Goralatide (AcSDKP), a negative growth regulator, protects the stem cell compartment during chemotherapy, enhancing the myelopoietic response to GM-CSF.

The aim of our study was to investigate the protection afforded to the bone marrow by Goralatide (AcSDKP), an inhibitor of hemopoietic stem cell proliferation, when administered alone or in combination with a growth factor (granulocyte/macrophage colony-stimulating factor [GM-CSF]) during iterative cycles of Ara-C (cytarabine) treatment. In control mice receiving the inhibitor alone without Ara-C, the number of granulocytes was reduced during treatment, and a surge in number of peripheral blood cells was observed after its completion. Peripheral hematological responses were monitored during 3 consecutive cycles of Ara-C chemotherapy and the resultant nadir and recoveries. Analysis of variance of the treatment effects pooled over the 3 cycles showed that a treatment regimen in which the inhibitor was administered during the myelotoxic periods of chemotherapy confirmed the existence of a surge after completion of administration of the inhibitor and showed a significant protective effect. When the cycles of chemotherapy plus Goralatide were followed by GM-CSF, the recovery from leukopenic nadirs was accelerated and the white blood cells and granulocyte levels were markedly increased over those observed in control mice and in mice treated either with Goralatide alone or with GM-CSF alone. The differences were highly significant. A consistent and significant increase (p < 0.001) in platelet count was also noted in animals given Goralatide in conjunction with Ara-C or Ara-C + GM-CSF. After three treatment cycles, this response to the CSF was far better in mice treated by the inhibitor than when CSF was given alone, suggesting a protection of the stem cell pool.

AcSDKP serum concentrations vary during chemotherapy in patients with acute myeloid leukaemia.

AcSDKP is a physiological negative regulator of cell proliferation in mammals. In Ara-C-treated mice its plasmatic concentrations decrease while the CFU-S start cycling. Infusion of AcSDKP protects these animals from death by blocking the proliferation of primitive haemopoietic cells. We measured AcSDKP serum concentrations in 20 AML patients during the course of high-dose cytoreductive treatment. We observed an early and sharp increase of AcSDKP during the induction treatment in 12 patients, reaching a peak during the initial 3 d of treatment in nine of them. These results are contrary to those observed in mice treated with high doses of Ara-C. They encourage further clinical investigation, and suggest that treatments with synthetic AcSDKP (Seraspenide) will perhaps have to be adjusted to the type of disease and the schedule of chemotherapy in order to optimize its myeloprotective effect.

The concentrations of AcSDKP, a physiological inhibitor of cell proliferation, vary during oogenesis and early development in Xenopus laevis.

The endogenous tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP) is present in the circulation and cells of mammals. Extracellular AcSDKP is a negative regulator of cell proliferation inhibiting the S phase entry of numerous cell types such as hematopoietic progenitors, hepatocytes, lymphocytes and several continuous cell lines. In contrast, the biological role of cellular AcSDKP remains unknown. We have recently reported a decrease of cellular AcSDKP concentrations by 50% accompanying the initiation of DNA synthesis in mitogen-induced proliferation of human lymphocytes. In the present study we measured the variations of cellular AcSDKP concentrations during oogenesis and early development of Xenopus. Cellular AcSDKP concentrations peaked at stage IV of oogenesis and then continuously decreased during the final oocyte maturation and during the initial 15-h period of exponential cell division of the embryo. Altogether, our present and previously published data suggest that cellular AcSDKP may be involved in the regulation of cell proliferation in eukaryotic species.

Ways of minimising hematopoietic damage induced by radiation and cytostatic drugs--the possible role of inhibitors.

