Strain Of Friend Virus Research Articles

Gp55 from Friend Erythroleukemia Virus Induces Oligomerization and Cell Surface Localization of a Truncated Form of the STK Receptor Tyrosine Kinase.

Friend disease is an acute erythroleukemia induced by various strains of Friend virus and provides an excellent model to study the multistage progression of carcinogenesis. In the first stage of the disease, the virus infects erythroid progenitor cells and a viral glycoprotein, gp55, interacts with both the EpoR and a truncated form of the stem cell derived tyrosine kinase (STK), Sf-stk. This results in Epo independent expansion of erythroid progenitor cells. Companion with proviral integration, inactivation of p53 and activation of ets gene family members (Spi-1/PU1, Fli-1) in the late stage of disease will cause leukemic transformation. The majority of gp55 is retained on the ER membrane, with only 5% transported to the cell surface. Previous data has demonstrated that the activation of EpoR by gp55 through the dualtropic and transmembrane binding domain on the cell surface is critical for the early stage Friend disease. However, the mechanism by which gp55 leads to Sf-stk activation remains largely unknown. Consistent with previous reports, we find that gp55 immunoprecipitates with Sf-stk under nonreducing conditions and that coexpression of Sf-stk with gp55 results in enhanced tyrosine phosphorylation of Sf-stk. Moreover, we find that Sf-stk forms homo-oligomers when coexpressed with gp55 and, by introducing point mutations in the four cysteine residues in the gp55 ecotropic domain, the cysteine 338 mediates homo-oligomer formation of Sf-stk. Furthermore, mutation of cysteine 338 to alanine inhibits the ability of gp55 to induce Epo independent polyclonal expansion of erythroid precursor cells. We also demonstrate that, in the absence of gp55, Sf-stk is primarily located in the ER, but coexpression of gp55 induces cell surface localization of the truncated receptor. Further, we show that by adding a 6aa ER retention signal to the C-terminal of Sf-stk (Sf-stk-ER), Sf-stk-ER cannot be processed to cell membrane. However, gp55 retains the ability to bind and induce the tyrosine phosphorylation of Sf-stk-ER, and Grb2 is recruited to both Sf-stk and Sf-stk-ER when gp55 is present. Despite active signaling, our preliminary work failed to detect Epo independent colonies from BM cells transfected with Sf-stk-ER followed by Friend virus infection, indicating that a productive interaction with EpoR at the surface may be required for the differentiation of infected progenitors in vitro. Taken together, our data supports a model in which gp55 interaction with Sf-stk through cysteine 338 in the ecotropic domain of gp55 leads to oligomerization of the receptor, resulting in enhanced autophosphorylation and recruitment of Grb2, as well as relocation of active Sf-stk to the cell surface.

Mutation of the Lyn tyrosine kinase delays the progression of Friend virus induced erythroleukemia without affecting susceptibility

During the initial phase of Friend virus (FV) induced erythroleukemia, the interaction between the viral envelope glycoprotein gp55, the Erythropoietin receptor (EpoR) and the naturally occurring truncated version of the Mst1r receptor tyrosine kinase, called Sf-Stk, drives the polyclonal expansion of infected progenitors in an erythropoietin independent manner. Sf-Stk provides signals that cooperate with EpoR signals to effect expansion of erythroid progenitors. The latter phase of disease is characterized by a clonal expansion of transformed leukemic cells causing an acute erythroleukemia in mice. Signaling by Sf-Stk and EpoR mediated by gp55 renders erythroid progenitors Epo independent through the activation of the EpoR downstream pathways such as PI3K, MAPK and JAK/STAT. Previous work has shown that Src family kinases also play an important role in erythropoiesis. In particular, mutation of Src and Lyn can affect erythropoiesis. In this report we analyze the role of the Lyn tyrosine kinase in the pathogenesis of Friend virus. We demonstrate that during FV infection of primary erythroblasts, Lyn is not required for expansion of viral targets. Lyn deficient bone marrow and spleen cells are able to form Epo independent FV colonies in vitro. In vivo infection of Lyn deficient animals also results in a massive splenomegaly characteristic of the virus. However, we observe differences in the pathogenesis of Friend erythroleukemia in Lyn−/− mice. Lyn−/− mice infected with the polycythemia inducing strain of FV, FVP, do not develop polycythemia suggesting that Lyn−/− infected erythroblasts have a defect in terminal differentiation. Furthermore, the expansion of transformed cells in the spleen is reduced in Lyn−/− mice. Our data show that Lyn signals are not required for susceptibility to Friend erythroleukemia, but Lyn plays a role in later events, the terminal differentiation of infected cells and the expansion of transformed cells.

