Introduction

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The development of drug resistance in tumor cells is a major obstacle to clinical response in cancer chemotherapy. Pgp, a 170-kDa plasma membrane protein, is one of the most widely studied of the proteins that are responsible for the phenomenon of multidrug resistance in mammalian cells. The protein acts as an energy-dependent pump that extrudes a broad range of hydrophobic cytotoxic drugs from the cell (reviewed by Gottesman and Pastan, 1993). Human Pgp, encoded by the MDR1 gene, consists of 1280 amino acids organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids. Each repeat consists of an amino-terminal hydrophobic domain containing six potential TM domains, followed by a hydrophilic domain containing a nucleotide-binding site. Transmembrane domains 5/6 and 11/12 have been demonstrated previously by photoaffinity labeling with substrate analogs to be substrate interaction sites (Bruggemann et al., 1992; Greenberger, 1993; Morris et al., 1994). Mutagenesis studies further reveal that changes of several different amino acids throughout Pgp (especially in the predicted transmembrane domains 5/6 and 11/12 of Pgp) change its drug resistance pattern (Germann et al., 1993; Loo and Clarke, 1993, 1994). Pgp, which is encoded by mouse mdr1a (mdr3), confers different degrees of resistance to known MDR drugs (Tang-Wai et al., 1995). At similar levels of protein expression, the mouse mdr1a isoform seemed to be a more efficient drug efflux pump and showed levels of resistance that were superior to mdr1b or MDR1 for all drugs tested.

The reversal of MDR by inhibitors or modulators of Pgp may improve the outcome of cancer chemotherapy (Dalton et al., 1989; Solary et al., 1992; Yahanda et al., 1992). Cyclosporin A, its analog PSC 833, verapamil, and other MDR modulators have been shown to increase cellular drug accumulation and reverse MDR through competitive binding to Pgp. Mouse mdr1a shows different sensitivity to the reversal effect of human MDR1 inhibitors (Yang et al., 1990; Tang-Wai et al., 1995). The mouse mdr1a was much less sensitive to modulators than the human MDR1.

We have initiated studies to select for Pgp mutants with altered drug resistance profiles as a potential tool in cancer gene therapy because those mutants could be used to protect bone marrow from the systemic toxicities of chemotherapy. Moreover, the localization of specific segments and amino acids implicated in substrate specificity and response to modulators in chimeric and mutant proteins, would facilitate the structure/function analysis of Pgp.

Chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling or sexual PCR (Stemmer, 1994a; Lorimer and Pastan, 1995). Because of fragment exchange and error-prone PCR, overall sexual PCR is an effective method to create recombinant molecules which resemble the process of molecular evolution.

An advantage of DNA shuffling is that it can optimize the function of genes without first determining which gene product is rate limiting. This process has been shown to yield functional optimization of different genes (Stemmer, 1994b; Crameri et al., 1996, 1997).

In this study, we describe DNA shuffling between TM domains 5 and 6 of human MDR1 and mouse mdr1a. We analyze here one novel chimeric Pgp (clone 3-4) with an altered spectrum of cross-resistance to cytotoxins and specific insensitivity to modulators.

	Materials and Methods

Top Summary Introduction Materials & Methods Results Discussion References

Substrates preparation for DNA shuffling. The substrates for the shuffling reaction were 300-bp double-strand DNA PCR products derived from pFRCMV-mdr1a and pSXLC-MDR with the primer sequences 5'-GAAGAAGCTAAGCGAATTGG-3' and 5'-GTGCCCACTCTTCGAATAGC-3'. The MDR1 and mdr1a sequences used here have 86% DNA sequence identity. The primers contain BlpI and BstBI restriction sites for subcloning into the pHaMDR-DHFR vector (Zhang et al., 1998) (Fig. 1). PCR products were purified with Wizard PCR (Promega, Madison, WI).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   The bicistronic retroviral expression vector pHaMDR-DHFR containing the Harvey virus long terminal repeat (LTR) followed by the human MDR1 gene, the IRES from encephalomyocarditis virus, and a mutant form of the mouse DHFR gene (dihydrofolate reductase) (Zhang et al. 1998). Unique restriction sites flanking MDR1 and DHFR are labeled.

DNaseI digestion. PCR product (4 µg) was digested with 0.15 units of DNase I (Sigma, St. Louis, MO) in 100 µl of 50 mM Tris·HCl, pH 7.4, 10 mM MnCl₂ for 1.5 min at 15° and terminated by heating at 90° for 10 min. The digestion products were passed through Centri-Sep Columns (Princeton Separations, Adelphia, NJ).

