(Received for publication, August 22, 1996, and in revised form, December 10, 1996)
From the Divisions of Oncology and Clinical Pharmacology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5306
A variant of the multidrug-resistant human sarcoma cell line Dx5 was derived by co-selection with doxorubicin and the cyclosporin D analogue PSC 833, a potent inhibitor of the multidrug transporter P-glycoprotein. The variant DxP cells manifest an altered phenotype compared with Dx5, with decreased cross-resistance to Vinca alkaloids and no resistance to dactinomycin. Resistance to doxorubicin and paclitaxel is retained. The multidrug resistance phenotype of DxP cells is not modulated by 2 µM PSC 833 or cyclosporine. DxP cells manifest a decreased ability to transport [3H]cyclosporine. DNA heteroduplex analysis and sequencing reveal a mutant mdr1 gene (deletion of a phenylalanine at amino acid residue 335) in the DxP cell line. The mutant P-glycoprotein has a decreased affinity for PSC 833 and vinblastine and a decreased ability to transport rhodamine 123. Transfection of the mutant mdr1 gene into drug-sensitive MES-SA sarcoma cells confers resistance to both doxorubicin and PSC 833. Our study demonstrates that survival of cells exposed to doxorubicin and PSC 833 in a multistep selection occurred as a result of a P-glycoprotein mutation in transmembrane region 6. These data suggest that Phe335 is an important binding site on P-glycoprotein for substrates such as dactinomycin and vinblastine and for inhibitors such as cyclosporine and PSC 833.
The development of drug resistance in tumor cells is a major
obstacle to clinical response in cancer chemotherapy. A well characterized cellular phenotype termed multidrug resistance
(MDR)1 is mediated by the multidrug
transporter P-glycoprotein (P-gp), which functions as an
ATP-dependent drug efflux pump of broad substrate
specificity (1-3). The mdr1 gene, which encodes P-gp, is
expressed in some normal and malignant tissues and has been associated
with a poor prognosis in several types of cancer (4-9). The reversal
of MDR by inhibitors or modulators of P-gp may improve the outcome of
cancer chemotherapy (4, 9-15). Cyclosporine, its analogue PSC 833 (PSC; Refs. 13 and 14), verapamil, and other MDR modulators have been
shown to increase cellular drug accumulation and reverse MDR through
competitive binding to P-gp (reviewed in Refs. 2, 4, and 9). Current
clinical trials using antitumor agents combined with MDR modulators to
circumvent MDR have raised the issue of whether the suppression of P-gp
function might result in the selection of alternative mechanisms that
could confer resistance to multiple agents. These mechanisms include changes in topoisomerase (Topo) II or II
(reviewed in Refs. 16-17 and 18) and increased expression of the gene for the multidrug resistance-associated protein, mrp (19, 20), or the p110
major vault protein, LRP-56 (21).
In this study, we utilized co-selection to investigate the mechanisms conferring resistance to both doxorubicin (DOX) and PSC. The parental cells expressed high levels of P-gp. We hypothesized that the suppression of P-gp function might favor the emergence of an altered form of P-gp or an alternative mechanism of resistance (22, 23). Our data reveal the expression of a novel mutant P-gp in variant cells with an altered spectrum of cross-resistance to cytotoxins and resistance to modulation by cyclosporins. The resistant cells and mutant P-gp were characterized in terms of their patterns of drug resistance, modulation by inhibitors of P-gp, and transport of drugs and their P-gp expression, gene sequence, and transfection of the mutant mdr1.
Doxorubicin was obtained from Adria Laboratories (Columbus, OH), etoposide from Bristol-Myers (Evansville, IN), and vinblastine from Eli Lilly and Co. (Indianapolis, IN). PSC was provided by Sandoz Pharmaceutical Corporation (Basel, Switzerland). Rhodamine 123 (Rh-123) was purchased from Molecular Probes (Eugene, OR). All other anti-cancer agents were obtained from the NCI (National Institutes of Health), and all other chemicals were from Sigma.
