A chloroquine-resistant Swiss 3T3 cell line with a defect in late endocytic acidification

Abstract

Abstract. To investigate the role of acidification in cell proliferation, several cell lines resistant to chloroquine were isolated with the expectation that some would express altered endocytic acidification. The preliminary characterization of one of these lines, CHL60-64, is described. In contrast to endocytic mutants described previously, the initial phase of endocytic acidification, as measured by transferrin acidification, is normal in this cell line. However, a difference in subsequent endocytic acidification was observed in CHL60-64. In the parental cells, internalized dextran was fully acidified to approximately pH 5.5 within 1 h. In CHL60-64, the pH in the endocytic compartment was only 6.1 after 1 h and remained as high as 5.8 for at least 4 h. After an 8-h incubation, the pH decreased to 5.5, indicating that the second phase of acidification is only slowed in CHL60-64, and not blocked. Consistent with this retarded acidification, ATP-dependent acidification in vitro (as measured by acridine orange accumulation) was reduced in both the lysosomal fraction and the endosomal fraction isolated from CHL60-64. A decrease in the in vivo rate of acridine orange accumulation after perturbation with amine was also observed. In addition to amine resistance and defective acidification, CHL60-64 was found to be resistant to vacuolation in the presence of chloroquine and ammonium chloride, and was resistant to ouabain. Further studies on this new class of endocytosis mutant, in combination with existing mutants, should help to clarify the mechanisms responsible for the regulation of endocytic acidification. M^Nv viruses and toxins require internalization and exposure to an acidic pH in order to penetrate into the cytosol (8,20,24). A number of cell lines that are defective in endocytosis and/or endocytic acidification have been isolated by taking advantage of this fact. Robbins et al. (6,15,16,19) have described a number of cell lines, selected for diphtheria toxin resistance and impaired mannose-6-phosphate receptor activity, that are resistant to infection by Sindbis virus and vesicular stomatitis virus, as well as protein toxins. Some mutants were found to be defective in the uptake of iron from transferrin, a phenotype linked to their defect in vacuolar acidification, and some exhibited altered Golgi apparatus functions as well. Selection for resistance to both modeccin and diphtheria toxin enabled Draper and co-workers (7, 22) to isolate Chinese hamster ovary cell lines that were temperature sensitive for viability, resistance to protein toxins (diphtheria toxin, modeccin, Pseudomonas exotoxin A), and ATP-stimulated acidification of endocytic vesicles. The temperature-sensitive lesion in viability could be overcome by the addition of FeSO4 to the growth media. Merion et al. (9) have characterized Chinese hamster ovary acidification mutants resistant to Pseudomonas exotoxin A, which were found to be cross-resistant to diphtheria toxin and several animal viruses. Monensin-resistant mutants have been isolated th...

PIKfyve Regulates the Endosomal Localization of CpG Oligodeoxynucleotides to Elicit TLR9-Dependent Cellular Responses

“…Interestingly, CpG-induced phosphorylation of Erk1/2 was not inhibited by YM201636 (Figure 7A), which suggested that it is not dependent on the endosomal localization of CpG. Chloroquine, which inhibits endosome acidification [21], showed similar effects to YM201636 on the TLR-induced phosphorylation of signaling molecules (Figure 7A). CpG-induced Stat3 phosphorylation but not LPS-induced Stat3 phosphorylation, was inhibited by both YM201636 and by the knockdown of PIKfyve (Figure 7B).…”

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

“…AO is a weak base which accumulates into acidic compartments (32)(33)(34). When present at high concentrations, it forms complexes, resulting in a shift in fluorescence from green to red.…”

Lysosomal Sequestration of Amine-Containing Drugs: Analysis and Therapeutic Implications

“…Most agree that accumulation of amines into lysosomes occurs predominantly according to a process referred to as pH‐partitioning; however, it is important to realize that not every cell line has normally acidified lysosomes and endocytic compartments and as a result, such accumulations might not pertain to all cells 10–12. A simplified model of this mechanism is depicted in Figure 1.…”