Bcl-2 expression in F-MuLV-induced erythroleukemias: a role for the anti-apoptotic action of Bcl-2 during tumor progression

Erythroleukemias induced by various strains of Friend virus are multistage malignancies that result from the accumulation of genetic mutations, including the activation of proto-oncogenes and the inactivation of tumor suppressor genes. In this study, we demonstrate that Bcl-2 expression is activated in the majority of F-MuLV-induced erythroleukemia cell lines. In contrast, Bcl-2 was not expressed in any of the FV-P-induced erythroleukemia cell lines and protein levels were low or negligible in FV-A-induced erythroleukemia cell lines examined. In vivo, Bcl-2 expression levels gradually increased in F-MuLV-induced erythroleukemic cells prior to adaptation to culture. High expression of Bcl-2 in F-MuLV-induced erythroleukemic cells was shown to proceed the emergence of p53 mutation suggesting that Bcl-2 expression may delay p53 mutation in the leukemic cells. This is further supported by the demonstration that the majority of F-MuLV-induced erythroleukemia cell lines established from primary tumors induced in p53 mutant mice express low to negligible levels of Bcl-2. We have shown that the high levels of Bcl-2 expression in FV-P-induced erythroleukemic cells inhibited apoptosis induced by etoposide, low serum and p53 expression. Similarly, ectopic Bcl-2 expression within these cells also provided protection from apoptosis induced by etoposide and growth in low serum. These results suggest that the anti-apoptotic action of Bcl-2 may confer a selective in vivo and in vitro growth advantage to F-MuLV-induced erythroleukemic cells, which is not shared by FV-P/FV-A-induced erythroleukemic cells. The observed induction of Bcl-2 expression in vivo constitutes a novel but late oncogenic event associated with the progression of F-MuLV-induced erythroleukemias.

C-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-Myc and resultant depression of proliferation.

Potential role of protein kinase C on the differentiation of erythroid progenitor cells

The effect of protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinyl sulfonyl)-2-methyl piperazine(H7) onin vitro differentiation of erythroid progenitor cells which were isolated from spleens of mice infected with the anemia-inducing strain of Friend virus were examined. Erythropoietin-mediated differentiation of erythroid progenitor cells, as determined by the incorporation of59Fe into protoporphyrin, was inhibited by staurosporine and H7 in a concentration-dependent manner. Scatchard analysis of the3H-phorbol-12, 13-dibutyrate binding to erythroid progenitor cells revealed that at the high affinity sites the dissociation constant was 22nM and the maximum number of3H-phorbol-12,13-dibutyrate binding sites per cell was approximately 3.7×105. Cytosolic protein kinase C was isolated from erythroid progenitor cells and then purified by sequential column chromatography. Two isoforms of protein kinase C were found. Photoaffinity labeling of the purified protein kinase C samples with3H-phorbol 12-myristate 13-acetate followed by analysis of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autofluorography showed radiolabeled 82-KDa peptides. Radiolabeling of the 82-kDa peptides with3H-phorbol 12-myristate 13-acetate was almost completely blocked by excess unlabeled phorbol 12-myristate 13-acetate. Results of phorbol 12-myristate 13-acetate-promoted phosphorylation with the purified protein kinase C samples showed that the phosphorylation of 82-kDa peptides was increased as the concentration of phorbol 12-myristate 13-acetate was increased from 10−8 M to 10−4M. In light of the findings that erythroid progenitor cells possessed an abundance of protein kinase C and that staurosporine and H7 inhibited erythroid differentiation, it seemed likely that protein kinase C would play a role in the erythroid progenitor cell development.

Receptor binding of asialoerythropoietin.

The interaction of 125I-asialoerythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross-linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 125I-asialoepo with two classes of affinity constant: one with Kd = 0.02-0.03 nM and 300-400 per cell, the other with lower affinity (Kd = 0.9-1.2 nM) and 1,000-1,200 per cell. The Kd value for the high affinity site is one-third of that for the binding of native 125I-erythropoietin (125I-epo) to the same FVA cells (Kd = 0.08-0.1 nM). Using 125I-asialoepo or 125I-epo affinity cross-linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I-epo. These results indicate that 125I-asialoepo binds to the same receptors as 125I-epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross-linking experiment indicates that the two components do not represent the two classes of receptor.

