ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.     

Activation of the Jun N-terminal kinase pathway by friend spleen focus-forming virus and its role in the growth and survival of friend virus-induced erythroleukemia cells

Members of the mitogen-activated protein kinase (MAPK) family, including Jun amino-terminal kinase (JNK) and extracellular signal-related kinase (ERK), play an important role in the proliferation of erythroid cells in response to erythropoietin (Epo). Erythroid cells infected with the Friend spleen focus-forming virus (SFFV) proliferate in the absence of Epo and show constitutive activation of Epo signal transduction pathways. We previously demonstrated that the ERK pathway was constitutively activated in Friend SFFV-infected erythroid cells, and in this study JNK is also shown to be constitutively activated. Pharmacological inhibitors of both the ERK and JNK pathways stopped the proliferation of primary erythroleukemic cells from Friend SFFV-infected mice, with little induction of apoptosis, and furthermore blocked their ability to form Epo-independent colonies. However, only the JNK inhibitor blocked the proliferation of erythroleukemia cell lines derived from these mice. The JNK inhibitor caused significant apoptosis in these cell lines as well as an increase in the fraction of cells in G(2)/M and undergoing endoreduplication. In contrast, the growth of erythroleukemia cell lines derived from Friend murine leukemia virus (MuLV)-infected mice was inhibited by both the MEK and JNK inhibitors. JNK is important for AP1 activity, and we found that JNK inhibitor treatment reduced AP1 DNA-binding activity in primary erythroleukemic splenocytes from Friend SFFV-infected mice and in erythroleukemia cell lines from Friend MuLV-infected mice but did not alter AP1 DNA binding in erythroleukemia cell lines from Friend SFFV-infected mice. These data suggest that JNK plays an important role in cell proliferation and/or the survival of erythroleukemia cells.     

Spontaneous regression of Friend virus-induced erythroleukemia. III. The role of macrophages in regression.

The spontaneous regression of erythroleukemia induced by the RFV strain of Friend virus is a macrophage-dependent process. Functional suppression or elimination of the macrophage population in leukemic mice with silica, carrageenan, anti-macrophage serum, or trypan blue inhibited regression. Prior protection of the macrophages with PVNO allowed regression in silica or carrageenan-treated mice. Macrophage phagocytic activity was inhibited in about half the RFV-induced leukemic mice at 25 to 30 days post virus inoculation. Those animals with normal macrophages regressed, whereas whereas those with inhibited macrophages did not. Progressor mice could be induced to regress by inoculation with normal syngeneic macrophages; other cell types were ineffective. The inhibition of macrophage function in leukemic mice was the result of infection of the macrophages by virus. Removal of the infected cells by cytolysis with anti-virus antiserum and C restored the phagocytic activity of the population. Inhibited macrophages were less capable of responding to immobilized antigen-antibody complexes than normal macrophages, suggesting that the loss of function was due to a change in their Fc receptor.     

Persistence of infectious Friend virus in spleens of mice after spontaneous recovery from virus-induced erythroleukemia.

Persistent infectious virus was detected in the majority of spleens of (C57BL/10 X A.BY)F1 mice after spontaneous recovery from Friend virus-induced erythroleukeima. The Friend murine leukemia helper virus (F-MuLV) was detected in titers up to 3 X 10(5) PFU/g of spleen. The defective spleen focus-forming virus (SFFV) was present in much lower titers and could be detected in cell-free spleen homogenates only after amplification of virus titer by growth of virus in vitro on SC1 cells. The incidence of cells producing F-MuLV alone in spleens after recovery from leukemia was 0.003 to 0.3%, and the incidence of cells producing both F-MuLV and SFFV was less than 0.0001 to 0.01%. In most recovered mouse spleens there appeared to be a selective reduction of SFFV relative to F-MuLV.     

