Identification of a regulatory peptide distinct from normal granulocyte-derived hemoregulatory peptide produced by human promyelocytic HL-60 leukemia cells after differentiation induction with retinoic acid.

It has been found that leukemia cells can be induced by various agents [e.g., by retinoic acid (RA)] to mature to a nonproliferative end stage. It has also been found that normal mature granulocytes produce a chalone-like "hemoregulatory peptide (HP)" which seems to be involved in the inhibitory proliferation control of myelopoietic cells. In view of the intended use of maturation induction treatment as an alternative to current antileukemic therapy it appeared to be of interest to know if granulocytes, obtained by RA treatment of the promyelocytic leukemia cell line HL-60, would produce normal HP or if their transformed phenotype would cause production of deviant regulatory peptide(s). It was found that conditioned media from RA-treated HL-60 cells inhibited myeloid proliferation but strongly stimulated the growth of erythroid and lymphoid cells. A low molecular weight thiol-containing peptide was isolated which inhibited colony formation by normal granulocyte-macrophage committed stem cells but unlike HP had no effect on (untreated) HL-60 cells themselves. It was also shown that the HL-60 RA peptide is chemically different from HP in terms of molecular size, electrophoretic mobility, composition, and NH2-terminal sequence, which was determined as glutamine-aspartic acid-proline. It is concluded that differentiated HL-60 cells produce hemoregulatory factor(s) with properties different from those of normal HP. The implication of a possible abnormal regulatory behavior of induced leukemic populations is discussed with respect to leukemia therapy by differentiation induction.

Prevention of hematotoxic side effects of cytostatic drugs in mice by a synthetic hemoregulatory peptide.

The application of certain cytostatic drugs causes the recruitment of pluripotent hemopoietic stem cells (CFU-S) into active proliferation. Further application of the drug(s) may then lead to severe and long lasting disturbances of hemopoiesis. We investigated if the hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys (HP5b) could be used to inhibit stem cell recruitment and consequently to protect mice against the toxicity of repeated high doses of 1-beta-D-arabinofuranosylcytosine (ara-C). CFU-S recruitment (induced by injecting a single dose of 900 mg/kg ara-C) was prevented by either treating the bone marrow of these mice in vitro with 1 x 10(-7) M/liter HP5b, or by injecting 0.6 microgram HP5b (10(-9) mol, 30 micrograms/kg) at -2, +2, and +6 h relative to the ara-C injection. Multiple high dose ara-C applications (4 x 900 mg/kg at 0, 7, 24, and 30 h) lead to proliferative activation of CFU-S and resulted in the death of 90% of the mice within 7-9 days. Reconstitution of the hemopoietic system by a bone marrow transplant given after ara-C application decreased the mortality to about 45%, indicating the nonhematological component of ara-C toxicity. A single injection of HP5b (30 micrograms/kg at 26 h, when few CFU-S were found in S phase) decreased the mortality to 59%, not significantly different from the transplanted group. Inactive peptides given instead of HP5b had no protective effect. HP5b did not change the ara-C sensitivity of transformed cell lines (HL-60, Raji, Friend), even not in such cases (myeloid cell lines) where it had a direct inhibitory effect on the cells (e.g., HL-60). These results suggest that HP5b may be used as a myeloprotector in cancer chemotherapy by keeping hemopoietic stem cells out of cycle during the most hazardous treatment phase. Its lack of species specificity, its low toxicity, its high selectivity for hemopoiesis, the small size, as well as the availability through standard synthetic techniques may be of advantage for its clinical use.

Haemoprotection against cytostatic drugs by stem cell inhibition.

Cytostatic drug-induced haematopoietic damage is a major problem in tumour chemotherapy, due to the intensive proliferation of many bone marrow constituents and to the drug-induced recruitment of immature pluripotent haematopoietic cells (spleen colony-forming units, CFU-S). Marie-Hélène Moser and Walter Paukovits discuss how it should be possible to minimize such proliferation-associated damage by inhibiting CFU-S during the most dangerous treatment phases, with factors such as transforming growth factor beta, tumour necrosis factor alpha, macrophage inflammatory protein 1 alpha, and the CFU-S-inhibitory peptides N-acetyl-Ser-Asp-Lys-Pro and pyroGlu-Glu-Asp-Cys-Lys (pEEDCK). Clinically relevant data are available for pEEDCK, showing that application of this peptide leads to a delayed, shorter, and less severe neutropenia, combination of pEEDCK with a stimulator avoids neutropenia, and stem cell preservation with pEEDCK improves long-term reconstitution of the haematopoietic system. Stem cell inhibition by synthetic peptides like pEEDCK may provide a useful strategy for bone marrow protection.

