Changes in protease during differentiation of mouse myeloid leukemia cells.

The protease activities of mouse myeloid leukemia cells Ml were examined using fluorescein isothiocyanate-labeled albumin as substrate. Protease activity in Ml cells was greatest at alkaline pH values with a maximum at pH 11.0, and only slight activity was seen at neutral and acidic pHs. When Ml cells were induced to differentiate into mature cells by lipopolysaccharide, their alkaline protease activity decreased greatly with marked increase in acid protease activity. Moreover, in a variant cell line Mml with the properties of differentiated Ml cells, no protease activity was found at alkaline pH values.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells.

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducers including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polyacrylamide slab gel electrophoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with L-[14C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells. P180 was solubilized from 125I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Transport of vitamin B12 into mouse leukemia cells.

Transport of [⁵⁷Co]cyanocobalamin (vitamin B₁₂) into L1210 murine leukemia cells, mediated by transcobalamin-II, is a biphasic process. The primary step, which occurs very rapidly (within the first minute), appears to involve binding of the B₁₂-transcobalamin complex to the external membrane of the cells; at 37 °C and pH 7.3, apparent K_m and V values are 180 pM and 5.9 pmol/min/10⁹ cells, respectively. This step is relatively insensitive to temperature, has an apparent pK_a of 6.0, and is inhibited by EDTA; Ca²⁺ or Mg²⁺ can reverse the inhibition. The slower secondary step appears to involve translocation of the vitamin into the cell; V for this step is 0.4 pmol/min/10⁹ cells. Temperature and pH optima are 30 ° and 6.5?7.0, respectively. This step is stimulated by glucose and inhibited by cyanide, arsenite, arsenate, p-chloromercuriphenylsulfonate and dinitrophenol. Labeled B₁₂ in an amount corresponding to that taken up in the initial step can be released by: (a) resuspending the cells in a B₁₂-free medium; (b) addition of unlabeled B₁₂-transcobalamin-II; and (c) addition of EDTA. The material released is chromatographically and functionally identical with B₁₂-transcobalamin-II.     

Characterization of lysozyme synthesized by differentiated mouse myeloid leukemia cells.

Lysozyme was induced by dexamethasone during normal differentiation of cultured mouse myeloid leukemia cells (M1) to macrophages and granulocytes. A large amount of lysozyme was produced by macrophage-like line cells (Mm-1), established from spontaneously differentiated macrophage-like cells from a clonal line of M1 cells. Lysozyme purified from the culture medium of these Mm-1 cells (Mm-1 lysozyme) had a molecular weight of 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and showed maximal activity at pH 6.6 with an optimal NaCl concentration of 0.04 M. Its mobility on polyacrylamide gel electrophoresis at pH 4.5 was distinctly lower than those of lysozymes from hen egg white and human urine. Rabbit anti-Mm-1 lysozyme serum inhibited the activities of lysozyme preparations from peritoneal macrophages of normal mice and rats and dexamethasone-induced differentiated M1 cells, but not those of preparations from hen egg white and human urine. Lysozyme was also purified from normal mouse lung, which is rich in alveolar macrophages and was found to be similar to lysozyme purified from the culture medium of Mm-1 cells in size and electrophoretic mobility and in its pH optimum, trypsin peptide map, and antigenicity. Thus the molecular structure of the lysozyme induced in differentiated mouse myeloid leukemia cells is similar to that of lysozyme produced by normal cells.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.     

Amphotropic host range of naturally occuring wild mouse leukemia viruses.

Seven murine leukemia virus field isolates (uncloned) from wild mice (Musmusculus) of four widely separated areas in southern California show an unusually wide in vitro host range. They replicate well in human, feline, canine, guinea pig, rabbit, rat, and mouse cells, whereas bovine, hamster, and avian cells are resistant. Since this host range includes that of both mouse tropic (ecotropic) and xenotropic murine leukemia viruses, they are designated as "amphotropic". No purely xenotropic virus component is detectable in these field isolates. They may represent the "wild" or ancestral viruses from which the ecotropic and xenotrophic murine leukemia virus strains of laboratory mice have been derived.     

