scholarly journals Hypomethylation of DNA during differentiation of friend erythroleukemia cells

1980 ◽  
Vol 86 (2) ◽  
pp. 366-370 ◽  
Author(s):  
JK Christman ◽  
N Weich ◽  
B Schoenbrun ◽  
N Schneiderman ◽  
G Acs
Keyword(s):  
Dimethyl Sulfoxide ◽  
Mammalian Cells ◽  
Dna Methyltransferases ◽  
Methyl Groups ◽  
Close Link ◽  
Erythroleukemia Cells ◽  
Chemically Induced ◽  
Methyl Cytosine ◽  
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.

scholarly journals Biosynthesis of spectrin and its assembly into the cytoskeletal system of Friend erythroleukemia cells.

1986 ◽  
Vol 103 (1) ◽  
pp. 103-113 ◽  
Author(s):  
S R Pfeffer ◽  
T Huima ◽  
C M Redman
Keyword(s):  
Dimethyl Sulfoxide ◽  
Soluble Fraction ◽  
Insoluble Fraction ◽  
Cell Fraction ◽  
Erythroleukemia Cells ◽  
Alpha Spectrin ◽  
Membrane Bound ◽  
Friend Erythroleukemia Cells ◽  
Cytoskeletal System ◽  
In Cells

Friend erythroleukemia cells, grown in the presence of dimethyl sulfoxide for 3 d, synthesize unequal amounts of the two chains (alpha and beta) of spectrin with approximately 15-30% more beta than alpha spectrin. When cells were ruptured by nitrogen cavitation, nascent alpha and beta spectrin were found to be associated with a membranous cell fraction and were not detected in the soluble cytoplasmic cell fraction. Nascent membrane-bound spectrin appeared not to be protected by membranes, since it was susceptible to trypsin degradation in the absence of detergent. On fractionation of cells with 1% Triton X-100, more (1.75-fold) nascent spectrin was found in the Triton-soluble fraction than in the Triton-insoluble fraction (cytoskeleton). In the Triton-soluble fraction, there was 55% more nascent beta spectrin than alpha spectrin, while the cytoskeleton contained nearly equal amounts of alpha and beta spectrin. Cells were pulse-labeled with L-[35S]methionine for 2 min and chase incubated for varying periods of time from 15 to 90 min with nonradioactive L-methionine. Radioactive spectrin accumulated in the Triton-soluble fraction for the first 15 min of chase incubation and then dropped by 25% in the next hour. By contrast, the amount of radioactive spectrin in the Triton-insoluble fraction rose gradually for 1 h of the chase period. This indicates that, in Friend erythroleukemia cells, a pool of membrane-bound spectrin containing an excess of the beta polypeptide is used to form the cytoskeletal system which is composed of equal molar amounts of alpha and beta spectrin. The location of spectrin was determined by immunoelectron microscopy. Small amounts of spectrin were detected in cells not treated with dimethyl sulfoxide and in these cells it was located on the surface membrane and within the cytoplasm. On treatment with dimethyl sulfoxide, complex vacuolar structures containing viruses appeared in the cells. In cells treated with dimethyl sulfoxide for 3 d 30% of the spectrin was near the outer membrane and 25% was associated with vacuolar structures, whereas in cells treated for 5 and 7 d the majority of spectrin (57-61%) was located in the vacuolar areas.

scholarly journals Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

1992 ◽  
Vol 12 (7) ◽  
pp. 2967-2975 ◽  
Author(s):  
S Schuetze ◽  
R Paul ◽  
B C Gliniak ◽  
D Kabat
Keyword(s):  
Transcription Factor ◽  
Dimethyl Sulfoxide ◽  
Nuclear Localization ◽  
Erythroid Differentiation ◽  
Polyclonal Antiserum ◽  
Cellular Genes ◽  
Erythroleukemia Cells ◽  
Chemically Induced ◽  
Preleukemic Stage

Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.

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