Specific binding of 125 i‐rerythropoietin to friend polycythemia virus‐transformed erythroleukemia cells purified by centrifugal elutriation

We have used countercurrent centrifugal elutriation (CCE) to determine the distribution of cells with respect to cell volume and buoyant density for an erythroleukemia cell line (JG6) transformed by the polycythemia strain of Friend virus (FV-P), and to determine the effect of inducing the cells to differentiate with dimethylsulfoxide (DMSO) on this distribution. CCE made it possible to obtain suspensions of modal JG6 populations virtually free of dead cells and uniform with respect to volume and buoyant density. These modal populations were assayed for specific binding of erythropoietin (Epo). Between 500 and 550 Epo receptors per cell were detected. These belonged to a single class having a dissociation constant of 0.36 nM. DMSO induction of differentiation of the JG6 cells had no effect on the number of Epo receptors expressed.

Erythroid Cell Fusion in the Early Phase of Friend Virus Leukemogenesis<xref ref-type="fn" rid="FN2">2</xref>

Infection of susceptible strain mice with the oncogenic Friend erythroleukemia virus initially results in fulminant erythroid hyperplasia. Several weeks later a frank erythroid leukemia develops. At the earliest stages of Friend disease there is extensive cell fusion involving erythroid cells but not platelets and granulocytes. Fusion was detected in experiments with allophenic (chimeric) mice whose component strains express electrophoretically distinct forms of the dimeric enzyme glucose phosphate isomerase (GPI). Infection of such animals with the polycythemic strain of Friend virus resulted in the rapid development of Friend disease. Concomitant with the appearance of early disease symptoms was the appearance in the red cells of the heterodimeric form of GPI, an unequivocal consequence of cell fusion. Platelet and granulocyte samples from the same infected animals failed to exhibit the hybrid GPI form. Furthermore, no hybrid dimer was evident in red cells from chimeric mice in which blood formation had been stimulated by phenylhydrazine treatment. These observations suggest that the occurrence of cell fusion early after infection by Friend virus is a significant aspect in the rapid development of neoplastic disease.

In Vivo Distinction Between a Target Cell for Friend Virus (FVI) and Murine Hematopoietic Stem Cells2

Busulphan (BU) treatment of DBA/2 mice with hypertransfusion (HT)-induced polycythemia resulted in an ablation of detectable hematopoietic stem cells (CFUS) in pooled marrow from the long bones. Daily injections of erythropoietin (EP) stimulated an EP-responsive population of cells in the absence of detectable CFUS. Mice treated with BU and EP and having HT-induced polycythemia were inoculated with the polycythemia-inducing strain of Friend virus (FVP) and determinations were made for the presence of tumor colony-forming units (tCFU). No change in CFUS/10(6) bone marrow cells was detected as a result of EP treatment. However, tCFU were increased more than 100-fold in HT-BU-EP-treated mice compared with saline-treated controls. The demonstration of tCFU in mice in which CFUS were not detectable indicated that this leukemogenic effect of FVP could occur in the absence of the pluripotent stem cell. Furthermore, the increased numbers of this FVP target cell in the EP-stimulated, BU-treated mice with HT-induced polycythemia supported the model licating the target for this effect in the EP-responsive cell population.

Erythropoietin-independent erythroid colony formation in vitro by hemopoietic cells of mice infected with friend virus.

We have investigated the production of erythroid colonies in plasma culture by bone-marrow and spleen cells taken form C3Hf/Bi mice previously infected with a polycythemic strain of Friend virus (FV). Inclusion of erythropoietin (Epo) in the medium was found unnecessary for erythroid colony formation in vitro by these cells, although it was essential for the production of erythroid colonies by hemopoietic cells from normal animals. Development of erythroid colonies also proceeded umimpeded when cells from FV-infected animals were cultivated in medium pretreated with rabbit anti-serum that was shown to inactivate Epo. Thus, the hemopoietic tissues of FV-infected mice contained erythroid colony-forming units (CFU-Es) which appeared to be Epo-independent. When spleen cells from FV-infected mice were exposed to antiserum directed against syngeneic FV-infected spleen cells and complement, and then cultured with or without Epo, the number of erythroid colonies that developed was drastically reduced, indicating that the CFU-Es in these animals carried FV-induced antigen(s), and must themselves have been infected with virus. Electron microscopy of erythroid colonies produced by cells from FV-infected mice revealed the presence of budding and abundant free type-C virus particles. The efficiency of erythroid colony formation in vitro either with or without Epo by hemopoietic cells from FV-infected mice was substantially increased over that of cells from normal mice. The increase in the number of CFU-Es in these animals was due mainly to an increase in the number of Epo-independent CFU-Es. Epo-independent CFU-Es were first detected in bone marrow and spleen as early as 3 days after FV infection; thereafter their numbers progressively increased for at least 9 days. Hypertransfusion with red blood cells prior to FV infection reduced, while bleeding greatly increased, the efficiency of erythoid colony formation without Epo by cells from the spleens of the infected mice. The phenomenon of erythroid colony formation in plasma cultures lacking Epo provides a sensitive and reliable means of detecting Epo-independent CFU-Es, which appear to play a fundamental part in pathogenesis of the disease resulting from infection with the polycythemic strain of FV.

