP5b) with an isosteric methylene group yields a chemically stable compound, SK&F108636. In this study, we describe the effects of SK&F108636 on highly enriched Lin–Sca1+ hematopoietic stem cells. SK&F108636 significantly reduced the fraction of cycling progenitor cells, granulocyte macrophage colony-forming cells (GM-CFC), in vitro and in vivo. There was no effect on GM-CFC or Mix-CFC colony formation. SK&F108636 significantly inhibited proliferation of high proliferative potential (HPP)-CFC in semisolid agar cultures stimulated by stem cell factor + interleukin 3 (IL-3) + IL-1, but had no effect in cultures stimulated with M-CSF + IL-3 + IL-1. SK&F108636 was shown to act directly on the stem cells since SK&F108636 inhibited proliferation of Lin–Sca1+ cells in single cell assays. Administration of SK&F108636 to lethally irradiated mice transplanted with 2000 Lin–Sca1+ cells significantly inhibited proliferation/differentiation of cells developing into colony forming units-spleen (CFU-S) (preCFU-S) and the reconstitution of HPP-CFC and GM-CFC. There was no effect of SK&F108636 on CFU-S colony formation or mature cell regeneration in bone marrow, spleen and blood. Hence, the hemoregulatory peptide monomer SK&F108636 is a potent primitive stem cell inhibitor in vivo and in vitro. Inhibition of stem cell proliferation by small specific inhibitors may protect hematopoiesis from myelotoxic side effects during chemotherapy treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Although the concept of inhibitory growth control [1] predates the discovery of stimulatory growth factors, most studies of cell proliferation have been directed toward molecules which promote growth and differentiation. There is now increasing awareness that regulation of hematopoiesis involves the action of inhibitory as well as stimulatory molecules and several inhibitors of hematopoiesis have been described [2]. Transforming growth factor-ß (TGF-ß) was the first inhibitor to be cloned and expressed in mammalian cells and has shown both stimulatory and inhibitory activities on hematopoietic cells [3–5]. Tumor necrosis factor-{alpha} (TNF-{alpha}) is a highly pleiotropic cytokine that plays a central role in immunologic and inflammatory responses [6,7]. The effects of TNF-{alpha} on hematopoietic cells have also been shown to be both stimulatory and inhibitory [8–12]. The macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) was purified and characterized for its ability to inhibit hematopoietic stem cell (HSC) proliferation in vitro [13]. Later, it was shown to be a potent inhibitor of both primitive and committed progenitor cell populations [14]. The interferons (IFN) were originally described by their antiviral activity, but several investigators have reported an inhibitory effect of IFN on hematopoietic progenitor cell proliferation [15, 16]. Other negative regulators of hematopoiesis include inhibin [17], prostaglandin E [18], the Toronto negative regulatory protein [19] and AcSDKP (seraspenide) [20].

Since 1971, Paukovits et al. have published several reports on the purification and chemical properties of a low molecular weight peptide that inhibited myelopoiesis [21–23]. This factor was extracted from mature granulocytes and from bone marrow cell suspensions. In 1982, Paukovits and Lærum published the chemical structure of the factor, pyroGluGluAspCysLys (pEEDCK) [24]. Both the natural factor and the synthetic peptide (HP5b) had inhibitory effects on myelopoietic progenitor cells and granulocyte macrophage colony-forming cells (GM-CFC) in vitro [25]. Later, it was shown that HP5b also inhibited hematopoiesis in vivo by reducing the blood cell counts and the progenitor cell levels in bone marrow [26]. However, the formation of a disulfide bridge between two monomeric HP5b peptides led to the formation of a stimulator of both human and murine myelopoiesis [27]. To circumvent dimerization, the thiol (-SH) group of HP5b was replaced with an isosteric methyl (-CH3) group. This modification provided a simple and chemically stable molecule, SK&F108636 (pGlu-Glu-Asp-Abu-Lys) [28].

