Concurrent activation of granulocytes and osteoclasts in busulfan-suppressed bone marrow in response to transplantation of a mammary carcinoma in mice

Transplantation of CE mammary adenocarcinoma (CE maca) into normal mice produces both neutrophilia and hypercalcemia due to osteoclastic bone resorption. In order to explore the physiology of osteoclast formation in vivo, the time course of neutrophilia and osteoclast development was examined in mice that had been pretreated with busulfan prior to the CE maca implantation. Busulfan-treated tumor-bearing mice (BUTUM), busulfan-treated control mice (BUCON), tumor-bearing mice with no busulfan (TUM), and normal controls (CON) were sacrificed on days 4, 7, 11, 14, and 17 after tumor implantation. Leukocyte counts, serum calcium levels, marrow cellularity, and marrow colony-forming units (CFU) were determined. Osteoclasts were quantified histologically by the osteoclast: endosteum ratio (OER). BUCON bone marrow was hypoplastic with CFU remaining significantly lower than that of controls over the course of the experiment. In contrast, BUTUM marrow CFU increased dramatically with the growth of the tumor. The most predominant increase was observed in neutrophilic CFU. Development of hypercalcemia closely paralleled neutrophilia in both TUM and BUTUM mice, although these changes were significantly delayed in the BUTUM group. The neutrophil count and serum calcium levels remained within normal control levels for BUCON mice. The OER correlated with serum calcium, and it closely paralleled the neutrophil count in TUM and BUTUM mice. These results clearly indicated the stimulation of bone marrow neutrophilic granulocyte progenitors and osteoclasts by the CE maca, indicating that the bone marrow is the primary target of this tumor. There may be a closely related mechanism in osteoclast and granulocyte stimulation by one or more CE maca factors.     

Mechanisms of tumor-induced neutrophilia: constitutive production of colony-stimulating factors and their synergistic actions

Transplantation of a murine mammary carcinoma (CE maca) into mice induces marked granulocytosis and hypercalcemia secondary to excessive bone resorption. Such responses are not induced by another murine mammary carcinoma Bc66. In order to understand the mechanisms of these unique phenomena, we analyzed mRNA of tumor cells for expression of murine granulopoietic growth factors and studied interactions of tumor-derived factors using antiserum to a growth factor in vitro and in vivo. The Northern blot analysis of CE tumor clones revealed the expression of granulocyte colony stimulating factor (G-CSF) and macrophage colony stimulating factor (M-CSF), but no other CSF genes, while the Bc66 clone expressed only M-CSF. The G-CSF and M-CSF gene expression in CE tumor clones was accompanied by secretion of these proteins in culture. The granulocyte stimulating activity of CE tumor-derived G-CSF or recombinant human G-CSF was markedly enhanced by purified M-CSF in vitro. Significant but variable neutrophilia was observed in mice inoculated with CE tumor clones. Anti-M-CSF treatment of CE tumor-bearing mice significantly reduced neutrophilia, but did not affect hypercalcemia. These studies document that G-CSF and M-CSF are produced constitutively from the CE maca, and G-CSF is likely responsible for granulocytosis induced by this tumor. G-CSF and M-CSF function synergistically in granulocyte stimulation in vitro and this synergism may also play a role in marked granulocytosis of tumor-bearing animals, providing further evidence of the effect of CSFs in vivo.     

In vivo and in vitro suppression of primary B lymphocytopoiesis by tumor-derived and recombinant granulocyte colony-stimulating factor

