Hemopoietic Colony-forming Cells Research Articles

Role of damage-sensing/processing genes in the radiation response of haemopoietic in vitro colony-forming cells

Purpose : To characterize the role of various cellular damagesensing, processing and survival genes in the in-vitro radiosensitivity of haemopoietic colony-forming cells. Materials and methods : Bone marrow cells from a range of different gene-knockout mice were irradiated in vitro with graded radiation doses and assayed for colony-forming efficiency. Results : Colony-forming efficiency in the nulls was often lower by up to threefold compared with the wild-types. This was noticeable in particular for the atm, bax and p21 nulls. Radiosensitivity was markedly increased in the scid mouse (about 2.3-fold), more than in the atm null mouse (about 1.7-fold). There was resistance in the p53 nulls compared with the wild-types, using two different background strains, that gave similar results. There was slight sensitization in the p21 nulls. In the bcl-2 nulls, there was sensitization at low dose, but not at high dose. In contrast, in the bax nulls, there was protection at low dose, but again not at high dose. The heterozygotes for p53, bcl-2 and bax responded similarly to the wild types, so that no gene dosage effects were identified. Conclusions : These studies are the first to elucidate the role of as many as six relevant genes in the radiosensitivity of a single cell type. They show the greater importance of 'survival' genes at lower cytotoxic doses of radiation compared with the greater importance of 'damage-sensing' genes at higher doses.

111 oral The role of damage-sensing/processing genes in the radiation response of haemopoietic colony-forming cells

111 oral The role of damage-sensing/processing genes in the radiation response of haemopoietic colony-forming cells

Gamma-Ray-Induced Cell Killing and Chromosome Abnormalities in the Bone Marrow of p53-Deficient Mice

Resistance to the lethal effects of ionizing radiation has been demonstrated in a wide variety of cell types with defects in the p53 gene (thymocytes, splenic B and T cells, in vitro hemopoietic colony-forming cells and intestinal cells of the mouse, embryo cells of the rat, and human Burkitt's lymphoma cells). In contrast, Slichenmeyer et al. (Cancer Res. 53, 4164-4167, 1993) found no evidence of resistance in fibroblasts derived from p53 null mice. The aim of our study was to compare the radiation response of hemopoietic colony-forming cells (in vitro CFC) and of fibroblastoid colony-forming cells or units (CFU-F) within the same tissue (marrow) in p53 null mice (-/-), heterozygotes (+/-) and wild-type animals (+/+). We have also tested the hypothesis that, in proliferating cells, radiation-induced cell killing is mediated through chromosome damage by examining the relationship between these end points in hemopoietic cells of the three mouse types. Both in vitro CFC and CFU-F of -/- mice were resistant to cell killing compared with +/+ and +/- mice whose cellular sensitivities were indistinguishable. The resistance was characterized by a broader "shoulder" on the cell survival curve, i.e. a higher extrapolation number but similar D0 values using the multitarget model or a lower alpha coefficient using the linear-quadratic model. The frequency of chromosomally abnormal marrow cells after irradiation was similar for the three genotypes. However, marrow cells with aberrations carried more aberrations in -/- mice than in +/+ or +/- mice such that the total number of aberrations per 100 cells was higher in -/- mice. Since there were no differences in the yields of aberrations between genotypes in spleen lymphocytes or in CFU-F (both noncycling at the time of irradiation) and less mitotic inhibition in -/- marrow cells than in +/+ or +/- cells, the chromosomal radiosensitivity of -/- marrow hemopoietic cells might be related to reduced cell cycle delay allowing insufficient time for repair, but other explanations have been considered. We postulate that the radiation resistance of both hemopoietic CFC and CFU-F in -/- mice is a consequence of the failure of DNA/chromosome damage to trigger apoptosis or permanent cell cycle arrest to the same extent as in the +/+ or +/- mice: hence the lack of correlation between chromosome damage and cell death in the three mouse types.