Acute and chronic bone marrow toxicities are the major limiting factors in the treatment of cancer. They are related to two factors. (i) The first is a decrease in the number of hematopoietic stem cells and progenitors caused by both a lethal effect of cytotoxic agents on these cells and by differentiation of stem cells provoked by a feed-back mechanism, itself induced by the depletion of more mature marrow compartments. (ii) The second factor is a reduction in self-renewal capacity of stem cells, which is also related to both direct (mutation) and indirect (ageing of stem cell population) effects. Stimulators and inhibitors of bone marrow kinetics play a prominent role in the induction of damage and recovery patterns. Acute effects can be circumvented by an increase in the number of cell divisions in the more mature compartments. This amplification is enlarged by the administration of hemopoietic growth factors which enhance regeneration and shorten the duration of blood aplasia. However, these stimulators may contribute to the exhaustion of the stem cell pool and they may increase the severity of late effects. Protection against chronic effects is difficult; however, the ability to 'switch on' and 'switch off' proliferation opens new avenues which are currently being explored. In particular, inhibitors may protect stem cells against early and late damage by maintaining them in a quiescent state during a course of radiotherapy or chemotherapy. Several inhibitors of hematopoietic stem cell proliferation have been identified during the past 5 years. AcSDKP (Seraspenide) was the first to be isolated and its protective effects against cytotoxic agents were described over a decade ago in mice. Its physiological role is now well established in mouse and man. Preliminary results of a Phase I-Phase II clinical trial strongly suggest that it may have a useful clinical role. Further research is necessary to assess the long-term protective effects of this new family of regulators.

Serum levels of a negative regulator of cell proliferation (AcSDKP) are increased in certain human haemopathies.

One of the first known effects of the endogenous peptide N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) is to inhibit entry into DNA synthesis of pluripotent haematopoietic stem cells (CFU-S) in mice. A specific anti-AcSDKP polyclonal antibody allows the level of the tetrapeptide by to be determined by enzyme immunoassay with good sensitivity and specificity. We present results demonstrating the presence of AcSDKP in humans: serum levels of 34 healthy controls were found to be between 0.7 and 2.5 pm/ml, regardless of age and sex. High levels were found in 44% of asymptomatic controls but only in 8% of AIDS patients out of a total of 37 patients with HIV. Subsequently, studies of serum levels were performed before treatment in 121 subjects with disorders of the nonlymphoid and the lymphoid lineages. Our results did not demonstrate any decrease in serum levels, however a moderate or marked increase was noted in one-third of the subjects, which was greater in disorders of the non-lymphoid lineages (48% of 72 patients) than the lymphoid lineage (21% of 50 patients). The most significant differences were observed between controls versus patients with myeloproliferative disorders (MPD, 24 patients: p < 0.001), controls versus patients with acute myelogenous leukaemia (AML, 15 patients: p < 0.02), as well as patients with AML versus patients with primary myelodysplastic syndromes (PMDS, 10 patients: p < 0.05). The pathophysiology of these abnormalities is discussed.

Inhibitory effects of AcSDKP on the mixed lymphocyte reaction (MLR). Part I. MLR with mouse spleen cells.

AcSDKP (inhibitor of entry into cycle of pluripotent hemopoietic stem cells) is able to decrease mixed lymphocyte reaction intensity when H-2 incompatible allogeneic spleen cells are used as stimulators. This is a first approach to determining whether AcSDKP has potential therapeutic value for clinical bone marrow transplantation.

Inhibitory effects of AcSDKP on the mixed lymphocyte reaction (MLR). Part II. Human whole blood cells.

AcSDKP is a physiological negative regulator of hematopoietic stem cell proliferation. To investigate the applicability of AcSDKP in the prevention of graft-versus-host disease, this tetrapeptide was tested in mice and showed an inhibitory effect on the mixed lymphocyte reaction (MLR). In this paper we report MLR using human whole blood cells. The maximum inhibitory effect (50%) was obtained at 2.5 ng/ml AcSDKP. All experiments showed a constant dose response. Experiments are now being conducted to elucidate the mechanism of this inhibition.

Biological activities of tetrapeptide AcSDKP on hemopoietic cell binding to the stromal cell in vitro.

