Gain- and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage.

To investigate the role of the Lyn kinase in establishing signaling thresholds in hematopoietic cells, a gain-of-function mutation analogous to the Src Y527F-activating mutation was introduced into the Lyn gene. Intriguingly, although Lyn is widely expressed within the hematopoietic system, these mice displayed no propensity toward hematological malignancy. By contrast, analysis of aging cohorts of both loss- and gain-of-function Lyn mutant mice revealed that Lyn(-/-) mice develop splenomegaly, increased numbers of myeloid progenitors, and monocyte/macrophage (M phi) tumors. Biochemical analysis of cells from these mutants revealed that Lyn is essential in establishing ITIM-dependent inhibitory signaling and for activation of specific protein tyrosine phosphatases within myeloid cells. Loss of such inhibitory signaling may predispose mice lacking this putative protooncogene to tumorigenesis.

Pretreatment with cytosine arabinoside does not protect against the delayed effects of irradiation on thrombopoiesis and erythropoiesis.

We have examined the capacity of the thrombopoietic and erythropoietic systems to respond to challenge with the cytotoxic drug 5-fluorouracil (5-FU) following sublethal doses of irradiation. Normal adult mice respond to 5-FU with a mild thrombocytopenia followed by a marked rebound thrombocytosis. One month after 2 Gy whole-body radiation, platelet counts took longer than normal to reach a nadir after 5-FU, and the rebound thrombocytosis was reduced. A normal response was seen when the interval after radiation was extended to 4 months. Increasing the radiation dose to 6.5 Gy resulted in a much smaller rebound thrombocytosis when 5-FU was given 1 month later. Extending the interval before the drug was given resulted in a normal response being regained between 4 and 7 months. Erythrocyte levels were temporarily depressed after 5-FU in all mice, and it took longer for recovery to occur if they had been irradiated. In another series of experiments, we investigated the effect of priming the mice before irradiation to see if this resulted in radioprotection. An injection of cytosine arabinoside (Ara-C) 2 days before a dose of 6.5 Gy resulted in the expected earlier recovery in platelet counts. To see if cells of the megakaryocyte lineage were protected from the delayed effects of irradiation by this treatment, mice were given 5-FU 1 month after Ara-C plus irradiation. The period of thrombocytopenia was followed by only a small rebound thrombocytosis, and platelet counts were indistinguishable from those found for mice not primed with Ara-C before irradiation. These experiments revealed a delayed effect of irradiation on the thrombopoietic and erythropoietic systems, which was long-lasting but not permanent at the doses used. The effects were not eliminated by priming mice with Ara-C before irradiation.

The resolution, enrichment, and organization of normal bone marrow high proliferative potential colony-forming cell subsets on the basis of rhodamine-123 fluorescence.

Cell sorting on the basis of rhodamine-123 (Rh123) fluorescence has been used in conjunction with negative immunomagnetic selection to analyze the high proliferative potential colony-forming cell (HPP-CFC) compartment of normal murine bone marrow and to resolve and enrich HPP-CFC subpopulations responsive to different combinations of the hemopoietic growth factors interleukin 1 alpha (IL-1 alpha), interleukin-3 (IL-3), and colony-stimulating factor 1 (CSF-1). HPP-CFC with a specific requirement for IL-1 alpha plus IL-3 plus CSF-1 in order to proliferate were resolved and enriched on the basis of their low Rh123 retention (Rh-dull), whereas HPP-CFC that grew in the presence of IL-3 plus CSF-1, IL-3 alone, or CSF-1 alone were Rh-bright. Further addition of IL-1 alpha to IL-3 plus CSF-1 stimulated few additional HPP-CFC in the Rh-bright fraction. Our data confirm the value of Rh123 as a probe for the dissection and analysis of the primitive hemopoietic stem cell (PHSC) compartment. These data also show that the Rh123 staining characteristics of IL-1 alpha plus IL-3 plus CSF-1-responsive HPP-CFC are consistent with the hypothesis that these HPP-CFC are closely related to PHSC with long-term reconstituting capacity in vivo and that they are among the most primitive progenitors yet detected in clonal agar culture.

