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Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines.

Chauhan SS, Liang XJ, Su AW, Pai-Panandiker A, Shen DW, Hanover JA, Gottesman MM - Br. J. Cancer (2003)

Bottom Line: In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells.Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF.Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20842-4254, USA.

ABSTRACT
We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

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Killing curves of KB-3-1 and KB-CP20 cells with unconjugated PE (A), and human Pseudomonas exotoxin-resistant cell lines ET-12, ET-22 and ET-28 to cisplatin in comparison to their wild-type KB-3-1 cells (B) were determined as described in Materials and Methods.
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fig7: Killing curves of KB-3-1 and KB-CP20 cells with unconjugated PE (A), and human Pseudomonas exotoxin-resistant cell lines ET-12, ET-22 and ET-28 to cisplatin in comparison to their wild-type KB-3-1 cells (B) were determined as described in Materials and Methods.

Mentions: The killing curves shown in Figure 7AFigure 7


Reduced endocytosis and altered lysosome function in cisplatin-resistant cell lines.

Chauhan SS, Liang XJ, Su AW, Pai-Panandiker A, Shen DW, Hanover JA, Gottesman MM - Br. J. Cancer (2003)

Killing curves of KB-3-1 and KB-CP20 cells with unconjugated PE (A), and human Pseudomonas exotoxin-resistant cell lines ET-12, ET-22 and ET-28 to cisplatin in comparison to their wild-type KB-3-1 cells (B) were determined as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747565&req=5

fig7: Killing curves of KB-3-1 and KB-CP20 cells with unconjugated PE (A), and human Pseudomonas exotoxin-resistant cell lines ET-12, ET-22 and ET-28 to cisplatin in comparison to their wild-type KB-3-1 cells (B) were determined as described in Materials and Methods.
Mentions: The killing curves shown in Figure 7AFigure 7

Bottom Line: In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells.Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF.Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20842-4254, USA.

ABSTRACT
We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Show MeSH
Related in: MedlinePlus