Assuntos
Hematopoese , Leucemia Experimental/fisiopatologia , Proteínas de Neoplasias/biossíntese , Proteínas Virais/biossíntese , Animais , Linhagem Celular , Dimetil Sulfóxido/farmacologia , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/fisiopatologia , Leucemia Experimental/microbiologia , Leucemia Experimental/ultraestrutura , Microscopia Eletrônica , Infecções Tumorais por Vírus/fisiopatologia , Replicação Viral/efeitos dos fármacosRESUMO
The factors that control oncornavirus formation were analyzed in Friend leukemia cells that undergo hematopoiesis when treated with dimethyl sulfoxide. Suspension cultures of Ostertag FSD-1 cell line were found to enter a G or resting state at the end of their proliferative phase and to simultaneously cease producing helper and dependent components of Friend virus. Whereas the decline in virus production is at least 100-fold, rates of cellular RNA and protein synthesis are only slightly lower in resting than in growing cells. Both resting and growing cells contain similarly large concentrations of the viral proteins P(30) and P(12). Dimethyl sulfoxide induces hemoglobin synthesis in growing cells, but its effects on virus production appear to be indirect results of its action to inhibit cell growth and thus to delay entry of cells into the G resting state. Furthermore, variant cell lines were obtained with differing abilities to synthesize virus or hemoglobin. Some lines no longer produce infectious virus, although they all harbor murine leukemia virus genes which are expressed to varying extents. The major internal protein of these oncornaviruses, P(30), is synthesized in large amounts by all of the cell lines. These results suggest that Friend virus production is not coinduced with erythroid differentiation, as had been proposed, but rather is controlled by a cellular growth cycle.
Assuntos
Vírus da Leucemia Murina de Friend/crescimento & desenvolvimento , Hemoglobinas/biossíntese , Divisão Celular , Linhagem Celular , DNA de Neoplasias/biossíntese , Dimetil Sulfóxido/farmacologia , Vírus da Leucemia Murina de Friend/metabolismo , Variação Genética , Vírus Auxiliares/crescimento & desenvolvimento , Leucemia Eritroblástica Aguda , Proteínas Virais/biossíntese , Replicação ViralAssuntos
Hemoglobinas/biossíntese , Leucemia/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/metabolismo , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , Meios de Cultura , Citidina/metabolismo , DNA de Neoplasias/análise , Dimetil Sulfóxido/farmacologia , Hematopoese , Camundongos , Proteínas de Neoplasias/análise , RNA Neoplásico/análise , RNA Ribossômico/metabolismo , Fatores de Tempo , Uridina/metabolismoRESUMO
We have studied synthesis of specific proteins in two permanent lines of Friend virus-induced erythroleukemia cells (Friend line 745 and Ostertag line FSD-1, both derived from DBA/2 mice). By 96 hr following treatment with 1-2% dimethyl sulfoxide (Me2SO), up to 25% of the protein being synthesized by both these cultures is hemoglobin. At that time, hemoglobin constitutes up to 10% of the cellular soluble protein. Both lines synthesize heme and globin coordinately, and alpha and beta globin chains in a nearly balanced 1:1 ratio. However, the ratio of betaMajor:betaMinor chains synthesized by these induced Friend leukemia (FL) cells is approximately 9 in the FSD-1 line and 1.3 in the Friend Clone 745 line, whereas it is 4 in normal adult DBA/2 mouse erythrocytes. Evidence for the latter conclusion was obtained by electrophoresis of FL hemoglobins on cellulose acetate membranes, and also by chromatographic separation of alpha, betaMajor, and betaMinor globins on carboxymethylcellulose in 8 M urea at 20 degrees C. Carbonic anhydrase activity per mg protein is 3 times higher in induced than in control cultures. 2,3-diphosphoglyceric acid is not found in induced FL cells. Induced and control FL cells agglutinate strongly and equally with Phaseolus vulgaris phytohemagglutinin. The developmental process in these cultured leukemia cells appears to be an aberrant erythropoiesis.
Assuntos
Linhagem Celular , Eritrócitos , Hemoglobinas/biossíntese , Leucemia Eritroblástica Aguda/metabolismo , Aglutinação , Animais , Proteínas Sanguíneas/biossíntese , Anidrases Carbônicas/metabolismo , Transformação Celular Neoplásica , Cromatografia , Células Clonais/metabolismo , Dimetil Sulfóxido/farmacologia , Ácidos Difosfoglicéricos/análise , Eletroforese em Acetato de Celulose , Vírus da Leucemia Murina de Friend , Globinas/biossíntese , Heme/biossíntese , Hemoglobinas/análise , Cinética , Lectinas/farmacologia , Camundongos , Camundongos Endogâmicos DBAAssuntos
Fígado/análise , Proteínas Musculares/isolamento & purificação , Proteínas/isolamento & purificação , Ribossomos/análise , Animais , Centrifugação com Gradiente de Concentração , Centrifugação Zonal , Eletroforese em Gel de Poliacrilamida , Escherichia coli/citologia , Estudos de Avaliação como Assunto , Concentração de Íons de Hidrogênio , Fígado/citologia , Magnésio , Masculino , Métodos , Proteínas Musculares/análise , Músculos/citologia , Especificidade de Órgãos , Potássio , Proteínas/análise , RatosAssuntos
Fígado/análise , Proteínas Musculares/isolamento & purificação , Músculos/análise , Proteínas/isolamento & purificação , Ribossomos/análise , Animais , Centrifugação com Gradiente de Concentração , Centrifugação Zonal , Cromatografia por Troca Iônica , Diálise , Eletroforese em Gel de Poliacrilamida , Estudos de Avaliação como Assunto , Fígado/citologia , Masculino , Métodos , Músculos/citologia , Organoides/análise , Ratos , Espectrofotometria UltravioletaAssuntos
Ribossomos/análise , Animais , Centrifugação Zonal , Cães , Estudos de Avaliação como Assunto , Células HeLa/citologia , Humanos , Insetos/citologia , Fígado/citologia , Métodos , Músculos/citologia , Especificidade de Órgãos , Concentração Osmolar , Tonsila Palatina/citologia , Pâncreas/citologia , Plasmocitoma/patologia , RNA Ribossômico/análise , Coelhos , Ratos , Reticulócitos/citologia , Especificidade da Espécie , Espectrofotometria UltravioletaRESUMO
Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.