Progenitor cells in murine bone marrow stimulated by growth factors produced by the AF1-19T rat cell line.

The AF1-19T rat cell line has been found to produce an activity that acts synergistically with colony-stimulating factor 1 (CSF-1) to stimulate primitive high proliferative potential colony-forming cells (HPP-CFC) in mouse bone marrow (BM) that appear to be the same as those stimulated by the combination of 5637-cell-conditioned medium (CM) plus CSF-1 or recombinant human (rh) interleukin 1 (IL-1) plus recombinant murine (rm) interleukin 3 (IL-3) plus CSF-1. AF1-19T also produced granulocyte-macrophage colony-stimulating factor (GM-CSF), which could be separated from this synergistic activity by gel filtration followed by hydroxylapatite chromatography. Results obtained from the mouse thymocyte costimulation assay for IL-1, the hybridoma growth factor assay for interleukin 6 (IL-6), the ability to stimulate HPP-CFC, and the ability to block this stimulation with an antibody to murine IL-1 alpha suggest that the synergistic activity in AF1-19T-CM is probably a mixture of IL-1 activity and IL-6 or an IL-6-like activity. Other workers have described a progenitor cell population in mouse BM (CFU-A) that forms large colonies in response to AF1-19T-CM plus CSF-1 or GM-CSF plus CSF-1. Experiments involving the kinetics of recovery after 5-fluorouracil treatment and generation of progenitors suggest that the GM-CSF-plus-CSF-1-responsive progenitors, and hence CFU-A, are a more mature cell type than the more primitive HPP-CFC, responsive to 5637-cell-CM plus CSF-1 or rhIL-1 plus rmIL-3 plus CSF-1.

Radiation sensitization by an iodine-labelled DNA ligand.

An iodinated DNA ligand, iodo Hoechst 33258, which binds in the minor groove of DNA, enhances DNA strand breakage and cell killing by UV-A irradiation. The sites of UV-induced strand breaks reflect the known sequence specificity of the ligand.

High proliferative potential colony forming cells.

High proliferative potential colony forming cells (HPP-CFC) in mouse bone marrow (BM) were defined functionally by their ability to form large colonies, greater than 0.5 mm diameter, and containing an average of 5 × 10(4) cells, in low-cell-density nutrient agar cultures after 10-14 d of incubation (1).

In vivo effects of interleukin-1 alpha on regenerating mouse bone marrow myeloid colony-forming cells after treatment with 5-fluorouracil.

Injection of a single dose of recombinant human interleukin-1 alpha (r-hu-IL-1 alpha) into mice 24 hr after 5-fluorouracil (FU) treatment resulted in an increased rate of recovery of three types of colony-forming cells (CFCs) in the bone marrow. Myeloid progenitors with high proliferative potential (responsive to CSF-1 + IL-3 + IL-1 alpha), low proliferative potential (responsive to CSF-1), megakaryocyte progenitors, and total nucleated cells per femur increased up to 5-fold, 7-fold, 3-fold, and 3-fold, respectively, in a dose related fashion compared with the control FU treated marrows. The kinetics of FU kill and recovery of these CFCs are shown.

The relation of radionuclide uptake by bone to the rate of calcium mineralization. I: Experimental studies using 45Ca, 32P and 99Tcm-MDP.

The study of the uptake of radionuclides by bone has been undertaken in a mouse tail graft model using 45Ca, 32P and a routine bone scanning agent 99Tcm-MDP, together with serial calcium determinations. The model provided an experimental system in which the calcium mineral content and the rate of mineralization both changed progressively throughout its development. A significant linear correlation was found between 45Ca and 32P uptake and the rate of calcium mineralization, which held for all stages of the graft's growth. Both radiotracers therefore accurately reflected the calcium mineral deposition. In the case of 99Tcm-MDP, the correlation with mineralization rate only applied for the most active growth period of the graft when the rate was increasing. For all radiotracers, the peak in bone uptake corresponded to the maximum in mineralization rate.