In the present study, we describe the effects of SK&F108636 on HSC. Due to the low frequency of HSC in the bone marrow, studies on immature HSC populations need to be performed on enriched cells. Major advances in the purification and enrichment of pluripotential HSC have been made recently. Lin–Sca1+ cells in murine bone marrow have been demonstrated to be highly enriched in primitive HSC since it has been shown that these cells can efficiently long-term reconstitute all cell lineages of the blood and proliferate in vitro in response to defined cytokine combinations [29–32].

We show here that SK&F108636 potently inhibits HSC proliferation/differentiation in vivo and in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Specific pathogen-free 8 to 12-week-old female C57BL/6J mice (Bomholt Gaard; Denmark) were used. The animals were kept five per cage in rooms with a 12 h light/darkness rhythm (6:00 am to 6:00 pm) with controlled temperature (22° C) and humidity (50%). They had free access to commercial rodent chow (EWOS R34, B&K Universal; Oslo, Norway) and acidified water (pH 3). The animals were allowed one week to acclimatize to the environment prior to experimentation.

Reagents
The peptide was synthesized in manual shaker vessels. Standard solid-phase procedure using Boc protection scheme was used. All couplings were carried out with a three-fold excess of protected amino acid derivative, three equivalents of N,N'-dicyclohexyl carbodiimide (DCC) and 1-hydroxybenzotriazole hydrate (HOBt) in dimethylformamide (DMF)/CH2CI2 (1:1). Deprotection and cleavage from the resin were performed using anhydrous HF. The crude peptide was extracted in 0.1% aqueous trifluoroacetic acid (TFA). The crude product was purified on a preparative C18 reverse phase HPLC (Vydac® column, 2.2 x 25 cm, 5µ particle size). A linear gradient of 0.1% aqueous TFA with increasing concentration of acetonitrile (containing 0.1% TFA) at a flow rate of 5 mL/min was used for elution. The fractions containing the product (monitored at 210 nm) were pooled and lyophilized. The purified peptide was homogeneous as assessed by both isocratic and gradient C18 reverse phase HPLC analysis (Vydac® column, 0.46 x 25 cm, 5µ particle size). The structure was confirmed by both amino acid analysis (Dionex Auto Ion 100 analyzer and a Nelson chromatographic data analysis program) and mass spectroscopy (FABMS: VG Zab high-resolution mass spectrometer with fast atom bombardment technique).

Monoclonal antibodies specific for CD2 (clone RM2-5), CD4 (clone RM4-4), CD8 (clone 53-6.7), Mac-1 (clone M1/70), B220 (clone RA3-6B2), GR-1 (clone RB6-8C5) (all from Pharmingen; San Diego, CA) and TER-119 (kindly provided by Dr. Kina, Kyoto University; Kyoto, Japan) were included at predetermined optimal concentrations in a Lin– cocktail. Anti Ly6A/E (fluorescein isothiocyanate [FITC] conjugated) and IgG2a isotype control (FITC conjugated) were purchased from Pharmingen. Secondary anti-rat IgG (phycoerythrin [PE] conjugated) was purchased from Southern Biotechnology Associates, Inc.; Birmingham, AL.

Recombinant murine interleukin 1ß (IL-1ß), IL-3 and GM-CSF were purchased from Peprotech EC, Ltd.; London, UK. Recombinant murine stem cell factor (SCF) was purchased from Genzyme Corp.; Cambridge, MA. Recombinant human erythropoietin (Epo) was purchased from Boehringer-Mannheim; Mannheim, Germany. As a source for M-CSF, conditioned medium from the murine fibroblastic cell line L929 was used [33].

