Endocytic recycling compartments altered in cisplatin-resistant cancer cells

Abstract

The clinical utility of cisplatin to treat human malignancies is often limited by the development of drug resistance. We have previously shown that cisplatin-resistant human KB adenocarcinoma cells that are cross-resistant to methotrexate and heavy metals have altered endocytic recycling. In this work, we tracked lipids in the endocytic recycling compartment (ERC) and found that the distribution of the ERC is altered in KB-CP.5 cells compared with parental KB-3-1 cells. A tightly clustered ERC is located near the nucleus in parental KB-3-1 cells but it appears loosely arranged and widely dispersed throughout the cytoplasm in KB-CP.5 cells. The altered distribution of the ERC in KB-CP.5 cells is related to the amount and distribution of stable detyrosinated microtubules (Glu-alpha-tubulin), as previously shown in Chinese hamster ovary B104-5 cells that carry a temperature-sensitive Glu-alpha-tubulin allele. In addition, B104-5 cells with a dispersed ERC under nonpermissive conditions were more resistant to cisplatin compared with B104-5 cells with a clustered ERC under permissive conditions. We conclude that resistance to cisplatin might be due, in part, to reduced uptake of cisplatin resulting from an endocytic defect reflecting defective formation of the ERC, possibly related to a shift in the relative amounts and distributions of stable microtubules.

Figure 1.

Altered ERC structures in CP-r cells as shown by labeling with lipid probes. A, DiIC₁₆ labeling of KB-3-1 cells and KB-CP.5 cells. The cells were labeled for 1 min at 37°C with DiIC₁₆ loaded on fatty acid free BSA, rinsed and chased for 30 min at 37°C. Cells were imaged live using wide field microscopy. Bar, 10 μm. B DiIC₁₂ labeling of KB-3-1 cells and KB-CP.5 cells. DiIC₁₂ labeling is similar to that of DiIC₁₆ mentioned above. Bar, 10 μm. C, Colocalization of Alexa488-Tf and DiIC₁₂ in KB-CP.5 cells. The cells were labeled for 5 min at 37°C with 10 μg/ml Alexa488-Tf, rinsed, labeled for 1 min with DiIC₁₂ loaded on fatty acid free BSA, rinsed and chased for 30 min at 37°C. Cells were imaged live using wide field microscopy. Arrows point to individual endosomes or clusters of endosomes, which are colocalized. Bar, 10 μm.

Figure 2.

Indirect fluorescence to localize transferrin with fluorescently-labeled cisplatin. Cells were labeled with Texas Red-Transferrin (red color) and Alexa 488-cisplatin (green color) as described in “Materials and Methods”, Labeled cells were excited at 488 nm and 568 nm provided by a krypton-laser as indicated, and fluorescence emissions at 520 nm and 598 nm were used for collecting green and red fluorescence, respectively, while DIC images of the same cells were collected in the third channel using a transmitted light detector. After sequential excitation, red and green fluorescent images of the same cells were merged for co-localization analysis. Colocalization of Texas Red-Transferrin (Red) and Alexa 488-cisplatin (Green) is shown in yellow.

Figure 3.

Sphingomyelin trafficking and metabolism measured in KB-3-1 and KB-CP.5 cells. A and B: NBD-SM labeling of KB-3-1 (panel A) and KB-CP.5 (panel B) cells. The cells were labeled with NBD-SM at 37° for 1 minute, chased for 30 min to achieve steady state distribution, back exchanged on ice to remove excess cell surface label, and then imaged using wide field microscopy. Bar, 10 μm. C and D: KB-CP.5 cells double-labeled with Alexa546-Tf (panel C) and NBD-SM (panel D). Negligible colocalization is observed. Bar, 10 μm. E: Ratio of NBD-Ceramide to NBD-SM in KB-3-1 cells (black bars) and KB-CP.5 cells (white bars), after excess NBD-SM on the plasma membrane has been removed by back exchange. The NBD lipid ratio thus represents primarily the NBD-lipid proportions in the ERC (since most of the NBD-SM from the plasma membranes is removed by the back exchange procedure). The protocol used for the NBD lipid ratio assay is explained in Materials and Methods. The first three sets of bars in the figure show three independent measurements, while the fourth set of bars show the average of these measurements, along with the standard deviation.

Figure 4.

Frequency distribution of the ratios of Alexa546 to Fluorescein (R/F ratio) in Tf-labeled KB-3-1 cells (black bars) and KB-CP.5 cells (grey bars). The cells were labeled for 30 min at 37°C with Tf double labeled with Alexa546 (red fluorescence; pH insensitive) and Fluorescein (green fluorescence; pH sensitive). They were then rinsed and chased for an additional 10 min at 37°C in the presence of deferroxamine (an iron chelator) and 10-fold excess unlabeled Tf, to prevent rebinding of labeled Tf. The cells were then fixed with 3% paraformaldehyde for 2 min at room temperature, and imaged using wide field microscopy. The Alexa 546/Fluorescein (R/F) ratio was measured after background correction. Increasing R/F ratio indicates increasing pH values.

Figure 5.

Expression of Glu-α-tubulin and Tyr-α-tubulin by Western blotting and confocal microscopy. A, Expression of Glu-α-tubulin and Tyr-α-tubulin in B104-5 cells under permissive and non-permissive condition. B104-5 cells were incubated under permissive 32°C and non-permissive 39°C conditions for 3 days in 5% CO₂, then cells were fixed and permeabilized as described in “Materials and Methods”. Several fields of cells were captured and one representative field of each is shown. Scale bar, 20 μm. B, Detection of Glu-α-tubulin and Tyr-α-tubulin in isolated fractions of KB-3-1 and KB-CP.5 cells. Fractions (W, whole cell lysis; M, plasma membrane enriched fraction; C, cytosol fraction) were isolated by different types of ultracentrifugation as described in “Materials and Methods.” C, Immunofluorescence pictures of KB-3-1 and KB-CP.5 cells stained with antibodies against Glu-α-tubulin and Tyr-α-tubulin. A pronounced aggregation of Glu-α-tubulin near the nuclei in KB-3-1 cells is observed, compared with KB-CP.5 cells. No changes in the cytoplasmic distribution pattern of the Tyr-α-tubulin are detectable. A representative image from at least 6 is shown.

Figure 6.

Cytotoxicity of cisplatin measured in cells with different ERC structures. A, The cytotoxicity of cisplatin in B104-5 cells incubated at permissive or non-permissive temperature was determined by colony forming assays, 4 weeks after continuous exposure to CP at different concentrations. The results are expressed as the percent of viable CP-treated cells compared with untreated cells. Each value represents the mean ± SEM of triplicate measurements. B, Colony forming assay to measure the cisplatin resistance of parental TRVb-1 CHO cells. After the cells were incubated in medium with various concentrations of CP, cells were treated and counted as described in Materials and Methods.