Synthesis of tetrahydrobiopterin in friend erythroleukemia cells and its modulator effect on cell proliferation

The induction of the enzymes in the tetrahydrobiopterin pathway by dimethyl sulfoxide (DMSO) was investigated in subclones F4N and B8/3 of the proerythroblastoid Friend erythroleukemia cell line (MEL). GTP-cyclohydrolase, the initial enzyme in the biosynthetic pathway, is virtually absent in both clones, but expression increases during 3 days of DMSO treatment. The final enzyme levels show 12-fold (subclone B8/3) and 40-fold (subclone F4N) increases compared to initial values. Enhancement of 6-pyruvoyl tetrahydropterin synthase activity is detectable 6 h after exposure to DMSO and continues to increase in the 3-day time period to 2.4-fold and 1.8-fold levels in subclones B8/3 and F4N, respectively. Sepiapterin reductase is present in unstimulated F4N cells and absent in B8/3 cells. The enzyme activity is not affected by DMSO treatment in either cell line. This explains why DMSO treatment causes accumulation of tetrahydrobiopterin in the MEL subclone F4N, but not in subclone B8/3. MEL cells are devoid of phenylalanine hydroxylase for which tetrahydrobiopterin serves as cofactor. In F4N, but not in B8/3, tetrahydrobiopterin modulates the rate of [3H]thymidine incorporation, thus being functionally linked with cell proliferation rather than with differentiation. In contrast to T lymphocytes, periods of tetrahydrobiopterin synthesis and of modulator function are uncoupled in MEL cells.     

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor.     

Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.     

A 2.4-kilobase-pair fragment of the Friend murine leukemia virus genome contains the sequences responsible for friend murine leukemia virus-induced erythroleukemia.

Friend murine leukemia virus (F-MuLV) is a replication-competent, ecotropic, NB-tropic retrovirus which produces a rapidly fatal erythroleukemia in susceptible strains of mice. We previously molecularly cloned the entire F-MuLV genome. Transfection of this cloned DNA into NIH 3T3 mouse fibroblasts produces a virus with the same leukemia-inducing characteristics as F-MuLV. To identify which portion of the F-MuLV genome is responsible for causing leukemia, we made recombinant viruses between subgenomic fragments of F-MuLV DNA and another retrovirus--Amphotroph clone 4070. Amphotroph clone 4070 is a replication-competent, amphotrophic, N-tropic virus which does not produce any detectable malignancy in mice. A 2.4-kilobase-pair fragment of F-MuLV DNA was isolated. This DNA fragment encompassed approximately 700 base pairs from the 3' end of the F-MuLV pol gene and 1.7 kilobase pairs of the env gene including all of gp70 and the N-terminal four-fifths of p15E. A molecularly cloned fragment of Amphotroph DNA was ligated to the 2.4-kilobase-pair F-MuLV DNA, and an 8.3-kilobase-pair hybrid F-MuLV-Amphotroph DNA was subcloned into a new plasmid (p5a25-H). Transfection of p5a25-H DNA into fibroblasts resulted in the production of a replication-competent, ecotropic, N-tropic retrovirus--5a25-H virus. Inoculation of this virus into newborn NIH Swiss mice caused leukemia within 4 to 6 months. The disease caused by 5a25-H was pathologically and histologically indistinguishable from the disease caused by F-MuLV. We conclude that the F-MuLV sequences needed to cause disease are contained in these 2.4 kilobase pairs of DNA.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Induction of differentiation of friend erythroleukemia cells with L histidinol

Addition of an analog of histidine, histidinol, together with lowering the level of histidine in the medium, can induce hemoglobin synthesis in murine erythroleukemia cells.     

Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia.

Resident peritoneal macrophages from normal mice were refractory to infection with the RFV or conventional strains of Friend virus (FV). Stimulation of DNA synthesis in the macrophage population by induction of an exudate in vivo or treatment in vitro with macrophage colony-stimulating factor resulted in productive infection following exposure to virus. Similarly, normal resident macrophages did not become infected in vivo following transfer to leukemic mice, while exudate macrophages did become infected. Bone marrow macrophage stem cells were stimulated to replicate and mature in clonal agar cultures in the presence of colony-stimulating factor. These replicating stem cells could be infected with RFV, as shown by virus production in the resultant progeny macrophages. Transfer of normal resident peritoneal macrophages to leukemic progressor mice caused regression of the disease. In contrast, transfer of normal bone marrow cells was ineffective in causing leukemia regression. During erythroleukemogenesis induced by RFV, macrophage precursor cells in all of the mice became infected with virus. In mice with a progressive and lethal leukemia, mature end-stage macrophages were produced which were also infected with virus. In mice in which the leukemia would later spontaneously regress, the infected stem cells were eliminated and the marrow became repopulated with uninfected cells. The resultant progeny macrophages which appeared in the peritoneal cavity were uninfected and thus capable of participating in or causing leukemia regression.     

Spontaneous regression of Friend virus-induced erythroleukemia. VI. Structural and antigenic differences between the regressing and conventional strains of virus.

