Inhibition of the discharge of endocytosed protein from phagosomes into lysosomes in hepatoma cells exposed to dimerized ribonuclease A

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Abstract
The mechanism of the cytostatic action of dimerized RNase A toward cultured rat HCT hepatoma cells was investigated. A decrease in mitotic index, modifications of adsorptive properties of the pericellular membrane and inhibition of the degradation of 2 different proteins taken up by endocytosis are the 1st cell functions to be affected by the dimer. This effect on protein digestion is not due to an inhibition of proteolytic enzymes. The intracellular localization of exogenous protein and of RNase dimer was studied by cell fractionation. When proteins (horseradish peroxidase or rabbit immunoglobulin G) are taken up by control hepatoma cells, they are 1st associated with phagosomes equilibrating at a lower density than lysosomes. Their density distribution gradually becomes similar to that of lysosomes. When cells are pre-exposed to RNase dimer, this modification of the density distribution as a function of time no longer occurs, although these proteins are still intracellular, as indicated by fractionation by differential centrifugation. During the 1st h after addition of RNase dimer, kinetic studies show an increased fixation of peroxidase to the cell membrane. Protein release into the culture medium is also increased. Either fusion between phagosomes and lysosomes is absent or the discharge of peroxidase adsorbed to the phagosomal membrane after fusion is inhibited.