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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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Kinetics of human procathepsin L secretion and processing in KB-3-1 (A) and KB-CP20 (B) cells. Cells were radiolabelled briefly for 30 min with Trans-35S-label and chased for varying time periods (0 – 240 min, as shown on the top of the gel panels A and B) in serum-free medium. At the end of each time period, medium was saved and cells were lysed after washing with ice-cold PBS. The radiolabelled human procathepsin L was immunoprecipitated using a polyclonal antibody. The immunoprecipitates were resolved on SDS – PAGE and subjected to fluorography. The values given on the left side of the panels indicate the molecular weight of different forms of cathepsin L in kDa.
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fig4: Kinetics of human procathepsin L secretion and processing in KB-3-1 (A) and KB-CP20 (B) cells. Cells were radiolabelled briefly for 30 min with Trans-35S-label and chased for varying time periods (0 – 240 min, as shown on the top of the gel panels A and B) in serum-free medium. At the end of each time period, medium was saved and cells were lysed after washing with ice-cold PBS. The radiolabelled human procathepsin L was immunoprecipitated using a polyclonal antibody. The immunoprecipitates were resolved on SDS – PAGE and subjected to fluorography. The values given on the left side of the panels indicate the molecular weight of different forms of cathepsin L in kDa.

Mentions: Malignantly transformed cells are known to secrete a large amount of the 42 kDa procathepsin L into the culture medium (Miesenbock et al 1998). To understand if cellular secretion of this protease is affected by the development of the CP-r phenotype in KB-3-1 cells, we studied the kinetics of procathepsin L secretion into the culture medium by KB-3-1 and KB-CP20 cells. Our results demonstrate that both KB-3-1 and KB-CP20 cells secrete procathepsin L into the culture medium in comparable amounts (Figure 4A, BFigure 4


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)

Kinetics of human procathepsin L secretion and processing in KB-3-1 (A) and KB-CP20 (B) cells. Cells were radiolabelled briefly for 30 min with Trans-35S-label and chased for varying time periods (0 – 240 min, as shown on the top of the gel panels A and B) in serum-free medium. At the end of each time period, medium was saved and cells were lysed after washing with ice-cold PBS. The radiolabelled human procathepsin L was immunoprecipitated using a polyclonal antibody. The immunoprecipitates were resolved on SDS – PAGE and subjected to fluorography. The values given on the left side of the panels indicate the molecular weight of different forms of cathepsin L in kDa.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Kinetics of human procathepsin L secretion and processing in KB-3-1 (A) and KB-CP20 (B) cells. Cells were radiolabelled briefly for 30 min with Trans-35S-label and chased for varying time periods (0 – 240 min, as shown on the top of the gel panels A and B) in serum-free medium. At the end of each time period, medium was saved and cells were lysed after washing with ice-cold PBS. The radiolabelled human procathepsin L was immunoprecipitated using a polyclonal antibody. The immunoprecipitates were resolved on SDS – PAGE and subjected to fluorography. The values given on the left side of the panels indicate the molecular weight of different forms of cathepsin L in kDa.
Mentions: Malignantly transformed cells are known to secrete a large amount of the 42 kDa procathepsin L into the culture medium (Miesenbock et al 1998). To understand if cellular secretion of this protease is affected by the development of the CP-r phenotype in KB-3-1 cells, we studied the kinetics of procathepsin L secretion into the culture medium by KB-3-1 and KB-CP20 cells. Our results demonstrate that both KB-3-1 and KB-CP20 cells secrete procathepsin L into the culture medium in comparable amounts (Figure 4A, BFigure 4

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