Organization and stability of endogenous xenotropic murine leukemia virus proviral DNA in mouse genomes.

In a series of blot hybridization experiments, using a xenotropic envelope probe and restriction enzymes known to cut xenotropic proviral DNA a single time (EcoRI) or not at all (HindIII), we have studied the organization and relationship of endogenous xenotropic env-related sequences in various mouse strains. Multiple copies (18 to 28) of xenotropic env-reactive fragments were found in all mouse DNAs after digestion with either HindIII or EcoRI, and the majority of fragments were of sizes compatible with their origin from full-length proviral DNA. Five HindIII and five EcoRI restriction fragments were common to all inbred mouse DNAs tested. In addition, each strain exhibited unique characteristic xenotropic env-reactive bands; these bands were remarkably stable during many years of inbreeding. The cleavage patterns characteristic of each strain were also useful for showing genealogical relatedness among the various inbred mice.     

Detection of polymorphism in BALB/c leukemia viruses with mouse antisera.

Antisera produced in mice recognize primarily type-specific antigenic determinants on both the major core protein, p30, and the major envelope proteins, gp70 and p15(E), of the endogenous leukemia viruses (MuLV) of BALB/c mice. Three different mouse sera were investigated in detail. (i) Antisera prepared in C57BL/6 mice against the AKR leukemia K36 reacted with the gp70, p15(E), and p30 proteins of MuLV. Certain pools of the C57BL/6 anti-AKR K36 serum contained antibodies which serologically distinguished the p30 proteins of N-ecotropic, B-ecotropic, and xenotropic BALB/c MuLV. (ii) Antisera prepared in BALB/c mice against the BALB/c sarcoma 1315 contained antibodies that reacted with a type-specific antigen of the 1315 MuLV gp70 that is not found on other BALB/c MuLV. (iii) The normal sera of multiparous BALB/c mice contained antibodies that reacted with gp70 and p15(E) proteins of ecotropic MuLV. Sera from some of these mice contained antibodies that serologically distinguished the gp70 of N-ecotropic and B-ecotropic BALB/c viruses. These results emphasize the utility of mouse antisera in the serological typing of MuLV. Furthermore, the antigenic differences observed in the p30 and gp70 proteins should be of particular use in the future analysis of recombinant BALB/c MuLV.     

Adenosine transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes.

The intracellular accumulation of free [³H] adenosine was measured by rapid kinetic techniques in P388 murine leukemia cells in which adenosine metabolism (phosphorylation and deamination) was completely prevented by depletion of cellular ATP and by treatment with deoxycoformycin. Nonlinear regression of integrated rate equations on the data demonstrate that the time courses of labeled adenosine accumulation at various extracellular adenosine concentrations in zero-trans and equilibrium exchange protocols are well described by a simple, completely symmetrical, transport model with a carrier:substrate affinity constant of about 150 μM. Adenosine transport was not affected by 1 mM deoxycoformycin indicating that this analog has a low affinity for the nucleoside transport system. The transport capacity of dog thymocytes and peripheral leukocytes was similar to that of P388 cells. Transport was not inhibited by deoxycoformycin and remained constant during the first two hours after mitogenic stimulation with concanavalin A. In untreated, metabolizing P388 cells transport was found to be the major determinant of the rate of intracellular metabolism, regardless of the extracellular adenosine concentration (up to at least 160 μM), but the long-term accumulation (longer than 30–60 seconds) of radioactivity from extracellular adenosine strictly reflected the rate of formation of nucleotides (mainly ATP). The metabolism of adenosine by whole cells was entirely consistent with the kinetic properties of the transport system and those of the metabolic enzymes. At low exogenous adenosine concentrations (1 μM and below) transport was slow enough to allow direct phosphorylation of most of the entering adenosine. The remainder was deaminated and rapidly converted to nucleotides via inosine, hypoxanthine, and IMP. At concentrations of 100 μM or higher, on the other hand, influx exceeded the maximum velocity of adenosine kinase about 100 times so that most of the entering adenosine was deaminated. But since the maximum velocity of adenosine deaminase exceeded those of nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase about 5 and 100 times, respectively, hypoxanthine and inosine rapidly exited from the cells and accumulated in the medium. A 98% reduction of adenosine transport (at 100 μM), caused by the transport inhibitor Persantin, inhibited adenosine deamination by whole cells to about the same extent as transport, whereas adenosine phosphorylation was relatively little affected; thus in the presence of Persantin, transport and metabolism resembled that occurring at the low adenosine concentration. These and other results indicate that adenosine deamination is an event distinct from transport, which occurs only subsequent to adenosine's transport into the cell.     