The relation of radionuclide uptake by bone to the rate of calcium mineralization. II: Patient studies using 99Tcm-MDP.

The relation of radionuclide uptake by bone and rate of calcification has been studied in normal vertebrae and in vertebral metastases from cancer of the prostate. Specifically, the determination of 99Tcm-MDP uptake by radionuclide scanning and the estimation of calcium concentration of trabecular bone by dual-energy computed tomography have provided the means of obtaining a relation between these parameters which was similar to that found in an animal model, in which the dependence of radionuclide uptake on the rate of mineralization was established. This relationship has enabled the experimental findings to be extrapolated to those in patient studies and has shown that in sclerotic bone lesions, the increase in 99Tcm-MDP uptake accompanying the progression of the metastases was proportional to the rate of calcification.

Changes in cell surface antigen expression during murine bone marrow cell regeneration in vivo and proliferation in vitro.

Modulations in surface antigen expression during marrow regeneration in vivo, and proliferation in vitro in response to haemopoietic growth factors, were studied using a panel of monoclonal antibodies recognizing antigenic determinants expressed by primitive multipotential progenitor cells (Thy-1, Qa-m7), or lineage antigens restricted to committed progenitors and differentiated cells of the neutrophil/macrophage (7/4) and B lymphocyte (B220) lineages. These two categories of antigen exhibited differing responses to marrow perturbation and proliferation. Following administration of a cytotoxic dose of 5-fluorouracil, or lethal irradiation and transplantation of normal donor marrow, the levels of Thy-1 and Qa-m7 antigen expression rapidly increase, reaching a peak at the onset of regeneration: the nadir of marrow cellularity. Expression of these antigens returns to normal as regeneration proceeds and marrow is reconstituted. 7/4 and B220 antigen expression reflect the presence or absence of maturing cells bearing these markers: antigen expression declining following perturbation, and re-emerging during the course of regeneration. In vitro, when marrow cells taken from mice 8 days following treatment with 5-FU are grown in liquid culture in the presence of colony-stimulating factor-1 plus bladder cell carcinoma cell line 5637 conditioned medium, marrow cells are stimulated to proliferate and differentiate along the neutrophil/macrophage lineage. 7/4 antigen expression increases throughout the culture period, and B220 antigen is undetectable after the fifth day of culture. Thy-1 antigen expression also rises and remains elevated, and Qa-m7 antigen expression remains stable.

Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.

The concentration and resolution of primitive hemopoietic cells from normal mouse bone marrow by negative selection using monoclonal antibodies and Dynabead monodisperse magnetic microspheres.

High proliferative potential colony-forming cells (HPP-CFC) detected in clonal agar culture in the presence of the combined stimulus of colony-stimulating factor 1 (CSF-1) + interleukin 3 (IL-3) + interleukin 1 alpha (IL-1 alpha) are closely related to developmentally early progenitor cells capable of reconstituting the hemopoietic system of lethally irradiated mice following transplantation. Flow cytometric analysis and sorting of normal, unperturbed bone marrow has shown that HPP-CFC are B220- and 7/4-, whereas the committed progenitors of the macrophage lineage responsive to CSF-1 alone (CSFCSF-1) are B220- and 7/4+. Negative immunomagnetic selection using an anti-7/4, anti-B220 antibody cocktail and second-antibody-coupled Dynabead microspheres to replace flow cytometry results in the highly reproducible and specific enrichment of HPP-CFC, and simultaneous resolution of HPP-CFC from CFCCSF-1. The tenfold enrichment of HPP-CFC compared with unfractionated bone marrow cell suspensions was comparable to that obtained by fluorescence-activated cell sorting. Enrichment was achieved with negligible loss of HPP-CFC at the immunomagnetic bead selection step, and 65% of HPP-CFC were recovered. The method is rapid, highly reproducible, and efficient, and has wide application to the separation of rare hemopoietic cells from normal bone marrow.

Critical DNA target size model of ionizing radiation-induced mammalian cell death.

