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Experimental Therapeutics

Immunotoxin Resistance in Multidrug Resistant Cells

Melissa S. McGrath, Michael G. Rosenblum, Mark R. Philips and David A. Scheinberg
DOI:  Published January 2003

Abstract

Multidrug resistance (MDR) can be mediated, in part, by overexpression of P-glycoprotein (P-gp) and is characterized by broad resistance to several structurally, chemically, and pharmacologically distinct chemotherapeutic compounds. It has been hypothesized that immunological approaches to cytolysis may be used to overcome drug resistance. RV+ is a P-gp-expressing variant of the human myeloid leukemic cell line HL60 that displays a typical MDR phenotype. MDR RV+ cells displayed relative resistance to the immunotoxin (IT) HuM195-gelonin and to free rGelonin. K562 leukemia cells retrovirally infected to overexpress P-gp are also resistant to HuM195-gelonin. In addition, a monoclonal antibody capable of inhibiting the function of P-gp was able to partially reverse resistance to the IT. These data indicated that the expression of P-gp may contribute to IT resistance in RV+. Resistance to the IT was not mediated through decreased binding to cells, nor reduced internalization into the cell because the IT displayed similar kinetics of binding and internalization for both the parental HL60 and MDR RV+ cell lines. Comparison of the cytotoxicity of other ribosome-inactivating toxins indicated that RV+ cells were not universally resistant to toxins: RV+ cells were sensitive to the actions of ricin A chain, which acts on precisely the same RNase target as gelonin. Sensitivity of the MDR RV+ cells to the protein synthesis inhibitor cycloheximide, saponin, and Pseudomonas exotoxin A additionally confirmed that the resistance was not mediated through the ribosome and that pathways downstream from the inactivation of protein synthesis leading to cell death were not substantially perturbed in the MDR cells. Resistance could be partially abrogated by bafilomycin A, which inhibits lysosomal function. Moreover, direct visualization by confocal microscopy of the intracellular trafficking route of the IT showed that the IT accumulated preferentially in the lysosome in MDR RV+ cells but not in sensitive cells. These observations implicated the process of increased lysosomal degradation as the most likely basis for resistance. Such pathways of resistance may be important in the therapeutic applications of ITs, now becoming available for human use.

INTRODUCTION

The existence of multidrug resistant tumor cells remains a major obstacle for effective chemotherapy of cancer. Studies of MDR 2 have demonstrated that decreased accumulation of drug (1, 2, 3, 4, 5, 6) , overexpression of the MDR-1 gene product, P-gp (7) , and increased intracellular pH (8, 9, 10, 11, 12) are associated with the phenotype. It has been hypothesized that immunological approaches may be used to overcome the MDR induced by chemotherapy (13) . We and others, however, showed previously that MDR cells appear to be resistant also to some forms of immunotherapeutic attack, including complement-mediated cytotoxicity (14 , 15) , interleukin 2 (16) , and drug immunoconjugates (17) .

Gelonin is a Mr 30,000 type I single polypeptide chain RIP originally isolated from the seeds of Gelonium multiflorum. Gelonin irreversibly inhibits protein synthesis by inactivating the 28S ribosomal subunit. Protein synthesis is arrested by cleavage of RNase adenine N-glycosidic linkages, resulting in cell death (18) . Lack of a cell surface binding domain renders gelonin substantially less toxic to intact cells (19) and allows it to be specifically targeted to tumor cells via conjugation to a mAb. Gelonin’s activity is similar to that of ricin A chain in that both toxins share cleavage specificity but only 33% sequence homology (20) .

The IT HuM195-gelonin consists of a humanized mAb specific for CD33 (21, 22, 23) conjugated to a recombinant form of the gelonin toxin (20 , 21 , 24 , 25) . CD33 is expressed on 85% of acute myelogenous leukemias and is a therapeutically useful target because of its lack of expression outside of the hematopoietic lineage and its absence from CD34+ pluripotent stem cells of the bone marrow (26 , 27) . Recently, an anti-CD33 antibody drug conjugate was approved for human use in the treatment of acute leukemias (28) .

