Release of gelonin from endosomes and lysosomes to cytosol by photochemical internalization.
Gelonin, a type I ribosome-inactivating plant toxin, executes N-glycosidase activity on eukaryotic ribosomes. However, on intact cells, gelonin is relatively non-toxic, due to an incapability to penetrate cell membranes. Recently, a novel method, photochemical internalization (PCI), was invented for the translocation of membrane-impermeable molecules including gelonin to the cytosol [K. Berg et al., Cancer Res. 59 (1999) 1180-1183]. The combination of gelonin and photoactivation of endosomal and lysosomal localizing photosensitizers gives strong synergistic cytotoxic effects. In this study, we have evaluated the intracellular transport and stability of gelonin. By fluorescence microscopy, it was shown that gelonin co-localizes with the endosomal and lysosomal localizing photosensitizer, aluminum phthalocyanine with two sulfonate groups on adjacent phenyl rings, and both molecules re-localized to cytosol subsequently to light exposure. Gelonin accumulated in endosomal compartments by incubation at 18 degrees C was released to cytosol by PCI with concomitant inhibition of protein synthesis indicating that PCI can be executed through rupture of endosomal vesicles. The cathepsin inhibitor L-trans-epoxysuccinyl-leucyl amido(4-guanido)butane increased the cytotoxic effect of gelonin after PCI when gelonin was provided as a 2 h pulse followed by 4 h chase before PCI. Thus, although gelonin can enter the cytosol from lysosomes, lysosomal degradation is a limiting factor for the outcome of PCI of gelonin.
Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway. p.k.selbo@labmed.uio.no
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D015853:Cysteine Proteinase Inhibitors; D003600:Cytosol / Q000737:chemistry*; D011992:Endosomes / Q000737:chemistry*; D005453:Fluorescence; D007211:Indoles; D007425:Intracellular Membranes / Q000737:chemistry; D007930:Leucine / Q000031:analogs & derivatives*; D008247:Lysosomes / Q000737:chemistry*; D008858:Microscopy, Phase-Contrast; D009942:Organometallic Compounds; D010777:Photochemistry; D010940:Plant Proteins / Q000737:chemistry*; D011166:Porphyrins; D011838:Radiation-Sensitizing Agents; D054789:Ribosome Inactivating Proteins, Type 1; D013696:Temperature; D014407:Tumor Cells, Cultured
D015853:Cysteine Proteinase Inhibitors; D007211:Indoles; D009942:Organometallic Compounds; D010940:Plant Proteins; D011166:Porphyrins; D011838:Radiation-Sensitizing Agents; D054789:Ribosome Inactivating Proteins, Type 1; C016136:tetraphenylporphine sulfonate; C060570:aluminum phthalocyanine; C025674:GEL protein, Gelonium multiflorum; D007930:Leucine; C024974:E 64