Haemopoietic cell responses in the blood and bone marrow of sheep infected with the abomasal nematode Telodorsagia circumcincta

The generation of bone marrow and blood haemopoietic progenitor colony-forming cells (CFCs) in sheep given primary or challenge infections with the nematode parasite Telodorsagia circumcincta is described. Ten days after a primary infection, the frequency of early multipotential-CFCs, eosinophil-CFCs, macrophage-CFCs and mast cell or basophil-CFCs was greater than in controls. These frequencies then declined to pre-infection levels by day 21. Blood CFCs (mainly macrophage-CFCs and eosinophil-CFCs) also increased after infection, indicating a migration of CFCs, presumably to the site of infection. Ten days after challenge infection there was less marked myelopoiesis than in the primary infection on day 10, though both eosinophil-CFCs and mast cell or basophil-CFCs were significantly above control values. Blood CFC output (mainly macrophage-CFCs and eosinophil-CFCs) reached a peak 2-6 days after challenge, evidence of rapid recruitment to the site of infection. Telodorsagia circumcincta infection is therefore associated with an increase in myelopoiesis, particularly for the cell types characteristic of the local inflammatory response to abomasal nematodes. There was no correlation between any of the haemopoietic cell responses measured and worm burdens in individual animals after either primary or challenge infection.

Haemopoietic colony-forming cells from peripheral blood stem cell harvests: cytokine requirements and lineage potential.

We have measured the in vitro growth requirements of progenitor cells released into the blood of cancer patients following administration of chemotherapy and cytokines. In order to distinguish the direct effects of cytokines on progenitors from those activating accessory cells, we have compared clonogenic growth before and after CD34-positive selection of progenitors, in serum-free conditions. CD34 selection had little effect on the cytokine requirements of erythroid colony-forming cells and single cytokines, particularly interleukin-3, could support considerable colony growth in both mononuclear and CD34+ cell suspensions. Optimal erythroid colony growth, however, usually required the addition of a combination of stem cell factor and interleukin-3, in addition to erythropoietin, which was always required. Maximal numbers of granulocyte-monocyte progenitors in mononuclear cell cultures, could be achieved with a mixture of stem cell factor, interleukin-3 and granulocyte-monocyte colony stimulating factor. However, after CD34 selection, full myeloid colony growth was only achieved when granulocyte colony stimulating factor was added to the above mixture. This presumably reflects loss of accessory cells, during CD34 selection, which produce this cytokine. When transplanted after 8 d of culture, 16/22 myeloid colonies from erythropoietin-free cultures of peripheral blood stem cell harvests, could generate secondary erythroid colonies implying that these progenitors are multipotential. However, surface marker analysis of individual erythroid colonies revealed only the occasional presence of granulocytes and monocytes. These data demonstrate that cytokine mixtures are required for optimal colony growth, particularly after CD34 selection, and that most mobilized, blood clonogenic cells are multipotential.

Purification and adhesion receptor phenotype of ovine bone marrow-derived haemopoietic colony-forming cells

Ovine haemopoietic progenitor cells that form colonies (CFC) in soft agar cultures were compared to more mature bone marrow cells for their level of expression of the adhesion receptor molecules ovine (ov) CD44, ov CD11a (LFA-1) and ov CD58 (LFA-3) as well as the 175-antigen using specific monoclonal antibodies. Ov CD44, ov CD11a and ov CD58 were expressed on all CFC of the myeloid (non-erythroid) series, whereas ov CD44 and ov CD11a expression was very low or absent from a small number of blast and erythroid series CFC. Within the mature non-erythroid population of myeloid cells, neutrophils retained a low level of expression of ov CD11a. Most CFC representing all lineages strongly expressed the ov CD44 antigen. In contrast, the majority of CFC lacked the 175-antigen, as did bone marrow lymphocytes, basophils and mast cells. This property of CFC was exploited in a negative selection technique using panning and immunomagnetic beads to select CFC from other bone marrow cells with a 116–125-fold enrichment, 12–14% purity and 29–40% yield. These results demonstrate that ovine CFC express some of the molecules necessary to allow adhesion to haemapoietic stromal cells and vascular endothelium in the tissues. Future studies will concentrate on the function of the adhesion receptor molecules in medullary and extra-medullary haemopoiesis and inflammatory cell development in sheep.

Precursors (pre-CFCmulti) of multilineage hemopoietic colony-forming cells quantitated in vitro. Uniqueness of IL-1 requirement, partial separation from pluripotential colony-forming cells, and correlation with long term reconstituting cells in vivo.