The cytotoxic effects of HuM195-gelonin and free rGelonin were tested on the CD33+ human myeloid leukemic cell line HL60 and their P-gp-expressing variant RV+, which is daunorubicin resistant (14 , 29) . RV+ cells were selected on the chemotherapeutic drug vincristine to overexpress P-gp. In addition, we tested the effects of the IT on the CD33-myeloid leukemic cell line K562, and its variant K562 i/s9 that had been retrovirally infected with mdr1. Although HuM195-gelonin can effectively kill parental HL60 and K562 cells, MDR RV+ and K562 i/s9 cells were shown to be resistant. Therefore, this article seeks to explain this observation. To determine the mechanism(s) of resistance of MDR RV+ cells to HuM195-gelonin, we divided the functional pathway of the actions of the IT into discrete steps for individual study. These were IT binding to the cells, initial internalization, metabolism, cellular trafficking/lysosomal accumulation, ribosome inactivation, and apoptotic pathways leading to cell death. We were able to show that lysis of the conjugate linkage, altered binding of the IT, changes in internalization, ribosome inactivation, and apoptotic pathways did not appear to be responsible for mediating resistance to the IT. However, the trafficking pattern of the IT was altered in the MDR RV+ and the IT preferentially accumulates within the lysosomes.

MATERIALS AND METHODS

Cells and Reagents.

HL60 cells (acute myeloid leukemia, CD33+) and K562 cells (chronic myeloid leukemia) were maintained at the Memorial Sloan-Kettering Cancer Center. RV+ cells (MDR-overexpressing HL60 variant) that have been selected to overexpress P-gp through exposure to chemotherapeutic drug were the generous gift of Dr. Melvin Center (Kansas State University, Manhattan, KS) and were provided by Dr. Ellen Berman (Memorial Hospital). RV+ cells were maintained in 120 μl (0.1 mg/ml) vincristine (Eli Lilly, Indianapolis, IN) to maintain the MDR phenotype. K562 i/s9 cells (retrovirally infected with the mdr1 gene) were the gift of Dr. Kathleen Scotto (Memorial Sloan-Kettering, New York, NY). HuM195-gelonin and rGelonin were produced by Dr. Michael Rosenblum (M. D. Anderson Cancer Center, Houston, TX; Ref. 21 ). UIC2, an IgG2a antibody, was provided by Memorial Sloan-Kettering Monoclonal Antibody Core Facility. Fluoresceinated, affinity-purified goat antimouse and goat antihuman antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). IgG2a isotypic control and T9 (anti-CD71) antibodies were purchased from Coulter (Hialeah, FL). Cycloheximide, ricin A chain, DT, saponin, and Pseudomonas exotoxin A (PE) and bafilomycin A were purchased from Sigma (St. Louis, MO). Bioporter protein delivery reagent was purchased from Gene Therapy Systems (San Diego, CA). Alexa-Fluor 594 protein labeling kit, Alexa-Fluor 488 Transferrin, LysoTracker yellow 123, and LysoSensor yellow were purchased from Molecular Probes (Eugene, OR).

Inhibition of [3H]Thymidine or [3H]Leucine Incorporation.

Cells were washed and resuspended in complete RPMI media to a concentration of 5 × 105 cells/ml unless otherwise stated. A total of 100-μl aliquots of cells was plated in 96-well plates in the presence of various amounts of HuM195-gelonin, rGelonin, or stated toxin. This mixture was then incubated for 2–5 days at 37°C and 5% CO2, followed by a 4–6 h incubation in [3H]thymidine or [3H]leucine. Cells were harvested using a Combi-Cell Harvester (Skatron, Sterling, VA) and read on a 1205 Beta Plate Liquid Scintillation Counter (Wallac, Turku, Finland).

Flow Cytometric Analysis.

RV+ and HL60 cells were washed and resuspended in 2% rabbit serum (Pel Freeze, Rogers, AK) to reduce nonspecific binding. Samples containing 2 × 105 cells in a final volume of 0.1 ml were then incubated on ice for 45 min in the presence of primary antibody (UIC2 or HuM195-gelonin). Cells were washed twice in ice-cold PBS then incubated on ice with a secondary FITC-labeled goat antimouse or goat antihuman immunoglobulin (Jackson ImmunoResearch Laboratories). Cells were again washed twice in PBS and then fixed in 0.5% paraformaldehyde. FITC fluorescence intensity was measured on a FACSCalibur (Becton Dickinson, San Jose, CA).

Radioiodination and Radioimmunoassays.

HuM195-gelonin was radiolabeled with Na-125I (NEN-Dupont) using chloramine-T to start and sodium metabisulfite to stop the reaction as described previously (27) . Specific activity was 44,300 cpm/ng. Binding and internalization assays were performed in tubes that had been blocked with PBS/BSA. RV+ and HL60 cells were washed and blocked in 2% rabbit serum on ice for 20 min. Cells were then washed and resuspended in ice-cold media. A total of 150 μl of 125I-HuM195-gelonin was added to cells. Immediately following the addition of the IT, 200 μl of aliquots were removed and placed in ice-cold media and washed three times. One ml of stripping solution [50 mm glycine/150 mm NaCl (pH 2.8)] was added, and tubes were incubated at room temperature for 10 min with occasional vortexing. The internalized (acid resistant) radioactivity and surface bound activity (stripped) were measured (23) .