The regressing and conventional strains of Friend virus were compared by neutralization assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping of the individual viral components. Neutralization rates of the two viruses differed in the presence of monospecific anti-gp70 antiserum and sera from regressed or immunized mice. Neutralization of regressing Friend virus, but not conventional Friend virus, occurred when the viruses were incubated with anti-p15(E) and complement. Human serum inactivated conventional Friend virus more rapidly than regressing Friend virus, probably as a result of virolysis induced by the reaction of viral p15(E) with human complement component C1. Structural differences between the viruses were detected in their gp70 viral glycoproteins and p15(E) and p12 proteins. Analysis of different stocks and clonal isolates of the viruses showed that the differences between the gp70 and p15(E), but not the p12 proteins, were associated with the regressing phenotype of the regressing strain of Friend virus.     

Membrane-bound redox proteins of the murine Friend virus-induced erythroleukemia cell

We have obtained and studied a 105,000-g pellet from T-3-Cl-2 cells, a cloned line of Friend virus-induced erythroleukemia cells. By difference spectrophotometry, the pellet was shown to contain cytochrome b5 and cytochrome P-450, hemeproteins that have been shown to participate in electron-transport reactions of endoplasmic reticulum and other membranous fractions of various tissues. The pellet also possesses NADH-cytochrome c reductase activity which is inhibited by anti-cytochrome b5 gamma-globulin, indicating the presence of cytochrome b5 reductase. This is the first demonstration of membrane-bound forms of these redox proteins in erythroid cells. Dimethyl sulfoxide-treated T-3-Cl-2 cells were also shown to possess membrane-bound cytochrome b5 and NADH-cytochrome c reductase activity. We failed to detect soluble cytochrome b5 in the 105,000-g supernatant fraction from homogenates of untreated or dimethyl sulfoxide-treated T-3-Cl-2 cells. In contrast, erythrocytes obtained from mouse blood were shown to possess soluble cytochrome b5 but no membrane-bound form of this protein. These findings are supportive of our hypothesis that soluble cytochrome b5 of erythrocytes is derived from endoplasmic reticulum or some other membrane structure of immature erythroid cells during cell maturation.     

Transferrin receptors and iron utilization in DMSO-inducible and -uninducible friend erythroleukemia cells

Dimethylsulfoxide (DMSO) induces hemoglobin synthesis and erythroid differentiation of Friend erythroleukemia cells in vitro. Induction is accompanied by increased transferrin-binding activity which is necessary for the cellular acquisition of iron from transferrin for hemoglobin synthesis. There are Friend cell variants in which hemoglobin synthesis is not induced by DMSO unless exogenous hemin is also present. In this study we have compared the inducibility of transferrin receptors and iron incorporation in DMSO-inducible (745) and -uninducible (M-18 and TG-13) Friend cell lines. Cellular transferrin-binding sites were estimated by Scatchard analysis of data obtained from specific binding of [125I]transferrin by the cells. Our results show that unlike 745, DMSO treatment of the variant cell lines M-18 and TG-13 does not result in increased transferrin-binding activity. The number of transferrin-binding sites and the rate of iron uptake is similar in uninduced 745 and DMSO-treated M-18 and TG-13 cells. Although exposure of M-18 cells to DMSO and hemin induces hemoglobinization, this treatment does not cause induction of transferrin receptors. These results indicate that the primary defect in M-18 cells may be the uninducibility of transferrin receptors. We have also shown that exposure of 745 cells to hemin during DMSO treatment prevents the induction of transferrin receptors, suggesting that hemin may control the expression of transferrin receptors in erythroid cells.     

Hemoglobin synthesis in cell hybrids formed between teratocarcinoma and friend erythroleukemia cells

Viable hybrid cells have been isolated following fusion of Friend erythroleukemia cells and undifferentiated teratocarcinoma cells. The hybrids formed between near-diploid parental cells resembled Friend cells in their ability to grow in suspension and to synthesize hemoglobin in the presence of the chemical inducers dimethyl sulfoxide (DMSO) and ouabain. Erythropoietin (EPO) was effective in inducing hemoglobin synthesis in some of the hybrid cell lines. The hemoglobins synthesized by the hybrids were of the adult forms, but were quantitatively different from those hemoglobins synthesized by the parental Friend cells, suggesting that the fusion event modulated the expression of the hemoglobin chain genes.