Cells and Tissue CultureDetails of the development and characterization of the human cell line MES-SA and its MDR variant MES-SA/Dx5 (Dx5), which were derived from sarcoma elements of a uterine mixed Mullerian tumor, have previously been described (24, 25). MES-SA/DxP5002 (DxP) cells were derived from Dx5.05 cells (Dx5 cells selected and maintained at a DOX concentration of 0.5 µM) by stepwise selection in the presence of increasing DOX concentrations (from 40 nM to 0.5 µM) and a constant PSC concentration at 2 µM over a 1-year period.
Cytogenetic Analysis and Fluorescence in Situ HybridizationMetaphase chromosome preparations were examined for the presence and structure of chromosome 7, where the human mdr1 gene is normally located, by karyotyping and in situ hybridization with a chromosome 7-specific probe (Oncor, Inc., Gaithersburg, MD). The hybridized chromosome was visualized by the method of Sasai et al. (26) using fluorescence microscopy.
Growth Inhibition Assay and Reversal of MDR by ModulatorsGrowth inhibition and doubling times were evaluated by the MTT colorimetric assay in triplicate or quadruplicate as described previously (22, 23). The modulation of resistance to DOX, paclitaxel, vinblastine, and etoposide was also determined by MTT assays in the presence of the MDR modulator PSC (2 µM) or verapamil 6 µM. These concentrations of the modulators are noncytotoxic and completely reverse resistance to DOX in our cellular MDR models. The modulation ratio was obtained by comparing IC50 values in the presence and absence of modulators (22).
Cellular Accumulation of [3H]Daunorubicin, [3H]Vinblastine, and [3H]CyclosporineIntracellular drug accumulation was quantified using radiolabeled drugs as described previously (23). All values were normalized to protein content.
Flow Cytometric AnalysisThe flow cytometric analysis of Rh-123 retention and P-gp expression were determined by a dual laser flow cytometer (FACS-IITM; Becton-Dickinson Corp., Mountain View, CA). Double labeling was performed with Rh-123 and the monoclonal antibody UIC2 Immunotech, Inc. (Westbrook, ME).
Amplimers Used for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)The oligonucleotides used as amplimers in this
study were synthesized by Operon Technologies (Alameda, CA) and Ana-Gen
Inc. (Palo Alto, CA). The sets of amplimers specific for Topo II and II
protein and mrp have been previously documented (22,
23). A panel of mdr1 primers for mRNA sequence
amplification was designed according to the published sequence (27) and
is summarized in Table I. 28 S ribosomal cDNA was
used as an endogenous control for PCR (22, 23).
|
The isolation of total RNA and procedures of RT-PCR were performed as previously reported (22, 23). PCR conditions for each pair of primers were determined by evaluating the linearity of the generation of PCR products with serial dilutions of cDNA (5, 25, 50, and 100 ng per reaction). Reactions were determined to be in the linear range of amplification by comparison of at least two dilutions of each sample and two cycle end points. All PCR experiments were performed on RNAs from several different preparations.
DNA Heteroduplex Analysis and PCR Sequencingmdr1 PCR products were subjected to heteroduplex analysis using the mutation detection enhancement system (J.T. Baker Inc.). An 8-12% MDETM Gel (J.T. Baker) was used and stained with ethidium bromide, and the desired products were located on an ultraviolet transilluminator and photographed. The results were further assessed by DNA sequencing of the PCR products (Amersham Life Science Inc.). Sequences were obtained from several preparations of cDNA, and these were compared with the parental cell line, Dx5 and the published human mdr1 sequence (27).
Genomic DNA PCR, Heteroduplex, Subcloning, and SequencingIn order to identify the presence of the mutation in
genomic DNA of DxP cells, we isolated genomic DNA from MES-SA, Dx5, and DxP cells. The PCR products were obtained by amplifying the target region using genomic specific primers located in introns 9 and 10, which are adjacent to exon 10 of mdr1: forward primer,
5-ATGGATCCTCTTCACATTCCTCAGGTAT-3
; reverse primer,
5
-CTCTCGAGGGCCAACTCAGACTTACATTAT-3
(27). Heteroduplex analysis was
performed, and heteroduplex bands were subjected to PCR
reamplification. The bands were purified from 8% polyacrylamide gel
and subcloned into the pGEM-T vector (Promega). Individual clones were
selected for sequencing.