Reversal of Resistance with UIC2 mAb.

RV+, HL60, K562, and K562 i/s9 cells were washed and resuspended in complete RPMI media to a concentration of 5 × 105 cells/ml. 100-μl aliquots of cells were then incubated at 37°C in a final concentration of 20 μg/ml UIC2 mAb for 2 h before the addition of HuM195-gelonin. A [3H]thymidine incorporation was then performed as described.

Inhibition of Protein Synthesis.

Inhibition of protein synthesis by rGelonin in a cell-free assay was performed using TNT T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI) as described by the manufacturer.

Cell Viability Assay.

Cells were washed and then resuspended in complete RPMI media to a concentration of 0.1–0.3 × 106 cells/ml and plated in 96-well plates. Various amounts of HuM195-gelonin or other toxin were then added for a final volume of 100 μl and allowed to incubate at 37°C and 5% CO2 for 2–3 days. Twenty μl of ProCheck Cell Viability Reagent (Intergen, Purchase, NY) were added to the cells for a total volume of 120 μl. Cells were incubated in the dark at 37°C and 5% CO2 for 1–4 h, and the absorbance was read at 475 nm.

Staining with PI.

A total of 5 × 105 RV+ or HL60 cells was incubated in 1 μg/ml HuM195-gelonin for 0–72 h. Staining with PI was then performed as described previously (30) .

Confocal Microscopy.

HuM195-gelonin was labeled with Alexa-Fluor 594 according to the manufacturer’s instructions (Molecular Probes). RV+ and HL60 cells were washed and then plated at 1 × 105 in 96-well plates. Alexa-Fluor-labeled IT was then added to the cells with or without Hoecsht nuclear stain, Alexa-Fluor Transferrin, LysoTracker, or LysoSensor dyes and incubated for 1, 4, 12, 24, and 48 h. Cells were then plated on microfrost slides, allowed to dry for 1–2 h, and fixed in 2% paraformaldehyde for 10 min at room temperature. Slides were kept in the dark at 4°C until read on a confocal microscope (LSM-510 Confocal System).

Bioporter Protein Delivery.

The reagent was dissolved in 250 μl of methanol and vortexed for 20 s on high speed. One μl of the Bioporter reagent was then transferred to an Eppendorf tube and allowed to dry in a laminar flow hood for 2–4 h. A total of 10–100 μg/ml free rGelonin was diluted in PBS [20 mm sodium phosphate, 150 mm NaCl (pH 7.4)] and then added to the Eppendorf tube. The mixture was vortexed and incubated at room temperature for 5 min. After the addition of 40 μl of serum-free medium, the bioporter reagent/free rGelonin mixture was added to 50 μl of cells plated in a 96-well plate at 2 × 104 cells/well. Cells were allowed to incubate 37°C and 5% CO2 for 4 h at 37°C and 5% CO2 in serum-free media. One hundred μl of complete media was then added to each well, and cells were allowed to incubate for an additional 20 h followed by a [3H]thymidine incorporation assay.

RESULTS

RV+ and K562 i/s9 cells Are Resistant to HuM195-Gelonin and Free rGelonin.

RV+ cells have been selected to overexpress P-gp through exposure to chemotherapeutic drugs, whereas K562 i/s9 cells have been retrovirally infected with MDR. Both the RV+ and K562 i/s9 cells expressed significant amounts of cell surface P-gp, and sensitive parental cells did not express P-gp above background levels as determined by the use of flow cytometry for expression the protein (Table 1) ⇓ . The cytotoxic effects of HuM195-gelonin and free rGelonin were tested on these CD33+, RV+, and HL60 human myeloid leukemic cell lines. The P-gp expression correlated with resistance to the IT in the two cell types. MDR RV+ cells had a LD50 in excess 1 μg/ml, whereas the parental HL60 had a LD50 of ∼0.001 μg/ml (Fig. 1A) ⇓ . Sensitivity and resistance of the cells to death from the IT, rather than simply a reduction in DNA synthesis, was confirmed by performing parallel experiments with trypan blue, PI staining, and ProCheck cell viability assays (data not shown). RV+ cells were found to be resistant to free rGelonin as compared with parental HL60 cells (Fig. 1B) ⇓ . Because RV+ cells were resistant to the free rGelonin, an inability to lyse the covalent bond between the HuM195 IgG and the gelonin could not be the cause of resistance. P-gp-expressing K562 i/s9 cells (CD33 low) were also shown to be resistant to HuM195-gelonin as compared with parental K562 (Fig. 1C) ⇓ . The LD50 of the IT is higher on cells that have low CD33 expression. The LD50 of free rGelonin is higher on all cells because of the lack of a receptor.

