Emilia Frindel

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.

Distribution of a negative regulator of haematopoietic stem cell proliferation (AcSDKP) and thymosin, β4 in mouse tissues

A competitive enzyme immunoassay using acetylcholinesterase as tracer for thymosin β4, 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.

Amelioration of Chemotherapy‐Induced Toxicity by Cotreatment with AcSDKP, a Tetrapeptide Inhibitor of Hematopoietic Stem Cell Proliferation

Acute myelosuppression as a consequence of cytotoxic chemotherapy is well recognized as a dose-limiting factor in cancer treatment. Although other normal tissues may be adversely affected, bone marrow is particularly sensitive to the phase-specific agents. Self-renewing, pluripotent hematopoietic progenitor cells, or stem cells, as measured by spleen colony-forming units (CFU-S) are responsible for generation of the hematopoietic system. In normal mice, such stem cells are mainly quiescent, and renewal of mature cells is provided by the partially differentiated, lineage-committed hematopoietic progenitors. At this stage, the stem cells are relatively invulnerable to phase-specific cytotoxic drugs that affect (i.e., kill) only those cells that are already in proliferation or about to begin proliferation. The stem cell compartment of the bone marrow that guarantees a sufficient supply of cells bound for differentiation, though mainly quiescent, still has approximately 10% of its cellular component in some state of mitosis.* During chemotherapy the first treatment kills proliferating malignant cells in the tumor as well as proliferating hematopoietic cells in the bone marrow, but has no effect on the quiescent CFU-S.

Some effects of chemotherapeutic drugs on bone marrow stem cells

SummaryTwo phase-specific drugs, cytosine arabinoside and hydroxyurea, were studied with regard to their effects on various murine hematologic cell compartments of the same mouse.Effects of single and multiple injections of Ara-C were compared.Following a significant decrease in the first few days, and a subsequent overshoot of pluripotential stem cells (CFU-S), colony-forming cells (CFC), bone marrow nucleated cells, and leukocytes, the number of these cells returned to normal values with a time sequence that varied with the cell type. During the 6-month observation period the number of these cells oscillated around control values after both drugs and both types of protocols.

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.

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.

Some effects of chemotherapeutic drugs

Summarycis-Platinum is a relatively new active anticancer drug. In the study described in this paper, its toxicity was tested in the hematopoietic and renal systems of mice after six injections of 3 mg per kg body weight at 10-day intervals.Acute hematopoietic toxicity was studied by determining the survival of pluripotent (CFU-S) and granulo-macrophagic unipotent (GM-CFC) stem cells. The number of nucleated cells in the bone marrow and in the spleen and the number of granulocytes in the blood were determined.Renal toxicity was studied by histological examination of kidneys from treated mice compared with control animals.The number of stem cells in the bone marrow and in the spleen decreased during the treatment. One year after treatment, the autorepopulating ability of CFU-S was still diminished in spite of normal numbers of these cells.No renal damage could be demonstrated by light microscopy when the protocol described was used.

Study of an inhibiting factor of epidermal proliferation in plucked skin and various tumours of mice

The aim of our research was to investigate, by autoradiographic meAods, the presence of an inhibitory factor (IF) in normal skin, in plucked skin and in five experimental skin tumours of the mouse, capable of inhibiting epidermal proliferation. The experimental model used was that of BuUough & Laurence (1960), i.e. a wound in the ear of a mouse. After tritiated thymidine administration the measurement of the labelling index confirmed the existence of an inhibitory substance in normal skin. The plucked skin had no inhibitory effect when it was taken 24 h after plucking, but after 4 days its effect had returned to normal. The skin tumours, whether epithelial or mesenchymal, showed the same inhibitory effect as normal skin whereas a non‐cutaneous tumour tested had no inhibitory effect.

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.