Transplantation of a granulocytosis-inducing murine CE mammary carcinoma into mice suppresses primary B lymphopoiesis in the marrow. The mechanisms of this tumor-induced B-cell suppression were investigated using Whitlock-Witte-type lymphoid cultures. When seeded with normal marrow progenitors, stromal cells of tumor-bearing mice supported the production of B220+ cells as well as did either stomal cells derived from control mice or the stromal cell line S17. Cultured over normal stroma, marrow cells of tumor-bearing mice depleted of adherent cells and B220+ cells generated B220+ cells as effectively as a similar cell population from control mice. However, interleukin-7- responsive progenitors, were completely depleted from the marrow of tumor-bearing mice. When conditioned medium (CM) of cloned CE tumor cells known to produce granulocyte colony-stimulating factor (G-CSF) and macrophage-CSF, or recombinant murine G-CSF was added to the cultures established with S17 cells, B220+ cell production was significantly diminished. Antiserum to murine G-CSF blocked these effects. These in vitro observations were corroborated by the elimination of marrow B220+ cells in mice injected with G-CSF. These in vitro and in vivo studies suggest that G-CSF plays an inhibitory role in primary B lymphopoiesis by blocking stromal cell-mediated differentiation of early B-cell progenitors into phenotypically recognizable B220+ pre-B cells.     

The effect of syngeneic peripheral blood cells on the formation of colonies by normal human bone marrow cells in diffusion chambers in mice.

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2-5 x 105 cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 105 cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 x 105 1500 R-irradiated syngeneic light density blood cells (less than 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 x 105 1500 R-irradiated high density blood cells(greater than 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.     

Polycythemia vera terminating in chronic neutrophilic leukemia: studies on in vitro growth of hematopoietic progenitor cells]

A 57-year-old man, diagnosed as Polycythemia vera (PV), had been treated with administrations of Busulfan since 1984. Three years later, the number of neutrophils in peripheral blood increased to 50,000/microliters with progression of splenomegaly, and the case was diagnosed as Chronic neutrophilic leukemia (CNL) based on the criteria by Miura et al, in November, 1989. In spite of 6MP and Busulfan therapy, marked neutrophilia and splenomegaly progressed, and the patient died due to liver dysfunction in June of 1991. To clarify the pathophysiology of PV and CNL, we studied the in vitro growth kinetics of hematopoietic progenitor cells in bone marrow of this unique case and made a comparison with those of 4 cases of PV and 4 normal volunteers employing methylcellulose culture. As in other cases of PV, erythroid colonies were formed in culture of bone marrow from this patient without addition of erythropoietin. Furthermore, spontaneous colonies derived from CFU-GM and CFU-Mix increased remarkably in this case only. The results suggest that the hematopoietic abnormalities in this case involve the multipotent stem cells as well as erythroid and granuloid-macrophage progenitors.     

Demonstration of granulopoietic factor(s) in the plasma of nude mice transplanted with a human lung cancer and in the tumor tissue.

A human lung cancer (OTUK-tumor) was transplanted serially to nude mice, which invariably developed a marked neutrophilia. In order to analyze this phenomenon, in vitro agar culture studies were carried out. A three- to fourfold increase of colony-forming units in culture was observed in the femurs of these nude mice. Moreover, the plasma of nude mice bearing this tumor, as well as the tumor extract, had a significantly higher colony-stimulating activity on mouse bone marrow cells than appropriate controls. This activity had the property to stimulate granulocyte and/or mixed cell type colonies rather than mononuclear cells. This activity was also demonstrated, in a dose-dependent way, on normal human bone marrow cells. These findings indicated that the OTUK-tumor produced human granulopoietic colony-stimulating activity, which may have stimulated granulopoiesis in vivo as well.     

Effects of recombinant murine granulocyte colony-stimulating factor on granulocyte-macrophage and blast colony formation

We performed the present study to define the in vitro hemopoietic activity of murine recombinant (r) granulocyte colony-stimulating factor (G-CSF) using murine hemopoietic culture systems of normal bone marrow cells, fetal liver cells, and spleen cells of 5-fluorouracil (FU)-treated mice. Recombinant G-CSF supported only neutrophil and/or macrophage colony formation by normal bone marrow cells. It did not enhance the formation of erythroid bursts in the fetal liver cell assay, but interleukin-3 (IL-3) did. Paradoxically, rG-CSF could support the colony formation of multilineage colonies as well as blast colonies from the spleen cells of 5-FU-treated mice, while r-granulocyte-macrophage colony-stimulating factor (GM-CSF) and r-erythropoietin (Ep) did not. When blast colonies, formed in the presence of G-CSF, were replated to dishes containing IL-3, they were able to differentiate along multilineage pathways. However, when they were replated to dishes containing rG-CSF, they could differentiate only into neutrophils and macrophages. Single cells transferred from blast colonies formed only neutrophil-macrophage colonies. These data indicate that rG-CSF had a direct effect on the growth and development of GM progenitors at a late stage and a significant effect on multipotential hemopoietic precursors. Although it remains to be clarified how G-CSF acts on multipotential stem cells, this unique effect is important in the understanding of its pluripotent hemopoietic activity in vivo.     

Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit.

Mice with W mutations characterized by hypopigmentation, sterility, anemia, and mast cell deficiency have abnormalities in c-kit, a receptor with tyrosine kinase activity. Recently, the ligand for c-kit was cloned by investigators in several laboratories. Zsebo et al identified and cloned a gene for a cytokine termed stem cell factor (SCF) in the medium conditioned by buffalo rat liver cells, and this cytokine proved to be c-kit ligand. We have examined the effects of recombinant rat SCF (rrSCF) on colony formation from primitive hematopoietic progenitors in culture. rrSCF and erythropoietin (Ep) supported formation of granulocyte/macrophage (GM) colonies as well as a small number of multilineage and blast cell colonies from marrow cells of normal mice. We then examined the effects of rrSCF using marrow and spleen cells of mice that had been treated with 150 mg/kg 5-fluorouracil (5-FU). Unlike single factors, combinations of factors such as rrSCF plus interleukin-3 (IL-3), rrSCF plus IL-6, and rrSCF plus granulocyte colony-stimulating factor (G-CSF) markedly stimulated the growth of multilineage colonies. In contrast to these factor combinations and a combination of IL-3 and IL-6, a combination of rrSCF and IL-4 did not support multilineage colony formation. Mapping studies of the development of multipotential blast cell colonies further indicated that rrSCF, like IL-6, G-CSF, and IL-11, shortens the dormant period in which the stem cells reside. When we tested the effects of rrSCF using pooled blast cells, which are highly enriched for progenitors and are devoid of stromal cells, rrSCF plus Ep supported formation of only a few multilineage colonies, indicating that rrSCF itself is ineffective in support of the proliferation of multipotential progenitors. However, rrSCF supported formation of a significant number of neutrophil and neutrophil/macrophage colonies from pooled blast cells, indicating that rrSCF is able to support directly the proliferation of progenitors in neutrophil/monocyte lineages. c-kit ligand may play important roles in adult hematopoiesis.     

The Effect of Syngeneic Peripheral Blood Cells on the Formation of Colonies by Normal Human Bone Marrow Cells in Diffusion Chambers in Mice

This paper describes the influence of cells capable of releasing colony stimulating activity (CSA) in vitro on the formation of granulocytic colonies by normal human bone marrow in diffusion chambers in mice. A carbonyl iron method was used to remove phagocytic cells from normal human bone marrow. This treatment prevented spontaneous colony and cluster formation when the cells were cultured in agar in vitro at initial concentrations of 2–5 times 10⁵cells per ml. However, non-phagocytic bone marrow cells formed granulocytic colonies when inoculated into diffusion chambers at 10⁵cells per chamber and cultured in 450 R-irradiated or non-irradiated mice. The formation of granulocytic colonies by carbonyl iron treated marrow in diffusion chamber cultures was not consistently enhanced by the admixture of 1.4 times 10⁵1500 R-irradiated syngeneic light density blood cells (< 1.077 g/ml) to the chamber inoculum. In contrast, these cells induced cluster or colony formation when added in the same proportion to the marrow cells in agar cultures in vitro. Addition of 1.4 times 10⁵1500 R-irradiated high density blood cells (> 1.077 g/ml) to the inoculum resulted in a slight, non-significant decrease in the number of colonies in diffusion chambers. The stimulating effect of host irradiation on neutrophilic colony formation was independent of the presence of CSA releasing cells in the chamber inoculum.     