Fig. 1.
Fig. 1.

RV+ and MDR K562 i/s9 cells are resistant to HuM195-gelonin and free rGelonin. A, MDR RV+ (▪) and parental HL60 cells (○) were incubated with free HuM195-gelonin for 5 days at 37°C and 5% CO2 followed by [3H]thymidine incorporation as described in “Materials and Methods.” Data shown represent a single experiment performed in triplicate taken as a percentage of the control of cells and media alone. B, experiment performed using free rGelonin as described above. C, MDR K562 i/S9 cells (♦), K562 parental cells (⋄) were incubated with HuM195-gelonin for 3 days followed by a [3H]thymidine incorporation. Samples that have no error bars have SDs that are too small for representation on the graph. A–C are representative of >10 experiments.

View this table:
Table 1

Flow cytometric analysis of P-gp Expressiona

Cells were prepared as described in “Materials and Methods.” Briefly, cells were incubated in UIC2 mAb for 45 min. Cells were washed twice in ice-cold PBS and then incubated on ice with secondary FITC-labeled goat-antimouse antibody. Cells were fixed and read by flow cytometry.

Inhibition of Protein Synthesis by Free rGelonin and IT (Biological Activity).

Functional analysis of both free rGelonin and HuM195-gelonin were performed to show that the mechanism of action of the IT involved inhibition of the ribosomes. A cell-free coupled transcription/translation assay in which rGelonin inhibits protein synthesis in a dose-dependent manner is illustrated in Fig. 2A ⇓ . To confirm that HuM195-gelonin was capable of inhibiting protein synthesis in whole cells, we performed a [3H]leucine incorporation in RV+ and HL60 cells. We found that the IT was able to inhibit protein synthesis only in parental HL60 cells (Fig. 2B) ⇓ . HuM195 antibody alone has no activity (23) .

Fig. 2.
Fig. 2.

Inhibition of protein synthesis by free rGelonin (in vitro) and HuM195-gelonin (in vivo). A, an in vitro-coupled transcription translation assay performed as described in “Materials and Methods.” The data shown are a mean of triplicate measurements with fmr used as the template DNA. B, MDR RV+ cells (▪) and HL60 cells (○) were incubated with varying amounts of HuM195-gelonin for 3 days at 37°C and 5% CO2 followed by a [3H]leucine incorporation. Data shown represent a mean ± SD of experiments performed in triplicate as described above. Samples that have no error bars have SDs that are too small for representation on the graph.

Resistance to HuM195-Gelonin Can be Reversed with UIC2 mAb.

To further support the role of P-gp expression in the mediation of resistance to the IT and to argue against another form of resistance that might have been acquired by these cells during their cultivation, cytotoxicity assays were performed in the presence of UIC2, a monoclonal specific for P-gp. UIC2 can partially reverse the resistance to daunorubicin (14) . UIC2 was also able to substantially reverse resistance to HuM195-gelonin in RV+ cells (Fig. 3A) ⇓ . Incubation with control antibody, which binds to the surface of the cells (anti-CD71, T9) or UIC2 antibody alone, did not sensitize the cells to the IT and did not effect the percentage incorporation of [3H]thymidine > 3.5% in either direction. Similar experiments were performed using MDR K562 i/s9 cells in which we also observed a partial reversal of resistance to the IT (Fig. 3B) ⇓ .

Fig. 3.
Fig. 3.

UIC2 can reverse resistance to HuM195-gelonin in MDR-expressing cells. A, RV+ cells pretreated for 2 h in a final concentration of 20 μg/ml UIC2 mAb, RV+ cells untreated, HL60 cells pretreated with UIC2, and HL60 cells untreated were incubated with HuM195-gelonin for 5 days at 37°C and 5% CO2 followed by a [3H]thymidine incorporation. B, MDR K562 i/s9 cells pretreated with UIC2, untreated MDR K562 i/s9 cells, and untreated parental K562 cells were incubated with HuM195-gelonin for 3 days at 37°C and 5% CO2 followed by a [3H]thymidine incorporation. Data shown represent a mean ± SD of experiments performed in triplicate as described above. UIC2 antibody alone and isotypic control antibodies alone were not toxic to the cells.