Stem cell stimulation in vitro by the dekapeptide (pEEDCK)2: a single-factor alternative for multifactor cocktails.

Insertion of foreign genes into cellular DNA requires (at least one round of) DNA replication. Since hemopoietic stem cells do not divide rapidly, numerous semi-empirically designed multifactor cocktails have been used to stimulate them. In an attempt to find an alternative to this approach we have investigated the effects of the stem cell stimulatory peptide (pGlu-Glu-Asp-Glys-Lys)2, (pEEDCK)2, on progenitor output in murine long-term bone marrow cultures (LTBMC). (pEEDCK)2 may act by inducing growth factor production in stromal cells. Addition of (pEEDCK)2 to LTBMCs resulted in a three-fold increase in CFU-GM production. For showing an effect of (pEEDCK)2 on primitive hemopoietic cells (long-term-culture initiating cells, (LTC-IC)) LTBMCs were depleted of rapidly dividing progenitors by 5-Fluoro-Uracil (5-FU). LTC-IC survive and repopulate the culture with new CFU-GM. (pEEDCK)2 greatly enhanced this process (eight-fold in the second week after 5-FU). Enhanced progenitor production was observed for several weeks even after discontinuation of (pEEDCK)2 additions to the cultures (100-fold, five weeks after 5-FU, three weeks after end of peptide additions). This increase in progenitor production resulted in increased numbers of total nucleated cells. Our results suggest that (pEEDCK)2 may be a useful alternative for multifactor cocktails when proliferation of primitive stem-cell-like cells is required, as in gene therapy and transplantation. Our experiments also indicate that the redox equilibrium between (stem cell inhibitory) monomeric pEEDCK and (stem cell stimulatory) dimeric (pEEDCK)2, which are both endogenous constituents of LTBMCs may play a role in physiological stem cell regulation.

Inhibitory effects of a synthetic pentapeptide on hemopoietic stem cells in vitro and in vivo.

A synthetic analogue of a pentapeptide associated with mature granulocytes has been investigated for biological effects on stem cell activity in vitro and in vivo. When tested on bone marrow cells from female C3H mice, a short incubation in vitro in doses from 10(-9) to 10(-5) M inhibited myelopoietic colony formation (CFU-C). A maximum of 80% reduction of colony yield was found at 10(-7) M. An oxidized form of the molecule had a stimulatory effect on colony formation, but the inhibitory effect was restored by treatment with a reducing substance (mercaptoethanol). The peptide was nontoxic during continuous exposure of liquid cultures of bone marrow cells for up to 24 h at a dose range of 10(-11) to 10(-4) M. When injected into mice, a dose-dependent inhibitory effect on CFU-C was seen. Maximal effect was obtained by continuous infusion of 1.4 micrograms/h for six days, where only one-fifth of the normal CFU-C number per femur could be retrieved. Prolonged exposure to the same dose level resulted in less reduction. A significant, but less pronounced reduction of spleen colony formation (CFU-S) per femur was seen in the same dose range. Inhibition of both CFU-S and CFU-C was in all cases reversible and mostly accompanied by an overshoot of up to 75% above normal level. In addition, primarily noninhibitory doses led to a secondary increase in the numbers of CFU-S. The total cell number per femur was moderately but significantly reduced, and a prolonged reduction of granulocyte numbers in peripheral blood resulted. No direct toxic effects were seen in vivo on 271 mice given up to 9 mg of the peptide. The results indicate that the peptide may have a regulatory function for stem cell activity in vivo.

Activated granulocytes oxidize the endogenous stem cell inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK) to the stimulatory dimer: a redox-mediated mechanism for demand-induced hematopoietic regulation.