Enrichment of Bone Marrow Lin–Sca1+ Cells
Bone marrow Lin–Sca1+ cells were isolated by the method described previously by Spangrude and Scollay [30]. Briefly, Lin– cells were obtained by removing Mac-1, GR-1, B220, CD2, CD4, CD8 and Ter119 positive cells with magnetic beads coated with sheep anti-rat IgG (Dynal; Olso, Norway). The purity of the Lin– cells was checked by staining with anti-rat IgG-PE and analyzed in a flow cytometer. Optimal concentrations of Ly6A/E-FITC or an equal amount of isotype matched control were added and incubated for 30-45 min on ice. Cells positive for Ly6A/E were sorted in a Coulter Epics Elite Cell Sorter (Coulter Electronics; Hialeah, FL) equipped with a 488 nm tuned argon ion laser set to give an effect of 15 mW with a rate of 1500-2000 cells/second. The final recovery of Lin–Sca1+ cells was 0.05-0.1% of the unfractionated bone marrow. Routinely, the purity of the Sca1+ fraction was >95% as assessed by re-analysis.

Irradiation and Transplantation
Recipient animals were exposed to 2 x 445 Rad (x-ray source, Siemens Stabilipan, dose rate of 100 rad/min) given 3.5 h apart. Irradiated recipients were transplanted i.v. with Lin–Sca1+ cells within 6 h after the first exposure. The primary recipients received 2000 sorted cells only without addition of any filler cells. Groups of mice were injected i.p. with daily doses of SK&F108636 or dilution buffer, phosphate-buffered saline ([PBS], Biowhittaker; Walkersville, MD), for 11 days starting the day after transplantation (day 1). On day 12, the animals were sacrificed by cervical dislocation. Spleens were removed and fixed in Bouins solution before macroscopic colonies, colony forming units-spleen (CFU-S) were counted. Femurs were flushed with 1.5 ml of McCoys 5A medium (GIBCO BRL Life Technologies; Paisley, Scotland) with 20% fetal bovine serum ([FBS], Hyclone Sterile Systems, lot # 11112080; Logan, UT). Bone marrow cells were seeded in GM-CFC and high prolifertive potential (HPP)-CFC agar colony formation assays as described below. The number of cells that had developed into CFU-S at day 12 (preCFU-S) was determined by transplanting 5 x 105 bone marrow cells from the primary transplanted animals into secondary lethally-irradiated syngeneic recipients. After another 12 days, the secondary transplanted animals were sacrificed and their spleens were removed. After being fixed in Bouins solution, macroscopic colonies were counted.

HPP-CFC Assay
HPP-CFC colony formation was obtained by seeding 400 Lin–Sca1+ cells in {alpha}-minimal essential medium ([{alpha}-MEM] GIBCO; Grand Island, NY) supplemented with 200 mM L-Gln, Penicillin/Streptomycin (BioWhittaker; Walkersville, MD), 20% FBS and either stimulated with IL-1 (10 ng/ml) + IL-3 (10 ng/ml) + SCF (40 ng/ml) or IL-1 (10 ng/ml) + IL-3 (10 ng/ml) + M-CSF (as 10% L929 conditioned medium). Growth factors were seeded into a 1 ml 0.6% agar (Difco; Detroit, MI) in culture medium ({alpha}-MEM/20% FBS) bottom layer while cells were plated on top in 0.3% agar in culture medium. Colonies >0.5 mm in diameter were scored after 14 days at 37°C in a humidified atmosphere containing 10% CO2 and 5% O2.

GM-CFC Assay
Briefly, cells were seeded in {alpha}-MEM supplemented with 200 mM L-Gln 20% FBS and 0.3% bacto-agar. GM-CFC were stimulated with a predetermined optimal dose of recombinant murine GM-CSF and IL-3 (20 ng/ml of each). Cultures were grown at 37°C for 7 days in a humidified atmosphere containing 7.5% CO2. Colonies containing more than 50 cells were scored using an inverted microscope.

CFC-Mix Assay
Mix-CFC were determined by seeding 104 Lin– in {alpha}-MEM supplemented with 200 mM L-Gln 20% FBS and 0.3% agar. Mix-CFC colony formation was stimulated by SCF (40 ng/ml), IL-3 (20 ng/ml) and Epo (1 U/ml). The cultures were grown at 37°C for 10 days in a humidified atmosphere containing 7.5% CO2. Colonies containing cells of granulocyte, erythroid and macrophage lineages were determined as Mix-CFC.

