Transformation parameters in chicken fibroblasts transformed by AEV and MC29 avian leukemia viruses.

Infection of chicken fibroblasts with avian erythroblastosis virus (AEV) strain ES4 or with avian myelocytomatosis virus strain MC29 leads to a rapid morphological transformation of most cells. AEV-transformed fibroblasts are similar to Rous sarcoma virus (RSV)-transformed fibroblasts in that they exhibit microvilli at their surface, show a disappearance of actin cables, are agglutinable by lectins, and show a decrease in LETS protein and an increase in the rate of hexose uptake. They also elicit slightly increased levels of cell-associated proteolytic activity, but show no increase in the fibrinolytic activity of the harvest fluids. In addition, as shown previously, they are capable of anchorage-independent growth and of sarcoma induction.In contrast, MC29-transformed fibroblasts express a different pattern of transformation parameters. They are similar to both RSV- and AEV-transformed fibroblasts in that they are morphologically transformed, show a disappearance of actin cables and are agglutinable by lectins. They also elicit surface alterations which consist of bleb-like protrusions rather than of microvilli, and are capable of anchorage-independent growth. They are strikingly different from RSV- and AEV-transformed cells, however, in that they express normal levels of LETS protein and elicit no increase in the rate of hexose uptake or in proteolytic activity. They are not sarcomagenic although they show an accelerated growth rate in culture.In conjunction with the finding that MC29 and AEV do not contain sequences related to the fibroblast-transforming src gene of RSV, these results raise the possibility that MC29 and perhaps also AEV transform fibroblasts by a mechanism different from RSV.     

Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus.

The gag gene-related, nonstructural proteins of three avian acute leukemia viruses (namely, myelocytomatosis viruses MC29 and CMII and avian erythroblastosis virus) and of avian Fujinami sarcoma virus (FSV) isolated by immunoprecipitation from cellular lysates with anti-gag serum were shown to be phosphoproteins in vivo. The specific 32P radioactivity of the nonstructural proteins of MC29, CMII, and FSV was significantly higher than that of helper viral, intracellular gag proteins. Two of these proteins, i.e., the 140,000-dalton FSV and the 110,000-dalton MC29 proteins, were also phosphorylated in vitro by a kinase activity associated with immunocomplexes. This kinase activity is either separated from these proteins or inactivated by incubation of cellular lysates with normal serum followed by adsorption to staphylococcal protein A or sedimentation at 100,000 x g or both. It remains to be resolved whether the 110,000-dalton MC29 and 140,000-dalton FV proteins, in addition to being substrates for phosphorylation, also have intrinsic kinase activity.     

Response of hemopoietic cells to avian acute leukemia viruses: effects on the differentiation of the target cells.

Chicken bone marrow cells were infected with three avian acute leukemia viruses (ALV)--avian myeloblastosis virus (AMV), myelocytomatosis virus strain MC29 and Mill Hill 2 virus (MH2)--and then cultured in agar in the presence of conditioned medium. Under these conditions, it was found that very few cells served as target cells for these three viruses. Density gradient separation showed that ALV target cells were found primarily in the light density fractions and might be represented by cells committed to the mononuclear phagocyte pathway. Separation of bone marrow cells on the basis of their sedimentation velocity at unit gravity suggested that MC29 and AMV did not share the same target cells. In addition, the analysis of surface receptors and functional markers characteristic of macrophages (Fc and complement receptors, phagocytosis and immune phagocytosis) indicated that the ALV-transformed cells were blocked during their differentiation. These results indicate that the transforming ability of ALV interferes with the differentiation of their target cells.     

Characterization of the hematopoietic target cells of AEV, MC29 and AMV avian leukemia viruses

The hematopoietic target cells of the three prototype strains of replication defective avian leukemia viruses (DLVs) were studied, using a newly developed, quantitative in vitro transformation assay. Our results show that the target cells of avian erythroblastosis virus (AEV) belong to the erythroid lineage while those of myelocytomatosis virus 29 (MC29) and avian myeloblastosis virus (AMV) belong to the myeloid lineage. As judged from suicide experiments using BUdR incorporation and irradiation with visible light, a higher proportion of AEV- and AMV-target cells are in cycle than MC29-target cells. Using differentiation specific antisera directed against cell surface antigens, we could demonstrate that the target cells of AEV express erythroblast-specific antigen(s) and less intensively erythrocyte-specific antigen(s), while those of MC29 and AMV express myeloblast-specific antigen(s). In addition, MC29-target cells express macrophage-specific antigen(s). None of the AEV-target cells are adherent or phagocytic, while a small proportion of the AMV-target cells are adherent and about half of the MC29-target cells are both adherent and phagocytic. Our results support the concept that DLVs specifically transform certain types of committed erythroid and myeloid progenitor cells. The target cells of AEV and AMV appear to resemble the respective transformed cells in their state of differentiation, whereas those of MC29 appear to be more immature than the corresponding transformed cells.     

Altered protein kinase activities of lymphoid cells transformed by Abelson and Moloney leukemia viruses

Five different types of protein kinase activities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A-MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukemia virus (M-MuLV), lacking oncogene sequences in its genome. While cAMP-dependent protein kinase and casein kinase-1 do not undergo significant changes, casein kinase-2 rises in both A-MuLV and M-MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase-C on the other hand is unchanged in M-MuLV transformed cells but it undergoes a 2-3-fold increment in both soluble and particulate fractions of A-MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity.     