The biological activity of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) on hemopoietic cell binding to the stromal cells was studied by using a rosette formation technique that quantitatively represents the specific hemopoietic cell binding to the stroma. Marrow hemopoietic cell binding to the stroma was enhanced by AcSDKP. This enhancement was completely neutralized by addition of anti-AcSDKP polyclonal antibody. Furthermore, preincubation of stromal cells with AcSDKP increased hemopoietic cell binding to the stroma, whereas preincubation of hemopoietic cells with AcSDKP showed no increment of the binding. These findings suggest that AcSDKP enhanced hemopoietic cell binding through the activation of stromal cells.

Correlation of endogenous acetyl-ser-asp-lys-pro plasma levels in mice and the kinetics of pluripotent hemopoietic stem cells entry into the cycle after cytosine arabinoside treatment: fundamental and clinical aspects.

A decrease of endogenous acetyl-ser-asp-lys-pro (AcSDKP) levels in murine plasma was observed after Ara-C treatment. This decrease preceded the entry of pluripotent hemopoietic stem cells (CFU-S) into the cell cycle. This suggests a correlation between CFU-S kinetics and levels of endogenous AcSDKP. The subsequent increase of AcSDKP levels seem to indicate a feedback mechanism which should permit the reestablishment of homeostasis in the stem cells. Therefore, the expulsion of the physiological brake may be the response to a signal (stimulatory factors) to start dividing and the retention of the physiological brake may the mechanism for a return to normal values of cell proliferation.

[Study of the role of negative regulator, AcSer-Asp-Lys-Pro (AcSDKP) in the phenomena of cellular proliferation]. / L'étude du rôle d'un régulateur négatif, l'AcSer-Asp-Lys-Pro (AcSDKP) dans les phénomènes de prolifération cellulaire.

The intra and extracellular kinetics of AcSDKP levels in cell culture have been studied. A close correlation was observed between the minimal level of intracellular AcSDKP (a negative regulator of cell proliferation) and the initiation of DNA synthesis. The return to initial levels of intracellular AcSDKP when the rate of DNA synthesis decreases, suggests a role for the tetrapeptide in homeostasis during cell growth. The return to normal values was not observed in preliminary studies on cell lineages during uncontrolled proliferation.

Distribution of a negative regulator of haematopoietic stem cell proliferation (AcSDKP) and thymosin beta 4 in mouse tissues.

A competitive enzyme immunoassay using acetylcholinesterase as tracer for thymosin beta 4, has been developed. Using this assay and a previously described EIA for AcSDKP, a negative regulator of pluripotent haematopoietic stem cell proliferation, the levels of these two peptides were determined in mouse tissue extracts. The combination of EIAs with different HPLC procedures validated these methods and clearly demonstrated the ubiquity of these peptides in mouse tissues. Similar results are reported for rabbit thymus which suggest different hypotheses for AcSDKP biosynthesis.

Human placental low molecular weight factors inhibit the entry into cell cycle of murine pluripotent stem cells.

In 1977, Frindel et al. reported the presence in fetal calf bone marrow of a low molecular factor, the tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP), capable of inhibiting in vivo the hematopoietic pluripotent stem cell (CFU-S) recruitment into DNA synthesis and to increase the survival of mice which had received lethal doses of cytosine arabinoside (AraC), a phase-specific drug. Considering the potential clinical importance of CFU-S proliferation inhibitor during anticancer chemotherapy and the importance of monitoring the inhibitor by immunological methods, we tried to determine if a similar inhibitor is present in humans. Preliminary results indicate the presence in human placenta of an inhibitor coeluted with AcSDKP and which is effective in inhibiting murine CFU-S.

Negative regulator of pluripotent hematopoietic stem cell proliferation in human white blood cells and plasma as analysed by enzyme immunoassay.

