Effects of overexpression of Ran/TC4 mammalian cells in vitro.

We investigated the effect of overexpression of Ran/TC4 on cell cycle progression. Ran/TC4 (ras-related nuclear protein) is a highly conserved 25-kDa GTP-binding protein that, in concert with its guanine-nucleotide-exchange factor RCC1, is involved in signal transduction. Ran and RCC1 act on nuclear transport of RNA and protein, cell cycle regulation at the G1/S interphase, chromatin decondensation after mitosis, and chromosome stability. These two proteins are essential for the coupling of DNA synthesis with the onset of mitosis. The cDNA for rabbit Ran/TC4 was identified in a cDNA library using degenerate oligonucleotide probes devised on the basis of deduced protein sequence data. This cDNA was cloned into pCDM8 expression vector to yield a plasmid, pTC4, in which Ran/TC4 expression is driven by the cytomegalovirus intermediate early promoter. Both a human tumor cell line, MCF7, and a normal rabbit fibroblast line, RK-13, were tested. Following transfection with pTC4 we observed an increase in Ran/TC4 transcript levels. Transfection with pTC4 prolonged the duration of S phase in both MCF7 and RK-13 cells and led to reduced cell proliferation and decreased total cell numbers. DNA fragmentation was seen in pTC4-transfected cultures but not in control cultures. These findings underscore the function of Ran/TC4 as a molecular switch that guides the cell to completion of DNA synthesis before it enters mitosis and suggest that its overexpression may greatly alter cell cycle kinetics and cell viability.