Control of nucleic acid synthesis in human diploid cells undergoing contact inhibition

Abstract

In order to define the control mechanisms operative in nucleic acid synthesis in diploid cells in vitro, the synthesis of the nucleic acids of embryonic, human fibroblasts was compared during replication and contact inhibition. Nucleic acid synthesis was measured by the rate of incorporation of isotopically labeled precursors and during a short time interval (4 per cent of doubling time) the nucleic acids were separated by column chromatography. Autoradiography was used as an adjunct to assess the heterogeneity of synthesis in the cell population. Several days after the formation of a monolayer, which induces a state of contact inhibition of replication, the following changes in nucleic acid synthesis ensued: 1. 1. DNA synthesis virtually ceased. 2. 2. The synthesis of tRNA and ribosomal RNA were coordinately repressed to a level which was 1 10 that of the replicating cell. 3. 3. The synthesis of DNA-like RNA, including mRNA, was much less reduced, to a level 1 4 that in replicating cells. 4. 4. Use of the quantitative data on rates of cell replication and assuming that ribosomal RNA was synthesized at a basal level in the contact inhibited cell, the half-life of ribosomal RNA in these cells was calculated as 5.75 days. A similar calculation using in addition the distribution of isotope between DNA-like RNA and rRNA at 1 h, yielded a value for the half-life of the average DNA-like RNA molecule, of 7.9 h. These values are similar to those derived from other mammalian systems. 5. 5. During prolonged maintenance of the diploid cell culture differentiation and slow cell replication occurred. This resulted in the formation of a primitive connective tissue. No significant differences were detected in the patterns of nucleic acid synthesis of the cells in early contact inhibition or in the late stage of an organized fibrous tissue membrane. 6. 6. The stimulation of RNA synthesis and subsequent release from contact inhibition of a small proportion of cells induced by fresh medium was examined. The stimulation of precursor incorporation into RNA was most marked with tRNA, and the decreasing order of response was 5s RNA and other low molecular weight RNA fractions, rRNA, and the least was DNA-like RNA. The stimulatory effect was marked in the 1st hour following the changing of the medium. Since the effect was not abolished by inhibition of protein synthesis its mechanism resembled a derepression of the cistrons involved. The selectivity for different RNA types ruled out a precursor pool effect for anything more than a minor role. There was little change in the sedimentation characteristics of the DNA-like RNA in fresh medium stimulated cells. 7. 7. The subsequent cell replication involved only a small proportion of the cells and showed no obvious relationship to the initial stimulation of RNA synthesis. 8. 8. The study has thus defined several control systems operative in tRNA and rRNA synthesis in diploid cells in vitro.