We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.
When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.
Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.
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