We have previously shown that the pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which is associated with mature leukocytes, maintains pluripotent hematopoietic stem cells (colony-forming units-spleen [CFU-S]) in a quiescent state under physiological conditions. It is also known that its oxidation product, the disulfide-bonded homodimer (pEEDCK)2, is a growth factor for CFU-S in vivo. In this paper we report on the combined actions of the monomer and dimer in inducing rapid changes in stem cell proliferation in vivo. A single injection of 20 microg/kg synthetic dimer into mice stimulated CFU-S proliferation (60% in S-phase after 9-11 hours) and population expansion. Stimulated CFU-S traversed one cell cycle, with an estimated S-phase time of 5.5 hours, and then become quiescent again. Proliferation of CFU-S in response to dimer showed no sensitivity to the inhibitory effects of monomeric pEEDCK, whereas CFU-S proliferation did display sensitivity to inhibition after injection of cytosine arabinoside or doxorubicin. Products of mature granulocytes undergoing an oxidative burst reaction rapidly oxidized monomeric pEEDCK to the dimer. The suppressive effect of endogenous pEEDCK monomer on stem cell proliferation was thus converted within minutes to a stimulatory signal (dimer). Because many in vivo situations (e.g., infection) requiring increased hematopoiesis involve granulocyte and macrophage activation, the formation of dimer from endogenous pEEDCK monomer may provide an almost instantaneous demand-induced emergency signal for increasing stem cell proliferation and blood cell production.

The dimer of hemoregulatory peptide (HP5B) stimulates mouse and human myelopoiesis in vitro.

A synthetic analogue of a pentapeptide associated with mature granulocytes has been described earlier and shown to suppress myelopoietic colony formation in vitro in concentrations from 10(-13) to 10(-6) M. By oxidation of the peptide, a dimer will rapidly occur by formation of disulfide bridges between cysteine residues. We here demonstrate that this dimer has the opposite effects of the monomer. For both mouse and human granulocyte-macrophage colony-forming units (CFU-GM), a dose-dependent enhancement of colony formation was observed in the dose range 10(-16) to 10(-5) M, where a saturation level was reached above 10(-8) M. At low doses of colony-stimulating activity (CSA) and in the linear stimulating phase, an up to ten times increase of colony formation was seen, whereas at higher doses the effect was less pronounced. Also at the plateau level of CSA stimulation an increased colony yield was seen. All types of colonies were stimulated. The dimer itself had no colony-stimulating factor activity and was not toxic to bone marrow cells in suspension cultures up to 24 h. Upon reduction of the dimer by use of sulfhydryl compounds, inhibitory effects on CFU-GM were restored. The peptide had no effect on the phagocytic process in human granulocytes, including attachment and internalization of bacteria or Zymosan particles. The monomerdimer equilibrium of hemoregulatory peptide may constitute a new mechanism for proliferative regulation of myelopoietic cells.

The use of haemoregulatory peptides (pEEDCK monomer and dimer) for reduction of cytostatic drug induced haemopoietic damage.