Pluripotential colony-forming cells (CFCmulti) from mouse marrow can expand significantly in number during 4 days of suspension culture with IL-1 and IL-3. In this study, the cells ("pre-CFCmulti") which originate this response are characterized in terms of their frequency, progeny number, factor requirements, buoyant density, and extent of restoration following marrow transplantation. Parallel measurements of both CFCmulti and cells providing long term marrow reconstitution in vivo allowed direct comparisons to be made with pre-CFCmulti. The proliferative response of pre-CFCmulti was found to depend uniquely on the combination of IL-1 and IL-3, and neither of these regulators was replaceable by any of IL-4, IL-6, IL-7, GM-CSF, G-CSF, M-CSF or LIF. After separation on density gradients, pre-CFCmulti were recovered in fractions of lower density than most of the CFCmulti, but in the same fractions that contained most of the in vivo reconstituting cells. After irradiation and marrow transplantation, marrow CFCmulti were restored to near normal levels, while both pre-CFCmulti as well as reconstituting stem cells remained profoundly depressed. These results show pre-CFCmulti to be distinct from most CFCmulti and to represent the closest approach to quantitative detection of reconstituting stem cells so far achieved in vitro.

Responses of Hemopoietic Precursors to 13-cis Retinoic Acid and 1,25 Dihydroxyvitamin D3 in the Myelodysplastic Syndromes

To determine the effects of the "maturation-inducing" agents 13-cis retinoic acid and 1,25 dihydroxyvitamin D3 on marrow cells from normal individuals and patients with myelodysplastic syndromes (MDS), we assessed marrow hemopoietic clonogenicity and differentiation response patterns to these agents. These vitamins caused increased proliferation in vitro of normal clonogenic marrow myeloid precursor cells (CFU-GM), decreased erythroid precursors (BFU-E), and no change in multipotent stem cells (CFU-GEMM). Marrow hemopoietic colony-forming cell incidence was generally subnormal in the 22 MDS patients evaluated. In vitro exposure to both agents caused various patterns of alteration of MDS hemopoietic colony and cluster formation, with similar but more pronounced effects evoked by retinoic acid. In the vast majority of MDS patients, enhanced marrow clonal granulocyte-monocyte differentiation and decreased BFU-E growth were noted after in vitro exposure to these vitamins. Correlation of biological effects was demonstrated between in vivo changes of peripheral neutrophil counts and in vitro responses of myeloid precursors for ten MDS patients treated with an eight-week therapeutic course of retinoic acid. Cytogenetic analyses indicated persisting aneuploidy or coexisting normal and aneuploid karyotypes in the cultured MDS myeloid cells and (with one exception) in native marrow cells from the treated patients. The varying responses of the MDS cells may monitor differing proportions of normal versus leukemic marrow cells susceptible to proliferative and differentiative expression on exposure to these agents.

Responses of hemopoietic precursors to 13-cis retinoic acid and 1,25 dihydroxyvitamin D3 in the myelodysplastic syndromes

To determine the effects of the “maturation-inducing” agents 13-cis retinoic acid and 1,25 dihydroxyvitamin D3 on marrow cells from normal individuals and patients with myelodysplastic syndromes (MDS), we assessed marrow hemopoietic clonogenicity and differentiation response patterns to these agents. These vitamins caused increased proliferation in vitro of normal clonogenic marrow myeloid precursor cells (CFU-GM), decreased erythroid precursors (BFU-E), and no change in multipotent stem cells (CFU-GEMM). Marrow hemopoietic colony-forming cell incidence was generally subnormal in the 22 MDS patients evaluated. In vitro exposure to both agents caused various patterns of alteration of MDS hemopoietic colony and cluster formation, with similar but more pronounced effects evoked by retinoic acid. In the vast majority of MDS patients, enhanced marrow clonal granulocyte-monocyte differentiation and decreased BFU-E growth were noted after in vitro exposure to these vitamins. Correlation of biological effects was demonstrated between in vivo changes of peripheral neutrophil counts and in vitro responses of myeloid precursors for ten MDS patients treated with an eight-week therapeutic course of retinoic acid. Cytogenetic analyses indicated persisting aneuploidy or coexisting normal and aneuploid karyotypes in the cultured MDS myeloid cells and (with one exception) in native marrow cells from the treated patients. The varying responses of the MDS cells may monitor differing proportions of normal versus leukemic marrow cells susceptible to proliferative and differentiative expression on exposure to these agents.