Binding and Internalization Kinetics Are Similar for Parental HL60 and MDR RV+ Cells.

One simple explanation for IT resistance would be reduced of binding or subsequent internalization of the IT into the resistant cells. The kinetics of 125I-HuM195-gelonin binding and internal accumulation in MDR RV+ and HL60 cells were studied (Fig. 4A) ⇓ . The binding kinetics and internalization curves are similar for both cell lines, indicating similar initial processing of the IT. Moreover, experiments performed on ice demonstrated equal binding of the 125I-HuM195-gelonin to both MDR RV+ and parental HL60. To confirm these results, binding and internalization studies analyzed by flow cytometry were also performed. The binding of HuM195-gelonin indirectly labeled with goat antihuman-FITC antibody was followed >4 h on ice (data not shown) and 37°C (Fig. 4B) ⇓ to promote modulation of the IT. Comparable curves illustrate similar binding and initial internalization kinetics of HuM195-gelonin immune complex in both cell lines. Controls using goat anti-human Ig-FITC (GAH-FITC) alone (Fig. 4B) ⇓ and IgG2a isotypic control plus GAH-FITC (data not shown) showed no significant binding to either of these cell lines.

Fig. 4.
Fig. 4.

RV+ and HL60 cells do not differ in binding and internalization kinetics of HuM195-gelonin. A, RV+ cells and HL60 cells were incubated at 37°C with 125I-HuM195-gelonin for up to 4 h, followed by the removal of 200 μl-aliquots and stripping of bound activity from the surface. The same experiment was conducted at 0°C with RV+ cells and HL60 cells. These data are representative of ≥3 experiments. B, RV+ and HL60 cells were incubated with HuM195-gelonin at 37°C for 0, 1, 2, and 4 h and then indirectly labeled with GAH-FITC. RV+ and HL60 were also labeled with GAH-FITC alone at 37°C for 0 and 4 h. Data represents a percentage of indirectly labeled GAH-FITC HuM195-gelonin or GAH-FITC alone binding to HL60 and RV+ cells in ≥3 experiments.

RV+ Cells Are Sensitive to other Toxins.

To determine whether changes in the ribosome of the MDR RV+ cells could account for resistance to the IT, we examined the cytotoxicity of other inhibitors of protein synthesis, including DT, ricin A chain, PE, and cycloheximide. Ricin A chain is a RIP which hydrolyzes a specific N-glycosidic linkage in 28S rRNA similar to gelonin (31, 32, 33, 34) . Both toxins cleave an adenine in position 4324 (35 , 36) . Surprisingly, MDR RV+ cells were sensitive to the effects ricin A; despite relative resistance to free rGelonin and HuM195-gelonin IT (Table 2) ⇓ . Because these two toxins work on precisely the same target in the ribosome, we concluded that the ribosome target was unaltered in the MDR RV+ cells and was not responsible for conferring resistance to the IT. We then examined the cytotoxicity of PE and DT, which work via the same mechanism by inactivating EF-2, resulting in inhibition of protein synthesis (37) . Interestingly, MDR RV+ cells were sensitive to PE as compared with parental HL60 cells while remaining slightly resistant to DT (Table 2) ⇓ . Cycloheximide, which works by inhibiting the peptidyl transferase of the 60S ribosomal subunit (38) , was also tested. RV+ cells were found to be equally as sensitive to cycloheximide as parental HL60 cells (Table 2) ⇓ . These data support our conclusion that the ribosome itself is not involved in the resistance to the IT and that these agents have somewhat different mechanisms of action. In addition, because the RV+ cells were sensitive, in particular to ricin, which has the same known mechanism of action as gelonin, it shows that pathways downstream from the ribosomal inactivation (e.g., cell death pathways) were likely to be intact in the resistant cells. It does not rule out alternative pathways, however.

View this table:
Table 2

Toxin cytotoxicitya

Cells were plated into 96-well plates and incubated with various amounts of toxins or ITs for 2–5 days at 37 C and 5% CO2 followed by [3H]thymidine incorporation or ProCheck cell viability assay.

Time-Course of Inhibition of Protein Synthesis.