We have previously shown that the stem cell inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK monomer) leads to a good tolerance of otherwise lethal multiple ara-C doses and an increased survival of ara-C + peptide treated mice. This effect was due to the prevention of drug-induced CFU-S proliferation, thus keeping stem cells in a quiescent state insensitive to ara-C. Here we show that the pEEDCK monomer also inhibits stem cell proliferation after clinically relevant (non-lethal) ara-C doses. This leads to a sustained (100%) stem cell number in the femoral bone marrow, which was greatly reduced without protective peptide treatment (27%). We have measured the kinetics of influx of CFU-S into the empty S-phase (after two consecutive ara-C injections). This influx reached peak levels of 60-70%; pEEDCK treatment reduced it to 25-30%. Due to its cysteine content the pEEDCK monomer is easily oxidized and forms a symmetric disulfide-bonded dimer (pEEDCK)2. This dimer is a potent stimulator of haemopoiesis. Various modes of protective peptide treatment (monomer and dimer) were investigated in conjunction with a standardized protocol of 2 x 300 mg/kg ara-C given 12 h apart. (a) ara-monomer-ara: The administration of pEEDCK-monomer 2 h before the second ara-C injection retarded the onset of neutropenia, shortened its duration and improved recovery after depression. The degree of short-term neutropenia was not changed. (b) ara-ara-HN2-dimer: Post chemotherapy infusion of the stimulatory (pEEDCK)2 dimer led to considerable increases of progenitor levels (6.8 CFU-GM/1000 bone marrow cells vs. 1.2 CFU-GM/1000 in normal mice) 2 days after cytostatic treatment when CFU-GM were not detectable in unprotected mice. This increase was followed by greatly elevated granulocyte counts (8000 PMN/mm3 vs. 750 PMN/mm3 in normal mice). In the dimer-treated mice, up to 75% of the peripheral leukocytes were mature PMN (normal, 10%). (c) ara-monomer-ara-dimer: ara-C and monomer treatment as above (a) followed by dimer infusion led to complete protection of haemopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression seen in unprotected animals. Our results show that the haemoregulatory peptide monomer and dimer can be used to improve the haematological status of mice treated with clinically relevant doses of cytostatic drugs (anti-metabolite and alkylating, alone and in combination). The pEEDCK monomer and dimer are equally active also on human haemopoietic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Peripheral blood leukocyte alterations in mice induced by a hemoregulatory pentapeptide (HP 5b).

The effects of different doses of a synthetic hemoregulatory pentapeptide, analoguous to an inhibitory factor associated with human granulocytes, on the numbers of peripheral blood leukocytes have been investigated in female C3H mice. Following single injections as well as continuous infusion for 6 days, the numbers of granulocytes in peripheral blood were reduced to about one half of the normal. Maximal effect with single injection was seen with 120 micrograms (10(-5) M), while 1.2 mg (10(-4) M) as one dose only had a slight, temporary effect on the granulocyte numbers in peripheral blood. Upon continuous infusion of 14 micrograms/h (less than 10(-5) M) for 19 days, a stimulation with doubling of the granulocyte numbers was seen instead. The substance also decreased the monocyte numbers in peripheral blood. With prolonged exposure, a relative monocytosis was seen instead. After both single injection and continuous infusion, the pentapeptide increased the lymphocyte numbers in peripheral blood. The thrombocyte numbers were not altered. The decreased granulocyte numbers seemed to be due to a dual mechanism, one reducing proliferation in the maturing compartment which gives a rapid cell reduction, and one mediated through inhibition of the committed stem cells for myelopoiesis, giving a long-lasting reduction in the granulocyte numbers. It is postulated that the pentapeptide has a complex regulatory effect in vivo.

[Specificity and mechanism of action of the granulocyte chalon and its role in the regulatory system of granulopoiesis]. / Spezifität und Wirkungsmechanismus des Granulozytenchalons und seine Rolle im Regulationssystem der Granulopese

An inhibitor of granulopoiesis is described which, on the basis of its cellular origin and its specificity of action, can be identified as the granulocytic chalone. A control system is discussed which is based on the interplay of leukocytosis inducing factors, colony stimulating factors and the granulocytic chalone. The granulocytic chalone inhibits all immature myeloid cells of the bone marrow by interacting with specific receptor sites on the cell membrane.

The regulation system of granulopoiesis.

Extracts containing the granulocytic chalone (GCh) have been prepared from bone marrow conditioned medium by ultrafiltration and column chromatography. This preparation selectively inhibits myeloid cells and does not affect other, e.g., lymphoid cells. The action on myelopoiesis seems to include all mitotically competent immature granulocytes as indicated also by an inhibitory effect on bone marrow colony formation in agar. Results obtained by this technique and by applying mild trypsinization of bone marrow cells prior to testing chalone responsiveness indicate that GCh acts via membrane receptors. Finally the apparent antagonistic role of colony stimulating factor and GCh as regulators of granulopoiesis is discussed and a model of the regulation system is described.

Modification of mouse hemopoietic cell proliferation in vivo by a hemoregulatory pentapeptide (HP 5b). Relation to circadian rhythms.