Single Cell Assay
Lin–Sca1+ cells were isolated as described above and deposited in each well of 60-well Terasaki plates (Nunc; Kamstrup, Denmark) with an Autoclone device attached to the Coulter Epics Elite cell sorter. The Autoclone was calibrated with fluorescent beads (Immunocheck from Coulter Electronics). Routinely, >99% of the wells contained one bead only. Cells were incubated for 10-12 days in 20 µl/well {alpha}-MEM supplemented with 200 mM L-Gln, Penicillin/Streptomycin, 20% FBS and growth factors (IL-1 [20 ng/ml], IL-3 [20 ng/ml] and SCF [80 ng/ml]). Wells containing >10 cells on day 10-12 were scored as positive. Proliferation was divided into four groups depending upon the fraction of the well that was covered with cells.

Determination of GM-CFC Proliferation Rate
The fraction of GM-CFC in S-phase after in vitro incubation or after injection into normal untreated animals was determined by the 3H-Thymidine suicide assay as previously described [34,35]. Briefly, Lin– cells or freshly isolated bone marrow cells were resuspended in culture medium at a concentration of 2 x 106 cells/ml and incubated with 80µCi/ml of 3H-Thymidine (Amersham Life Science; Buckinghamshire, UK) or equal amounts of cold thymidine (Sigma; St. Louis, MO) as control for 30 min at 37°C. Incubation was terminated by placing the cells on ice and adding excess cold thymidine. The cells were washed with culture medium containing 100 µg/ml cold thymidine and seeded into GM-CFC assays as described above. The fraction of cells in S-phase was determined by the calculation:



   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of SK&F108636 on HPP-CFC, Mix-CFC and GM-CFC Colony Formation
The Lin–Sca1+ cells are highly enriched in HPP-CFC and approximately 20% of these cells form HPP-CFC colonies under optimal growth conditions and multiple cytokine stimulation (data not shown). When 400 Lin–Sca1+ cells were plated and stimulated with either SCF + IL-1 + IL-3 or M-CSF + IL-1 + IL-3, 62 ± 6 and 50 ± 5 colonies were obtained, respectively. The colonies formed with SCF + IL-1 + IL-3 contained dense centers with a large number of satellite cells indicating multilineage differentiation, while the colonies obtained with M-CSF + IL-1 + IL-3 were macrophage-like colonies without dense centers (not shown). Thus, the two factor combinations seemed to stimulate colony formation of different subpopulations of the Lin–Sca1+ cells. In order to evaluate the effects of SK&F108636 on HPP-CFC, the peptide was added together with the growth factors in the bottom layer agar. Interestingly, SK&F108636 significantly inhibited SCF + IL-1 + IL-3-induced colony formation but did not inhibit M-CSF + IL-1 + IL-3 -induced colony formation (Fig. 1Go). The inhibition was dose-dependent with maximum inhibition at 10 and 100 ng/ml of SK&F108636. The effect of SK&F108636 was also studied on the committed stem cells, GM-CFC and Mix-CFC. GM-CFC colony formation was induced by either GM-CSF, IL-3, M-CSF (L929 CM) or IL-3 + GM-CSF. SK&F108636 did not inhibit GM-CFC colony formation in either factor combinations. Results from IL-3 + GM-CSF-stimulated cultures are shown in Table 1Go. Mix-CFC were stimulated by either IL-3 + Epo or SCF + IL-3 + Epo, but SK&F108636 did not affect multilineage colony formation with any of the factor combinations tried. These results indicate that SK&F108636 inhibits immature cell colony formation, but does not have any effect on committed stem cell colony formation.