The period of time required for the IT to bind, internalize, traffic, and finally reach the ribosome where it inhibits protein synthesis was established through a series of [3H]leucine incorporations. These experiments were performed to suggest the window of time in which the resistance phenomenon occurred. Effective inhibition of protein synthesis occurred by 18 h (Fig. 5) ⇓ . The previous internalization studies showed that sustained internalization occurred within the first 4 h of incubation (Fig. 4A) ⇓ . These data suggest that a significant amount of processing occurs within the cell before the IT reaching its final target and affecting measurable ribosomal activity.

Fig. 5.
Fig. 5.

Time course of inhibition of protein synthesis by HuM195-gelonin. RV+ cells and HL60 cells were incubated with 1 μg/ml HuM195-gelonin for the time period indicated followed by [3H]leucine incorporation. Each data point represents an individual experiment.

Resistance to HuM195-Gelonin Can be Reversed with Bafilomycin A.

Degradation within the lysosomal compartment may be a potential cause of resistance to the IT in MDR RV+ cells. To determine whether the resistance to the IT was mediated by increased degradation within the lysosomal compartment, we preincubated both RV+ and HL60 cells with an inhibitor of lysosomal acidification. Bafilomycin A is a potent inhibitor of the vacuolar ATPase pump that prevents acidification and thereby inactivates lysosomal proteases in addition to preventing cellular trafficking to the lysosome (Refs. 39, 40, 41, 42, 43, 44 ; Fig. 6 ⇓ ). Preincubation of cells in bafilomycin A partially reversed the resistance to the IT in RV+ cells. Cytotoxicity of the IT in the parental HL60 cells was only moderately affected; interestingly, the HL60 cells were more resistant.

Fig. 6.
Fig. 6.

Bafilomycin A treatment can reverse resistance in RV+ cells. RV+ cells pretreated with 5 nm bafilomycin A (□), HL60 cells pretreated with 5 nm bafilomycin A (○), untreated RV+ cells (▪), and untreated HL60 cells (•) were incubated with HuM195-gelonin for 5 days followed by a [3H]thymidine incorporation. Data shown represent a mean ± SD of experiments performed in triplicate as described above. Samples that have no error bars have SDs that are too small for representation on the graph.

Confocal Microscopy and Cellular Trafficking Model.

The bafilomycin experiments implied that lysosomal degradation might be mediating resistance, and other steps before this stage of processing appeared similar between the sensitive and resistant lines; therefore, lysosomal trafficking of the IT became the focus of the experiments. The binding, internalization, and trafficking of Alexa-Fluor 594-labeled HuM195-gelonin were studied. RV+ and HL60 cells were incubated with Alexa-Fluor 594-labeled IT on ice for 1 h to determine the extent of binding. As before, similar binding of the labeled IT to the cell surface of both RV+ and HL60 cells was shown (Fig. 7, A and B) ⇓ . There was slightly more binding of Alexa-Fluor-labeled IT to RV+ cells, supporting our previous binding data (Fig. 4) ⇓ . Similar internalization of the Alexa-Fluor-labeled IT into a transferrin containing endosome in both parental and resistant cells was also demonstrated (data not shown). There was no significant difference in kinetics or distribution between the parental and MDR line. However, when HL60 and RV+ cells were incubated with Alexa-Fluor-labeled IT and the LysoTracker probe for 24 h (Fig. 7, C–F) ⇓ , the IT is preferentially accumulated in the lysosome (indicated by the orange color which is a result of the colocalization of both probes; Fig. 7H ⇓ ). In contrast, the IT does not appear to accumulate in the lysosome of the parental HL60 cells (Fig. 7G) ⇓ .

Fig. 7.
Fig. 7.

Confocal microscopy of Alexa-Fluor-594-HuM195-gelonin (AF-IT) and LysoTracker (LT). A, HL60 cells were incubated with AF-IT on ice for 1 h. Cells were washed twice in PBS, fixed in 2% paraformaldehyde, and imaged with a confocal microscope at ×63. B, a similar experiment was performed with RV+ cells. HL60 cells were incubated at 37° with AF-IT (C) and LysoTracker (E) for 24 h and read as described above. RV+ cells were incubated with AF-IT (D) and LysoTracker (F) for 24 h and read as described above. G, composite of C and E. H, composite of D and F. Each experiment was done in triplicate with hundreds of cells analyzed.

Bioporter Delivery of Free rGelonin into RV+ Cells.

