The hemoregulatory peptide pEEDCK may inhibit stem cell proliferation via hydropathic binding to antisense sequence motifs in interleukin-11 and other growth factors.

In undisturbed bone marrow, most hemopoietic stem cells are nonproliferating despite the presence of multiple growth factors. Endogenous inhibitory factors are responsible for maintenance of this quiescence. Previously we sequenced and synthesized the inhibitory pentapeptide pGlu-Glu-Asp-Cys-Lys (pEEDCK), which originally derives from granulocytes, and investigated the role of this peptide in stem cell quiescence. To provide some mechanistic insight, in the present work we studied the structural relationship of this peptide to specific growth-factor-derived sequence motifs. In the murine system in vivo as well as in long-term bone marrow, antiserum to pEEDCK produced a significant stimulation of formation of colony-forming units-granulocyte/macrophage. Binding of peptides to proteins often takes place at hydropathically complementary sites. Therefore, we searched for peptides corresponding to the complementary sequence to pEEDCK. We identified antisense sequences in the genes of various cytokines and cytokine receptors including interleukin-11. The corresponding peptide Val-Leu-Leu-Thre-Arg (VLLTR) and several other peptides hydropathically complementary to pEEDCK were synthesized. We found that pEEDCK binds specifically to these peptides as well as to complete interleukin-11. Dissociation constants were in the 10 microM range. The peptide hydropathically corresponding to pEEDCK (VLLTR) was found to stimulate colony-forming units-granulocyte/macrophage formation. Our data suggest that pEEDCK could exert a coordinating function in the hemopoietic cytokine network by binding to multiple regulatory proteins and modulating their activity.

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.

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.

Pre-CFU-S quiescence and stem cell exhaustion after cytostatic drug treatment: protective effects of the inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK).

Pre--CFU-S are characterized by their ability to generate spleen colony-forming cells (CFU-S) and by their ability to repopulate the hematopoietic system after damage. We have investigated their response to three consecutive injections of cytosine arabinoside (ara-C), given at t = 0, 12, and 20 hours. Nine hours after treatment, the number of CFU-S and pre--CFU-S was reduced to 10% or 30%, respectively. No pre--CFU-S were in S-phase at this time, indicating that the pre--CFU-S losses were not caused by direct drug killing. Up to 1 year after treatment, pre--CFU-S were still depleted to 10% of normal, indicating that their proliferative quiescence was permanent. We have previously shown that inhibition of CFU-S recruitment with pGlu-Glu-Asp-Cys-Lys (pEEDCK) makes them ara-C resistant and prevents their decimation. We now found that this also prevented the excessive drainage of the pre--CFU-S pool, suggesting that pre--CFU-S allocation into active hematopoiesis is triggered by the CFU-S deficit. pEEDCK may thus be applicable as a protector of the hematopoietic repopulation potential against cytostatic drug-induced aplasia. Postchemotherapeutic stimulator treatment with (pEEDCK)2-dimer did not ameliorate pre--CFU-S losses. Long-term culture-initiating cells (LTC-ICs) showed a similar pattern of irreversible reduction after cytostatic drug treatment, which could be prevented by pEEDCK. Our results suggest, that certain subclasses of hematopoietic stem cells (pre--CFU-S) are permanently quiescent and exhaustible and that the capacity for self-renewal is not a necessary property of all stem cell-like cells.

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.

Protection from arabinofuranosylcytosine and n-mustard-induced myelotoxicity using hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys monomer and dimer.

We have previously shown that the synthetic peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK monomer) inhibits the cytostatic drug-induced proliferation of hematopoietic stem cells CFU-S. Keeping CFU-S quiescent by pEEDCK treatment renders them insensitive to cycle-specific cytostatic drugs and leads to reduced toxicity. Here we show that pEEDCK application during repeated (twice) administration of clinically relevant (nonlethal) 1-beta-D-arabinofuranosylcytosine (Ara-C) doses reduced the percentage of CFU-S in S-phase from 60%-70% to 25%-30% and led to a sustained stem cell number in the bone marrow (BM), whereas unprotected mice had lost about 75% of their CFU-S population. Owing to its cysteine content, the pEEDCK monomer is easily oxidized. The resulting dimer (pEEDCK)2 is a potent stimulator of hematopoiesis. As we show, it can be used for postchemotherapy acceleration of hematologic recovery, similar to the use of recombinant hematopoietic growth factors. A single injection of 30 micrograms/kg pEEDCK monomer to mice 2 hours before the second Ara-C injection retarded onset of neutropenia (by 2 to 3 days) and improved recovery after depression. The quantitative degree of neutropenia was not changed. Postchemotherapy (Ara-C administered twice, followed by N-mustard) infusion of the stimulatory (pEEDCK)2 dimer (1.4 micrograms/kg/d) produced a 4.6-fold increase of progenitor levels (6.7 CFU-GM/1,000 BM cells v 1.45 CFU-GM/1,000 in normal mice) 2 days after the end of the cytostatic treatment when CFU-GM were not detectable in unprotected mice. This increase was followed after several days by strongly elevated granulocyte counts, which remained high for approximately 1 week. Up to 75% of the peripheral leukocytes were mature polymorphonuclear leukocytes (PMN) during this phase. Ara-C (twice) and monomer treatment as above followed by dimer infusion resulted in the complete protection of hematopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression noted in unprotected animals. The inhibitory monomer appears to keep the stem cell population numerically and qualitatively intact, thus providing optimum target cell conditions for the subsequent stimulator (dimer) treatment. Our results show that the hemoregulatory peptide monomer and dimer can be used for improving the hematologic status of mice treated with clinically relevant doses of cytostatic drugs (antimetabolite and alkylating, alone and in combination). Combining both peptides can prevent occurrence of neutropenia completely. Both peptides can be obtained easily by chemical synthesis and are also active on human cells. They are thus highly promising candidates for application as multilevel hemoprotectors in cancer chemotherapy.