PCR without primers. The purified fragments (10 µl from mdr1a digestion plus 10 µl from MDR1 digestion) were added to the PCR mixture (0.4 mM each deoxynucleoside triphosphate, 2 mM MgCl₂, 2.5 units of Perkin Elmer AmpliTaq DNA polymerase per 100 µl of reaction mixture). A PCR program of 94°, 3 min; 94°, 1 min; 55°, 1 min; 72°, 1 min (40 cycles); and 72° for 7 min was used.

PCR with primers. After 1:25 dilution of this primerless PCR product into the PCR mixture with the primers described above and 25 additional cycles of PCR, a single product of the correct size was obtained (Fig. 2E).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Reassembly of 300 bp PCR product from domains 5 and 6 of human MDR1 and mouse mdr1a. A, PCR products before digestion. B, DNA after 1.5-min digestion with DNase I. C, Fragments after purification on spin column. D, Reassembly of fragments in the absence of primers. E, A single PCR product of the correct size after addition of primers and additional cycles of PCR.

Cloning and sequencing. After digestion of the PCR product with terminal restriction enzymes (BlpI and BstBI) and gel purification, the reassembled fragments were ligated into the pHaMDR-DHFR bicistronic expression vector digested with BlpI and BstBI. Ten randomly chosen plasmids containing inserts were purified and sequenced on a 373A DNA Sequencing System (Applied Biosystems, Foster City, CA) using the Taq Dyedeoxy Cycle Sequencing kit. Sequences were analyzed using the SeqEd 675 Sequence Editor software supplied with this system.

Bacterial transformation library. DNA from the above ligation was electroporated into D10B competent cells (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. Bacterial transformants (5 × 10⁵) were collected from plates containing ampicillin.

Mammalian cell transfection library. DNA isolated from the bacterial library was electroporated into KB-3-1 cells (Akiyama et al., 1985) with an electroporator (Bio-Rad, Hercules, CA). For a 0.4-cm cuvette, 10⁷ cells and 10 µg of DNA resuspended in 0.8 ml of phosphate-buffered saline. The transfection conditions were 350 V and 960 mF.

Selection for methotrexate resistance. Twenty-four hours after electroporation, cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% dialyzed bovine calf serum and selected in 30 ng/ml MTX. Three thousand methotrexate-resistance colonies were pooled 3 weeks later. The mammalian library was maintained in MTX.

Selection and counting of drug-resistant colonies. The dose-response survival curves of the mammalian library, the normal pHaMDR1-DHFR transfected KB-3-1 cells, and the selected colonies were determined by plating 1000 exponentially growing cells per 100-mm dish in the absence of drug. After a 16-hr incubation at 37°, the appropriate concentrations of stock solutions of colchicine, daunorubicin, taxol, vinblastine, and vincristine dissolved in dimethylsulfoxide (<0.5% of total volume/dish) were added to each cell line. Either cyclosporin A or verapamil at concentrations of 0.5 or 1 µg/ml was added to each cell line immediately after the addition of the cytotoxic drug. After incubating the cells at 37° for 10 days, colonies from the library that were found to be resistant to high drug concentrations were isolated and were grown in methotrexate-containing medium for further characterization. For counting, colonies were stained with 0.5% methylene blue in 50% ethanol and counted with a Manostal colony counter.

Immunofluorescence analysis. The level of cell-surface Pgp was determined by FACS analysis using MRK-16 (Hamada and Tsuruo, 1986), and UIC2 (Mechetner and Roninson, 1992), which react with external surface domains of Pgp. The staining was performed as described previously (Germann et al., 1996).

Drug accumulation assays. Cells were harvested by trypsinization, washed, and resuspended in Iscove's modified minimal essential medium supplemented with 5% fetal bovine serum. For accumulation measurements, 500,000 cells were incubated at 37° in 5 ml of Iscove's modified minimal essential medium containing 5% fetal bovine serum and the fluorescent substrates 0.5 µg/ml rhodamine 123, 0.5 µM calcein AM, 5 µM daunorubicin, 0.5 µM bodipy-verapamil or bodipy-Taxol with or without 20 µM verapamil, 5 µM cyclosporin A, or 2.5 µM PSC 833. After 40 min, cells were pelleted and resuspended in 300 µl of phosphate-buffered saline and immediately analyzed by FACS, using a Becton-Dickinson instrument equipped with CellQuest software.

