Identification of cells with lowered sensitivity to cytostatic agents by the use of fluorescent dyes

With a help of stepwise increase of vincristine concentrations in culture medium several lines of mouse myeloma X63 Ag 8.863 cells resistant to low concentrations of vincristine (6-35-fold) were selected. Rhodamine 123 stained resistant cells and wild-type cells with an equal intensity. However, resistant cells differ significantly from the sensitive ones by the rate of rhodamine efflux. The rate of the efflux was in proportion to the degree of resistance. The efflux of the dye could be blocked by the addition to reserpine, the inhibitor of multidrug resistance. Thus, fluorescent dyes can be used for the detection of cells with low levels of multidrug resistance.     

The membrane transport system responsible for multidrug resistance is operating in nonresistant cells

Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.     

Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.

Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

Expression of hamster P-glycoprotein and multidrug resistance in DNA-mediated transformants of mouse LTA cells.

The overexpression of a plasma membrane glycoprotein, P-glycoprotein, is strongly correlated with the expression of multidrug resistance. This phenotype (frequently observed in cell lines selected for resistance to a single drug) is characterized by cross resistance to many drugs, some of which are used in cancer chemotherapy. In the present study we showed that DNA-mediated transformants of mouse LTA cells with DNA from multidrug-resistant hamster cells acquired the multidrug resistance phenotype, that the transformants contained hamster P-glycoprotein DNA sequences, that these sequences were amplified whereas the recipient mouse P-glycoprotein sequences remained at wild-type levels, and that the overexpressed P-glycoprotein in these cells was of hamster origin. Furthermore, we showed that the hamster P-glycoprotein sequences were transfected independently of a group of genes that were originally coamplified and linked within a 1-megabase-pair region in the donor hamster genome. These data indicate that the high expression of P-glycoprotein is the only alteration required to mediate multidrug resistance.     

New Djungarian hamster cell lines with selective cytoplasmic and nuclear genetic markers

Two new Djungarian hamster cell lines which are resistant to chloramphenicol (CAP) are described. The clonal DMCAP subline was obtained by incubation of HPRT-deficient DM-15 cells for 6 months in the medium containing 50 micron/ml of CAP. Resistance to CAP is determined in DMCAP cells by the cytoplasm: cytoplasts from these cells could transmit resistance to CAP into sensitive cells, such as L or DMCH-2/1 cells by hybridization. However, after transplantation of DMCAP nuclei into L cytoplasts, the resulting hybrid cells lost resistance to CAP to a great extent. Using the capacity of DMCAP cytoplasts to transfer CAP-resistance, we obtained a line of hybrids (cyt. DMCAP X DMCH-2/1) which was resistant to 8-azaguanine, CAP and colchicine. As in the original DMCH-2/1 cell line, colchicine-resistance in the cybrid line appeared to be associated with gene amplification. Thus, chromosomal analysis showed that the karyotype of the hybrids was identical to that of DMCH-2/1 cells. Both contained marker chromosomes with homogeneously staining regions (HSRs) and, during incubation in the colchicine-free medium, lost resistance to colchicine. The loss of resistance was accompanied by a decrease in the number of cells containing chromosomes with HSRs and an increase in the number with double minutes (DMs). Many cells containing small chromatin bodies in their cytoplasm also appeared. These chromatin bodies may be DMs lost from the nucleus during mitosis. These new sublines with cytoplasmic and nuclear genetic markers may be useful in the further study of cytoplasmic-nuclear interactions, particularly, in the analysis of possible activities of the DNA fragments which appear in the cytoplasm during reversion to colchicine sensitivity.     

Molecular mechanism of multidrug resistance in tumor cells

The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy. This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells. This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed. Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance. The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport. P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells. The functions of the mdr system in normal cells and its potential clinical implications are discussed.     

Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins

We have established four cell lines derived from the human KB carcinoma cell line which express high-level multiple drug resistance. One of these lines was selected for resistance to colchicine, one was selected for resistance to colchicine in the presence of the tumor promoter, mezerein, one for resistance to vinblastine, and one for resistance to adriamycin. All of these cell lines are cross-resistant to the other selective agents. The development of multidrug resistance in these cultured human carcinoma cells is associated with a limited number of specific protein alterations revealed by high resolution two-dimensional gel electrophoresis and Western blot analysis. These protein alterations in multidrug-resistant lines include the decreased prevalence of members of a family of proteins of molecular mass 70,000 to 80,000 daltons, pI 4.8-5.0, the increased synthesis of a protein of molecular mass 21,000 daltons, pI 5.0, in the colchicine-resistant cell lines only, and the increased expression of a 170,000-dalton protein in membrane preparations from all of the resistant cells. The loss of the 70,000- to 80,000-dalton proteins in the multidrug-resistant lines, which can also be demonstrated by immunoprecipitation of these proteins with specific antisera, is associated with a loss of translatable mRNA for these proteins. These studies suggest that only a limited number of protein changes occur in multidrug-resistant cell lines.     

Sensitization to the cytotoxicity of adriamycin by verapamil and heat in multidrug-resistant Chinese hamster ovary cells.

Development of multidrug resistance to anticancer agents is a major limitation for the success of cancer chemotherapy. The chemosensitizer verapamil increases intracellular accumulation of drugs such as adriamycin in certain multidrug-resistant cell lines. When combined with verapamil, hyperthermia should be able to alter membrane permeability to adriamycin and to enhance the cytotoxicity of the drug. Verapamil increased the cytotoxicity of adriamycin in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) but not in drug-sensitive cells (AuxB1). Hyperthermia (42 degrees C) alone clearly increased the cytotoxicity of adriamycin in AuxB1 cells. There was also a small increase in CH(R)C5 cells at 42 and 43 degrees C. In drug-resistant cells, the cytotoxicity of adriamycin increased considerably when verapamil was combined with heat. This effect was dependent on temperature and increased with time of incubation. At 37 degrees C, verapamil increased the uptake of adriamycin in CH(R)C5 cells, while drug efflux decreased. When verapamil was combined with hyperthermia, drug efflux decreased even further. These results led to an overall increase in intracellular accumulation of the drug. In drug-sensitive cells, hyperthermia increased both the uptake and efflux of adriamycin, but verapamil had no effect. Verapamil plus heat increased the cytotoxicity of adriamycin in drug-resistant cells, and this was accompanied by altered permeability of the membrane to the drug. Hyperthermia combined with verapamil could be beneficial by increasing the effectiveness of adriamycin in the elimination of multidrug-resistant cells in a localized target region.     

Resistance against cycloheximide in cell lines from Chinese hamster and human cells is conferred by the large subunit of cytoplasmic ribosomes

Summary Cell lines from Chinese hamster ovary [CHO-K1-D3] and human fibroblast cells [46, XX, 18p-] were mutagenized with N-nitrosomethylurea followed by a selection for cycloheximide resistance. Two mutants resistant against the durg were selected from either wildtype. 80S ribosomes and their ribosomal subunits were isolated from all mutant and wildtype cells. 80S ribosomes reassociated from the isolated subunits were as active as isolated 80S couples in the poly (U) dependent poly (Phe) synthesis. Hybrid 80S ribosomes constructed from subunits of the various cell lines of the same species were fully active, whereas the interspecies 80S hybrids were not active at all in poly (Phe) synthesis.Hybrid 80S ribosomes from subunits of mutant and the ocrresponding wildtype cells were tested in the poly (U) assay in the presence and absence of cycloheximide. The results strikingly indicate that in all four mutant cell lines the resistance against cycloheximide is conferred by the large subunit of cytoplasmic ribosomes.Abbreviations CHM Cycloheximide - CHO Chinese hamster ovarien - FBS foetal bovine serum - Eagle MEM Eagle minimal essential medium - EMS Ethyl-metansulfonate - NMU N-nitrosomethylurea     

Chromosome-mediated gene transfer of multidrug resistance.

Multidrug resistance can be transferred from drug-resistant LZ Chinese hamster cells to drug-susceptible mouse LTA cells by chromosome-mediated gene transfer. Analysis of genomic DNA demonstrated the transfer of multiple copies of a DNA domain which is amplified in the donor multidrug-resistant cells. The transfer of 10 to 15 copies of the Chinese hamster gene was sufficient to produce a multidrug-resistant phenotype. Chromosome transferents exhibited overexpression of an mRNA of approximately 5 kilobases which has previously been demonstrated to be encoded by the amplified DNA domain of the donor LZ cells. Phenotypic analysis of individual clones selected in adriamycin showed the resistance to be pleiotropic. All clones tested demonstrated similar levels of cross-resistance to the drugs daunorubicin and colchicine. These results indicate that the DNA sequences transferred confer the complete multidrug-resistant phenotype on recipient cells and suggest that multidrug resistance is due to overexpression of the protein encoded by the 5-kilobase mRNA.     

Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.     

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.     

Differential amplification and disproportionate expression of five genes in three multidrug-resistant Chinese hamster lung cell lines.

At least five linked genes are amplified in the multidrug-resistant Chinese hamster ovary cell line CHRC5, selected with colchicine (A. M. Van der Bliek, T. Van der Velde-Koerts, V. Ling, and P. Borst, Mol. Cell. Biol. 6:1671-1678, 1986). We report here that only a subset of these, encoding the 170-kilodalton P-glycoprotein, are consistently amplified in three different multidrug-resistant Chinese hamster lung cell lines, selected with vincristine, daunorubicin, or actinomycin D. Within each cell line, genomic sequences homologous to the P-glycoprotein cDNA probe were amplified to different levels. The pattern of differential amplification was consistent with the presence of at least two and possibly three P-glycoprotein genes. In the actinomycin D-selected cell line, these genes were disproportionately overexpressed relative to the associated levels of amplification. These results underline a central role for P-glycoprotein in multidrug resistance. In the daunorubicin-selected cell line, another, as yet uncharacterized, gene was amplified but disproportionately underexpressed. Its amplification was therefore fortuitous. We present a tentative map of the region in the hamster genome that is amplified in the multidrug-resistant cell lines which were analyzed.     

From amplification to function: the case of the MDR1 gene.

This review describes the features of gene amplification associated with the selection of multidrug-resistant cell lines. Some of these lines carry multiple copies of the MDR1 gene that encodes P-glycoprotein, a broad specificity efflux pump. The MDR1 gene was initially identified as the common component of the amplicons found in multidrug-resistant cell lines selected with different drugs. Subsequent studies have established that increased MDR1 expression is sufficient for the multidrug-resistant phenotype. MDR1-containing amplicons may include a number of additional transcribed genes that do not appear to contribute to multidrug resistance. MDR1 amplification is associated with specific chromosomal changes and apparently non-random recombinational events. Increased expression of the MDR1 gene, however, does not necessarily require gene amplification. Although amplification of the MDR1 gene has not been found in clinical tumor samples, increased expression of this gene is commonly observed in different types of cancer and appears to be a significant marker of clinical drug resistance.     

The fluorescent probe Bodipy-FL-verapamil is a substrate for both P-glycoprotein and multidrug resistance-related protein (MRP)-1.

Several fluorescent probes have been used in functional studies to analyze drug transport in multidrug-resistant cells by fluorescent microscopy. Because many of these molecules have some drawbacks, such as toxicity, nonspecific background, or accumulation in mitochondria, new fluorescent compounds have been proposed as more useful tools. Among these substances, Bodipy-FL-Verapamil, a fluorescent conjugate of the drug efflux blocker verapamil, has been used to study P-glycoprotein activity in different cell types. In this study we tested by fluorescent microscopy the accumulation of Bodipy-FL-Verapamil in cell lines that overexpress either P-glycoprotein (P-gp) or multidrug resistance-related protein 1 (MRP1). Expression of P-gp and MRP1 was evaluated at the mRNA level by RT-PCR technique and at the protein level by flow cytometric analysis using C219 and MRP-m6 monoclonal antibodies. Results indicate that Bodipy-FL-Verapamil is actually a substrate for both proteins. As a consequence, any conclusion about P-gp activity obtained by the use of Bodipy-FL-Verapamil as fluorescent tracer should be interpreted with caution.     

Relationship between multidrug resistance,hypersensitivity to resistance modifiers and cell volume in Chinese hamster ovary cells.

We have previously shown that very high levels of hypersensitivity to several resistance modifiers are correlated with increasing multidrug resistance in a series of Chinese hamster ovary cell lines. We have now selected a new member of the series which is an exception to this correlation in that although it is almost twice as multidrug resistant as the cell line from which it was derived, it shows much less hypersensitivity to resistance modifiers. Level of resistance modifier hypersensitivity correlated with the level of reduction of verapamil accumulation in these cells, and with the density of P-glycoprotein, but since the selection of this cell line has involved a doubling of cell volume, it was not correlated with total amount of P-glycoprotein.     

Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference

ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [¹²⁵I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC₅₀ value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.     

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.