Modulation of in utero total body irradiation induced newborn mouse growth retardation by maternal manganese superoxide dismutase-plasmid liposome (MnSOD-PL) gene therapy.

To determine the effects of manganese superoxide dismutase (MnSOD) plasmid liposome (PL) maternal radioprotection on fetal mice, timed pregnant female mice (E14 gestation) were irradiated to 3.0 Gy total body irradiation (TBI) dose, and the number, weight and growth and development over 6 months after birth of newborn mice was quantitated compared with irradiated controls. Maternal MnSOD-PL treatment at E13 improved pup survival at birth (5.4±0.9 per litter) compared with non-irradiated 3.0 Gy controls 4.9±1.1. There was no statistically significant difference in newborn abnormalities, male to female ratio in newborn litters, or other evidence of teratogenesis in surviving newborn mice from MnSOD-PL treated compared with irradiated controls. However, E14 3 Gy irradiated pups from gene therapy-treated mothers showed a significant increase in both growth and overall survival over 6 months after birth (P=0.0022). To determine if transgene product crossed the placenta pregnant E13 mice were injected intravenously with hemagglutinin-epitope-tagged MnSOD (100 µg plasmid in 100 µl liposomes), then after 24 h, fetal mice, placentas and maternal tissues were removed and tested by both immunohistochemistry and reverse transcriptase-PCR for transgene and product. There was no evidence of transgene or product in placenta or any fetal tissue while maternal liver was positive by both assays. The data provide evidence for fetal radioprotection by maternal MnSOD-PL gene therapy before irradiation, which is mediated by an indirect bystander effect and is associated with a significant improvement in both survival at birth and growth and development of newborn mice.

Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis.

Adhesion molecules are probably required for retention of maturing lymphocyte precursors in bone marrow, where they closely interact with and are dependent on stromal cells. Lymphomyeloid cell lines avidly adhere to cloned stromal cell lines in culture and screening pairs of these resulted in a selection strategy for a new monoclonal antibody to a leukocyte adhesion molecule. Immunoprecipitation analyses and comparison to a previously described antibody showed that it recognizes the alpha 4 chain of the integrin, VLA-4. This antibody totally inhibited lymphopoiesis and retarded myelopoiesis in long-term bone marrow cultures. A similar selection strategy resulted in two additional antibodies which define a single 100-kD species on stromal cells. This stromal cell adhesion molecule is a potential counter-receptor/ligand for VLA-4 on murine lympho-myeloid cells. Our findings suggest a new role for VLA-4 in lymphoid progenitor-microenvironment interactions. Recognition molecules that function in cell migration and inflammation in peripheral tissues may be important for steady-state lymphopoiesis within bone marrow.

Dissociation of opsonized particle phagocytosis and respiratory burst activity in an Epstein-Barr virus-infected myeloid cell line.

A continuous tissue culture cell line (Karpas line 120), derived from a patient with acute myeloblastic leukemia, not only demonstrates myeloblastic morphology and in vitro expression of several myeloid-specific biochemical markers but also contains Epstein-Barr virus (EBV) nuclear antigen. The present studies demonstrate EBV-genome-specific DNA within the total cellular DNA by molecular hybridization, thus establishing the presence of stable viral genome integration. The cells demonstrate complex coordinated myeloid functions including ingestion, degranulation, and respiratory burst activity. Line 120 cells show a respiratory burst (superoxide and hydrogen peroxide generation and hexosemonophosphate shunt activity) in response to soluble (phorbol myristate acetate) and particulate (latex beads) stimuli, as do normal granulocytes. They ingest complement-opsonized particles (lipopolysaccharide-oil droplets, zymosan, and bacteria), and degranulate in response to them. However, unlike normal granulocytes, the line 120 cells do not demonstrate respiratory burst activity in response to these complementopsonized particles. The dissociation between ingestion of complement-opsonized particles and activation of oxygen-dependent bactericidal activity severely impairs bacterial killing as compared with normal polymorphonuclear phagocytes.

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation.

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of beta-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.

Alkaline phosphatase in Epstein-Barr viral nuclear antigen--positive cell lines.