Radiosensitivity of Murine Hemopoietic Colony-Forming Units Assayed in Situ in the Rib and in Other Marrow Sites

The radiosensitivity of murine hemopoietic colony-forming cells, which produce colonies in situ and which were counted at Day 8 after irradiation in sections of the femur, humerus, sternum, and spleen, is characterized by a D0 value of 91 +/- 9 cGy. The radiosensitivity of such cells in the rib was assessed using a new technique measuring regeneration or ablation of marrow in transverse sections of ribs observed at Day 8 after irradiation. The mean D0 value over a range determined using several different criteria was 108 cGy. These results provide evidence for the common assumptions that radiosensitivity measured using conventional transplantation assays reflects radiosensitivity in situ, and that the radiosensitivity of stem cells in different medullary marrow sites is similar. The techniques could be used with other species where assays for stem cells are not available.

4-Hydroperoxycyclophosphamide inhibits proliferation by human granulocytemacrophage colony-forming cells (GM-CFC) but spares more primitive progenitor cells

Despite its considerable toxicity to haemopoietic colony-forming cells, 4-hydroperoxycyclophosphamide (4-HC) has successfully been used to purge marrow of leukaemic cells before it is used to rescue patients from high-dose chemoradiotherapy. These conflicting observations indicate that haemopoietic progenitor cells that are not detected by the established colonyforming assays survive exposure to 4-HC and repopulate the marrow. The recent finding that murine spleen colony-forming cells (CFU-S) are resistant to 4-HC [Porcellini A, et al. (1983) Expl Hemat. 11 (suppl 14) 331 (abstract)] [14] also indicates that sensitivity to 4-HC can be used to distinguish primitive progenitor cells from committed progenitor cells. As part of a study on the nature of a population of blast colony-forming cells in human bone marrow, we tested their sensitivity to 4-HC to see whether they also are spared by the drug. We found that 4-HC had much less effect on the blast colony-forming cells than on the granulocyte-macrophage colony-forming cells (GM-CFC). This result suggests that the blast-colony-forming cells may be early human haemopoietic progenitor cells.

Enzymatic Treatment of Long-Term Human Marrow Cultures Reveals the Preferential Location of Primitive Hemopoietic Progenitors in the Adherent Layer

AbstractRecent studies with long-term mouse marrow cultures have indicated the importance of the adherent layer as a primary reservoir of the most primitive stem cells, from which derivative stem cells and more differentiated progenitors are continuously generated. We have now examined the role of the adherent cell layer in long-term human marrow cultures from this point of view. Prerequisite to such an undertaking was the development of a nontoxic and reproducible method for detaching the adherent layer and making it into a single-cell suspension suitable for characterization by colony assays. Both trypsin and collagenase could be used to obtain suspensions that met these criteria. Lack of toxicity was demonstrated by the preservation of CFU-E, BFU-E, and CFU-C plating efficiency in fresh human marrow cell suspensions exposed to the same enzymatic treatments. Collagenase treatment of long-term marrow culture adherent layers was considered superior because it freed all hemopoietic colony-forming cells but left some of the other cells still adherent. Using this method, we found that CFU-C, BFU-E, and CFU-G/E were consistently detectable in the adherent layer for at least 8 wk, with the majority of the BFU-E and CFU-G/E being located in the adherent layer (70%-75% after 2–3 wk and more than 90% by 7–8 wk). Although corresponding numerical differences in adherent and nonadherent CFU-C populations were not observed, the colonies derived from them showed marked differences in the size they achieved; the adherent layer being the exclusive site of CFU-C, with a very high proliferative capacity. These findings emphasize the importance of assessing the progenitor content of the adherent layer of long-term human marrow cultures and provide an appropriate methodology.

Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer

Recent studies with long-term mouse marrow cultures have indicated the importance of the adherent layer as a primary reservoir of the most primitive stem cells, from which derivative stem cells and more differentiated progenitors are continuously generated. We have now examined the role of the adherent cell layer in long-term human marrow cultures from this point of view. Prerequisite to such an undertaking was the development of a nontoxic and reproducible method for detaching the adherent layer and making it into a single-cell suspension suitable for characterization by colony assays. Both trypsin and collagenase could be used to obtain suspensions that met these criteria. Lack of toxicity was demonstrated by the preservation of CFU-E, BFU-E, and CFU- C plating efficiency in fresh human marrow cell suspensions exposed to the same enzymatic treatments. Collagenase treatment of long-term marrow culture adherent layers was considered superior because it freed all hemopoietic colony-forming cells but left some of the other cells still adherent. Using this method, we found that CFU-C, BFU-E, and CFU- G/E were consistently detectable in the adherent layer for at least 8 wk, with the majority of the BFU-E and CFU-G/E being located in the adherent layer (70%-75% after 2–3 wk and more than 90% by 7–8 wk). Although corresponding numerical differences in adherent and nonadherent CFU-C populations were not observed, the colonies derived from them showed marked differences in the size they achieved; the adherent layer being the exclusive site of CFU-C, with a very high proliferative capacity. These findings emphasize the importance of assessing the progenitor content of the adherent layer of long-term human marrow cultures and provide an appropriate methodology.