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  		Fig. 1. Inhibition of high proliferative potential - colony forming cell (HPP-CFC) colony formation by SK&F108636. HPP-CFC colony formation was obtained by seeding 400 Lin–Sca1+ cells in {alpha}-MEM supplemented with 200 mM L-Gln, Penicillin/Streptomycin, 20% fetal bovine serum and either stimulated with predetermined optimal concentrations of interleukin 1 (IL-1) (10 ng/ml) + IL-3 (10 ng/ml) + stem cell factor (40 ng/ml) (A) or IL-1 (10 ng/ml) + IL-3 (10 ng/ml) + M-CSF (as 10% L929 conditioned medium) (B). *Significantly different from phosphate-buffered saline control, p < 0.01 by Student's t-test. The graphs represent the mean ± SD of at least 10 experiments.

 

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  		Table 1. Effect of SK&F108636 on granulocyte-macrophage-colony forming cells (GM-CFC) and Mix-CFC colony formation
 
Effects of SK&F108636 on Single Lin–Sca1+ Cells
To see whether SK&F108636 could have direct effects on stem cell proliferation, single Lin–Sca1+ cells were plated in the presence of SCF + IL-1 + IL-3 and various concentrations of SK&F108636 or PBS. In this assay, as much as 40% of the cells can proliferate at various degrees under optimal conditions (not shown). SCF + IL-1 + IL-3 stimulated the proliferation of 63 ± 15 cells per 300 Lin–Sca1+ cells. Addition of 100 ng/ml or higher of SK&F108636 inhibited proliferation of 30-50% of the cells (Fig. 2Go). The most pronounced effects were seen on the largest colonies, that is wells containing cells covering >100% of the well. These results show that SK&F108636 directly inhibits early stem cell proliferation.



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  		Fig. 2. The effect of SK&F108636 on the proliferation of single Lin–Sca1+ cells. Single Lin–Sca1+ cells were plated in 20 µl culture medium with stem cell factor + interleukin 1 (IL-1) + IL-3 at predetermined optimal concentrations. Wells containing >10 cells after 10-12 days incubation were scored as positive. *Significantly different from phosphate-buffered saline control, p < 0.01, Student's t-test. The graphs represent the mean ± SD of three experiments, each with at least 300 wells.

 
Effects of SK&F108636 on Progenitor Cell Cycle Rate
Freshly isolated bone marrow cells or enriched Lin– cells were incubated with SK&F108636 for 2 h or overnight (ON) before performing 3H-Thymidine suicide assays. Approximately 40% of the GM-CFC population was in S-phase at time 0. There were no differences in cell cycle rate between the control and SK&F108636-treated cells immediately after adding SK&F108636. SK&F108636 significantly reduced the fraction of cycling GM-CFC after 2 h and ON incubation (Fig. 3Go). After 48 h of incubation with SK&F108636, the cycling rate was similar to that of the control group (not shown) indicating a reversible effect of SK&F108636 on stem cell proliferation. The fraction of cycling GM-CFC did not change significantly in the control group during the period studied. Reductions in the fraction of cycling GM-CFC were also found after treating normal mice with single daily injections of SK&F108636 for four days (Fig. 4Go). Mice treated with 0.1 or 1 ng/kg SK&F108636 had significantly fewer cells in S-phase compared to mice treated with diluent only. However, administration of SK&F108636 to normal mice did not reduce the number of GM-CFC in the bone marrow (data not shown). These results show that SK&F108636 inhibits committed stem cell cycling rate.



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  		Fig. 3. The fraction of granulocyte macrophage colony-forming cells (GM-CFC) in S-phase after incubation for 0 h, 2 h and 18-24 h (ON) with 1000 ng/ml of SK&F108636 (filled bars) or phosphate-buffered saline (open bars). The fraction of GM-CFC in S-phase was determined by the 3H-Thymidine suicide assay. Mean ± SD of three experiments is shown. * p < 0.001 as determined by Student's t-test.

 


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  		Fig. 4. Effect of SK&F108636 on marrow granulocyte macrophage colony-forming cell (GM-CFC) cycling rate. C57BL/6J mice were given daily i.p. injections of SK&F108636 or dilution buffer for four days. Two h after the last injection, marrow GM-CFC cycling rate was determined by the 3H-Thymidine suicide method. Mean ± SD from three separate experiments of at least three mice per group. *Significantly different from phosphate-buffered saline (PBS) control, p <0.01 using Student's t-test.