The production and nature of alkaline phosphatase were studied in Epstein-Barr viral nuclear antigen-positive, surface membrane immunoglobulin negative-cell lines established from two patients, one with acute myeloid leukemia and one with acute lymphoblastic leukemia. The acute myeloid leukemia-derived cells contained myeloid alkaline phosphatase, while the acute lymphoblastic leukemia-derived cells contained lymphoid alkaline phosphatase. The presence of the myeloid-specific enzyme in a surface membrane immunoglobin--negative cell line suggests that the line is composed of myeloid precursor cells and that such cells may be susceptible to infection with Epstein-Barr virus.

Signal transduction through the EGF receptor transfected in IL-3-dependent hematopoietic cells.

An expression vector for the epidermal growth factor (EGF) receptor was introduced into the 32D myeloid cell line, which is devoid of EGF receptors and absolutely dependent on interleukin-3 (IL-3) for its proliferation and survival. Expression of the EGF receptor conferred the ability to utilize EGF for transduction of a mitogenic signal. When the transfected cells were propagated in EGF, they exhibited a more mature myeloid phenotype than was observed under conditions of IL-3-directed growth. Moreover, exposure to EGF led to a rapid stimulation of phosphoinositide metabolism, while IL-3 had no detectable effect on phosphoinositide turnover either in control or EGF receptor-transfected 32D cells. Although the transfected cells exhibited high levels of functional EGF receptors, they remained nontumorigenic. In contrast, transfection of v-erbB, an amino-terminal truncated form of the EGF receptor with constitutive tyrosine kinase activity, not only abrogated the IL-3 growth factor requirement of 32D cells, but caused them to become tumorigenic in nude mice. These results show that a naïve hematopoietic cell expresses all of the intracellular components of the EGF-signaling pathway necessary to evoke a mitogenic response and sustain continuous proliferation.

Bone marrow as a potential source of hepatic oval cells.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

Gene therapy approaches for stem cell protection.

Cytotoxic exposure of bone marrow and other non-hematopoietic organs containing self-renewing stem cell populations is associated with damage to the supportive microenvironment. Recent evidence indicates that radical oxygen species resulting from the initial oxidative stress persist for months after ionizing irradiation exposure of tissues including oral cavity, esophagus, lung and bone marrow. Antioxidant gene therapy using manganese superoxide dismutase plasmid liposomes has provided organ-specific radiation protection associated with delay or prevention of acute and late toxicity. Recent evidence has suggested that manganese superoxide dismutase transgene expression in cells of the organ microenvironment contributes significantly to the mechanism of protection. Incorporating this knowledge into designs of novel approaches for stem cell protection is addressed in the present review.

Intraesophageal MnSOD-plasmid liposome enhances engraftment and self-renewal of bone marrow derived progenitors of esophageal squamous epithelium.

We evaluated whether the improved esophageal radiation tolerance following Manganese Superoxide Dismutase (MnSOD)-Plasmid Liposomes was explained by improved engraftment of bone marrow-derived progenitors. C57BL/6NHsd female mice pretreated with intraesophageal MnSOD-PL were irradiated to 29 Gy to the esophagus and intravenously transplanted with marrow from male B6. 129S7-Gt (ROSA) 26S OR/J ROSA (Lac-Z+, G418-resistant) mice. After 14 days, esophagi were removed and side population and non-side population cells evaluated for donor multilineage (endothelin/vimentin/F480) positive esophageal cells. Serial intravenous transplantability was tested in second generation 29 Gy esophagus-irradiated mice. Esophagi from recipients receiving swallowed MnSOD-PL 24 h prior to irradiation demonstrated significantly increased esophageal repopulation with donor bone marrow-derived Lac-Z+, G418+, Y-probe+ multilineage cells (37.8+/-1.8>50 cell Lac-Z+ foci per esophagus) compared to irradiated controls (19.8+/-1.8) P<0.0001. Serial transfer to second-generation irradiated C57BL/6NHsd mice of intravenously injected SP or NSP first generation recipient esophagus cells was also significantly enhanced by MnSOD-PL intraesophageal pretreatment (74.4+/-3.6 SP-derived Lac-Z+ foci per esophagus, 48.6+/-5.4 NSP-derived) compared to irradiation controls (23.4+/-1.8 SP, 6.0+/-3.0 NSP), P<0.0001. Thus, intraesophageal MnSOD-PL administration enhances engraftment of marrow-derived progenitors.

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria.

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.

Role of beta 1 and beta 2 integrins in the adhesion of human CD34hi stem cells to bone marrow stroma.