	Results

Top Summary Introduction Materials & Methods Results Discussion References

DNA shuffling between transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a. Fig. 2 shows a DNase I digest of 300-bp PCR products from domains 5 and 6 of human MDR1 and mouse mdr1a. The column purified fragments were reassembled (initially without primers) to a single PCR product of the correct size.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Recombination in 10 randomly selected plasmids containing inserts. Clones resulting from reassembly of a mixture of human MDR1 and mouse mdr1a fragments were sequenced. DNA sequences are shown schematically. Black, human MDR1 sequences; white, mouse mdr1a sequences. Crossovers are arbitrarily shown to be midway between characteristic bases of the two sequences. Point mutations are labeled in their position.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Analysis of the mammalian library cell surface expression of Pgp. KB-3-1 cells stably transfected with pooled DNA isolated from the bacterial library (A) or the wild-type pHaMDR1-DHFR (B) were subjected to FACS analysis after staining with human Pgp external surface epitope-specific monoclonal antibody MRK-16 (unshaded profiles) and control mouse IgG2a (shaded profiles), as described in Materials and Methods.

Isolation of clones with altered drug resistance pattern. The mammalian library and the wild-type pHaMDR1-DHFR transfected KB-3-1 cells were selected with different concentrations of unrelated antitumor agents including the microtubule-destabilizing agents colchicine and taxol; the anthracycline daunorubicin; and the Vinca alkaloid vincristine. The mammalian library showed a decrease in relative resistance to all the antitumor agents compared with the relative resistance of pHaMDR1-DHFR transfected KB-3-1 cells.

Expression of the wild-type and the recombinant Pgp. Fluorescence intensity by FACS analysis with anti-Pgp MoAbs MRK16 and UIC2 of the recombinant clone cells was compared with fluorescence intensity of the wild-type pHaMDR1-DHFR transfected KB-3-1. As shown in Fig. 5, A and B, the recombinant clone cells stained less strongly with MRK16 than the wild-type pHaMDR-DHFR cells. In contrast, UIC2 fluorescence intensity was higher than in the wild-type Pgp (Fig. 5, C and D), but the fluorescence distribution was biphasic and some cells expressed very low P-gp when detected with UIC2. Whole cell lysates from both cell lines were prepared and analyzed for their level of Pgp expression by Western blotting using the anti-P-glycoprotein monoclonal antibody C219. The levels of total cellular expression in the recombinant clone cells detected with this monoclonal antibody were somewhat less than in the wild-type MDR1 transfected cells (Fig. 6). Similar results were obtained with the anti-Pgp polyclonal antibody PEPG 13 (Bruggemann et al., 1992) by Western blotting.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Analysis of the fluorescence intensity of the recombinant clone cells by FACS using two anti-Pgp monoclonal antibodies. The recombinant clone cells (A and C) and the wild-type pHaMDR-DHFR cells (B and D) were subjected to FACS analysis after staining with MRK-16 (A and B, unshaded profiles) or UIC2 (C and D, unshaded profiles) monoclonal antibody, and control mouse IgG2a (shaded profiles) as described in Materials and Methods.

View larger version (57K): [in this window] [in a new window]   Fig. 6.   Protein expression of Pgp. Parental KB-3-1 (kb), high Pgp expressing MDR1 cells KB-V1 (kv), the recombinant 3-4 clone cells (3-4), and the wild-type pHaMDR-DHFR (MDR) cells were analyzed for protein expression. Cells were lysed, and total cell lysates (10 µg of protein/lane) were subjected to immunoblot analysis with anti-Pgp monoclonal antibody C219.

Drug resistance of recombinant clone 3-4. To assess the resistance of recombinant clone 3-4 to various cytotoxic drugs and reversing agents, growth of the recombinant clone cells, the wild-type MDR1 cells, and the nontransfected KB-3-1 cells were compared using clonogenic cell killing assays. Both recombinant clone cells and wild-type MDR1 transfected cells exhibited resistance to the selecting agent colchicine, Furthermore, in the presence of 1 µM of the reversing agent cyclosporin A, the growth of the wild-type MDR1-transfected cells was inhibited, whereas the growth of the recombinant clone cells was similar to growth without cyclosporin A. When both cells were selected with the natural drug product, taxol, the recombinant clone cells were somewhat less resistant to taxol overall compared with the wild-type MDR1-transfected cells. In the presence of 1 µM cyclosporin A, the growth of both cell lines was inhibited. These results are consistent with the initial ability of this clone to survive in taxol despite a reduced level of chimeric Pgp expression. To verify these results, the chimeric cDNA was recloned by PCR back into the bicistronic vector, and was retransfected into KB-3-1 cells. Similar results were obtained with the new clone (Fig. 7), showing that the phenotype was caused by the 3-4 mutations as opposed to variations in host cells.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Drug survival characteristics of the recombinant 3-4 clone cells, the wild type pHaMDR-DHFR cells, and the drug-sensitive KB-3-1 cells. Colony formation assays were performed as described in Materials and Methods to determine the drug sensitivity of the different cell lines. Drug survival was measured in increasing concentrations of colchicine (A), colchicine + 1 µM cyclosporin A (B), taxol (C), or taxol + 1 µM cyclosporin A (D). , 3-4; , MDR1; , KB-3-1. Points, average of three independent experiments.