Western blotting
with chemiluminescent detection (ECL kit, Amersham Corp.) was used for
the analysis of P-gp and Topo II proteins. Total cell lysates from
the exponentially growing cells were used for P-gp immunoblotting (22,
23), and a nuclear protein extract, prepared by the methods previously
described (23), was utilized in the Topo II assay.
Immunohistochemical analyses were performed using the monoclonal antibodies MRK16 and C219 for P-gp and the antibody LRP-56 for the p110 major vault protein (Caltag, S. San Francisco, CA) (20, 22, 23).
Photoaffinity Labeling with [125I]Iodoarylazidoprazosin and [3H]AzidopinePlasma membranes for photoaffinity labeling were prepared as described (28), followed by assays of displacement of the P-gp probes [125I]iodoarylazidoprazosin and [3H]azidopine (29). MES-SA, Dx5, and DxP cells (100 µg of protein) were incubated with [125I]iodoarylazidoprazosin (81.4 TBq/mmol; DuPont NEN) and [3H]Azidopine (Amersham) in the presence or absence of modulators in a 10 mM Tris-HCl buffer (pH 7.4) at a final volume of 50 µl. These preparations were incubated at 25 °C for 1 h in the dark, followed by a 20-min exposure to a 254-nm UV source (UVP, Inc., San Gabriel, CA). 50 µl of loading buffer was added, and proteins were separated on an 8% SDS-polyacrylamide gel, dried, and analyzed by autoradiography.
Clonal Analysis of DxP CellsSubcloning of cells was performed by limiting dilution. Both resistant and revertant clones were analyzed for the MDR phenotype, drug accumulation, and RT-PCR or heteroduplex as described above.
Mutant mdr1 Transfection and Cytotoxic AssayA cDNA library
was constructed from DxP cells in gt10 phage, and mutant
mdr1 clones were identified by hybridization, digested with
NotI, and subcloned into the expression vector
NotI site of pcDNA3 (Invitrogen). The plasmid pcDMDR1.4,
which codes for wild-type P-gp was obtained by subcloning a
BamHI-XhoI fragment of pMDR2000XS containing
mdr1 sequence from position
140 to position 4240 into
pcDNA3 multiple cloning sites. An additional mutant mdr1
plasmid, pcDMDR3.5, was derived by replacing a 211-base pair NsiI-SfuI fragment of mdr1 from
pMDR2000XS with a PCR fragment from DxP cells spanning the position
Phe335 deletion. Transfection of drug-sensitive MES-SA
cells and K562 cells was performed by electroporation (30) and
Lipofectin-mediated DNA transfer (Life Technologies, Inc.). Recipient
cells (2 × 107), were transfected with 10 µg of
plasmid. In a transient transfection assay, the MDR phenotype was
tested after 48 h of incubation. Stably transfected cells were
obtained by selection with G418 (800 µg/ml) for 14 days.
Stepwise selection of the MDR human sarcoma cell line Dx5 in the presence of increasing DOX concentrations and constant exposure to PSC (2 µM) resulted, over a 1-year period, in the stably DOX- and PSC-resistant cell line, DxP. Karyotypic analysis revealed 47 or 48 chromosomes in both the Dx5 and DxP cells with a similar G-banding pattern of chromosome 7 (data not shown). Fluorescence in situ hybridization demonstrated two copies of chromosome 7 in both Dx5 and DxP cells, whereas parental, drug-sensitive MES-SA cells have 45 chromosomes and one chromosome 7 (24, 25).
Multidrug Resistance PhenotypeThe drug resistance phenotype of DxP cells is shown in Table II and Fig. 1. Their level of resistance to the anthracycline DOX is similar to that of the parental Dx5 cells, with a slightly decreased cross-resistance to daunorubicin. The most notable alterations were found in levels of resistance to dactinomycin and Vinca alkaloids. Resistance to vinblastine decreased 17-fold, to vincristine 25-fold, and to vinorelbine 9-fold (to a level similar to that of drug-sensitive MES-SA cells). Resistance to amsacrine decreased 13-fold, and sensitivity to dactinomycin was completely restored. This cell line maintained high levels of resistance to colchicine and paclitaxel (Taxol®), and moderately decreased resistance to podophyllotoxins (etoposide and teniposide). There was no significant difference in cellular generation time (22 h) compared with parental Dx5 cells.