Hematopoietic stem cell interaction with elements of the underlying stroma is essential for sustained normal hematopoiesis. Here we have determined that adhesion receptors in the integrin family play a role in promoting adhesion of human hematopoietic stem cells to cultured human marrow stromal cells. Enriched CD34hi progenitor cells expressed VLA-4, VLA-5, and at least one or more beta 2 integrins. Homogeneous marrow stromal cell monolayers capable of supporting proliferation of cocultivated CD34hi cells expressed VCAM-1 and fibronectin (ligands for VLA-4 and VLA-5) as well as ICAM-1 (ligand for LFA-1 and Mac-1). Adhesion-blocking experiments indicated that VLA-4/VCAM-1, VLA-5/fibronectin, and beta 2-integrin/ICAM-1 pathways all are important for CD34hi cell attachment to stromal cells. Consistent with this suggestion, IL-1 stimulation of stromal cells caused both increased VCAM-1 and ICAM-1 expression and increased attachment by CD34hi bone marrow cells. In addition, CD34hi cells utilized VLA-4 to adhere to purified VCAM-1 and employed VLA-5 (and to a lesser extent VLA-4) to adhere to purified fibronectin. Together these results suggest that CD34hi stem cells may utilize multiple integrin-mediated adhesion pathways to localize within specialized microenvironmental niches created by marrow stromal cells.

Granulocyte-macrophage colony-stimulating factor and tetradecanoyl phorbol acetate induce a distinct, restricted subset of primary-response TIS genes in both proliferating and terminally differentiated myeloid cells.

Induction of early-response genes (tetradecanoyl phorbol acetate [TPA]-induced sequences, or TIS genes; R.W. Lim, B.C. Varnum, and H.R. Herschman, Oncogene 1:263-270, 1987) by granulocyte-macrophage colony-stimulating factor (GM-CSF) and TPA was examined both in a factor-dependent murine cell line, 32D clone 3, and in mature human neutrophils. When GM-CSF-deprived 32D clone 3 cells were exposed to GM-CSF or to TPA, four TIS mRNAs (TIS7, TIS8, TIS10, and TIS11) were rapidly and transiently induced. However, neither GM-CSF nor TPA could induce accumulation of TIS1 mRNA in 32D clone 3 cells, even under superinducing conditions. Both GM-CSF and TPA also elicited rapid, transient expression of TIS8 and TIS11 mRNA in postmitotic human neutrophils. However, neither agent could induce accumulation of TIS1 mRNA in human neutrophils. TIS1 is a member of the nuclear receptor supergene family that codes for ligand-dependent transcription factors. Cell-type restriction of inducible transcription factors may contribute to developmental specification.

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes.

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.

Phenotypically distinct target cells for murine sarcoma virus and murine leukemia virus marrow transformation in vitro.

An in vitro hematopoietic microenvironment was established from explained fragments of bone marrow from adult noninbred NIH Swiss mice with the use of corticosteroid-reconstituted horse serum. Infection with Kirsten murine sarcoma virus (Ki-MuSV) with either a Rauscher murine leukemia virus (R-MuLV) or Balb:virus-1 helper virus coat reduced proliferation of granulocytic and pluripotent hematopoietic stem cells and produced neoplastic transformation of both macrophages and preadipocytes in the adherent cell population within a 4-week period. Ki-MuSV-transformed, virus-releasing macrophages formed clusters of 4-49 cells in 0.8% methylcellulose-containing medium in the absence of added colony-stimulating factor (CSF), synthesized lysozyme, ASD-chloroacetate substrate-specific esterase-M, and CSF, and produced tumors following inoculation iv into adult NIH Swiss mice or ip into newborn NIH Swiss mice. In cultures infected with helper leukemia viruses R-MuLV or Balb:virus-1, gradual transformation of a distinct cell phenotype was observed over a 9-week period with generation of increasing numbers of atypical myeloblasts and promyelocytes which showed dyssynchronous nuclear-cytoplasmic maturation, basophilic granulation, cytoplasmic vacuolation, and formation of incompletely maturing CSF-dependent granulocyte-macrophage colonies in vitro and small spleen colonies in vivo. These data demonstrated that rapid biologic expression of the murine sarcoma virus genome in specific adherent "stromal" marrow cells prevents detection of a more subtle helper-virus-induced dysmyelopoiesis in a distinct nonadherent cell population.

Granulopoiesis longevity in continuous bone marrow cultures and factor-dependent cell line generation: significant variation among 28 inbred mouse strains and outbred stocks.

