Molecular cytogenetics of multiple drug resistance

The refractory nature of many human cancers to multi-agent chemotherapy is termed multidrug resistance (MDR). In the past several decades, a major focus of clinical and basic research has been to characterize the genetic and biochemical mechanisms mediating this phenomenon. To provide model systems in which to study mechanisms of multidrug resistance,in vitro studies have established MDR cultured cell lines expressing resistance to a broad spectrum of unrelated drugs. In many of these cell lines, the expression of high levels of multidrug resistance developed in parallel to the appearance of cytogenetically-detectable chromosomal anomalies resulting from gene amplification. This review describes cytogenetic and molecular-based studies that have characterized DNA amplification structures in MDR cell lines and describes the important role gene amplification played in the cloning and characterization of the mammalian multidrug resistance genes (mdr). In addition, this review discusses the genetic selection generally used to establish the MDR cell lines, and how drug selections performed in transformed cell lines generally favor the genetic process of gene amplification, which is still exploited to identify drug resistance genes that may play an important role in clinical MDR.     

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.     

Multidrug-resistant phenotype cosegregates with an amplified gene in somatic cell hybrids of drug-resistant Chinese hamster ovary cells and drug-sensitive murine cells.

Gene amplification has been associated with multidrug resistance (MDR) in several drug-resistant Chinese hamster ovary (CHO) cell lines which exhibit cross-resistance to other unrelated, cytotoxic drugs. In situ hybridization studies (Teeter et al., J. Cell Biol., in press) suggested the presence of an amplified gene associated with the MDR phenotype on the long arm of either of the largest CHO chromosomes (1 or Z1) in vincristine-resistant cells. In this study, somatic cell hybrids were constructed between these vincristine-resistant CHO cells and drug-sensitive murine cells to determine the functional relationship between the chromosome bearing the amplified sequences and the MDR phenotype. Hybrids exhibited primary drug resistance and MDR in an incomplete dominant fashion. Hybrid clones and subclones segregated CHO chromosomes. Concordant segregation between vincristine resistance, the MDR phenotype, the presence of the MDR-associated amplified sequences, overexpression of the gene located in those sequences, and CHO chromosome Z1 was consistent with the hypothesis that there is an amplified gene on chromosome Z1 of the vincristine-resistant CHO cells which is responsible for the MDR in these cells. A low level of discordance between CHO chromosomes Z8 and 2 and the drug resistance phenotype suggests that these chromosomes may contain genes involved with the MDR phenotype.     

Multiple drug resistance and conservative amplification of the H region in Leishmania major

Amplification of the H region has been previously observed in methotrexate (MTX)-resistant strains of Leishmania major and in unselected laboratory stocks of L. tarentolae. We now show that selection of L. major with the structurally unrelated drugs primaquine or terbinafine generated resistant lines exhibiting H region amplification and 23- and 12-fold cross-resistance to MTX, respectively. These and other drug-resistant lines bearing H region amplification also exhibited weak cross-resistance to primaquine and terbinafine, associating the amplified H region with pleiotropic resistance to MTX and other drugs. In contrast, lines selected for chloroquine or pentamidine resistance did not show H region amplification or this pattern of drug cross-resistance. The primaquine- and terbinafine-selected lines exhibited wild-type levels of dihydrofolate reductase-thymidylate synthase and normal uptake and accumulation of MTX, and the MTX resistance of these lines was not reversed by verapamil. These data suggest that the mechanism of MTX cross-resistance associated with H region amplification is novel and distinct from that mediated by overexpression of MDR genes in multidrug-resistant mammalian cells. Structural studies indicated that the amplified H region DNA in these L. major lines was largely (possibly exclusively) extra-chromosomal and consisted of circular inverted repeats joined at two DNA rearrangement junctions. Southern blot analyses showed that these rearrangement junctions were identical in four independent cell lines, suggesting that these sites are "hotspots" for DNA rearrangement. H region amplification in all of these lines was conservative, defined as retention of the chromosomal H region locus without structural alteration or reduction in copy number. This finding is consistent with an over-replication/recombination model for amplification of the H region.     

Comparative study of the effect of daunorubicin and its nitroxyl derivative on in vitro function of rat liver mitochondria

The effect of rubomycin (daunorubicin) and its nitroxyl derivative, ruboxyl, on respiration and oxidative phosphorylation of the rat liver mitochondria was comparatively studied. It was shown that ruboxyl had a more pronounced uncoupling effect than rubomycin, especially during respiration in the presence of the glutamate mixture with malate. Unlike rubomycin, ruboxyl in concentrations of 0.05 to 0.5 mM induced stimulation under metabolic conditions rather than respiration. When the antibiotic concentration increased ruboxyl started to inhibit the respiration as compared to the control and the inhibition level appeared to be higher than that induced by rubomycin. Possible mechanisms for decreasing rubomycin toxicity by its modification with the nitroxyl radical are discussed.     