 
Effects of SK&F108636 on Early Hematopoiesis in a Murine Bone Marrow Reconstitution Model with Lin–Sca1+ Cells
In agreement with previous observations [29,30] we found that as few as 100 Lin–Sca1+ cells can rescue 50% of lethally irradiated syngeneic mice (data not shown). In contrast, 3 – 4 x 104 unfractionated bone marrow cells are required to give the same degree of protection. The reconstitution of committed and primitive stem cells in lethally irradiated syngeneic animals transplanted with 2000 Lin–Sca1+ cells was assayed on day 12 after transplantation. A linear dose-response relationship between the number of enriched stem cells transplanted and the number of CFU-S, preCFU-S and colony forming cells (HPP-CFC and GM-CFC) per femur was observed (data not shown). This indicates that the stem and progenitor cells analyzed on day 12 after transplantation were derived from the stem cell population transplanted on day 0. In mice treated with daily injection of SK&F108636 for 11 days after transplantation there was a more than 50% reduction in the number of cells developing into the CFU-S compartment, HPP-CFC and GM-CFC (Table 2Go). However, there were no effects on the development of CFU-S colonies in the primary transplanted animals (Table 2Go) or on the cellularity in femur or peripheral blood (data not shown). These results indicate that SK&F108636 is able to inhibit hematopoietic stem cell proliferation/differentiation.


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  		Table 2. Effect of SK&F108636 on various progenitor and stem cell populations in lethally-irradiated C57BL/6J mice reconstituted with 2000 Lin–Sca1+ cells
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sustained production of mature blood cells throughout adult life requires continued activity of HSC. These cells are controlled by an unknown number of both stimulatory and inhibitory regulators [36]. There are various defined populations of HSC which broadly can be divided into primitive and committed HSC. The committed HSC, such as GM-CFC and Mix-CFC, are normally rapidly proliferating making these cells susceptible to cancer treatment with chemotherapeutic agents or radiation [37]. Shortly after cytotoxic drug treatment, cytopenia occurs and the normally resting primitive cells will respond to a demand situation and start to proliferate [38]. A great deal of research has been invested in trying to prevent or reduce the severity of chemotherapy-induced cytopenia by using stimulators of hematopoietic progenitor cells, such as G-CSF [39,40]. The use of potential inhibitors of stem cell proliferation in protecting stem cells from cycle-specific cancer therapy represents an interesting alternative.

In the present study, we show that a novel hemoregulatory peptide, SK&F108636, inhibits GM-CFC cycling rate both in vivo and in vitro. In vitro, the fraction of cells in cycle was reduced by approximately 70%. This is in agreement with Pelus et al. who in addition have demonstrated that SK&F108636 can reduce the cycling rate of human progenitor cells such as burst-forming units-erythroid and granulocyte/erythroid/macrophage/megakaryocyte-CFC [28]. In vivo, repetitive injections of SK&F108636 reduced the cycling rate of GM-CFC approximately 40%, which confirms previous findings of SK&F108636 and HP5b monomer [26,28]. Several of the known SCIs have shown the ability to inhibit stem cell cycling or entry into cycle induced by cytotoxic drugs in vivo [20,41,42]. Our results indicate a potential role for SK&F108636 in preventing committed progenitors from cycling and thus protecting these important cells during cancer therapy.

There was no effect on GM-CFC colony formation or colony size, although the proliferation rate was reduced for 24-48 h. This is in contrast to observations made with HP5b monomer [24,25,43]. However, these studies were not performed with the exact same protocols. The fact that no such effect was observed in our study could be explained by transient inhibition of proliferation or that the addition of growth factors in the agar dishes overrides the inhibitory effect of SK&F108636. Alternatively, SK&F108636 and HP5b might act in different manners.