Longevity of granulopoiesis in corticosteroid-supplemented continuous bone marrow cultures and generation of permanent WEHI-3 dialyzed conditioned medium (DCM)-dependent hematopoietic progenitor cell lines were evaluated for 28 inbred mouse strains and outbred stocks. Cultures from AKR/J, NZW, C58/J, DBA/2J, and C57BL/KsJ mice generated granulocytes for 45-58 weeks and released more than 10(5) murine sarcoma virus (MuSV) rescue units of endogenous retrovirus; nonadherent (NA) cells from 50% or more cultures produced permanent WEHI-3 DCM-dependent cell lines. Cultures from 129/J, BALB/cJ, SJL/J, RF/J, CD-1 Swiss, C57BR/cdJ, C57BL/Ka, CBA/J, RFM/Sn, NZB, C3H/HeJ, NIH Swiss [N:NIH(S)], C57BL/10J, C57L/J, SWR/J, HRS/J, and C57BL/6J mice generated granulocytes for 12-38 weeks; some released 10(1)-10(2) MuSV rescue units of retrovirus, and NA cells from less than 50% of cultures from all but SWR/J and C57L/J mice produced WEHI-3 DCM-dependent cell lines. Thus longevity of granulopoiesis in mouse continuous bone marrow cultures varies significantly among strains.

Murine leukemia viruses: induction of macrophage production of granulocyte-macrophage colony-stimulating factor in vitro.

Infection in vitro of freshly explanted N:NIH(S) mouse bone marrow with ectopic murine leukemia viruses produced an increase over control uninfected cultures in the 50 or more cell granulocyte-macrophage (GM) colonies and 10-49 cell clusters detected after 7 days of incubation in 0.3% agar at 37 degrees C and 7% CO2. This effect was observed only at plating densities above 5.0 X 10(4) cells/ml and was not observed with macrophage-depleted populations of colony-forming units of GM progenitor cells (GM-CFUc) purified by isopyknic density gradient centrifugation of nonadherent cells harvested from long-term bone marrow cultures. Fewer virus-infected, compared to uninfected, peritoneal exudate macrophages were required to stimulate the same number of GM colonies and clusters in a given number of purified GM-CFUc. In contrast, murine leukemia virus infection of T-lymphocytes or NIH/3T3 embryo fibroblasts did not stimulate release of GM-CFUc coloney-stimulating factor (CSF). Single Cell suspensions of virus-infected freshly explanted whole bone marrow grown in CSF concentrated from L929 or WEHI-3 cell-conditioned medium produced more GM-CFUc colonies and GM clusters/1 X 10(5) cells compared to single cell suspensions of uninfected marrow. This phenomenon suggests that the colonoy-forming cells responding to CSF from virus-infected marrow may have been different from those responding to L929 or WEHI-3 cell CSF. The data indicate that increased granulopoiesis observed following retrovirus infection in vivo or in long-term marrow cultures was attributable in part to virus stimulation of production of CSF by adherent marrow stromal cells including macrophages.

Central nervous system (CNS) relapse following chemotherapy of WF rat acute myelogenous leukemia: a model for human CNS leukemia.

The treatment of central nervous system (CNS) involvement in human acute myelogenous leukemia (AML) presents a serious therapeutic dilemma. In an attempt to study the pathophysiology of this diseases in an animal model, the incidence and sites of detection of CNS leukemia were evaluated in inbred rats receiving chemotherapy for the transplantable WF AML. Eight of 100 rats with untreated WF AML demonstrated CNS leukemia at death with concomitant widespread visceral infiltrates, ascites, bone marrow involvement, and peripheral blood leukemia. Similarly, 6 of 120 rats (5%) failing to attain a complete remission following adriamycin. Cytoxan, or cytosine arabinoside chemotherapy demonstrated CNS leukemia in addition to systemic disease at death. In contrast, 70 of 75 rats (93.3%) achieving a complete remission subsequently relapsed in the CNS, either in combination with widespread systemic disease in 48 (64.0%) or with minimal evidence of systemic relapse in 22 (29.3%). There was a greater frequency of detectable cerebral compared to spinal cord infiltrates in relapsing rats. The WF AML appears to be a valuable model for study of the mechanism and ultimately the prevention of CNS relapse following chemotherapy in human AML.

Pool size of pluripotential hematopoietic stem cells increased in continuous bone marrow culture by Friend spleen focus-forming virus.

Continuous mouse bone marrow cultures were infected with Friend murine leukemia virus. Production of nonadherent (NA) and adherent cells, granulocyte-macrophage colony-forming unit(s) of progenitor cells (GM-CFUc), pluripotential hematopoietic stem cells (CFUs), the self-renewal potential (Rs) of CFUs, and generation of factor-dependent (FD) multipotential and committed permanent stem cell cloned lines were measured. Uninfected marrow cultures from C57BL/6J, C57BL/6JUt, B6.S, C3H/HeJ, (C57BL/6J x DBA/2J)F1, CD- 1 Swiss, or N:NIH(S) mice generated NA cells, GM-CFUc, and CFUs for 20-41 weeks; cultures infected with Rauscher or other helper viruses generated them for 35-45 weeks. GM-CFUc and CFUs production in SFFV-positive cultures persisted for over 65 weeks and exceeded control levels by twentyfold to fiftyfold. The Rs of CFUs in SFFV-positive cultures was not detectably increased above control cultures. Multipotential (erythroid-neutrophil-mast cell-basophil-eosinophil) permanent FD cell clones were derived from control and SFFV-positive cultures. Thus SFFV amplifies the stem cell pool in vitro without detectably increasing the Rs capacity of CFUs.

Technique for preclinical evaluation of continuous infusion chemotherapy with the use of WF rat acute myelogenous leukemia.

A technique was developed for continuous iv infusion chemotherapy in an inbred rat model of acute myelogenous leukemia. Polyethylene tubing was inserted surgically into the internal jugular vein of adult WF rats, burrowed sc to the base of the tail, and connected to an infusion pump. A flexible spring was sutured at the base of the tail and fastened to the cage wall; it protected the infusion catheter and allowed movement of the rat within the cage. This technique was used to compare bolus with continuous infusion therapy with adriamycin, cytosine arabinoside, and neocarzinostatin. Only small differences were noted in host toxicity and in antitumor effect against tumor grown as a subcutaneous myeloblastoma. Nearly three times more neocarzinostatin was required by continuous infusion for an effect equivalent to that of bolus injection. In contrast, continuous infusion of methotrexate with concurrent thymidine infusion prevented toxicity, enhanced the antitumor effect, and prolonged survival. This infusion system should facilitate rapid preclinical evaluation of drugs considered for constant iv infusion therapy.

Virus and cell requirements for Friend virus granulocytic leukemogenesis in long-term bone marrow cultures of NIH swiss [N:NIH(S)] mice.

The effect of hematopoietic stem cell age on leukemogenesis in vitro was tested in nonrecharged, corticosterold-supplemented NIH Swiss [N:NIH(S)] mouse long-term bone marrow cultures infected with Friend murine leukemia virus of anemia-inducing strain (F-MuLV-A) or spleen focus-forming virus (SFFV) [Rauscher murine leukemia virus (R-MuLV)], a pseudotype virus derived by rescue of the SFFV genome from SFFV-Balb/3T3 clone A31 nonproducer cells with clonal helper R-MuLV. Cultures at 33 degrees C derived from 10-day-old or adult mouse marrow generated colony-forming unit culture granulocytic macrophage (CFUc) progenitor cells for over 20 weeks and colony-forming unit spleen cells for 14 weeks and generated permanent granulocytic leukemia cell lines after infection with F-MuLV-A at week 1, 2, or 4 but not at week 8. Leukemia lines were of granulocyte phenotype whether induced by F-MuLV-A or SFFV (R-MuLV) and synthesized myeloperoxidase and lysozyme but were restricted in ability to generate superoxide in response to phorbol myristate acetate stimulation. Cultures (31 degrees C) infected with temperature-sensitive (ts) helper virus mutant pseudotypes of SFFV as well as SFFV (R-MuLV) generated granulocytic leukemia lines, whereas only SFFV (R-MuLV) pseudotype virus-infected cultures became leukemic at 37 degrees C. R-MuLV wild type or ts mutant helper virus infection alone increased cell proliferation and numbers of CFUc but did not generate leukemia. These data indicated that gene(s) specific to F-MuLV-A or a virus rescued from SFFV-Balb/3T3 clone A31 nonproducer cells are required for transformation in vitro of a hematopoietic stem cell present in early but absent in late bone marrow cultures.