Multiple drug resistance of tumor cells: manifestations, genetic basis, clinical aspects

Data are reviewed concerning the results of study of multidrug-resistant (MDR) tumor cells. MDR often develops in the course of chemotherapy or in vitro selection of tumor cells by vincristine, adriamycin, actinomycin D, colchicine, etc. MDR cells are resistant to all these drugs though their targets and mechanisms of toxic action are quite different. Resistance is due to the decreased accumulation by MDR cells of these compounds. The genetic basis for MDR is amplification of a large genomic region that contains a number of genes coding for products and functions that are under extensive study. Specific karyotype and amplified DNA alterations occur during the development of MDR imitating the processes of appearance and variability of multigene families. The obtained data demonstrate the ways of overcoming of tumor multidrug resistance in clinic.     

Amplicon structure in multidrug-resistant murine cells: a nonrearranged region of genomic DNA corresponding to large circular DNA.

Multidrug resistance (MDR) in tumor cell lines is frequently correlated with amplification of one or more mdr genes. Usually the amplified domain also includes several neighboring genes. Using pulsed-field gel electrophoresis, we have established a restriction map covering approximately 2,200 kb in the drug-sensitive mouse tumor cell line TC13K. The mapped region is located on mouse chromosome 5 and includes the three mdr genes, the gene for the calcium-binding sorcin protein, and a gene with unknown function designated class 5. Long-range maps of the amplified DNA sequences in five of six MDR sublines that had been independently derived from TC13K generally displayed the same pattern as did the parental cell line. All six MDR sublines exhibited numerous double minutes, and one of them displayed a homogeneously staining region in a subpopulation. Large circular molecules, most likely identical to one chromatid of the double minutes, were detected in four of the sublines by linearization with gamma irradiation. The size of the circles was about 2,500 kb, which correlated to a single unit of the amplified domain. We therefore propose that in four independent instances of MDR development, a single unit of about 2,500 kb has been amplified in the form of circular DNA molecules. The restriction enzyme map of the amplified unit is unchanged compared with that of the parental cell line, whereas the joining sites of the circular DNA molecules are not identical but are in the same region.     

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

Changes in glutathione-S-transferase activity during induction of resistance of leukemia P 388 and Ehrlich ascitic tumor cells to doxorubicin]

We have studied by uridine short term test the level of resistance of murine leukemia cell lines P 388/Dx and ELD/Dx carcinoma cells with induced resistance to doxorubicin, P 388/Fp + Dx cells with induced resistance to combination of finoptOFF++ and doxorubicin in vivo. It was shown that the level of resistance was 6 fold for P 388/Dx cells, 4.5 fold for ELD/Dx cells and 2 fold for P 388/Fp + Dx cells. It was shown that the P 388/Dx cells and P 388/Fr + Dx cells had a 3.5 and 4.4 fold increase level of glutathione-S-transferase activity than P 388 cells. No increase in the activity of glutathione-S-transferase was detected in ELD/Dx cells. We conclude that increase of cellular glutathione-S-transferase activity is not associated with the development of resistance to doxorubicin.     

Medicarpin and millepurpan,two flavonoids isolated from Medicago sativa,induce apoptosis and overcome multidrug resistance in leukemia P388 cells

BackgroundHigh consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines.Hypothesis/purposeWe aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX).Study design/methodsCells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay.ResultsP-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin (IC₅₀ ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC₅₀ = 54 µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells (IC₅₀ = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan.ConclusionThese data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.     

Use of fluorescent dyes as molecular probes for the study of multidrug resistance

