Erythroid stem cells in Rauscher virusinfected mice

Summary Using the technique for erythroid colony formation in vitro, bone marrow and spleen cells from NMRI mice were studied after Rauscher virus (RLV) infection. There was a substantial decrease in CFU_E concentrations during the first days after infection, this being more pronounced in the marrow than in the spleen. In the marrow values gradually return to and are maintained at control levels, while in the spleen a 40–50-fold increase is seen between days 8 and 18. In normal and RLV infected animals the same dose dependence of CFU_E growth for erythropoietin was seen. For normal and RLV infected cells in the absence of erythropoietin there were only very few background colonies. In exhypoxic plethoric mice the increase in CFU_E concentration seen in normal mice in the spleen, is delayed by 2–3 days.

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Zusammenfassung Die Zahl erythropoetischer Zellkolonien in vitro (CFU_E-Technik) wurde bei Rauscher-virusinfizierten NMRI-Mäusen in Knochenmark und Milz untersucht. Es ergab sich ein starker Abfall der CFU_E-Konzentration während der ersten Tage nach der Infektion, deutlicher im Knochenmark als in der Milz. Im Knochenmark werden dann wieder Kontrollwerte erreicht, während es in der Milz zu einem 40–50fachen Anstieg nach 8–18 Tagen kommt. Bei Rauscher-virusinfizierten Tieren wird die gleiche Abhängigkeit des CFU_E-Wachstums von Erythropoetin gefunden wie bei Kontrollen. Ohne Erythropoetinzugabe zu den Kulturplatten treten in beiden Gruppen nur geringe Untergrundwerte auf. Bei plethorischen Mäusen ist der Anstieg der CFUE-Konzentration in der Milz um 2–3 Tage verzögert.

     

Studies on the erythropoietic cell system in CBA mice after Rauscher virus infection

Summary Erythropoiesis in CBA mice was studied in Rauscher leukemia virus infected mice using the incorporation of⁵⁹ Fe into spleen, bone marrow and peripheral blood. Beginning at day 4 an increased uptake into the spleen and a decrease in the bone marrow and the peripheral blood was observed. The increased uptake by the spleen was also found in plethoric mice. The erythropoietin responsive compartment was also enlarged in the spleen of these mice. The dose-response-curve for erythropoietin was altered 4 days after infection, there was a higher background level of⁵⁹Fe incorporation and the response to low doses was better in infected animals. The reticulocytopenia which is usually seen in these mice, was overcome by administration of high doses of erythropoietin. It is concluded that the Rauscher virus acts in a similar manner to erythropoietin, but the erythropoiesis induced is ineffective since the cells do not mature. This maturation deficiency is influenced by administration of exogenous erythropoietin.Research supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 112 (Zellsystemphysiologie).     

Erythroid progenitors in paroxysmal nocturnal haemoglobinuria

In vitro colony formation of bone-marrow erythroid progenitor cells in patients with paroxysmal nocturnal haemoglobinuria (PNH) was examined. The numbers of early and late erythroid progenitors (BFU-E and CFU-E) showed wide variations; two cases out of eight cases of PNH showed decreased erythroid colony formation, but other cases showed normal or rather increased colony formation of BFU-E and CFU-E. The number of erythroid progenitors in patients with PNH may be related to the marrow cellularity.     

Susceptibility of mice reconstituted with trisomy 19 hematopoietic cells to infection with Rauscher leukemia virus

Summary Radiation chimeras with trisomy 19 hematopoietic cells were constructed to test the sensitivity of the trisomic hematopoietic system to infection with Rauscher leukemia virus: Hematopoietic cells from livers of trisomic fetuses were rescued by transplantation into lethally irradiated adult mice. These Ts 19 radiation chimeras show a stable and sufficiently long-lived trisomic hematopoiesis to allow experimental induction of Rauscher leukemia.Rauscher leukemia virus (RLV) induced a marked proliferation of erythroblasts in the spleens of Ts 19 mice and control chimeras within 3 weeks. The onset of erythroblast proliferation was significantly delayed in the Ts 19 mice, suggesting a smaller number of target cells for the RLV and/or reduced susceptibility of the target cells to RLV.Both Ts 19 and control chimeras developed nonlymphocytic leukemia 2–4 months after RLV injection. The course of leukemogenesis was similar in the two experimental groups.No numerical chromosome abnormalities associated with leukemogenesis were detectable in Ts 19 or control cells. The numbers of chromosomal sister chromatid exchanges 2 weeks after RLV injection were elevated to the same degree in both Ts 19 and control cells. Thus, cells with constitutional trisomy do not show increased chromosomal instability due to leukemogenesis.Supported by the Deutsche Forschungsgemeinschaft, Gr 71/50-3Dr A. Gropp died on 22 October 1983leave of absence from Bar Ilan University     

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.     