Visualization of the IT through confocal microscopy implied that the mechanism of resistance involved trafficking to the lysosomal compartment in MDR RV+ cells. To further clarify the mechanism of resistance, we attempted to bypass the endocytosis and trafficking route normally taken by the IT with the use of an alternative penetration method. The Bioporter protein delivery system was used to deliver free rGelonin directly into the cytosol and circumvent the normal trafficking pathway. Sensitivity of MDR RV+ cells to rGelonin internalized via this method would indicate its failure to traffic and reach its final destination within the cell under normal circumstances. We found that 10 μg/ml free rGelonin alone and Bioporter reagent alone were not cytotoxic to RV+ cells with 96 ± 5% cell survival and 99 ± 2% cell survival, respectively. In contrast, viable RV+ cells were reduced by half (48 ± 4%) when 10 μg/ml rGelonin were delivered via Bioporter.

DISCUSSION

The overexpression of P-gp has been shown to cause resistance to a broad range of structurally and pharmacologically distinct cytotoxic compounds, including antibodies and drug immunoconjugates (17) . We describe the relative resistance of MDR RV+ and K562 i/s9 cells to both HuM195-gelonin and free rGelonin. The observation that RV+ cells are resistant to free rGelonin indicates the resistance is to the toxin itself and is not mediated through an inability to break the conjugation of the toxin to the antibody nor to CD33-specific receptor-mediated endocytosis. Free rGelonin and HuM195-gelonin could effectively inhibit protein synthesis in a cell-free system and [3H]leucine incorporation, respectively, confirming that the toxin and the IT were functional.

The resistance to HuM195-gelonin was partially reversed by preincubation with UIC2, a mAb specific for P-gp. This observation supports our hypothesis that overexpression of P-gp is mediating resistance, at least in part; this effect may be indirect. To further test if this resistance was mediated by an overexpression of P-gp and not because of RV+ cell selection on the chemotherapeutic drug vincristine, similar experiments examined the effects of the IT on another MDR, P-gp-expressing cell line. The MDR-expressing variant of K562 was prepared by retrovirally infecting it with mdr1, and cells were not selected on the drug. K562 i/s9 expressed significant amounts of MDR by flow cytometric analysis and exhibited resistance to the IT; this was also partially reversed by preincubation with UIC2. Because these cells express P-gp through retroviral infection and not through selection on chemotherapeutic drug, this confirms that overexpression of P-gp was responsible, at least in part, for mediating resistance to the IT.

The resistance of the cells to the IT could potentially originate from any of the several cellular compartments or processes responsible for interacting with the IT. Antigen density, affinity, degradation within the lysosome, and altered intracellular routing have all been attributed to decreases in IT cytotoxicity (45, 46, 47, 48, 49, 50) . Other possibilities included decreased binding, decreased endocytosis, modification of the ribosome target, or alteration of the downstream death pathways. We approached the problem of defining the mechanism of resistance by individually analyzing each step that had potential to inhibit the cytotoxicity of the IT.

Flow cytometric analysis and radioimmunoassays provided evidence that there were no significant differences in either binding or internalization, ruling out their possible roles in resistance. We also evaluated whether there was an alteration in the ribosome preventing the mechanism of action of the IT. To accomplish this, we compared the cytotoxicity of HuM195-gelonin of other RIPs. Interestingly, the RV+ cells are sensitive to ricin A chain while remaining resistant to both free rGelonin and HuM195-gelonin. This indicated first that RV+ cells are not universally resistant to all toxins. More importantly, this observation led us to the conclusion that an alteration of the ribosome was not involved in the resistance as both ricin A chain and gelonin work via the same molecular mechanism. Other inhibitors of protein synthesis, including cycloheximide, DT, and PE, which work through different mechanisms to inhibit protein synthesis were also tested. DT and PE work by inactivating EF-2, thereby inhibiting protein synthesis (51) . PE binds to the plasma membrane through the α2-macroglobulin receptor/low-density LRP (52) , whereas DT binds to pro-herapin-binding epidermal growth factor. RV+ cells were slightly resistant to DT while sensitive to PE. The mechanism of action of these two toxins is identical, and these data suggest the resistance in this case may also be caused by a failure of DT to be trafficked and delivered to the ribosomal target. In contrast, cycloheximide works via inhibition of peptidyl transferase of the large 60S ribosomal subunit (38) . MDR RV+ cells display equal sensitivity to cycloheximide as parental HL60 cells. These data additionally support our conclusion that the ribosome is not involved in resistance. Moreover, because MDR RV+ cells can be killed by these toxins, it is likely that the downstream effector death pathways remain relatively intact in these cells.