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells.

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.

Factors altering the ability of multipotential hemopoietic colony-forming cells to self-generate or form progenitor cells

Individual in vitro colonies grown from multipotential cells have been analysed for their content of colony-forming cells including BFU-E, Mix-CFC and non-erythroid CFC. The CFC content of mixed colonies was higher in colonies grown from spleen cells than in colonies grown from bone marrow cells and was unaffected by the addition of thymocytes or postendotoxin serum. When the incubation period was extended from 7 to 14 days, CFC content of mixed colonies increased. However, plate mapping and reculturing of individual mixed colonies demonstrated that different subsets of mixed colonies were present in day 7 and day 14 cultures. Although the number of mixed colonies developing decreased when stimulated by decreasing concentrations of pokeweed mitogen-stimulated spleen cell conditioned medium (SCM) the mean number of cells per colony remained unaltered and the content of CFC in such colonies increased. With the SCM concentration range tested, as the SCM concentration was decreased, the absolute number of mixed colonies containing CFC rose from 0.6 per culture to 2.4 per culture. Over this same SCM concentration range the mean number of CFC per mixed colony increased from 1.3 to 185.2. These data suggest that amplification within mixed colonies of the number of cells capable of colony formation may occur at the expenses of the production of differentiating cells.

Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors.

We report identification of a unique class of human hemopoietic colony-forming cells with extensive ability to generate progenitors for secondary colonies. Mononuclear cells isolated from human umbilical cord blood formed colonies consisting of 40-500 blast cells after 25 d of incubation in methylcellulose culture in the presence of erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes. Replating of these blast cell colonies revealed that 100% of the primary colonies had the ability to generate secondary colonies, including multipotential colonies. These colonies could be distinguished from other hemopoietic colonies in situ by the complete absence of signs of terminal differentiation. Replating of granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies, consisting of an average of 2 x 10(4) cells, revealed less capacity for secondary colony formation. This human blast cell colony assay may provide a method for quantitation of more primitive hemopoietic stem cells than progenitors for GEMM colonies (CFU-GEMM) in man.

The Production of Committed Hemopoietic Colony-Forming Cells From Multipotential Precursor Cells In Vitro

Murine fetal liver cells have been fractionated by fluorescence-activated cell sorting into two fractions termed. For convenience, the pre-CFC and CFC (colony-forming cell) fractions, which differ in their relative binding of the lectin, pokeweed mitogen. The CFC fraction contained a high frequency of CFC (26%) and in liquid cultures stimulated with colony-stimulating factors generated large numbers of differentiated progeny. Consequently, residual CFC could not be detected in such cultures after day 4. In contrast, the pre-CFC fraction contained relatively few CFC (1%) but contained the majority of CFU-S and was able to generate large numbers of new CFC after 5-7 days in liquid culture. This production of CFC from precursor cells was absolutely dependent on factors present in pokeweed-mitogen-stimulated spleen cell conditioned medium (PWM-SCM) or postendotoxin serum (ES) but was not stimulated by known granulocyte or macrophage colony-stimulating factors. CFC production occurred from multipotential precursor cells and all types of CFC were detected (excluding lymphocytes). Clonal analysis of pre-CFC showed that most of the generated CFC arose from relatively few multipotential precursors some of which could generate up to 500 CFC. The data suggested a differentiation sequence of the multipotential precursor cells in which differentiation potentials are successively restricted in the order macrophage, eosinophil, granulocyte-macrophage leads to granulocyte, megakaryocyte, erythroid. The frequency of CFC-generating cells in the pre-CFC fraction was significantly less (ten-fold) than that of CFU-S, suggesting that pre-CFC may be a more ancestral subset of CFU-S. The cell fractions and assay systems described should be of use in defining and purifying factors regulating early phases of hemopoietic differentiation and in defining the differentiation steps involved in the restriction of potentialities of multipotential cells.

