ABSTRACT
Total and viable cell counts, differential mitotic cell counts, and incorporation of tritiated thymidine were used to study the kinetics of suspension cultures of HeLa cells exposed to urea concentrations of 0.5-1.5%. Aside from any nonspecific osmotic effects, urea concentrations of 1.0-1.4% exhibited significant cytokinetic and cytotoxic effects. Most characteristically, mitotic cells arrested in metaphase began to accumulate 4-6 hours after addition of urea and reached a peak at 15-18 hours. Thus when the cells were in the S-phase or at the S/G2 boundary at the time of addition of urea, they exhibited metaphase arrest. Subsequently, cultures continuously exposed to urea showed a decline in the mitotic index, indicating that the entry rate of cells into mitosis is lower than the rate at which cells escape from the mitotic block. Such cultures exhibited numerous abnormal and abortive mitoses and a decrease in growth and viability of the cell populations. In contrast to the initial single wave of arrested mitosis seen with continuous exposure to urea, intermittent exposure on alternate days resulted in successive waves of arrested metaphases and had considerably more pronounced effects on the growth and viability of the cultures.
Subject(s)
HeLa Cells/drug effects , Urea/toxicity , Cell Count , Culture Media/toxicity , DNA/biosynthesis , Dose-Response Relationship, Drug , HeLa Cells/metabolism , Humans , Kinetics , Mitosis/drug effects , Suspensions , Thymidine/metabolism , Time FactorsABSTRACT
The intracellular concentrations of the adenine nucleotides were determined in suspension cultures of WRL-10A cells, a subline of the L-929 mouse fibroblasts, during the progression of the cells from exponential growth to high-density, nonproliferating populations. The development of the nonproliferating state was associated with a 50% reduction of the adenine nucleotide pool, whereas the energy charge remained at values above 0.90. This change was also observed in the early phase of starvation of low-density cultures and could be reproduced by selective simultaneous withdrawal of glucose and glutamine, which indicated interference with the de novo synthesis of purines. In this respect, therefore, nonproliferating populations of WRL-10A cells resemble purine-limited bacterial systems but not density-inhibited normal fibroblasts in which the size of the adenine nucleotide pool is known to remain unchanged. This difference in the physiologic state of nonproliferating normal and neoplastic cells is potentially significant for tumor chemotherapy.
Subject(s)
Adenine Nucleotides/metabolism , Cell Division , Neoplasms, Experimental/metabolism , Adenosine Triphosphate/metabolism , Animals , Cells, Cultured , Energy Metabolism , Mice , Neoplasms, Experimental/pathologyABSTRACT
We have previously reported the presence and regulation of an acetylcholine-hydrolyzing enzyme in high density suspension cultures of WRL-10A fibroblasts where its activity increases 100-fold when growth is arrested. Substrate specificity, substrate inhibition, and product identification studies indicate that this enzyme is acetylcholinesterase (AChE, EC 3.1.1.7). Treatment of whole cells with 5 mM diazotized sulfanilic acid revealed that most of the AChE is located on the external surface of the cell membrane. It was also found that the enzyme is released in the medium at a rate of 0.5 U/h/mg cell protein and that within a 24-h period the de novo synthesized and liberated AChE is equivalent to 90% of the activity associated with the cells. No similar synthesis of AChE was found in six order fibroblastic cell lines examined. These and related findings indicating that acetylcholine is also present in high density populations of WRL-10A cells suggest that this unique phenotype may be used profitably in exploring further the relationship between components of the cholinergic system and non-neuronal cell growth.