Fluorescence microscopy has shown that 18 different fluorescent dyes, staining various intracellular structures in transformed hamster fibroblasts (DM-15), did not stain or stained weakly multidrug-resistant cells selected from DM-15 by colchicine. Reduced staining by fluorescent dyes was characteristic also of five other tested multidrug-resistant cell lines of hamster and mouse origin, selected by actinomycin D, colcemid, rubomycin, and ruboxyl. The intensity of staining of two revertant cell lines was similar to that of parental sensitive cells. All tested inhibitors of multidrug resistance, including weak detergent, metabolic inhibitors, calcium channel blockers, calmodulin inhibitors, and reserpine, restored normal staining of multidrug-resistant cells. The dyes accumulated in resistant cells in presence of these inhibitors left the cells several minutes after the removal of the inhibitor from the incubation medium. Sensitive cells retained the dyes for several hours. The efflux of the dyes from resistant cells is an active process since it occurred even in the presence of the dyes in the incubation medium. The efflux could be blocked by all tested inhibitors of multidrug resistance and it is possibly a basic mechanism of the reduced staining of resistant cells. These data support the idea that multidrug resistance is based on active nonspecific efflux of the drugs and indicate that the simple procedure of cell staining can be used for the detection of resistant cells and further study of the phenomenon of multidrug resistance.     

"DNA clearing" from non-covalently bound agents in mammalian cells as a new mechanism of drug resistance.

Earlier, we have described the process of active dissociation or "DNA clearing" from non-covalently bound agents in living mammalian cells. The vital fluorescent bisbenzimidazole dye Hoechst 33342, which binds DNA in the minor groove tightly but non-covalently, was used for studying the interaction of non-covalently binding agents with DNA. Multiple drug resistance (MDR) in tumour cells is related to the expression of transport proteins that alter the cellular drug transport and distribution. Three different groups of genes (mdr, MRP, and LRP) and their products are implicated in MDR (A. Krishan, C. M. Fitz, and I. Andritsch, Cytometry 29:279-285 (1997)). To obtain new cell lines characterized by enhanced process of active dissociation of non-covalently bound agents from DNA or "DNA clearing", we carried out step-by-step selection with increasing concentrations of Hoechst 33342. The rodent cell lines hyperresistant to Hoechst 33342 and selected from AA8 were named AA8Hoe-R-1-AA8Hoe-R-10, and the cell lines selected from L cells were called LHoe-R-1-LHoe-R-10. The most resistant of them, AA8Hoe-R-6 and AA8Hoe-R-7, were able to grow in the presence of 80 microm/ml of Hoechst 33342 in the cell culture medium. All mutants were analyzed with the flow cytometric technique and were divided into two different groups. We conclude that the drug resistance of the first group of cell lines was due to changes in transport proteins. The second group of the resistant cell lines was characterized by an enhanced dissociation of the bisbenzimidazole dye-DNA complex. As we believe, the enhanced level of "DNA clearing" was caused by the amplification of some genes, because the gradual increase of Hoechst resistance in the same cell line resulted from the increase in the ability to remove the dye from DNA. These lines were shown to be also resistant to netropsin.     

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.     

Chromosomal imbalances associated with drug resistance and thermoresistance in human pancreatic carcinoma cells

Resistance to therapeutic treatment is the major obstacle to advances in the successful management of pancreatic cancer. To characterize chromosomal alterations associated with different phenotypes of acquired multidrug resistance (MDR) and thermoresistance, comparative genomic hybridization (CGH) was applied to compare human pancreatic carcinoma-derived cells. This panel of cell lines consists of the parental, drug- and thermosensitive pancreatic carcinoma cell line EPP85 - 181P, its atypical MDR variant EPP85-181RNOV, the classical MDR subline EPP85-181RDB, and their thermoresistant counterparts EPP85-181P-TR, EPP85-181RNOV-TR, and EPP85 - 181RDB-TR, respectively. CGH using genomic DNA prepared from these cell lines as probes successfully identified genomic gains and/or losses in chromosomal regions encoding putative genes associated with drug resistance and/or thermoresistance. These genes included 23 members of the family of ABC transporters, 27 members of the family of cytochrome P450 (CYP) monooxygenases, various molecular chaperones, DNA repair enzymes, and factors involved in the regulation of cell cycle and apoptosis. The importance of these cell variant-specific genomic imbalances in the development of MDR and thermoresistance is discussed and remains to be elucidated.     

Comparative accumulation of the Hoechst 33258 fluorescent probe in leukemia P388 cells sensitive and resistant to doxorubicin

The authors studied accumulation of the fluorescent probe Hoechst 33258 in leukemia P 388 sensitive (P 388/0) and resistant to doxorubicin (P 388/DOX) cells. It was shown that intensity of fluorescence of the dye increased after binding with nuclear DNA during 25 min for both lines of the cells. Intensity of fluorescence was 40% greater in sensitive than resistant cells. If Triton X-100 was added no difference between two lines of the cell was observed. When doxorubicin was added to the cells with dye, the intensity of fluorescence decreased. It was suggested to use Hoechst 33258 for assessment extent doxorubicin accumulation in nuclei of the cells.