The production of committed hemopoietic colony-forming cells from multipotential precursor cells in vitro

Murine fetal liver cells have been fractionated by fluorescence- activated cell sorting into two fractions termed. For convenience, the pre-CFC and CFC (colony-forming cell) fractions, which differ in their relative binding of the lectin, pokeweed mitogen. The CFC fraction contained a high frequency of CFC (26%) and in liquid cultures stimulated with colony-stimulating factors generated large numbers of differentiated progeny. Consequently, residual CFC could not be detected in such cultures after day 4. In contrast, the pre-CFC fraction contained relatively few CFC (1%) but contained the majority of CFU-S and was able to generate large numbers of new CFC after 5–7 days in liquid culture. This production of CFC from precursor cells was absolutely dependent on factors present in pokeweed-mitogen-stimulated spleen cell conditioned medium (PWM-SCM) or postendotoxin serum (ES) but was not stimulated by known granulocyte or macrophage colony- stimulating factors. CFC production occurred from multipotential precursor cells and all types of CFC were detected (excluding lymphocytes). Clonal analysis of pre-CFC showed that most of the generated CFC arose from relatively few multipotential precursors some of which could generate up to 500 CFC. The data suggested a differentiation sequence of the multipotential precursor cells in which differentiation potentials are successively restricted in the order macrophage, eosinophil, granulocyte-macrophage leads to granulocyte, megakaryocyte, erythroid. The frequency of CFC-generating cells in the pre-CFC fraction was significantly less (ten-fold) than that of CFU-S, suggesting that pre-CFC may be a more ancestral subset of CFU-S. The cell fractions and assay systems described should be of use in defining and purifying factors regulating early phases of hemopoietic differentiation and in defining the differentiation steps involved in the restriction of potentialities of multipotential cells.

Clonal analysis of early and late stages of erythroleukemia induced by molecular clones of integrated spleen focus-forming virus.

The integrated proviral DNA of the polycythemia-inducing isolate of Friend spleen focus-forming virus (SFFVp) has been identified in rat cell clones nonproductively infected with this replication-defective erythroleukemia virus and cloned in phage lambda vectors. These lambda SFFVp recombinants, lambda SFFVp502 and lambda SFFVp542, contain endonuclease EcoRI inserts of size 7.4 and 8.2 kilobases, respectively, and include full copies of the SFFVp genome, along with host flanking sequences. Infectivity of the cloned SFFVp genomes was tested by a two-step DNA transfer procedure involving transfection of the cloned DNA into 3T3 mouse fibroblasts or cotransfer of the cloned DNA into thymidine kinase-deficient 3T3 cells together with the cloned thymidine kinase gene of herpes simplex virus, followed by rescue of the transferred DNA by superinfection with a helper virus. Inoculation of the rescued virus into adult mice resulted in the appearance of spleen foci, rapid splenomegaly, and polycythemia. Early after infection, spleen cell populations contained large numbers of cells capable of forming small erythroid colonies in vitro (CFU-E) in the absence of erythropoietin. Late after infection, these mice contained cells capable of forming macroscopic colonies (CFU-FV) in vitro. These data indicate that molecular clones of SFFVp, in conjunction with a helper virus, induce the appearance of hemopoietic colony-forming cells characteristic of both the early and late stages of Friend leukemia.

63] Measurement of effects of interferon on hemopoietic colony-forming cells: Effects of mouse interferon

This chapter discusses the measurement of effects of interferon on hemopoietic colony-forming cells. Colony growth from routine GM-CFC is dependent upon the addition of a granulocyte-macrophage colony-stimulating factor (GM-CSF). The GM-CFC culture method provides an experimental system that allows (a) enumeration of GM-CFC in different organs, (b) assay of GM-CSF levels, (c) assay of colony-inhibiting factors, (d) studies on multiplication rates and patterns of differentiation of GM cells in culture, and (e) studies on the responsiveness of GM-CFC to GM-CSF after exposure to various agents in vivo and in vitro . Commonly used sources of GM-CSF are media conditioned by tissue culture cells, for example, embryo fibroblasts or L cells; media conditioned by tissue fragments, for example, lungs; and mouse serum, particularly after inoculation of bacterial endotoxin. With interferon, the degree of inhibition by a given concentration is inversely related to the concentration of GM-CSF in the culture, indeed, whenever high GM-CSF concentrations are used, the inhibitory effect of interferon may be masked. Two types of inhibitor are described. One type is thought to affect the GM-CFC or their immediate progeny and the other type is thought to exert its effect by modulating the production of GM-CSF by macrophages.