A series of copolymers were prepared containing 1,2:3,4-di-O-isopropylidene-6-O-methacryloyl-alpha-D-galactopyranose (0 to 99 mol %), methacryoyltyrosinamide and N-(2-hydroxypropyl)methacrylamide (99 to 0 mol %). The effect of galactose content on interaction with hepatoma cells in vitro was studied. Increased galactose content caused increased accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by two human hepatoma cell lines (Hep G2 and SAH), but accumulation by rat and mouse hepatoma (HTC and NCTC) was not galactose dependent. Accumulation of N-(2-hydroxypropyl)methacrylamide copolymers by Hep G2 was shown to be an active process, being inhibited by low temperature and by the metabolic inhibitor 2,4-dinitrophenol. Addition of N-acetylgalactosamine and polymer-galactose to the incubation medium resulted in a concentration-dependent inhibition of accumulation of galactose-containing polymers. Addition of fucose or galactose was without effect at the concentrations used. Polymers bearing galactosamine or fucosylamine residues and, in addition, daunomycin were evaluated for cytotoxicity against Hep G2 and SAH. N-(2-Hydroxypropyl)methacrylamide copolymer-bound daunomycin produced a dose-dependent inhibition of DNA synthesis (measured by incorporation of [3H]thymidine), and the galactose-containing polymer showed greatest inhibition.
When cells are incubated in the presence of a weak base such as chloroquine or methylamine, the base is accumulated to a high concentration in the lysosomes, owing to the lower internal pH of those organelles and the low permeability of the lysosome membrane to cations. Lysosomes are also known to be disrupted by detergents. Firestone et al. (1979) used these two observations as the basis for the novel and imaginative concept of inducing cytolysis by exposing cells to a 'lysosomotropic detergent'. It was reasoned that long-chain aliphatic derivatives of weak bases with appropriate pK would accumulate in lysosomes, where protonation would confer detergent properties and lead to lysosome membrane destabilization. Molecules such as N-dodecylimidazole and N-dodecylmorpholine were synthesized and shown to be cytotoxic to lysosome-containing cells, and in a subsequent publication (Miller et a/., 1983) further evidence was adduced for the lysosome as their intracellular target and the mediator of their cytolytic properties.We have recently reported some experiments that were expected to demonstrate directly that lysosomotropic detergents damage lysosome membranes (Forster et a/., 1087). Surprisingly, isolated intact lysosomes were unaffected, even when incubated with high concentrations of the detergents at a pH below the pK. We also obtained negative results with a test that should have revealed quite minor damage to the integrity of the lysosome membrane. Human fibroblasts with a pathologically high lysosomal cystine concentration (cells taken from a patient with cystinosis, a dysfunction of the lysosome membrane cystineporter) were incubated in vitro in the presence of Ndodecylimidazole. Although a cytotoxic concentration of the detergent decreased the cystine content of the cells to 50°/0 within an hour, a marginally cytotoxic concentration caused no decrease. These results offered no clear support for the hypothesis that lysosomal damage precedes and causes cell death.We now report the changes in cystine content and in lactate dehydrogenase release in the initial phase of exposure of cystinotic fibroblasts to a cytotoxic concentration of Ndodecylimidazole. Incubations were performed as previously described (Forster et a/., 1987), but with sampling at 15, 30, 45 and 60 min. Fig. l ( a ) shows the release of lactate dehydrogenase activity from the cells. The 15 min value of 670 is no higher than that in control incubations (no detergent) after 1 h. By 15 min, however, the cystine content of the cells ( 4 nmol/mg protein) was 50% of the value for control cells, but there was no further decrease during the remainder of the incubation (Fig. 1 b).In these experiments cystine depletion by the detergent is very rapid and precedes any evidence of cell death. Cystine depletion is, however, incomplete and fails to proceed further even when cells are manifestly dying. This puzzling result is most readily explained by postulating the presence in the fibroblast culture of susceptible and non-susceptible VOl. 16 " 0 15 30 45 60...
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