Erythrocyte production in mice infected by the polycythaemia-inducing Friend virus or by the anaemia-inducing Friedn virus.

Summary. The changes induced by infection with the anaemia‐inducing (FVA) or the polycythaemia‐inducing (FVP) strain of Friend virus on PCV, RBC, reticulocyte counts, red‐cell and plasma volume and reticulocyte maturation time were studied in Swiss mice for 7 weeks after viral infection. The data indicated that the increase in total blood volume is mainly due to the augmentation of the spleen and liver blood volume in FVA‐disease, whereas it results from the large numbers of red cells produced by the spleen in FVP‐disease.The daily output of reticulocytes was increased four‐fold in FVA infected mice, and thirty‐fold in FVP infected mice by the 50th day post‐infection. The total redcell production for the duration of the experiment was 3 times normal for the FVA group and 13 times normal for the FVP group. A theoretical estimate of the mean red‐cell lifespan in infected mice gave 8 1/2 days for the FVP‐disease and 22 days for the FVA‐disease as opposed to 40 days for the normal mouse.

The effect of histocompatibility-2 type on response to friend leukemia virus in mice.

Two types of quantitative response to the F-B strain of Friend virus in segregating generations of a cross involving a susceptible (DBA/2 or BALB/c; H-2(2)) and a resistant (C57BL/6; H-2(b)) mouse strain show a marked correlation with the H-2 type of the mice. Essential susceptibility, as determined by the splenomegalic response to high virus doses, is controlled by a single pair of alleles which segregates independently with respect to the H-2 locus. However, relative susceptibility, as determined by the incidence of the splenomegalic response at moderate or low levels of virus dosage, is significantly greater among mice homozygous or heterozygous for the H-2(d) allele than among H-2(b) homozygotes in these populations. In addition, the incidence of recovery from splenomegaly induced by a given level of virus dosage is significantly greater in H-2(b) homozygotes than in segregants of other H-2 types among their littermates. Possible mechanisms responsible for these effects are discussed.

Spleen Focus Formation by Polycythemic Strains of Friend Leukemia Virus

Several strains of Friend virus were tested for their ability to initiate focus formation in the spleens of susceptible mice. Strains which induce polycythemia had high focus-forming activity, while strains which induce anemia had little or no focus-forming activity. A positive correlation was also demonstrated between virus strains with focus-forming activity and those which initiate the resumption of erythropoiesis in plethorized mice. It is concluded that most Friend virus preparations contain at least 2 viruses: one which has spleen focus-forming activity (SFFV) and which probably is responsible for inducing polycythemia when present in high titers, and another which, when active in the presence of little or no SFFV, induces anemia. The role of either virus in initiating leukemia remains to be determined.

DETECTION OF AN AVIRULENT VIRUS APPARENTLY RELATED TO FRIEND VIRUS.

SUMMARYThe WM1‐B strain of Friend virus was originally recovered from mice which had been inoculated with WM1 virus. The question of relationship between the two strains was examined.Antisera prepared in stock C3H mice against either WM1‐B or WM1 virus neutralized WM1‐B virus but not WM1 virus. Q.I.M.R. mice infected with WM1 virus were resistant to superinfection with WM1‐B virus. These two findings indicated that a relationship existed between some component of the WM1 line and the leukaemogenic component of the WM1‐B line.By treatment of WM1‐B virus with C3H antiserum to WM1 virus, a strain of WM1 virus was obtained which did not produce antibodies to WM1‐B virus in C3H mice and did not protect Q.I.M.R. mice against challenge with WM1‐B virus, but which did produce the lymphoid hyperplasia typical of WM1 virus infection. This pure WM1 strain was still free of relationship to WM1‐B virus at the 8th passage.The original WM1 line therefore contained two viruses. One (WM1 virus) caused lymphoid hyperplasia in Q.I.M.R. mice, while the other (an avirulent variant of WM1‐B virus) caused no signs but was detectable by means of neutralization and protection tests.Seventeen wild mice were studied virologically. Strains of virus resembling pure WM1 virus were isolated from two wild mice, and an avirulent strain related to WM1‐B virus was obtained from a third, while both viruses were isolated from three other wild mice.These results support the view that WM1 virus and the avirulent form of WM1‐B virus are distinct.It was concluded that an avirulent virus, which is apparently related to Friend virus, occurs in wild mice in Queensland.