|
In Vitro Modulation of MDR by PSC and Verapamil
Modulation of resistance to several MDR-related cytotoxins by PSC (2 µM), cyclosporine (3 µM), and verapamil (6 µM) was examined by the MTT assay. PSC, the most potent of the modulators used, completely restored the sensitivity of the highly multidrug-resistant cell line Dx5 to DOX, vinblastine, paclitaxel, vincristine, and colchicine, relative to the drug-sensitive MES-SA cells. In contrast, DxP cells were almost completely resistant to the modulating effects of PSC, substantially resistant to cyclosporine, and somewhat less resistant to modulation by verapamil (Table III, Fig. 1).
|
Total RNA was extracted from isolated DxP clones and
analyzed by RT-PCR for the presence of mdr1, Topo II,
Topo II
, and mrp transcripts (Fig. 2). In
comparison with the parental Dx5 cells, DxP displayed levels of
mdr1 transcripts similar to the parental Dx5 cells. The
expression of mrp was compared with MES-SA and Dx5 cells and
with normal human lung tissue as a positive control. A similar level of
expression of mrp was observed in human lung tissue, MES-SA
sarcoma cells (data not shown), and Dx5 and DxP cells. While no
significant difference was seen in Topo II
transcripts, DxP cells
manifested a 2-fold elevation of Topo II
expression relative to Dx5
cells (Fig. 2).
P-glycoprotein Expression
P-gp expression and function were
analyzed by RT-PCR, immunoblotting with the C219 antibody, and flow
cytometry with the antibody UIC2 (Figs. 3 and
4). Dx5 and DxP cells displayed similarly high levels of
expression of P-gp. Immunohistochemical staining of cells with the
MRK16 and C219 antibodies confirmed that the expression of P-gp on DxP
cells was predominantly localized to the plasma membrane and similar in
amount to that observed in Dx5 cells (data not shown).
Topo II
Expression of Topo II
isoforms by immunoblotting revealed a slight increase in DxP compared
with Dx5 cells. Neither Dx5 nor DxP cells had detectable expression of
the p110 major vault protein, although the drug-sensitive MES-SA cells
from which Dx5 cells were originally derived are weakly positive for
p110 (data not shown).
The cellular accumulation of the fluorescent P-gp substrate Rh-123 was markedly decreased in Dx5 cells, as expected for MDR cells (Fig. 4). In contrast, DxP cells (although expressing abundant P-gp by UIC2 staining) manifested Rh-123 retention similar to the MDR-negative MES-SA cell line. PSC (2 µM) completely restored Rh-123 accumulation in the Dx5 cell line, to levels similar to MES-SA and DxP cells (Fig. 4).
3H-Labeled Drug AccumulationIntracellular
accumulations of [3H]daunorubicin,
[3H]vinblastine, and [3H]cyclosporine were
measured to compare the function of P-gp in Dx5 and DxP cells. Both Dx5
and DxP cells displayed similar decreases in
[3H]daunorubicin accumulation, relative to the MES-SA
cell line, while the accumulation of [3H]vinblastine was
approximately 3-fold higher in DxP than Dx5 cells (Fig.
5, A and B). The decreased drug
accumulation in DxP cells was not modulated by PSC, whereas complete
modulation was displayed by Dx5 cells (Fig. 5). Uptake of
[3H]cyclosporine was examined to assess the ability of
P-gp to transport the cyclosporins. The intracellular concentration of
cyclosporine in DxP cells was equal to that in MES-SA cells and 2-fold
higher than in Dx5 cells (Fig. 6). The decreased
accumulation of cyclosporine in Dx5 cells was completely modulated by
PSC to the levels achieved in DxP and MES-SA cells (data not
shown).
