Interferon and cell division. VIII. Effect of interferon on macromolecular synthesis in L1210 cells in vitro
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Abstract Interferon inhibits multiplication of murine leukemia L1210 cells and the effect is evident 18 h after subcultivation. At this time a significant decrease in the synthesis of total RNA and protein was observed in interferon‐treated cultures compared to control cultures. Likewise, the formation of polyribosomes was inhibited in interferon‐treated cultures although no effect was observed on the formation of the ribosomal subunits. As control cells and interferon‐treated cells continued to multiply and the cultures attained cell density saturation, the differences in macromolecular synthesis and polyribosome formation between the cultures became less marked. Confidence that interferon itself was responsible for the effects observed stemmed from the use of highly purified interferon preparations and the availability, as a cell control, of a subline of L1210 cells resistant to interferon action. The possibility that a single mechanism of action is common to the antiviral effect of interferon and its effect on cell division is discussed.Keywords:
L1210 cells
Polysome
Abstract Interferon inhibits multiplication of murine leukemia L1210 cells and the effect is evident 18 h after subcultivation. At this time a significant decrease in the synthesis of total RNA and protein was observed in interferon‐treated cultures compared to control cultures. Likewise, the formation of polyribosomes was inhibited in interferon‐treated cultures although no effect was observed on the formation of the ribosomal subunits. As control cells and interferon‐treated cells continued to multiply and the cultures attained cell density saturation, the differences in macromolecular synthesis and polyribosome formation between the cultures became less marked. Confidence that interferon itself was responsible for the effects observed stemmed from the use of highly purified interferon preparations and the availability, as a cell control, of a subline of L1210 cells resistant to interferon action. The possibility that a single mechanism of action is common to the antiviral effect of interferon and its effect on cell division is discussed.
L1210 cells
Polysome
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The antiproliferative effect of interferon was studied at various stages of pre- and postimplantation mouse embryo development. Although 3.4 × 103 reference units/ml of NDV induced mouse interferon did not inhibit development of DNA synthesis in preimplantation embryos, an inhibitory effect was first detectable in primary cell cultures derived from whole 8-day embryos. This effect reached a maximum of 36 to 44% inhibition of DNA synthesis in cells cultured from 10-day embryos and remained at the same level with cells cultured from 12, 14, and 17-day embryos. These results indicate that responsiveness to interferon is achieved in the mouse embryos by 10 days.
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Cells cultured from young (6-day) chicken embryos differ from those of older (13-day) embryos in having a greater susceptibility to infection by certain viruses and a considerably lesser sensitivity to the action of interferon. These circumstances parallel those observed in the intact embryo. The addition of a small percentage of cells from young embryos alters the response of cells cultured from older embryos by increasing viral susceptibility sevenfold and decreasing sensitivity to interferon 25-fold. We postulate that a repressor which inhibits the expression of interferon in older embryonic cells is elaborated by cells from young embryos.
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In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. Optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KC1 concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase. Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers.
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The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.
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