Transcription in early and late passage embryonic mouse fibroblasts in primary culture: A correlated biochemical, autoradiographical and ultrastructural study

Populations of embryonic mouse (BALB) fibroblasts undergo 10 ± 2 population doublings prior to the cessation of mitotic activity. The RNA polymerase activity of nuclei isolated from late passage fibroblasts is reduced by 50% compared to early passage cells. A strong inhibition of α-amanitin resistant RNA synthesis accounts for this, the α-amanitin sensitive (RNA polymerase II) activity remaining relatively constant with age. Autoradiography of cells labelled with tritiated uridine reveals a 70% reduction in nucleolar RNA labelling and 30% reduction in nucleoplasmic labelling, in late passage fibroblasts. Taken together, these results suggest a large decline in RNA polymerase I and III activity with increasing passage, but a relatively constant level of RNA polymerase II. An age-related repression of nucleolar activity is also suggested by ultrastructural studies showing a reduced number of nucleoli per nucleus and segregated nucleoli in old cells. Ribosomes are numerous in both young and old fibroblasts, but in the latter they tend to be unattached to membranes. There is a highly active free (unbound) RNA polymerase in young cell nuclei, but this is of much lower activity in late passage cells. As the labelling of the cytoplasm by autoradiography is only relatively slightly reduced (10%) in old cells, a perturbation in the processing, transport and/or half-life of RNA may also accompany the in vitro senescence of mouse fibroblasts. The changes in RNA synthesis found in nuclei isolated from late passage cells also occur in the nuclei of serum restricted, early passage fibroblasts, suggesting that these modifications result from, rather than cause, the repression of cell division in such cultures. These results are discussed in relation to cellular ageing in vivo and the significance of cell division.