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.
Milano
J
J
Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Strayer
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002455:Cell Division; D002460:Cell Line; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014407:Tumor Cells, Cultured; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C062521:RCC1 protein, human; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein