Ethanol and cycloheximide alter protein and RNA synthesis of Cox astrocytoma cells in culture
Authors: Fleming Eugene W. , Woodson Mark E. , Tewari Sujata 
Published: January 2005

Chronic ethanol ingestion or cycloheximide treatment results in alterations in the properties and synthesis of protein and RNA of polyribosomes in the whole brain. To analyze the effects on a homogeneous neural cell population, Cox astrocytoma (glioma) cells were grown in tissue culture media with 100 mM ethanol or 0.017 mg/ml of cycloheximide. When ethanol had been present for ten days, the cell densities remained unchanged but had markedly reduced RNA and protein contents. Furthermore, the ethanol treatment reduced the whole‐cell pulse‐labeling of RNA with (5–3H) orotic acid and protein with (14C) leucine in the postmitochondrial supernatant. These results suggest that chronic ethanol treatment reduced the whole cell synthesis of RNA and protein or increased their degradation. Analysis of the polyribosomes on sucrose density gradients showed that dense polyribosomal chains were decreased after the ethanol treatment, supporting the concept that the polyribosomes were degraded with an alteration in the metabolism of mRNA. The cell‐free incorporation of (14C) leucine into hot TCA precipitable protein by the purified polyribosomes in the presence of ATP, GTP, a heterologous source of soluble factors, and endogenous mRNA was also reduced following the ethanol treatment, further indicating that the previous chronic exposure to ethanol had inhibited the translation of mRNA. When the control cells were grown in the presence of cycloheximide for one hour prior to harvesting, the cell densities remained unchanged, but again, as with the ethanol treatment, the polyribosomal protein and RNA yields decreased. In contrast to ethanol, however, cycloheximide treatment caused increases both in the whole‐cell incorporation of labeled RNA and protein precursors into the supernatant fraction and in the cell‐free incorporation of (14C) leucine into protein. These results suggest that, like the ethanol effects, cycloheximide reduces the total polyribosomes, but unlike the ethanol effects, the remaining polyribosomes have stable mRNA and rapidly incorporate radioactive amino acids, even more than untreated controls. The one‐hour cycloheximide treatment also caused an increase in the ratio of dense polyribosomes to monosomes plus 40s and 60s ribosomal subunits of control cells. In addition, it increased the incorporation of the labeled precursor into protein in the polyribosomal region of the sucrose gradients of both control and ethanol treated cells, suggesting that cycloheximide inhibited the termination step of protein synthesis. When cycloheximide was present for 24 hours prior to harvesting, the ethanol treated cells, in contrast to the controls, still had increased cell‐free incorporation of amino acid into protein, indicating that the stimulatory effects of cycloheximide are prolonged to 24 hours when ethanol is present. Thus, while the ethanol treatment in general inhibits polyribosomal biogenesis in the cells, it alters the complex of stimulating and then inhibiting effects of cycloheximide by preserving cycloheximide's stimulating effects on the amino acid incorporation activities of the polyribosomes.