Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6.

Murphy, Robert F.; Powers, Scott; Cantor, Charles R.

doi:10.1083/jcb.98.5.1757

Cited by 269 publications

(177 citation statements)

References 30 publications

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“…In contrast, F-Dex and F-~t2M move to endocytic compartments that are more acidic, having an average pH of <6.0 as measured by the null point method. Similar lowering of the average endosomal pH has been found in BALB/c 3T3 cells (15).…”

Section: Discussionsupporting

confidence: 65%

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

Yamashiro

Maxfield

1987

The Journal of Cell Biology

103

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Section: Discussionsupporting

confidence: 65%

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

Yamashiro

Maxfield

1987

The Journal of Cell Biology

103

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“…If so, then the higher pH might be due to differences in pH regulation between phagosomes and phagolysosomes. In some cases, it has been reported that endocytic compartments undergo further acidification as iysosomal fusion occurs (27)(28)(29). However, in other cell types, it has been found that the pH of prelysosomal endocytic compartments is about the same as the pH of lysosomes (18,21,27).…”

Section: Nonactivated Monocytesmentioning

confidence: 98%

Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

Horwitz¹,

Maxfield²

1984

The Journal of Cell Biology

356

229

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We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P < 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coil, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes.To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of ~6.1 and sheep erythrocyte phagosomes had a pH of ~5.0 or below.This study demonstrates that L. pneumophila is capable of modifying the pH of its phagocytic vacuole. This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens.Legionella pneumophila, the agent of Legionnaires' disease, multiplies intracellularly in human mononuclear phagocytes within a membrane-bound cytoplasmic phagosome (1, 2).Live L. pneumophila inhibits fusion of the phagosome with monocyte lysosomes and induces the formation of a novel ribosome-lined phagosome. In contrast, formalin-killed L. pneumophila does not inhibit fusion or induce the formation of the specialized phagosome, and the dead bacteria are degraded within the phagolysosome (3, 4).The intraphagosomal pH has been measured or estimated in several bacteria and other particles that are phagocytized by mammalian phagocytes and later reside in phagolysosomes (5-11). These phagolysosomes become acidified. Since live and formalin-killed L. pneumophila have different intracellular destinies, we were interested in comparing the pH of phagosomes containing these two types of bacteria and exploring the possibility that alteration of intraphagosomal pH may play a role in L. pneumophila intracellular survival.In this paper, we used quantitative fluorescence microscopy to measure the pH of monocyte phagosomes that contain L.

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“…To study MF-T internalization, MF-T was labeled with a pH-sensitive fluorescent dye CypHer5E exhibiting fluorescence activity at acidic pH, that is, in late endosomes and lysosomes (40). Consequently, any detected fluorescent signal is derived from internalized antibodies.…”

Section: Preparation and Characterization Of A Mesothelin Adc (Bay 94mentioning

confidence: 99%

Anetumab Ravtansine: A Novel Mesothelin-Targeting Antibody–Drug Conjugate Cures Tumors with Heterogeneous Target Expression Favored by Bystander Effect

Golfier

Kopitz

Kahnert

et al. 2014

Molecular Cancer Therapeutics

190

166

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Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC 50 of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors. Mol Cancer Ther; 13(6); 1537-48. Ó2014 AACR.

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Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6.

Cited by 269 publications

References 30 publications

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

Anetumab Ravtansine: A Novel Mesothelin-Targeting Antibody–Drug Conjugate Cures Tumors with Heterogeneous Target Expression Favored by Bystander Effect

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