Friend Virus-transformed Cells Research Articles

Inactivation of Friend Erythroleukemia Virus and Friend Virus-TransformedCells by Merocyanine 540-Mediated Photosensitization

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus-transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.

Effect of serum from mice with dormant Friend leukemia viral infections on synthesis and modulation of erythroleukemia cell surface gp70.

Serum of mice with dormant Friend virus infections modulates the expression of cell surface antigens on Friend virus-transformed cells (FLC-745 cells). By radioimmunoassay, the modulated antigen was identified as virion gp70 of Friend leukemia virus. After two days of culture in medium containing 5% serum from mice with dormant Friend virus infections, the modulated cells expressed about 50% less surface gp70 than non-modulated cells cultured in 5% normal mouse serum. After 9 days in culture, the modulated cells expressed no surface gp70; however, these cells contained the same amount of internal gp70 as nonmodulated cells, indicating that gp70 was synthesized during the modulation period. Both modulated and nonmodulated cells grew equally well during the 9 days of culture. These results indicate that the effects of modulation of Friend virus-coded cell surface antigens are restricted to the cell surface.

Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture

Hemin treatment of mouse Friend virus-transformed cells in cultured caused a dose-dependent increase in hemoglobin synthesis. By the addition of radioactively labeled hemin and by the analysis of the radioactive heme in hemoglobin, only 60 to 70% of heme in the newly synthesized hemoglobin was accounted for by the exogenously added hemin. In keeping with this finding, hemin treatment increased the activity of two enzymes in the heme biosynthetic activity, i.e. delta-aminolevulinate (ALA) dehydratase and uroporphyrinogen-I (URO) synthase in these cells. Incorporation of [2(-14C)]glycine, [14C]ALA, and 59Fe into heme was also significantly increased in the cells treated with hemin, suggesting that essentially all enzyme activities in the heme biosynethetic pathway were increased after hemin treatment. These results indicate that heme in the newly synthesized hemoglobin in hemin-treated Friend cells derives both from hemin added to the culture and from heme synthesized intracellularly. In addition, these results suggest that the stimulation of heme biosynthesis by hemin in Friend virus-transformed cells is in contrast to the hemin repression of heme biosynthesis in liver cells.

Variations in viral gene expression in friend virus-transformed cell lines congenic with respect to the H-2 locus

The effect of H-2 genotype on the genetic expression of Friend oncovirus was studied using Friend virus-transformed cells differing genetically only at the H-2 locus. Seven lines and three sublines were examined with regard to expression of Friend virus and the murine leukemia virus envelope glycoprotein gp69/71, the structural proteins p30 and p15 (FMR), and reverse transcriptase. Our results confirm that H-2 b, H-2 g and H-2 kh cell lines were stable upon serial passage for production of infectious Friend virus, whereas the H-2 d and H-2 k cell lines ceased to release infectious virus after a relatively short time. Synthesis of viral proteins by these cell lines was independent of the synthesis of infectious virus in that some viral proteins continued to be synthesized by H-2 d and H-2 k cell lines in the absence of infectious virus. Reduction of viral proteins can be attributed to the loss of expression of individual viral genes. The temporal sequence of change suggests that the loss of protein expression may occur in a stepwise fashion; reverse transcriptase was reduced first, followed by p30 and p15, and gp69/71 last. The results confirm that p30 and p15 are coordinately expressed. In contrast, the expression of p30 and gp69/71 are noncoordinate, indicating that these proteins are regulated by independent genetic loci. Loss of viral genes did not occur, since cell lines which had previously ceased to synthesize viral proteins could be chemically induced to produce infectious NB tropic Friend viruses. These data suggest that for these H-2-congenic cell lines, there is an association of the H-2D b allele and the long-term production and release in vitro of both infectious virus and viral proteins.

Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells

Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells

Appearance of a chromatin protein during the erythroid differentiation of Friend virus-transformed cells.

Chromatin proteins from erythroleukemia cells transformed by Friend virus were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A new chromosomal protein, apparent molecular weight 25,000, was shown to appear during erythroid differentiation of the cells induced by dimethyl sulfoxide or hexamethylenebisacetamide. This protein is tightly bound to the chromatin and does not appear to be related to any of the known histones. It is also demonstrated that this protein is not induced in a dimethyl sulfoxide-resistant variant of these cells.

Studies of cellular differentiation by automated cell separation. Two model systems: Friend virus-transformed cells and Hydra attenuata.

The automated high-speed analysis and separation of cells on the basis of spectroscopic parameters has been applied to studies of cellular differentiation in two systems. The temporal changes following induction of differentiation by dimethylsulfoxide in the Friend virus-transformed erythroid cells were quantitated by multiparameter analysis leading to the separation of discrete subpopulations. Thus, following induction, cell size decreased as measured by light scattering, the number of H-2 histocompatibility antigen sites decreased as measured by indirect fluorescent antibody binding, the number of lectin-binding sites per cell increased as measured by fluorescein-labeled concanavalin-A and the microviscosity of the hydrocarbon region of the plasma membrane increased as determined by the fluorescence emission anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene. Cells were separated on the basis of several of these parameters and analyzed for their hemogloglobin content by benzidine staining. Examination of cells separated according to the anisotropy parameter showed that high anisotropy values were correlated with (a) small cell size, (b) positive staining with benzidine and (c) pronounced reactivity with fluorescent antibody to the erythrocyte protein spectrin. Disaggregated cells from Hydra attenuata were selectively stained with the dyes rhodanile blue, 7-(p-methoxybenzylamino)-4-nitrobenz-2-oxa-1,3-diazole and fluorescamine. Distribution analyses and preliminary separations indicated the feasibility of obtaining homogeneous classes of cell types in a viable state. The experiments with emission anisotropy represent the first analyses and separations of single cells on the basis of fluorescence polarization. Many other uses of this technique are anticipated.

Analysis by computer-controlled cell sorter of Friend virus-transformed cells in different stages of differentiation.

In most systems involving cellular differentiation and cellular transformation the biological process is non-synchronous and the sample heterogeneous. In order to answer some of the basic questions about the control mechanisms of cellular changes and the order in which they proceed one must have access to homogeneous classes of cells. Friend virus transformed erythroid cells which are stably maintained in tissue culture can be chemically induced to differentiate and are thus very advantageous for in vitro studies (1-3). With such a system the questions which we pose are a) the reversibility of the differentiation process; b) the order of steps in the production of specialized messenger RNA; c) the time of shut-off of undifferentiated messenger production; d) the relationship of viral RNA production to the differentiation process; e) the onset and extent of specific protein synthesis; f) the correlation of DNA metabolism with the timing or course of events. By using a computer-controlled cell separator we can select live cells on the basis of their macromolecular content, membrane properties (using a new parameter, fluorescence emission anisotropy), and size (4, 5, 34). Thus with proper probes as described here, we are able to select cells at different stages in their differentiation and can begin to attack the questions posed above.