This paper describes the analysis, by a highly sensitive and specific enzyme immunoassay (EIA), of AcSDKP, a tetrapeptide recently isolated from fetal calf bone marrow and subsequently purified and identified which substantially inhibits entry into cycle of hematopoietic pluripotent stem cells (CFU-S). This molecule has a marked protective effect in mice during anticancer chemotherapy with phase-specific drugs and plays an essential role in maintaining CFU-S out of cycle in normal mice. Using acetylcholinesterase-AcSDKP conjugate as tracer, rabbit specific antiserum and 96-well microtiter plates coated with a mouse monoclonal anti-rabbit IgG antibody, this EIA allows detection of AcSDKP at 15 fmol levels with a coefficient of variation less than 10% in the 50-500 fmol range. When combined with high-performance liquid chromatography, this assay clearly reveals the presence of this peptide in normal human white blood cells whereas in supernatant from cultured lymphocytes and in plasma the immunoreactive material is distinct from standard AcSDKP.

A one step cell sorting procedure for the enrichment of murine bone marrow CFU-S which is independent of their proliferative activity.

Murine bone marrow day 9 splenic colony-forming units (CFU-S) have been concentrated using a one step cell sorting technique. CFU-S were discriminated on the basis of their rather high forward light scatter intensity, their high affinity for the lectin WGA and the absence of a cell surface marker expressed by the majority of hematopoietic cells and recognized by the RA3-5B3 MoAb. This procedure permitted a 40-fold enrichment of quiescent or cycling CFU-S (obtained from normal and Ara-C-treated mice respectively) and did not alter their differentiation pathways towards the various myeloid lineages.

Thyroid hormones induce hemopoietic pluripotent stem cell differentiation toward erythropoiesis through the production of pluripoietin-like factors.

We have previously reported that E pluripoietins are produced in mice after a single 20-mg injection of cytosine arabinoside (Ara-C) and that they are able to initiate the determination of hemopoietic pluripotent stem cells (CFU-S) toward the erythrocytic lineage. However, the mechanism of E pluripoietin release is still unclear. Since the stimulating effect of thyroid hormone on erythropoiesis is well known, we postulated a link between this hormone and the E pluripoietins. In previous papers we demonstrated that L-triiodothyronine (LT3) exhibits the capacity of inducing CFU-S differentiation toward erythropoiesis in vitro. Two series of data presented here suggest that LT3 acts indirectly on CFU-S determination by promoting the release of E pluripoietin-like factors. First, the Ara-C injection which induces the production of E pluripoietins in mice also promotes an increase in the LT3 plasma level. Second, medium conditioned with bone marrow cells exposed in vitro for 90 min to LT3 (even though this medium does not contain LT3) has E pluripoietin-like effects, inducing CFU-S differentiation toward the erythrocytic lineage.

AcSDKP, an inhibitor of CFU-S proliferation, is synthesized in mice under steady-state conditions and secreted by bone marrow in long-term culture.

The peptide AcSDKP, isolated from fetal calf bone marrow, is able to prevent DNA synthesis in mouse CFU-S in vivo and in vitro. The molecule is demonstrated here to be constitutively produced in mice and synthesized by bone marrow cells in long term culture.

Further studies on the biological activities of the CFU-S inhibitory tetrapeptide AcSDKP. I. The precise point of the cell cycle sensitive to AcSDKP. Studies on the effect of AcSDKP on GM-CFC and on the possible involvement of T-lymphocytes in AcSDKP response.

In order to further investigate the mechanisms of spleen colony-forming unit (CFU-S) inhibition by the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), the following related subjects were studied: 1) the effects of AcSDKP on the kinetics of granulocyte and macrophage precursor cells (granulocyte-macrophage colony-forming cells; GM-CFC); 2) the precise point in the cell cycle of CFU-S that is sensitive to AcSDKP; and 3) the role of lymphocytes in the chain of events leading to the inhibition of CFU-S entry into the cell cycle. The effects of AcSDKP on CFU-S and GM-CFC were tested using the spleen colony assay and methylcellulose culture technique, respectively; the cell cycle kinetic status was determined by the tritiated thymidine suicide technique. Nude mice were studied to assess the role of T-lymphocytes in the inhibitory phenomenon. Our results indicate that: 1) there is no inhibitory effect of AcSDKP on GM-CFC in vitro or in vivo; 2) AcSDKP is active at only the G0 or early G1 phases; and 3) AcSDKP activity is not modulated by T cells.

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.