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Tumors in vivo usually exhibit deceleratory rather than exponential growth kinetics. Deceleration results from a negative feedback inhibition of growth the effectiveness of which increases progressively with tumor size. A major obstacle to understanding the fine mechanisms of deceleratory growth has been the lack of a tissue culture system proven to exhibit these kinetics. Rat C6 glioma cells in culture enter a prolonged phase of deceleratory growth about 72 hr after subculture. As with tumor growth in vivo, the kinetics of the C6 deceleratory phase can be described with high accuracy by Gompertz, logistic, inverse cube root, and power functions. The least-squares correlation coefficients for the linearized forms of the rate equations for these functions were typically in the range of 0.75 to 0.90. During the deceleratory phase, the equilibrium growth rate of C6 cells is a monotonically decreasing function of population density at all observable densities, whether sub- or supraconfluent. Any change in density produces a compensatory change of opposite direction in the growth rate. To account for these relationships, a density-equilibrium hypothesis of C6 growth regulation is presented. The fine mechanisms responsible for this regulation do not involve medium or substratum depletion, production of conditioned medium factors, or the extracellular matrix. The development of growth deceleration appears to correlate with the extent of contact interactions between cells and their neighbors, indicating that deceleratory growth regulation is probably mediated by contact interactions. However, these interactions are fundamentally different from those postulated by the traditional contact inhibition theory. Confluency is irrelevant to the contact modulation which produces C6 growth deceleration. Any degree of contact, even at very sparse subconfluent densities, appears capable of exerting some degree of growth inhibition.

Thymidine kinase activity in rat C6 glioma cells is inhibited by 50 to 70% after 4 hr incubation with 20 mM D-glucosamine. The inhibition is uncompetitive with respect to thymidine, reducing both the apparent Km and Vmax of the enzyme. The inhibition does not appear to be caused by the reversible combination of the enzyme with a cytoplasmic inhibitor, including D-glucosamine and its metabolites. The addition of D-glucosamine or its metabolites to cell-free thymidine kinase produced an inhibition which differed quantitatively and qualitatively from that which resulted from treatment of intact cells with D-glucosamine. The presence of a reversible cytoplasmic inhibitor of the enzyme was also excluded by mixing experiments. D-Glucosamine inhibited the incorporation of labeled uridine and amino acids into acid-precipitable material. The magnitude of inhibition of thymidine kinase activity and amino acid incorporation by D-glucosamine was comparable to that produced by cycloheximide, suggesting that the inhibition might arise from interference with enzyme synthesis. However, whereas the kinetics of recovery of amino acid incorporation from inhibition was rapid, thymidine kinase activity was depressed for at least 6 hr after drug washout. The results presented are best explained by assuming either that two forms of thymidine kinase are present in rat C6 cells and are differently affected by D-glucosamine or that D-glucosamine acts by two separate mechanisms to inhibit a single form of the enzyme.

The amino sugar, D-glucosamine, inhibits the preformed route of thymidine metabolism in rat C6 glioma cells. This inhibition results from a concatenation of several distinct effects, including the inhibition of thymidine uptake, the reduction of thymidine phosphorylation, and an increased leakage of thymidine to the extracellular space. Each of these effects, while ostensibly small in magnitude, is significant and contributes to the observed inhibition of acid-precipitable thymidine incorporation by D-glucosamine. These effects of D-glucosamine, together with its known ability to reduce uridine nucleotide pools, may contribute to its toxicity toward certain experimental animal tumors.