The Cell Cycle During Growth Inhibition of Human Embryonic Fibroblasts in Vitro

Abstract

Human embryonic fibroblasts cultivated in vitro have been found to accumulate in the G₂ period of the division cycle when the growth curve reaches a plateau (1, 2). Subsequently it has been claimed that stationary cultures of WI-38 cells are arrested in the postmitotic G₁ period (3). This phenomenon was reexamined using different methods which showed that although human fibroblasts stop dividing in G₁ they are delayed in the G₂ period when they approach stationary phase.

Materials and Methods. One human embryonic lung fibroblastic line was used in the present studies between the 5th and 10th passage. The methods used to start and maintain the cultures were described elsewhere (4). Eagle's MEM supplemented with 10% foetal calf serum and 50 μg/ml aureomycin was used. Before the experiments cells were pooled and evenly distributed into new plastic 60 mm Petri dishes. Cell counts were made with an electronic cell counter (5) after suspending the cells in phosphate buffered saline (PBS) free of Ca and Mg supplemented with 0.25% trypsine and 2% ED TA. The technique used for autoradiography has been previously described (1). Samples of 3000, 100, and 1500 cells/slide were used to determine mitotic indices, % labeled mitoses and % labeled interphases respectively. Under the experimental conditions used, cells spending the whole S period in the presence of 0.01 μCi/ml tritium labeled thymidine (H³-TdR) with a specific activity of 2 Ci/mM had 70 grains/cell. Microspectrophoto-metric analysis was performed as previously described (6).

Results. The amount of cells accumulating in G₂ was measured by three different methods: in the first experiment cell kinetics were analysed on various days after subcultivation, through exponential growth up to resting phase; the second and third experiments followed the events after the cultures were stimulated in resting phase either by medium change or by replating at lower density.