Distribution of endogenous murine leukemia virus DNA sequences among mouse chromosomes.

We used mouse-Chinese hamster somatic cell hybrids which lose mouse chromosomes to examine the distribution of murine leukemia virus DNA sequences in the genome of A/HeJ mice. We analyzed total cellular DNA from various hybrid clones for the presence of viral sequences by molecular hybridization and used the Southern blot hybridization procedure to identify viral DNA in cellular restriction endonuclease fragments. Our results show that murine leukemia virus DNA sequences are distributed among many mouse chromosomes in this strain. Chromosome 4 was shown to contain murine leukemia virus DNA sequences.     

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.     

Immunocytochemical detection of a murine leukemia virus-related nuclear antigen in mouse oocytes and early embryos.

The expression of murine leukemia virus (MuLV)-related antigens in oocytes and early embryos of Swiss mice was studied by the unlabeled antibody peroxidase-antiperoxidase method using an antiserum to the major core protein, p30, of AKR MuLV. This procedure resulted in an intense staining, at antibody dilutions up to 1:500, of the germinal vesicles of oocytes and the interphase nuclei of early embryos. Nuclear staining was restricted to a specific period of oocyte growth and embryo development. It developed gradually in oocytes having reached one third to one half the full-grown size and persisted until meiotic maturation. In early embryos, nuclear staining was present from the one-cell stage to the morula, but disappeared during transition from morula to blastocyst and was not seen in expanded blastocysts. Nuclear staining was abolished by absorption of the anti-p30 serum with detergent-disrupted virions of Gross(A), AKR, Kirsten and Moloney MuLV, and Kirsten MSV(MuLV), but not with the Friend and Rauscher strains of MuLV, the xenotropic BALB:virus 2, a mouse tropic and an amphotropic clone of wild mouse MuLV, or with FeLV and RSV. On the basis of these results, it is suggested that the nuclear antigen (termed germinal vesicle antigen) reacting with the anti-p30 serum is the product of a cellular gene having a normal function in early embryonic development, and that sequences related to this gene are incorporated into the genome of AKR-type MuLV.     

Dissociation of interferon effects on murine leukemia virus and encephalomyocarditis virus replication in mouse cells.

Two subclones of Swiss mouse cells infected with Moloney murine leukemia virus (M-MuLV) were tested for their response to interferon (IFN). Whereas M-MuLV production in the two subclones was inhibited to the same extent, one of the subclones was significantly more sensitive to IFN when the antiviral effect was measured by replication of encephalomyocarditis (EMC) virus. The same subclone was also more sensitive to the anticellular activities of IFN. Additionally, NIH 3T3 cells infected with M-MuLV were completely resistant to IFN actions when EMC virus replication or the anticellular activities were tested. However, under the same conditions, M-MuLV production was completely inhibited by IFN. These results indicate that IFN may affect cell growth functions and EMC replication through mechanisms different from those by which MuLV production is inhibited.