Salmonella typhimurium strains carrying independent mutations display similar virulence phenotypes yet are controlled by distinct host defense mechanisms

The outcome of Salmonella infection in the mammalian host favors whoever succeeds best in disturbing the equilibrium between coordinate expression of bacterial (virulence) genes and host defense mechanisms. Intracellular persistence in host cells is critical for pathogenesis and disease, because Salmonella typhimurium strains defective in this property are avirulent. We examined whether similar host defense mechanisms are required for growth control of two S. typhimurium mutant strains. Salmonella pathogenicity island 2 (SPI2) and virulence plasmid-cured Salmonella mutants display similar virulence phenotypes in immunocompetent mice, yet their gene loci participate in independent virulence strategies. We determined the role of TNF-alpha and IFN-gamma as well as different T cell populations in infection with these Salmonella strains. After systemic infection, IFN-gamma was essential for growth restriction of plasmid-cured S. typhimurium, while SPI2 mutant infections were controlled in the absence of IFN-gamma. TNFRp55-deficiency restored systemic virulence to both Salmonella mutants. After oral inoculation, control of plasmid-cured bacteria substantially relied on both IFN-gamma and TNF-alpha signaling while control of SPI2 mutants did not. However, for both mutants, ultimate clearance of bacteria from infected mice depended on alphabeta T cells.     

Microfilaments and intermediate filaments in epithelial cells transformed by murine sarcoma or leukemia viruses

The murine epithelial cell line MMC-E was used to study changes in the cytoskeletal organization associated with viral transformation of epithelial cells by two different viruses. The cells were transformed with Moloney mouse sarcoma virus (MSV) or murine leukemia virus (MuLV). The expression of actin, myosin and of intermediate filament proteins in the cells was then studied. In MMC-E cells actin and myosin were organized as belt-like structures at the edges of the border cells of the cell islands and also circumferentially in the cells inside the islands. The major change after transformation was the decrease of the actomyosin containing belt extending from cell to cell at the borders of the cell islands. Both MMC-E cells and the MSV-transformed cells contained keratin as a juxtanuclear granular aggregate whereas the MuLV-transformed cells showed bright fibrillar arrays of keratin. Both virus-transformed cell lines showed enhanced vimentin-specific fluorescence and analysis of their cytoskeletal polypeptides confirmed the result. Similar molecular forms of keratin polypeptides were seen in all cells by immunoblotting. Viral transformation of MMC-E epithelial cells thus leads to different changes in their cytoskeletal organization depending on the transforming viral or cellular gene.     

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.     

Granulocyte differentiation by Friend leukemia cells.

Friend leukemia cells growing in suspension culture are thought to represent a population of primitive erythroid cells which have undergone malignant transformation. We have found that when growing in vivo or in plasma clots in vitro, these suspension culture cells can exhibit morphologic and enzymatic properties which are characteristic of primitive granulocytic cells. The microenvironment in which the tumor cells grow plays a major role in determining the direction of differentiation of these leukemia cells. Hence it appears likely that the Friend cell is in fact a neoplastic pluripotent hematopoietic stem cell.     

Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42°C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42°C in semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42°C.     

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus.

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33 degrees C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39 degrees C and 40 degrees C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.     

Induction of transformed phenotypes in sheep fibroblasts by culture fluids from cells persistently infected with bovine leukemia virus

FLK cells are fetal lamb kidney cells persistently infected with bovine leukemia virus (BLV). 3178 cells, originating from calf-form bovine lymphosarcoma, also showed persistent production of BLV and alteration of cell morphology, after treatment with 5'-iodo-2'-deoxyuridine. In the present paper, the first in vitro transformation of sheep fibroblasts by inoculation with BLV materials from these two cell lines is described. In a few passages after inoculation with these viral materials, morphological alteration occurred. The morphologically altered cells were grown as stable cultures and showed such transformed phenotypes as growth in soft agar medium, increased uptake of 2-deoxy-D-glucose and tumorigenicity in athymic nude mice. This result, together with our previous observation of simultaneous induction of BLV expression and morphological alteration of 3178 cells, suggests the presence of some transforming capacity in these BLV materials similar to that in, for example, murine or avian acute leukemia viruses. The possible acquisition of such capacity during the prolonged passage is discussed.     

Induction of syncytia by Moloney murine leukemia virus in myoblasts defective in differentiation.

fu-1 cells, a nonfusing variant of the L8 line of rat myoblasts, form syncytia upon infection with murine leukemia virus (MuLV) or upon cocultivation with MuLV-infected cells; L8 cells do not form these syncytia, but do fuse into multinucleate myotubes. Syncytia of fu-1 cells form within 1 h after infection. The number of syncytia formed is proportional to the multiplicity of virus within a range of 4 to 16 and is maximum when the cell density is subconfluent. When either XC or fu-1 cells are productively infected with MuLV, they become resistant to syncytia formation by passage 3. The rapid formation of syncytia in fu-1 cells was found amenable for selection of temperature-sensitive mutants of MuLV and for screening additional variants of the L8 line.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.