Detection by means of cell fusion of macromolecular synthesis involved in the reconstruction of the nuclear envelope in mitosis

Using the cultured Chinese hamster cell line Don, G1 or S or a mixture of late-S/G2 cells were prepared by release from metaphase arrest. Metaphase (M) cells were also obtained by mitotic arrest of log-phase cultures with Colcemid and held in metaphase; such M cells remained untreated with any other compound and were termed standard M cells. When interphase (I) cells were fused at pH 8.0 and 37 degrees C with standard cells in the presence of Colcemid by means of UV-inactivated Sendai virus, binucleate interphase-metaphase (I-M) cells were obtained. In a given I-M cell there occurred within 30 min after fusion either prophasing of the I nucleus or formation of a nuclear envelope (NE) around the chromosomes. About 20% of early G1 cells, 35% of cells at the G1/S boundary, 50% of S cells, and 70% of late S/G2 cells could induce NE formation. If, before fusion, cycloheximide (CHE), an inhibitor of protein synthesis, was present during release from M arrest, the cells entered G1 but not S. About 20% of such early G1 cells, like the untreated early G1 cells, had the capacity to induce NE formation during subsequent fusion. If the cells were blocked in S with 5 mM thymidine (TdR), At least 80% of these cells could induce NE formation during subsequent fusion, but in the presence of both TdR and CHE only 35% could do so. It appeared, therefore, that protein synthesis in interphase was required for NE formation. Experiments with actinomycin D indicated that RNA synthesis was also necessary for acquisition of NE-inducing capacity. About 35% of G1 cells from confluent monolayers had the NE-inducing capacity, but prolonged exposure to CHE reduced their number to 8% . Removal of CHE restored the ability while the cells still remained in G1. This result indicated that continuing protein synthesis in the G1 cell was needed for NE formation subsequent to fusion. The fact that macromolecular synthesis must occur in the I cell before fusion if NE formation was to occur in the fused I-M cell lends further support to evidence adduced earlier that this phenomenon is a normal mitotic event. Prophasing of the I nucleus in I-M cells did not appear to be dependent on macromolecular synthesis in the I cell; earlier results from this laboratory showed, however, that protein synthesis in the prior G2 period of the M cell of the I-M pair was required for prophasing.