Inter- and intraclonal variability of polypeptides synthesized in a rat hepatoma cell line. Quantitative two-dimensional gel analysis.

To examine the degree of clonal heterogeneity in the synthesis of polypeptides in neoplastic cells, single-cell subclones from the rat hepatoma cell line H4-II-E were isolated. Polypeptides from the clones were resolved on high resolution two-dimensional polyacrylamide gels (PAGE), and quantitatively analyzed with a computerized two-dimensional PAGE analysis system developed in this laboratory. Only four qualitatively different spots were found which were synthesized in one of the subclones in four out of five experiments. In contrast, 5-20% of the spots showed statistically significant quantitative differences when any one subclone was compared to any other. These differences were generally quite small, averaging about 1.5-fold in intensity, although variations of fourfold or more were observed. Different cultures of the same subclone showed quantitative differences of the same order as seen in different subclones, indicating that this variability was primarily intraclonal in nature, i.e. associated with the cultures rather than the subclones. The distribution of quantitatively variable spots indicates that 50% or more of the polypeptides in these cells may display intraclonal variability. Similar results were obtained with a second set of subclones derived from these primary ones. Time course studies were conducted where cells were maintained continuously for 12 weeks, with samples taken for two-dimensional PAGE analysis once a week. The fraction of polypeptides that vary significantly generally increased with time between sampling points. Experiments with independent cultures grown in parallel indicate that about 4% of this variability can be correlated to the age of the culture media, although the majority appears due to uncontrolled and/or random differences that arise between cultures. These results indicate that independent cultures quickly develop detectable quantitative differences in the expression of a large fraction of their polypeptides. These differences cannot, at present, be associated with the observable biology of the cells and probably reflect time-associated variations in the balance of cellular macromolecular synthesis which arise in tissue culture cells.