Photoaffinity Labeling with [125I]Iodoarylazidoprazosin and [3H]Azidopine
DxP cells displayed enhanced
photoaffinity binding of P-gp by
[125I]iodoarylazidoprazosin in the presence of 0.1 and 10 µM PSC and vinblastine, in contrast to Dx5 cells in which
the photoaffinity labeling was effectively competed by PSC and
vinblastine (Fig. 7). The higher concentration (100 µM) of PSC or vinblastine abolished detectable P-gp
labeling by azidoprazosin in both cell lines. DxP cells were also
resistant to the displacement by PSC or vinblastine of
[3H]azidopine photoaffinity labeling (Fig.
8, A and B). Verapamil was
moderately active in both DxP and Dx5 cells in displacing [3H]azidopine.
RT-PCR, DNA Heteroduplex Analysis, and mdr1 DNA Sequencing
RT-PCR using primer sets spanning the P-gp coding
sequences (Table I) confirmed that the levels of expression of
mdr1 were similar and that the PCR products showed no
differences in size when Dx5 and DxP cells were compared. DNA
heteroduplex analysis revealed the formation of a heteroduplex with
primers spanning nucleotides 1194-1519 of mdr1 cDNA,
suggesting a sequence difference in transmembrane region 6 (TM6), Fig.
9A. Sequencing of this PCR product identified
a deletion of base pairs 1427-1429 in this region, which encode the
amino acid phenylalanine at position 335 of P-gp (Phe335)
(Fig. 9, B and C). The PCR and sequencing results
were reproduced in four different preparations of cDNA from DxP
cells. The deletion of Phe335 is the only functional
mutation in DxP cells compared with Dx5 and the published human
mdr1 sequence (27). There are other changes from the
published mdr1 sequence that do not alter the P-gp amino
acid sequence in DxP cells: from TCT (Ser) to TCC (Ser) in nucleotide
964 and from ATC (Ile) to ATT (Ile) in nucleotide 3859. These may be
polymorphisms of P-gp, since both DxP and Dx5 have these same
substitutions. Amino acid 185 was identified as Gly in both Dx5 and DxP
cells (data not shown).
Identification of the 1427-1429 TTC Deletion in Genomic Sequence of DxP Cells
In order to identify the presence of the mutation in
genomic DNA of DxP cells, we amplified genomic DNA using genomic
specific primers of mdr1. Heteroduplex analysis was
performed and revealed that a heteroduplex band existed in DxP but not
Dx5 cells. The TTC deletion at codon 335 was also verified by
sequencing of the genomic PCR product. Thus, the one-codon
deletion was confirmed in DxP cells (Fig. 10).
Subclonal Analysis of DxP Cells
Single clones were obtained to analyze the genetic heterogeneity in DxP cells. As shown in Table IV, 11 isolated clones derived from DxP cells showed a similar phenotype including resistance to DOX, resistance to modulation by PSC, and a lower degree of resistance to vinblastine relative to parental Dx5 cells. All tested clones expressed the mutant mdr1.
|
Plasmids
containing wild-type and mutant mdr1 cDNAs were
transfected into drug-sensitive MES-SA cells. Expression of the mutant mdr1 was confirmed in the appropriate transfectants by DNA
heteroduplex analysis. Both wild-type and mutant mdr1
conferred resistance to doxorubicin, paclitaxel, etoposide, and
colchicine. Representative data demonstrating resistance to doxorubicin
are shown in Fig. 11A. Exposure of
transfectants to both doxorubicin and PSC revealed that only the cells
transfected with the mutant mdr1 bearing the Phe335 deletion survived, Fig. 11B.
The development of resistance to anticancer drugs is a major
impediment to successful chemotherapy, and it is often mediated by the
membrane-bound drug-efflux pump, P-gp (1-4). Substances that inhibit
P-gp function and reverse the resistance phenotype in vitro,
termed MDR modulators, have been developed with the intention of
administering them in conjunction with MDR-related cytotoxins (9-15).