The mechanism of cytotoxicity of HuM195-gelonin is dependent on the IT being specifically targeted and bound to the cell surface via CD33, where it is then internalized and delivered to an endocytic compartment (53) . The IT is then sorted within intracellular compartments and translocated through the lipid bilayer. Once delivered into the cytosol, the IT may reach and inhibit the ribosome (54) . Although all toxins appear to be transported through the endocytic compartments, only DT has been shown to translocate through the endosome. DT undergoes a pH-dependent conformational change that exposes hydrophobic residues, allowing translocation of the toxin from the early endosome to the cytosol (54, 55, 56, 57, 58, 59) . Although the intracellular site of gelonin translocation remains unknown, study of the IT through confocal microscopy allowed direct visualization of the intracellular route of the IT through the cell. Both sensitive and resistant cells appeared to bind and initially internalize the IT into endosomes with similar kinetics; this is consistent with our previous biochemical data. However, the IT is preferentially accumulated in the lysosome in MDR RV+ cells, whereas it does not do this in HL60 cells. It has been shown that MDR cells display increased acidification of the endosomal and lysosomal compartments, causing accumulation of basic chemotherapeutic compounds in these areas by sequestering them. This is one mechanism that renders the cells relatively resistant. Bafilomycin A treatment of MDR cells can reverse this resistance (12) . Bafilomycin A inhibits the vacuolar ATPase pump of the endocytic pathway and is thought to prevent acidification of the lysosomal compartment, which in turn, inhibits lysosomal proteases (39 , 40) . In addition, bafilomycin A has been shown to inhibit cellular trafficking to the lysosome (41, 42, 43, 44) and inhibit degradation of ITs (60) . The resistance to the IT is partially reversed with incubation of the RV+ cells in bafilomycin A, supporting our conclusion that cytotoxicity of the IT is limited by accumulation in and degradation within the acidified lysosomal compartment and that this process appears altered in the MDR cells. A direct link from expression of P-gp to this observation is not proven. To further clarify the mechanism of resistance, we attempted to bypass the normal endocytosis and trafficking route taken by the IT with the use of another method of internalization. The Bioporter protein delivery system was used to deliver free rGelonin directly into the cytosol and circumvent the normal trafficking pathway to the lysosome. Resistance to free rGelonin in RV+ cells was overcome with Bioporter delivery of rGelonin into the cell suggests that the gelonin is not reaching to its cytoplasmic target. Therefore, altered trafficking, poor translocation, or increased accumulation within the lysosome are implicated as potential mechanisms of resistance. Taken together, these observations implicate the process of efficient sorting to the lysosome and possibly subsequent degradation as the primary source of resistance of RV+ to HuM195-gelonin. Alternatively, the altered milieu of the lysosome may prevent effective translocation of the toxin.

The mechanism by which MDR or P-gp expression leads to altered cellular trafficking is unclear. Several studies have concluded that the cytosolic pH of MDR-expressing drug-resistant cells is more alkaline than drug-sensitive cells and that this alkalinization may result in reduced trafficking to the lysosome (9 , 11 , 12 , 61) . Furthermore, pH of endosomal and lysosomal compartments of MDR cells are altered, which may contribute to resistance of the IT in this case. It is possible that pH changes may also play a role in the observations here and will be studied further.

Similar resistance mechanisms may have an important role in the clinical applications of ITs. The first Food and Drug Administration-approved IT, gemtuzumab ozogamicin, which is a conjugate of calicheamicin to an anti-CD33 antibody (analogous to the HuM195-gelonin construct described here), has been reported to be less active in MDR cells and displays reduced accumulation in and reduced activity against leukemia cells with the MDR phenotype (17) . Gemtuzumab ozogamicin is also not effective against RV+ cells (Table 2) ⇓ , probably because of other P-gp-mediated mechanisms. Therefore, understanding the various mechanisms by which the MDR phenotype is accomplished by cells may allow new strategies for more effective anticancer activity.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 To whom requests for reprints should be addressed, at Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-5010; Fax: (212) 717-3068; E-mail: d-scheinberg{at}ski.mskcc.org

  • ↵2 The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein; mAb, monoclonal antibody; RIP, ribosome-inactivating protein; IT, immunotoxin; PI, propidium iodide; DT, diphtheria toxin; PE, Pseudomonas exotoxin; LRP, lipoprotein receptor-related protein; EF-2, elongation factor 2; GAH, goat anti-human Ig.

  • Received July 8, 2002.
  • Accepted October 30, 2002.

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Cancer Research: 63 (1)
January 2003
Volume 63, Issue 1

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Immunotoxin Resistance in Multidrug Resistant Cells
Melissa S. McGrath, Michael G. Rosenblum, Mark R. Philips and David A. Scheinberg
Cancer Res January 1 2003 (63) (1) 72-79;
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