Regulation of the G1 ? S phase transition in chick embryo fibroblasts with ?-keto acids and L-alanine

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Abstract
Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum‐stimulated DNA synthesis in G1‐arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H‐thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4° C while exposed to the drug. Several α‐keto acids (pyruvate, oxaloacetate, α‐keto‐butyrate) at 0.5‐1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L‐alanine (D‐alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Clucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin‐stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for α‐keto acid or L‐alanine. A serum component(s) with a molecular weight of about 100 permitted insulin‐stimulated DNA synthesis in inhibited cells.