This experiment was designed to examine the resistance mechanisms that
arise in MDR cells during multistep selection with DOX, an MDR
substrate, and PSC, an effective modulator. A similar selection in
non-MDR cells suppressed the activation of mdr1 and resulted
in the emergence of mutants expressing alternative mechanisms of
resistance, notably decreased expression of Topo II (23). Under the
conditions of drug exposure in our present experiment, DxP cells
displayed cross-resistance to several MDR-related drugs including
anthracyclines (DOX and daunorubicin), epipodophyllotoxins (etoposide
and teniposide), colchicine, and paclitaxel. However, DxP differed from
the parental Dx5 cells in their decreased resistance to
Vinca alkaloids and lack of cross-resistance to dactinomycin (Table II, Fig. 1). Most notably, the MDR phenotype was not modulated by treatment with the P-gp inhibitor PSC (Table III, Fig. 1).
Although overexpression of mdr1 is the best characterized
mechanism of pleiotropic drug resistance, other mechanisms have been
identified. Decreased expression or altered structure of Topo II has
been observed in many models of resistance to epipodophyllotoxins, mitoxantrone, and anthracyclines such as DOX (16-18, 23). A membrane ATPase of 190 kDa, distinct from P-gp, has been termed the multidrug resistance-associated protein, encoded by the mrp gene,
which has been cloned and sequenced in a DOX-selected lung cancer cell line (19). Under our experimental conditions, overexpression of the
mrp mRNA or significant changes in Topo II and II
were not observed. DxP cells, like the parental Dx5 cells, were not found to express the p110 major vault protein, recognized by the LRP-56
antibody and associated with doxorubicin resistance in some cell models
(21). The apparent lack of an alternative resistance mechanism in DxP
cells, the residual high expression of P-gp, and the pleiotropic nature
of the resistance suggested that a mutant or modified P-gp with
decreased affinity for cyclosporins was responsible for the phenotype
of these cells.
The altered phenotype of DxP cells correlated very well with an altered functional activity of P-gp assessed by the cellular uptake of daunorubicin, vinblastine, and cyclosporine. DxP cells displayed a significant decrease in daunorubicin accumulation, which was insensitive to modulation by PSC (Fig. 5A). These cells also exhibited increased vinblastine accumulation compared with Dx5 cells, although the level of this drug was not as high as in drug-sensitive MES-SA cells and was not further increased by PSC (Fig. 5A). The accumulation of cyclosporine in DxP cells was equivalent to that of drug-sensitive MES-SA cells, which do not express P-gp, strongly suggesting an altered affinity of the multidrug transporter for cyclosporins and consistent with the data that cyclosporine and its analogue PSC did not modulate the MDR phenotype of DxP cells (Fig. 6).
As previously reported, compartmentalization or redistribution of P-gp leading to redistribution of cytotoxins may result in resistance to modulation (31). Our immunohistochemical experiments localized P-gp to the cell membrane in both Dx5 and DxP. Furthermore, the same amount of P-gp expression and the existence of equivalent daunorubicin accumulation defects in the two cell lines demonstrated that P-gp in DxP cells was capable of transporting some substrates as well as the P-gp in Dx5 cells (Figs. 3, 4, and 5A). The P-gps from the two cell lines have a similar electrophoretic mobility (Fig. 3). Thus, a redistribution or marked structural change in the P-gp expressed in DxP cells was not evident.
Our results identified a novel mutation, consisting of a single codon deletion (Phe335) in the TM6 region of P-gp in DxP cells. The drug resistance phenotype we observed is similar, in some respects, to that described in a previously published report of the functional consequences of a substitution for the phenylalanine at codon 335 by site-directed mutagenesis, which resulted in decreased dactinomycin resistance (32). The expression of this mutant P-gp in DxP cells is probably the result of selection of a spontaneously arising mutant or selective allelic expression of an mdr1 gene that confers a PSC-resistant MDR phenotype. Karyotyping and fluorescent in situ hybridization analysis of chromosome 7 revealed no obvious alterations in chromosome structure in DxP cells.
The deletion of codon 335 as a mechanism of resistance to modulation of
MDR by cyclosporins may be an extremely rare event. We have not found a
similar mutation in 13 other mutants separately derived from MES-SA
cells that were co-selected by DOX and PSC, with a mutation rate of
2.5 × 107 per cell generation (23). Those mutants
were resistant to DOX and PSC on the basis of a down-regulation of Topo
II
expression. The MES-SA cells used in that experiment do not
express mdr1, whereas the MDR variant Dx5 cells used to
select the DxP variant with DOX and PSC actively transcribed
mdr1, a factor that may have contributed to the selection of
a mutant P-gp rather than to an alternative mechanism of resistance
such as altered Topo II expression.