A new model of mammalian cell killing by ionizing radiation is presented. This model, termed the critical DNA target size model, postulates that DNA double-strand breakage is the critical radiation-induced lesion and that the dose-response for such breakage can be non-linear due to the action of a saturable chemical repair process. DNA double-strand breakage occurring within critical targets (proto-oncogene- or common fragile site-associated sequences) is postulated to initiate recombination events with undamaged sequences, leading to chromosomal aberrations. The subsequent loss of acentric fragments at mitosis is postulated to prevent the continuity of the genome and to produce cell death by the induction of chromatin structural changes. Experimental evidence contrary to other radiation action models is examined, and the hypotheses of the model are justified.

Multiparameter analysis of transplantable hemopoietic stem cells. II. Stem cells of long-term bone marrow-reconstituted recipients.

Marrow obtained from mice (referred to as [X + BM] mice) 3 months after gamma-irradiation (9 Gy) and bone marrow inoculation (0.1 femur equivalents) showed a reduced capacity to reconstitute hemopoiesis of irradiated mice and an increased sensitivity to 5-fluorouracil. Sorting of marrow from (X + BM) mice on the basis of low angle and 90 degrees scatter, and low rhodamine 123 fluorescence, showed that the set of cells that in normal mice is enriched for cells efficient at hemopoietic reconstitution manifested the greatest reduction in hemopoietic reconstituting ability. In spite of this reduction this fraction contained as many 13-day spleen colony-forming units (CFU-S13) and high proliferative potential colony-forming cells (HPP-CFC) as the equivalent fraction from normal littermate mice. This could be explained by postulating that neither CFU-S13 nor HPP-CFC are responsible for hemopoietic reconstitution, but that this is dependent on an earlier, pre-CFU-S13 cell. Alternatively only a subset of either CFU-S13 or HPP-CFC is responsible for long-term hemopoietic reconstitution after lethal irradiation. It would appear that at present there is no adequate method of predicting the hemopoietic reconstituting ability of a given marrow, other than to test it by injection into lethally irradiated hosts.

A colorimetric liquid culture assay of a growth factor for primitive murine macrophage progenitor cells.

Synergistic factors from media conditioned (CM) by human placentas or the 5637 human bladder carcinoma cell line (SFH-HPCM and SFH-5637 respectively) have the ability to stimulate early progenitor cells in mouse bone marrow to form large colonies in agar cultures after 12-14 days, in the presence of CSF-1. Culture conditions have been examined and a quicker and more convenient liquid culture assay has been developed for this factor, using a tetrazolium salt to quantitate cell proliferation. The use of flat-bottomed vessels, high cell density, supra-optimal doses of CSF-1 or the addition of WEHI-3-CM to these cultures, all resulted in a decrease in the required incubation time. In combination, these modifications reduced the assay time to 4 days.

Generation of murine hematopoietic precursor cells from macrophage high-proliferative-potential colony-forming cells.

High-proliferative-potential colony-forming cells (HPP-CFC) have been described as primitive murine macrophage progenitors. We have previously demonstrated the existence of two populations of HPP-CFC: one population, termed HPP-CFC-1, is stimulated by the combination of macrophage colony-stimulating factor (CSF-1) plus haemopoietin-1 (H-1) and actively generates a second population of HPP-CFC, termed HPP-CFC-2. HPP-CFC-2 are stimulated by CSF-1 plus interleukin-3 and generate macrophage CFC that differentiate to form mature macrophages. In this study, we have demonstrated that HPP-CFC-1, when stimulated by CSF-1 plus H-1, generate colony-forming cells (CFC) for the megakaryocyte and granulocyte lineages in addition to HPP-CFC-2 and M-CFC. No CFC were detected with erythroid potential. In addition, HPP-CFC-1 generated cells that formed day-13 spleen colonies, cells that repopulated the bone marrow, cells with platelet-repopulating ability, and cells with erythroid-repopulating ability in lethally irradiated mice. These data support previous data that the HPP-CFC-1 represent a primitive hemopoietic cell population and demonstrate the multipotentiality but not totipotentiality of these cells.