The effects on murine hemopoietic cell proliferation of hemoregulatory peptide 5b, a synthetic factor which inhibits granulopoiesis, have been studied in vivo by 3H-thymidine labeling, cell cycle analyses by flow cytometry and total cell counts per femur. Single injections of the peptide seemed to obliterate the normal circadian rhythm of bone marrow cell proliferation. By autoradiography, the main effect was an inhibition of myelopoietic cell proliferation, although erythropoiesis was also affected. This occurred in two ways: a) an immediate effect observed in the first 24 h after a single injection, and b) a delayed effect observed 6-12 days after a week of peptide infusion which could be a result of stem cell inhibition. A secondary accumulation of cells with G2 phase DNA content was seen regularly. Thus the hemoregulatory peptide seems to influence hemopoietic cell proliferation and has its main effect on myelopoiesis. However, inhibition is less marked than observed previously in stem cells.

Modulation of murine hemopoiesis in vivo by a synthetic hemoregulatory pentapeptide (HP 5b).

A synthetic pentapeptide analogous to an inhibitory factor associated with human granulocytes was tested in vivo on female C3H mice. The relative and absolute numbers of myelopoietic and erythropoietic cells in the bone marrow were measured following injections as well as the continuous infusion of the pentapeptide in dose ranges between 10(-8)M and 10(-4)M (0.12 micrograms to 1.2 mg/mouse). In low doses, the pentapeptide reduced the number of myelopoietic cells in the bone marrow, and this was accompanied by reduced numbers of granulocytes and monocytes and peripheral blood. Elevated doses also decreased erythropoiesis. In contrast, continuous infusion of 14 micrograms/h for 19 days seemed to make the myelopoietic cells refractory to further action. A regulatory function of the pentapeptide is proposed.

Structural investigations on a peptide regulating hemopoiesis in vitro and in vivo.

Clonal growth in agar of myeloid committed stem cells (CFU-c) is inhibited by a specifically acting "hemoregulatory peptide" which had been isolated from human leukocytes. The inhibitory peptide is composed of aspartic acid, glutamic acid, lysine, cysteine and possibly glycine. It was [3H]carboxymethylated with [3H]iodoacetic acid. From the electrophoretic behaviour of this derivative at pH 1.9 a single positive charge and a molecular mass of about 600-650 Da could be inferred. This positive charge is lost after acetylation and is identified as the epsilon-amino group in the side chain of a lysine residue since the N-terminal group is a pyroglutamic residue. Electrophoresis of the native peptide at pH 6.5 reveals the presence of three carboxyl groups, two of which were localized in the N-terminal part of the molecule, while cysteine and lysine were located in the C-terminal portion. Lysine was identified as the C-terminal residue by a double labeling technique applied to the Cys-[(3H]Cm)-containing fission peptide after partial hydrolysis. Uncertainties in the molecular mass determinations allow for the possible presence of a small residue without reactive side chain (glycine?) in the C-terminal portion of the molecule. The structure of the inhibitory peptide was thus determined as less than Glu-(Asp/Glu)-(Asp/Glu)-Cys-(Gly?)-Lys. This structure is supported by comparison with synthetic peptides. The analogue less than Glu-Glu-Asp-Cys-Lys has properties which are similar to those of the natural inhibitor.

Separation, identification and mechanism of action of the granulocytic chalone.

The preparation of highly purified granulocytic chalone from bone marrow conditioned medium is described. A sequence of gel-chromatographic steps on Sephadex G-25, G-15 and Biogel P-6 is applied. The end product has a sigmoid dose-response-curve with a plateau at 40-50% inhibition of thymidine incorporation. The inhibition is only observed with bone marrow cells as target cells whereas lymphoid cells are not affected. When bone marrow cells are treated for 1 min with 0.005% trypsin and subsequently used for assaying chalone activity in the presence of cycloheximide, no inhibition is found. These results may indicate that chalone specific receptor sites (of protein nature) are present on the surface of the target cells, which are destroyed by the trypsin treatment. Granulocytic chalone is soluble in aqueous solvents and in polar organic solvent mixtures. Its activity is destroyed by trypsin but only after prolonged treatment. The elution behaviour of chalone is discussed in detail. Chromatography on Sephadex G-75 and Ultrafiltration point to a molecular weight of several thousands, whereas chromatography on G-25 or G-15 or on Biogel P-6 would indicate a rather low molecular weight.