Effect of culture conditions on in vitro erythroid colony formation in myelodysplastic syndromes

In vitro colony formation by erythroid progenitors from the bone marrow was studied in 42 patients with a myelodysplastic syndrome using both a standard assay for growing erythroid colonies and an assay designed for growing megakaryocyte colonies. In the standard assay 5 patients had normal numbers of erythroid burst-forming unit (BFU-E) colonies, 8 showed reduced numbers of colonies, and 29 patients had no colony formation. Six patients with markedly reduced numbers of erythroid colonies or no colonies at all in the standard assay showed normal or increased numbers of erythroid colonies in the megakaryocyte assay. In three of these patients the erythroid colony morphology was normal, whereas the other three showed abundant diffuse growth of erythroid subclusters with no normal colonies. In the other patients, the erythroid culture results were similar in both assays. These results indicate that the impairment of erythroid colony growth seen in most patients with a myelodysplastic syndrome is, at least in a number of patients, not due to reduced numbers of erythroid progenitors but to unusual milieu requirements of abnormal progenitors.     

Aging and hematopoiesis. VI. Neutrophilia and other leukocyte changes in aged mice

The concentration of blood leukocytes rose progressively as mice aged. All blood leukocytes increased, although a greater degree of increase was seen in neutrophils and monocytes than in lymphocytes and eosinophils. The total number of nucleated cells per marrow cavity of the humerus was also higher in aged than in young adult mice, the increase primarily reflecting peroxidase-positive cells. Both blood and marrow neutrophils of aged mice responded to perturbations induced by bleeding or by endotoxin injection in a manner qualitatively similar to that seen in young adults. When hematopoietic chimeras were produced by marrow transplantation, blood and marrow neutrophils were characteristic of the age of the recipient, not the cells; i.e., young mice kept a "young" neutrophil pattern and old mice kept an "old" neutrophil pattern when given marrow from either old or young mice. Colonies of granulocytes and/or monocyte-macrophages were grown from young marrow cells placed in plasma clots in Millipore chambers in the peritoneal cavity. The number of colonies was the same in young and in old mice, suggesting that long-range humoral stimulation of cell growth was similar in young and old. Thus, neutrophilia due to increased neutrophil production appears to be a normal part of the aging process in the mouse. The increase in neutrophil production may be due to a changing hematopoietic microenvironment.     

Interleukin-6 enhances murine megakaryocytopoiesis in serum-free culture

We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL-6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL-6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage-megakaryocyte colonies. The combination of granulocyte colony-stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.     

Cytobiologic and clinical aspects in a patient with chronic neutrophilic leukemia after thorotrast exposure

A patient with fairly typical chronic neutrophilic leukemia, as represented by some two dozen such reported cases, had been given Thorotrast more than 20 years before. Typical myeloblastic crisis developed with remarkable terminal leukocytosis. Mature blood neutrophils had normal function with respect to phagocytosis, bacterial killing, metabolic activation, and chemotactic response. The number of cells producing colonies of neutrophils and monocytes in in vitro semisolid cultures was normal in the blood and increased in marrow. Colony size was smaller than is usually observed in normal patients or in typical patients with chronic myeloid leukemia. Termination in blast crisis, also seen in a few other patients with chronic neutrophilic leukemia, indicates that this is indeed a form of leukemia and not a "leukemoid" reaction of obscure cause. The differential diagnosis of extreme neutrophilia is discussed.     

Role of human immunodeficiency virus replication in defective in vitro growth of hematopoietic progenitors.

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, a group of 27 patients with HIV-1 infection was studied. In all patients, a marked reduction of in vitro colony formation by erythroid, granulomacrophagic, and megakaryocytic bone marrow progenitors was observed in comparison to normal donors. HIV-1 infection of marrow progenitors was investigated in studying individual colonies with the polymerase chain reaction (PCR) technique. No HIV-1 DNA could be detected in these colonies, suggesting either that marrow progenitors were not infected or that infected progenitors were not able to generate colonies in vitro. The addition of antisense oligonucleotides directed against HIV tat or nef sequences in the culture medium led to a significant increase in colony formation, suggesting that HIV replication in hematopoietic progenitors could be responsible for their defective growth. However, no HIV-1-infected colonies could be detected by PCR after the antisense treatment, indicating that the increase in colony number was not due to the proliferation and differentiation of infected progenitors but to an inhibition of HIV replication in an accessory cell. This last hypothesis was further confirmed by the absence of effects of antisense oligomers on the plating efficiency of hematopoietic progenitors grown from CD34+ cells. These data indicate that hematologic abnormalities of HIV-infected patients cannot be explained by a direct infection of hematopoietic progenitor cells and suggest that a defective modulation of progenitor cell growth by HIV replication outside these cells might play a role in these abnormalities.     