Erythroid progenitors in tumor-bearing mice: erythroid inhibitory factors produced by adenocarcinoma-755

Adenocarcinoma-755-bearing C57BL/6 mice developed anemia with the growth of the tumors. The numbers of granuloid and monocytoid progenitors (colony-forming unit in culture, CFU-C) of the bone marrow and spleen and the peripheral blood WBC counts increased in tumor-bearing mice. The erythroid progenitors (erythroid colony-forming units, CFU-E; erythroid burst-forming units, BFU-E) of the bone marrow showed a marked decrease, overcoming their increase in the spleen in tumor-bearing mice. Fractionation of conditioned medium of tumor cells led to the isolation of a protein of 80-kd molecular weight that stimulated murine CFU-C growth and inhibited the CFU-E and BFU-E growth in a dose-dependent manner. These results indicate that erythroid inhibitory factors produced by tumors exist in tumor-bearing mice. Erythroid inhibitory factors might also exist in non-hematological malignancies of humans, and they might be one of the mechanisms of anemia.     

Erythroid colony induction without erythropoietin by Friend leukemia virus in vitro.

Erythroid colonies could be produced without the addition of erythropeietin in plasma cultures seeded with bone marrow cells from normal C3Hf/Bi mice by exposure of the cells in vitro to medium from a cell line (IS) that continuously produces Friend leukemia virus in culture. The activity in the culture medium was viral rather than erythropoietin-like, since it was sedimentable by high-speed centrifugation and heat labile. Erythroid colonies did not develop when the bone marrow cells exposed to virus-containing medium were from mice genetically resistant to Friend virus. IS culture medium contained both Friend spleen focus-forming and XC-plaque-forming activities. No erythroid colonies were induced when genetically sensitive cells were exposed to a preparation from which the spleen focus-forming activity had been removed, but which contained XC plaque-forming activity in high concentration. Thus the spleen focus-forming component of Friend virus appeared to be responsible for inducing erythroid colony formation without erythropoietin in vitro. Some erythroid colonies were also found in control cultures to which neither virus nor erythropoietin had been added. Reduction in the concentration of fetal calf serum in the culture medium substantially decreased the number of these colonies but had only a minor effect on the number of virus-induced colonies. The number of erythroid colonies produced after 2 days of culture without erythropoietin or fetal calf serum was approximately proportional to the titer of Friend spleen focus-forming virus to whcih the bone marrow cells had been exposed. This system should prove useful for investigation in vitro of Friend virus--host cell interactions which lead to erythropoietin-independent erythropoiesis.     

Effect of a Rauscher leukemia virus vaccine upon chemical oncogenesis in the mouse.

The concept that type-C RNA viruses serve as determinants of chemically induced cancer would be supported if immunization against such viruses reduced the incidence of methylcholanthrene-induced sarcomas. A formalin vaccine was employed which was able to protect mice against the development of virus-induced Rauscher leukemia: 8/12 vaccinated mice survived versus 1/12 controls. When mice so immunized were challenged with near-threshold doses of chemical carcinogen, sarcoma incidence and death latency did not differ between vaccinated and control groups: within 10 months, a 320 mug dose of methylcholanthrene induced 100% sarcomas in both groups, while a 64 mug dose induced 62% and 65% tumors in vaccinated and control mice respectively. Thus, a relevant postulate of the oncogene hypothesis could not be supported by these studies. Formalin treatment neutralizes the oncogenic effect of mouse leukemia viruses yiwlding preparations that are immunogenic and can be successfully used as vaccines to protect against virus-induced leukemia (5). The finding of murine leukemia viruses in chemically induced tumors (1, 2) poses the question of whether these viruses are present in the tumors as passengers, or whether they are etiologically involved in the process of tumor induction. An approach to answering this question would be to challenge with chemical carcinogens mice which have been vaccinated with leukemia virus. If the virus were somehow involved in tumor induction, antiviral immunization might conceivably interfere with chemical carcinogenesis. Whitmire and Huebner (9) have reported that mice immunized with formalin-treated leukemia viruses, become resistant to sarcomagenesis by methylcholanthrene. Repetition of this experiment by Gericke and Chandra (6) gave non-significant differences between experimental and control groups. The present paper deals with the effect of immunization against Rauscher leukemia virus upon tumor induction by near-threshold doses of methylcholanthrene.     

The influence of macrophages on the leukemogenic activity of Rauscher murine leukemia virus

A single application of trypan blue 3 to 24 hours before or simultaneous with inoculation of the Rauscher leukemia virus (RLV) resulted in enhancement of leukemogenesis with an especially high viremia. Repeated administration of trypan blue after RLV infection resulted in reduced spleen weight. Viremia, however, was also increased compared with controls (no application of trypan blue), but in a lower rate than in mice treated with the virus plus trypan blue. Since trypan blue is known as an inhibitor of the functions of macrophages the results point at an important role of this cell type in preventing infection with this oncogenic retrovirus. In this regard there are differences in the role of macrophages in human immunodeficiency virus (HIV) infections.     