Analysis of the drug transport function of the recombinant Pgp. The abilities of the wild-type and the recombinant Pgp expressed in KB-3-1 cells to transport different fluorescent substrates were determined. As shown in Fig. 8, both Pgp-expressing cells accumulated considerably less bodipy-verapamil, daunorubicin, and calcein AM than the drug-sensitive KB-3-1 cells. The recombinant clone cells accumulated more rhodamine 123 compared with the wild-type pHaMDR-DHFR transfected cells.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   Accumulation of fluorescent substrates in the recombinant 3-4 clone cells (3-4), the wild type pHaMDR-DHFR cells (mdr1), the mammalian 3+1 library (3+1lib), and the drug-sensitive parental KB-3-1 cells (kb3-1). Substrate accumulation was determined by FACS: Rhodamine (A), daunorubicin (B), bodipy-verapamil (C), and calcein AM (D).

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Fluorescent substrate accumulation and the effect of reversing agents. A, Rhodamine 123 accumulation in the presence or absence of 20 µM verapamil in the wild-type pHaMDR-DHFR cells (left), or the recombinant 3-4 clone cells (right). B, Bodipy verapamil accumulation in the presence or absence of 2.5 µM PSC 833. C, Calcein AM accumulation in the presence or absence of 5 µM cyclosporin A.

Photoaffinity labeling of the recombinant Pgp. Photoaffinity labeling experiments with [¹²⁵I] IAAP in plasma membrane preparations were performed to determine the drug-binding properties of the recombinant Pgp. cis-(Z)-Flupentixol, an antipsychotic drug, has been shown to be a potent agent for reversing Pgp-mediated drug resistance and an enhancer of Pgp photoaffinity labeling with IAAP (Dey et al., 1997). Before labeling with IAAP, membranes from the wild-type MDR1-transfected cells, and from the recombinant clone cells were incubated with cis-(Z)-flupentixol for 3 min at 21°. As shown in Fig. 10, both the wild-type and the recombinant Pgp membranes were labeled with IAAP, but labeling of recombinant clone 3-4 was reduced several-fold compared with wild-type Pgp. Despite its relative inability to reverse colchicine resistance of the recombinant clone cells (Fig. 7), cyclosporin A competed for the [¹²⁵I]IAAP photoaffinity labeling of both the wild-type and the recombinant Pgp.

View larger version (65K): [in this window] [in a new window]   Fig. 10.   Effect of 10 µM cyclosporin A on [125I]IAAP labeling of the wild-type and recombinant Pgp cell membranes in the presence of cis-(Z)-flupentixol.

	Discussion

Top Summary Introduction Materials & Methods Results Discussion References

Characterization of a novel chimeric Pgp. In this study, we have found that random recombination between human MDR1 and mouse mdr1a, which changes five amino acids in the segment, including TM5 and TM6 and the adjacent extracellular loops in Pgp, decreases resistance to colchicine and taxol. This recombinant is also relatively insensitive to the ability of known Pgp modulators, such as verapamil, cyclosporin A, and PSC 833, to inhibit Pgp pumping of several fluorescent substrates and colchicine but not taxol. This new phenotype is unique to the recombinant and is not characteristic of either mouse mdr1a or human MDR1.

Use of the bicistronic vector pHaMDR-DHFR. The use of bicistronic retroviral vectors containing a therapeutic gene, an intercistronic IRES element, and a selectable MDR1 gene has been developed for the gene therapy of several disorders (Aran et al., 1994; Metz et al., 1996). Our present effort uses the bicistronic vector as a stable expression system for Pgp. Our results demonstrate that the use of the bicistronic vector for stable transfection by methotrexate selection guarantees that the majority of the methotrexate resistance clones also express Pgp. As shown in Fig. 4, a library of chimeric Pgp molecules can be selected in methotrexate without concern about pleiotropic cellular effects caused by selection in MDR drugs.

Implications of MDR shuffling. Stemmer (1994b) has demonstrated that by applying repeated cycles of DNA shuffling and selection, he can create a library of chimeras from a pair of homologous genes from different species with improved activity and a new function. Our results demonstrate that the shuffling between transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a generates a new phenotype that could be useful in selecting for MDR mutants with altered drug resistance and differential reversing effects as a potential tool for cancer gene therapy. Despite the large diversity in the bacterial library, after selection of the mammalian library with MDR drugs, the majority of the clones had the wild-type MDR1 sequence, which suggests that this region cannot tolerate most random recombination events. It should be useful in the future to shuffle larger regions from human MDR1 and mouse mdr1a that are less conserved to select for more MDR mutants with an improved drug resistance profile.