Detection of murine hemopoietin-1 in media conditioned by EMT6 cells.

We have previously reported replating experiments which demonstrated the existence of subpopulations of murine high-proliferative-potential colony-forming cells (HPP-CFC). One population of HPP-CFC, termed HPP-CFC-1, is stimulated by the combination of macrophage colony-stimulating factor (CSF-1) plus hemopoietin-1 (H-1), and actively generate a second population of HPP-CFC, termed HPP-CFC-2, which is responsive to CSF-1 plus interleukin-3 (IL-3). These reclonal experiments represent an assay system that discriminates between the two types of synergistic factors, namely H-1 and IL-3. To date H-1 has only been detected in medium conditioned by human cells. In this paper we have utilized these recloning experiments to study the synergistic factor(s) present in media conditioned by the murine mammary carcinoma cell line EMT6. Colony formation in secondary cultures containing cells picked up from primary cultures incubated in CSF-1 plus EMT6-conditioned medium was identical to that seen in secondary cultures containing cells picked up from primary cultures incubated in CSF-1 plus a source of H-1. Both sets of cultures demonstrated the generation of HPP-CFC-2 in the primary cultures, indicating the presence of a molecule in EMT6-conditioned medium that is the murine equivalent of H-1.

Increased Qa-m7 antigen expression is characteristic of primitive hemopoietic progenitors in regenerating marrow.

Developmentally early murine hemopoietic progenitor cells of high proliferative potential (HPP-CFC), which are detectable in clonal agar culture in the presence of the lineage-specific hemopoietic growth factor, colony-stimulating factor-1 (CSF-1) plus hemopoietin-1 (H-1), or interleukin 3 (IL 3), express relatively high levels of the Qa-m7 antigenic determinant. This determinant is progressively lost during differentiation, and the more committed progenitors which grow in the presence of CSF-1 alone are essentially devoid of Qa-m7. Significant increases in both the proportion of Qa-m7-positive myeloid cells and the level of Qa-m7 antigen expression have been observed in bone marrow cells regenerating after the administration of the cytotoxic agent 5-fluorouracil (5-FU). By exploiting this increase in Qa-m7 antigen expression during regeneration and the HPP-CFC-sparing properties of 5-FU, we have been able to enrich HPP-CFC from marrows 8 days post-5-FU treatment (FU8d) to purities of greater than 20%. Furthermore, discontinuous gradient centrifugation and fluorescence-activated cell sorting of FU8d bone marrow cells on the basis of their light-scattering properties and Qa-m7 expression has unmasked a further subset of HPP-CFC which strictly requires the combined stimulus of three hemopoietic growth factors (H-1, IL 3, and CSF-1) for clonal growth. These highly enriched subsets of HPP-CFC are either identical to or co-fractionate with transplantable multipotential hemopoietic progenitors capable of reconstituting the hemopoietic system of lethally irradiated mice. Up to one in three cells in these highly enriched fractions is an HPP-CFC, and up to one in two cells may be CFU-S assayed 13 days post-transplantation. In addition, these fractions contain progenitors capable of reconstituting the platelet, erythroid, and myeloid compartments of the marrow.

Effect of ploidy on the response of V79 cells to ionizing radiation.

The responses of diploid, tetraploid and near-hexaploid V79 cells to X-irradiation or DNA-associated 125I-decay were compared. When cell killing, following X-irradiation, was plotted against the induced level of DNA double-strand breakage (dsb) per unit length of DNA, there was no significant difference between the relationships for each cell line. This suggested that the number of X-ray-induced DNA dsb per cell required to produce a lethal lesion was proportional to ploidy. Consistent with the X-ray results, tetraploid cells required 121 +/- 4 and diploid cells 60 +/- 1 125I-decays to produce a lethal lesion. However, the hexaploid cells deviated from this relationship and required 137 +/- 5 decays. The relationship between relative elution and 125I decays/cell reflected cellular DNA content. It is concluded that current models of radiation action are unable to explain these findings satisfactorily.