Ultraviolet irradiation of fission yeast cells in G1 phaseinduced a delay in chromatin binding of replication initiation factors and, consistently, a transient delay in Sphase entry. The cell cycle delay was totally dependent on the Gcn2 kinase, a sensor of the nutritional status, and was accompanied by phosphorylation of the translation initiation factor eIF2␣ and by a general depression of translation. However, the G1-specific synthesis of factors required for DNA replication was not reduced by ultraviolet radiation. The cell cycle delay represents a novel checkpoint with a novel mechanism of action that is not activated by ionizing radiation.Supplemental material is available at http://www.genesdev.org.

The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.

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