Mouse fetal antigens in Rauscher leukemia.

Recently, various reports on fetal antigens associated with the plasma membranes of virus-induced tumor cells have appeared. Hanna et al. also suggested the presence of a fetal antigen on the cell membrane of Rauscher virus-induced leukemia cells. We were able to demonstrate the presence of cross-reacting antibodies against embryonic cells and Rauscher leukemia cells in the sera collected from C57BL/6 mice immunized with Rauscher leukemia cells, employing the indirect immunofluorescence test. Such cross-reacting antigenicity was also found in AKR lymphoma cells by the absorption test. The antibody was completely absorbed with embryonic cells, but not with adult spleen cells or adult C57BL/6 thymocytes. The absorption test showed that this antigen was different from Gross and Friend-Moloney-Rauscher antigens. The antigen detectable by the serum first appeared in 12-day-old embryos. It was found abundantly in the cells from the digestive tract and lungs, but not in the cells from the liver or brain. A natural antibody against the fetal antigen was also found to exist in the sera from a few aged C57BL/6 mice. The presence of the natural antibody, however, could not be related to the history of pregnancy of the mouse.     

A fucose-deficient glycoprotein precursor to Rauscher leukemia virus gp69/71.

Rauscher leukemia virus glycoprotein gp69/71 is synthesized in virus-infected cells by way of a 90,000 dalton glycoprotein precursor, termed Pr2a+b. This precursor could be labeled with radioactive glucosamine and methionine but not with fucose; whereas gp69/71 could be detected by labeling with radioactive glucosamine, fucose, or a mixture of amino acids but seemed to be deficient in methionine relative to Pr2a+b. Pr2a+b and gp69/71, were specifically precipitated by an antiserum prepared against phosphocellulose purified Rauscher gp69/71. Other virus-specific precursors, in addition to Pr2a+b, could be precipitated by antiserum prepared against detergent disrupted virus. Neither Pr2a+b nor gp69/71 was precipitated from cell extracts by antisera to Rauscher p30. Tryptic maps of Pr2a+b and gp69/71 showed that these glycoproteins share many tryptic peptides. Pulse-chase experiments with 14C-labeled amino acids indicated that gp69/71 was not radio-labeled during the pulse-labeling period but slowly appeared during the chase incubations. Pr2a+b, however, was rapidly labeled and tended to disappear during long chases. Furthermore, two nonglycosylated viral proteins, termed p15E and p12E, are structurally related to Pr2a+b. Viral p15E and p12E contained the same methionine-containing tryptic peptide fraction as Pr2a+b as determined by ion-exchange chromatography. These results provide evidence that Pr2a+b is a precursor to gp69/71 and establish a structural and possible precursor-product relationship between Pr2a+b, p15E, and p12E.     

Erythroid blast crisis in chronic myelogenous leukemia

Blood or bone marrow specimens from 22 patients with chronic myelogenous leukemia in blast crisis were studied for the surface expression of glycophorin-A, a marker for early erythroid differentiation. The leukemic blasts were stained with rabbit anti- glycophorin-A antiserum. The glycophorin-A molecules detected by the rabbit antiserum were identified by polyacrylamide slab gel electrophoresis of the immunoprecipitates from the membrane lysates of surface-labeled blasts. Blasts expressing surface glycophorin-A were found in 9 of the 22 patients. In 4 patients, almost all blasts were glycophorin-A positive, and in 5 patients, less than half of the blast population expressed glycophorin-A. The present study shows that when glycophorin-A is used as a marker for erythroid blasts, involvement of the erythroid lineage during blast crisis of chronic myelogenous leukemia seems to occur more frequently than previously recognized.     

Erythroid progenitors in anemic Belgrade laboratory (b/b) rats

The anemia of Belgrade b/b rats has been shown to be due to intracellular iron deficiency. The aim of this study of erythropoiesis at the progenitor cell level in these rats was to determine if a defect is present in the early phase of red cell production. Both erythroid colony-forming unit (CFU-E)- and erythroid burst-forming unit (BFU-E)-derived colonies were found to be few in untreated b/b rats and made up of a small number of poorly hemoglobinized erythroblasts of different size and irregular cell shape. Following treatment with iron, the anemia of the rats improved, and the number of CFU-E-derived colonies and the number of cells per colony increased, but the peculiar erythroblast morphology persisted. The high serum level of biologically active erythropoietin (Ep) in b/b rats rules out inadequate Ep production as a cause of their anemia. The results presented indicate, in addition to the earlier described defective transmembrane iron transport, a defect in erythroid progenitor cells. The effect of iron treatment in these rats detected in vitro on erythroid progenitors confirms the importance of iron for cellular proliferation.