Enhanced colony formation of human hemopoietic stem cells in reduced oxygen tension

In general, cell cultures, including hemopoietic stem cells, are produced in an atmosphere of various CO2 concentrations in air, although most cells in vivo proliferate and differentiate at lower oxygen tensions. We therefore investigated the effect of reduced oxygen tension on the in vitro colony growth of committed and multipotential hemopoietic progenitor cells from human bone marrow. All hemopoietic progenitor cells (CFU-mix, BFU-E, CFU-E, and CFU-GM) investigated showed enhanced colony growth at lower oxygen tension. CFU-E showed the highest enhancement, followed in order by BFU-E, CFU-mix and CFU-GM. At reduced oxygen tension, the sensitivity of early and late erythroid progenitor cells to erythropoietin was significantly increased, and this can be one of the mechanisms for the enhanced colony growth of erythroid progenitors. In the colony growth of CFU-GM, plating efficiency was also enhanced by the predominant increment of neutrophilic colonies. The lowering of oxygen tension would presumably reduce oxygen toxicity and result in the increased colony growth of human bone marrow stem cells, although the precise mechanisms of oxygen toxicity at the level of hemopoietic stem cells have yet to be elucidated. However, this clonal culture system, using a low oxygen tension, can be a useful means for elucidating the regulatory mechanisms involved in the proliferation and differentiation of hemopoietic progenitor cells in physiological and pathological conditions.     

The effect of recombinant murine interferon-gamma on the hematopoietic and immunological parameters of mice bearing metastatic Lewis lung carcinoma tumors

The in vitro and in vivo effects of murine recombinant interferon-gamma (rIFN-gamma) on hematopoietic and immune parameters of normal mice and of mice bearing metastatic variant Lewis lung carcinoma (LLC-C3) tumors were assessed. The in vitro addition of rIFN-gamma to bone marrow or spleen cells from normal and LLC-C3-bearing mice reduced their capacity to grow into colonies in soft agar (CFU) and minimized their immune suppressive activities. In vivo studies showed that when LLC-C3 tumor-bearing mice were injected with rIFN-gamma for 2 days prior to sacrifice, there was a reduction in femoral bone marrow cellularity, CFU, and suppressor cell activity. In contrast, spleen cells of tumor-bearing mice that were injected with rIFN-gamma showed reduced blastogenesis, and increased spleen cellularity, CFU, and suppressor cell activity. Thus, short-term rIFN-gamma treatment of LLC-C3-bearing mice may be beneficial with regard to the bone marrow because it caused a decrease in hematopoiesis and suppressor cell activity, whereas it may be detrimental in the spleen because it appeared to stimulate hematopoiesis and increase splenic suppressor cell activity. The dichotomy between the in vitro versus in vivo effects of rIFN-gamma on splenic hematopoiesis and suppressor activity may be due to the stimulation of production of colony-stimulating factor (CSF) activities by spleen cells of rIFN-gamma-treated mice. Our results suggest that the tumor stimulation of hematopoiesis and its associated appearance of immune suppressor cells can be both positively and negatively altered by rIFN-gamma.     

Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice.

Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18(+/+) and CD18(-/-) unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18(+/+) neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18(-/-) bone marrow cells. These data show that the neutrophilia in CD18(-/-) mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18(-/-), CD18(-/-)E(-/-), CD18(-/-)P(-/-), EP(-/-), and EPI(-/-) mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P <.05) or 70% (P <.01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.     

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.     

Factor-independent erythropoietic progenitor cells in leukemia induced by the myeloproliferative leukemia virus

The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform fibroblasts, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of growth factor requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (16%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few factor-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.     

The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2-4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.