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)

Hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys: a new synthetic derivative for avoiding dimerization and loss of inhibitory activity.

The hemoregulatory peptide (pGlu-Glu-Asp-Cys-Lys, pEEDCK) is a potent inhibitor of stem cell recruitment, which is a major source of hematological complications after cytostatic tumor therapy. By preventing recruitment, pEEDCK can keep hemopoietic stem cells in their normal nonproliferative state and in this way prevent damage by certain cell cycle-specific cytostatic drugs. pEEDCK could play a role as hemoprotector in tumor chemotherapy. As a thiol-containing peptide, pEEDCK is highly sensitive to oxidation, resulting in the formation of a dimer. Although monomeric pEEDCK is a strong inhibitor of colony-forming units-granulocyte/macrophage (CFU-GM) clonal growth, the dimer was previously found to enhance colony-stimulating factor-triggered CFU-GM colony formation. It seemed, thus, necessary to find methods that avoid undesired dimerization reactions. A solid phase strategy for pEEDCK synthesis is presented. The primary synthetic product, S-tert-butyl-sulfenyl-pEEDCK, was purified and stored with the thiol-protecting group remaining attached. Conversion to active monomeric pEEDCK was achieved by reductive treatment in situ before application and removal of tert-butyl-mercaptane in vacuo. The activation reagent (dithioerythritol) prevented reoxidation also in culture media, where unprotected peptide was oxidized rapidly (t1/2 less than 13 min). The purified synthetic peptide was found to be a potent inhibitor of CFU-GM colony formation (IC50 = 1.1 x 10(-12) M) in vitro. It was also found to inhibit colony formation of some leukemic cell lines (HL-60, RAJI) although at much higher concentrations (10(-8) to 10(-9) M). Friend leukemia cells were not inhibited in the dose range where CFU-GM were sensitive.

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.

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.

Deoxycytidine, insulin and epidermal growth factor overcome the effect of two natural inhibitors of the haemopoietic system.

The effect of 2'-deoxycytidine on a pluripotent haemopoietic stem cell proliferation inhibitor, as assayed in the mouse spleen colony technique and on the synthetic haemoregulatory pentapeptide (acting predominantly on myeloid determined cells) as assayed in the agar colony technique, was studied. 2'-deoxycytidine was able to overcome the effect of both inhibitors. The inhibition was also blocked by insulin or epidermal growth factor. Since these results are in agreement with those on other cells and other inhibitors, they may describe a more general phenomenon. It is suggested that the mode of action of the inhibitors involves, probably indirectly, interaction with the activity of ribonucleotide reductase.

A synthetic hemoregulatory peptide (HP5B) inhibits human myelopoietic colony formation (CFU-GM) but not leukocyte phagocytosis in vitro.

A synthetic analog of a hemoregulatory peptide associated with mature human granulocyte (HP5B) has been investigated for inhibitory effects on human myelopoietic stem cells in vitro. In addition, it has been tested for effects on phagocytosis by human granulocytes and monocytes by use of an automatic flow cytometric method. A dose-dependent inhibition of colony formation was found after preincubation of bone marrow cells for 1 h at 37 degrees C in the range 10(7) -10(-11) mol/l. Above or below these concentrations, no inhibitory effects were seen. The degree of inhibition varied from experiment to experiment, indicating variable responsiveness of the donor cells. Maximal effect was of magnitude 90% inhibition, and the optimal dose was 10(7) mol/l. The peptide had no effect on the kinetics of phagocytosis by measurements of the uptake of fluorescent Zymosan particles or Staphylococcus aureus. This may indicate a selective effect on the precursor cells, with no effect on the functional state of their progeny, the granulocytes and monocytes.

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.

Effects of a hemoregulatory peptide (HP5b) on erythroid and myelopoietic colony formation in vitro.

A hemoregulatory peptide (HP5b) associated with mature human granulocytes has been investigated for inhibitory effects on murine hemopoietic stem cells in vitro. Both highly purified peptide from natural sources and a synthetic analog peptide have been investigated in parallel. Strong inhibitory effects were found on myelopoietic colony formation in the dose range 10(-13)-10(-5) mol/l. On exceeding this dose, the inhibitory effect disappeared. Moderate to slight inhibitory effects on erythroid colony formation (BFU-E and CFU-E) from adult animals were only seen in 1,000 X the optimal doses for myelopoiesis. No effect was found on CFU-E from fetal liver. The peptide thus has a selective effect on myelopoiesis in a certain dose range. A regulatory mechanism for the peptide on hemopoiesis is proposed.

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.

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.

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.