SK&F108636 did inhibit colony formation of more immature cells both in vitro and in vivo. A significant reduction in the number of HPP-CFC was observed in vitro, but only when the cells were stimulated with a combination of SCF + IL-1 + IL-3. The receptor for SCF (c-kit) is expressed on primitive stem cells and stem cell populations proliferating in the presence of SCF in addition to other growth factors are considered as more immature than those proliferating without SCF [44]. This effect was dose-dependent showing a maximum inhibition with 10 and 100 ng/ml. The U-shaped dose-response curve may be due to indirect effects at higher concentrations of SK&F108636. Alternatively, SK&F108636 may aggregate at higher concentrations as has been shown for MIP-1{alpha} [45]. However, the results from the single cell experiments indicate that SK&F108636 directly inhibits stem cell proliferation. In addition, Lu et al. have demonstrated that HP5b acts directly on enriched progenitor cell populations [45]. Altogether, these data indicate that SK&F108636 exerts both direct as well as indirect effects on HSC.

In lethally irradiated syngeneic recipients of 2000 Lin–Sca1+ cells, SK&F108636 inhibited the proliferation/differentiation of transplanted stem cells into more mature stem cell compartments. On day 12 after transplantation, the cells developing into CFU-S, HPP-CFC and GM-CFC were inhibited, indicating an inhibitory effect on very primitive stem cells. There were no effects on the development of primary CFU-S colonies or bone marrow and peripheral blood cellularity, indicating that the effect of SK&F108636 was specific for the more primitive stem cell compartments. These data suggest that SK&F108636 inhibits, directly and/or indirectly, both committed and early stem cell proliferation/differentiation.

The combination of cytokines and growth factors present determines which type of CFC is promoted. This has a major influence on the response of the progenitor cells to TGF-ß, TNF-{alpha} and MIP-1{alpha}. TGF-ß inhibits the proliferation/differentiation of early HSC and IL-3-induced progenitor cell proliferation [46,47], but enhances progenitor cell proliferation in combination with GM-CSF or G-CSF [47,48]. Similarly, TNF-{alpha} inhibits the early stem cells and G-CSF-induced progenitor cell proliferation [12]. In contrast, TNF-{alpha} enhances IL-3 and GM-CSF-induced progenitor cell proliferation, and SCF and IL-7-induced macrophage proliferation from enriched stem cell populations [10,11]. MIP-1{alpha} inhibits primitive stem cells but enhances colony formation in combination with M-CSF and GM-CSF [49,50], while no effect is seen on GM-CFC colony formation in combination with G-CSF [49,51]. Thus, these cytokines exert bidirectional effects on HSC. Our results indicate that SK&F108636 seems to specifically act as an inhibitor of stem cell proliferation and does not exert bifunctional effects on stem cells such as TGF-ß, TNF-{alpha} and MIP-1{alpha}. In addition, the hemoregulatory peptide is distinguished from the cytokines by its size and synthetic nature, and as such represents a new class of regulatory factors.

In the present studies, SK&F108636 has for the first time been shown to be a specific inhibitor of primitive stem cell proliferation/differentiation. Inhibition of primitive stem cell proliferation can theoretically prevent chemotherapy-induced proliferation of stem cells and thus, protect these crucial cells from being killed by chemotherapeutic agents. Myelotoxic side effects of cancer therapy might be ameliorated if proliferating progenitors and stem cells could be protected during the action of cytotoxic agents. The marrow would have the potential to repopulate peripheral blood leukocytes faster, leading to reduced neutropenia and enhanced recovery. This would also lead to less pressure on the earlier stem cell populations and thus inhibit the irreversible exhaustion of repopulating stem cells [34]. Our data indicate that SK&F108636 has the potential of being used as a protective agent during cancer chemotherapy.


   Acknowledgments
 
The authors wish to acknowledge Anne Petersen, Stein Waagene and Lisa Weltzin-Eide for excellent technical assistance, Karin Haugen for taking care of the animals and Drs. Steinar Bergseth and Dagfinn Løvhaug for critically reviewing the manuscript.


   Footnotes
 
Provisionally accepted January 16, 1995.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 14, 1994; accepted for publication September 16, 1995.




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