Abstract
Abstract Ehrlich ascites tumor cells, grown in suspension cultures, could be cultivated for a period of time in serum-free medium, in which they stopped growing but remained viable. Their rate of protein synthesis was about ½ that of growing cells. Following serum addition protein synthetic rates in these cells rose to values characteristic of growing cells. These high rates were sustained and the cells went on to divide; thus stimulation of protein synthesis was the initial part of a growth response. Accompanying the increase in protein synthetic rate was a marked redistribution of ribosomes, with ribosomes in the inactive monomer pool entering the polyribosomes. The faster sedimenting polyribosomes increased to a greater extent than the slower sedimenting ones, suggesting that mRNAs became more completely loaded with ribosomes. During this process the pools of ribosomal sub-units remained constant. Rates of protein synthesis calculated as per polyribosomal ribosome increased; thus, both the rates of initiation of translation and the rates of peptide chain elongation were enhanced following serum addition. The role of increased RNA synthesis in serum stimulation of protein synthesis was investigated by testing the ability of cells previously treated with actinomycin, cordycepin, or 5-bromo-2'-deoxyuridine to respond to serum. The results are best interpreted as showing that in each case the mRNA pool was significantly diminished, but that cells stimulated with serum were able to translate it with about double the efficiency of serum-starved cells. The possible mechanisms by which serum growth factor or factors may regulate the efficiency of translation are discussed.
Highlights
Ehrlich ascites tumor cells, grown in suspension cultures, could be cultivated for a period of time in serum-free medium, in which they stopped growing but remained viable
The faster sedimenting polyribosomes increased to a greater extent than the slower sedimenting ones, suggesting that mRNAs became more completely loaded with ribosomes
The present study focuses on the initial response of nongrowing cells to serum by examining the mechanisms of serum-mediated stimulation of protein synthesis
Summary
Chemicals and Media-MinimalEssential Medium (Eagle) powder (Grand Island Biological Co.), calf serum (Microbiological Associates, Inc.), bovine serum albumin (Armour), L-[14C]lysine (New England Nuclear Corporation, specific activity258 mCi per mmole), cycloheximide, cordycepin, and 5-bromo-2’-deoxyuridine (Sigma), Whatman No 3MM 2.4-cm disks (H.Reeve Angel) were purchased commercially. Essential Medium (Eagle) powder (Grand Island Biological Co.), calf serum (Microbiological Associates, Inc.), bovine serum albumin (Armour), L-. 258 mCi per mmole), cycloheximide, cordycepin, and 5-bromo-. L-[14C]lysine was neutralized with 0.01 N NaOH before use. Calf serum was heat inactivated for 30 min at 56“. Dialyzed calf serum was prepared by thawing a bottle of frozen serum, dialyzing against double distilled demineralized water for 24 hours, and filtering through a Millipore 0.22 ~1 filter. Buffer A contained 20 mM triethanolamine, sufficient HCl to adjust the pH to 7.40, 2 mM magnesium acetate, and 25 MM KCI. Glucose, amino acids, and phenol red in the same concentrations as in Mimimal Essential Medium (Eagle), and 1 mg per ml of bovine serum albumin
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