Several point mutations or polymorphisms of mdr1 have been identified in different species including hamsters, mice, and humans (reviewed in Ref. 33; Refs. 34-38). Some of these have been associated with an altered phenotype, such as the substitution from Gly185 to Val185 in human P-gp, which confers preferential resistance to colchicine (36, 37). The colchicine selection of KB cells in this case may have favored overexpression of one allele of mdr1 and suggests that codon 185 in human P-gp may be subjected to DNA polymorphism. Both Dx5 and DxP cells express the Gly185. The impact of structural alterations of P-gp on modulation of MDR by inhibitors is poorly understood (9, 32, 33, 38).
The novel mutation we have identified in this study (deletion of Phe335) provides insight into the relationship between P-gp structure and modulation of MDR by cyclosporins. Our data support the theory that a specific ligand-receptor mechanism is involved in P-gp-mediated MDR. PSC and cyclosporine are known to bind to P-gp, and cyclosporine is a transport substrate for P-gp. Phenylalanine and other aromatic residues are preferentially located at the cytoplasmic boundaries, where they are thought to position the transmembrane segments. Several such residues, including Phe335, Phe777, and Phe978, are thought to be of functional importance in P-gp (32). Substitution of a nonaromatic residue for Phe335 substantially impairs the ability of the transporter to confer resistance to vinblastine and dactinomycin, while the ability to confer resistance to DOX and colchicine is preserved (32).
DxP cells showed enhanced iodoarylazidoprazosin labeling in the presence of both PSC and vinblastine and decreased ability of either PSC or vinblastine to replace the P-gp probe (Fig. 7). Photoaffinity experiments have localized azidopine binding domains in P-gp to TM5-6 or to TM6 and TM12 (reviewed in Ref. 33). Previous experiments have demonstrated that the two halves of P-gp come together to form a single site for drug binding, involving the TM5-6 and TM11-12 regions (33). Additional data suggest that azidopine and vinblastine may not bind to the same site (33, 39) and that conformational or allosteric effects could be responsible for the inhibition of labeling by azidopine in the presence of vinblastine. The collateral increased affinity to iodoarylazidoprazosin labeling in DxP cells may be due to an allosteric effect of the deletion of Phe335. The deletion of Phe335 in the P-gp expressed by DxP cells resulted in loss of the capacity to bind or transport cyclosporine, PSC, and vinblastine. Our results suggest that cyclosporine, PSC, vinblastine, Rh-123, and dactinomycin share at least one binding domain on P-gp (Tables II and III, Figs. 4, 5, 6, 7). These results indicate that this residue plays an important role in the interaction of P-gp with cyclosporine and PSC.
Our results do not completely rule out the existence of other modifying factors that may directly or indirectly affect the multidrug resistance phenotype in DxP cells, although no obvious alternative mechanism was observed. Several major known alternative mechanisms of resistance to DOX were not different between Dx5 and DxP cells. Moreover, the clonal analysis of the DxP cell population supported the association of the mutant P-gp expression with resistance to modulation by PSC and decreased cross-resistance to vinblastine in every subclone tested (Table IV). Finally, transfection of the mutant mdr1 gene conferred a drug resistance phenotype that was resistant to modulation by PSC (Fig. 11).
In summary, our data reveal a functionally important mutation of P-gp arising from co-selection of mdr1-positive cells with DOX and PSC. The resistance phenotype of the resulting DxP cell line may be attributed to the deletion of Phe335 from P-gp. Our data suggest that the phenylalanine residue at codon 335 may be important in the binding and transport of cyclosporins by P-gp and to their ability to modulate MDR. In addition, this mutation results in decreased resistance to Vinca alkaloids, lack of cross-resistance to dactinomycin, and markedly decreased ability to transport Rh-123.
We are grateful to Dr. Igor Roninson for valuable suggestions and comments. We thank Eva Pfendt, Mary Kovacs, Dana Bangs, and Dr. Yan Wang for excellent technical assistance.