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    Retroviral vector constructs and results of analysis of P-gp expression in COS-7 and MDCK cells. A—Schematic representation of pLEGFP-N1 and pLNCcMDR1 constructs. B—Immunoblot analysis of P-gp expression in COS-7 cells 48 hours after transfection with pLNC-cMDR1 as follows: lane 1, nontransfected COS-7 cells; lane 2, COS-7 cells transfected with the control plasmid (pLEGFP-N1); and lane 3, COS-7 cells transfected with pLNC-cMDR1 plasmid. C—Immunoblot analysis of P-gp expression in MDCK cells transduced with pLNC-cMDR1 as follows: lane 1, untreated MDCK cells; lane 2, MDCK cells transduced with the control retroviral vector (pLEGFP-N1); and lane 3, MDCK cells transfected with pLNC-cMDR1 plasmid. LTR = Long terminal repeat. MoMLV = Moloney murine leukemia virus. Neo = Neomycin resistance gene. CMV = Human cytomegalovirus immediate early promoter. EGFP = Enhanced green fluorescent protein gene.

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    Results of analysis of Rh123 efflux and retention in MDCK and MDCK/ cMDCR1 cells. A—Representative histogram of flow cytometric analysis of Rh123 efflux in MDCK and MDCK/cMDR1 cells. Fluorescence of MDCK and MDCK/ cMDR1 cells after 15 minutes of efflux of Rh123 are shown in comparison to the fluorescence of MDCK cells incubated with (Rh123+) and without Rh123 (Rh123+) as references. B—Percentage of Rh123 retention in MDCK and MDCK/cMDR1 cells calculated from the fluorescence intensity values of 3 independent experiments. Values are mean ± SD of 3 independent experiments.

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    Dose-response curves of the cytotoxic effects of doxorubicin (A) and vincristine (B) on nontransduced MDCK cells, MDCK cells transduced with the control retroviral vector (pLEGFP-N1), and MDCK/cMDR1 cells. Values represent the mean of 3 independent experiments.

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Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

Shinobu Matsuura DVM, PhD1, Hisao Koto DVM1, Kaori Ide DVM1, Yasuhito Fujino DVM, PhD1, Asuka Setoguchi-Mukai DVM, PhD1, Koichi Ohno DVM, PhD1, and Hajime Tsujimoto DVM, PhD1
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  • 1 Department of Veterinary Internal Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.

Abstract

Objective—To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1).

Sample Population—Madin-Darby canine kidney (MDCK) epithelial cell line.

Procedures—The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus–based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays.

Results—P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1.

Conclusions and Clinical Relevance—Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.

Abstract

Objective—To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1).

Sample Population—Madin-Darby canine kidney (MDCK) epithelial cell line.

Procedures—The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus–based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays.

Results—P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1.

Conclusions and Clinical Relevance—Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.

The major challenge in the management of lymphoid tumors in dogs is the dose-limiting myelotoxicity of the chemotherapeutic protocols. Increasing the intrinsic chemoresistance of normal bone marrow cells would allow dose-intensification of the chemotherapeutic regimens, and thus, better therapeutic responses are expected. One promising approach for induction of chemoresistance in normal bone marrow cells is a transfer of mdr1 in pursuit of its expression in hematopoietic cells.

The mdr1 was first identified as a drug-resistance gene that conferred multidrug resistance on tumor cells. The mdr1 encodes P-gp, a transmembrane protein responsible for efflux of various substrates in an energy-dependent manner driven by ATP.1 The P-gp effluxes the substrate drugs from the cells and reduces their intracellular concentrations, conferring drug resistance on the cells.2 Thereby, Galski et al3 reported a novel use of mdr1 to confer the drug resistance upon normal bone marrow cells, resulting in protection against myelosuppression by an antineoplastic agent.

In veterinary medicine, mdr1 was first linked to cancer multidrug resistance,4 but a remarkable finding later proved the central role of P-gp in cellular protection through the efflux of cytotoxic compounds from the cells. Dogs, mostly Collies, which harbor a homozygous mutation of mdr1 have high susceptibility to a neurotoxic agent, ivermectin, through the dysfunction of P-gp in the blood-brain barrier.5

For induction of chemoresistance in stem cells, preliminary clinical studies in humans made use of CD34+ cells as a source of hematopoietic stem cells. Retroviral vectors were used for stable integration of mdr1 into the genome.6 Transgene expression was detected in differentiated peripheral blood cells, indicating the feasibility of this approach to achieve engraftment of transduced cells and chemoprotection of the hematopoietic lineage.7–10

The purpose of the study reported here was to induce chemoresistance in a normal canine cell line through the transduction of canine mdr1. We constructed a retroviral vector inserted with canine mdr1 cDNA, prepared its vector virus, and then examined for the effect of transduction of mdr1 in a cultured canine cell line.

Materials and Methods

Construction of the retroviral vector—Peripheral blood was collected from a 5-year-old adult female Beagle. The dog was apparently healthy on the basis of physical examination and hematologic and serum biochemical analyses and was housed and treated at The University of Tokyo, Tokyo, in accordance to the institutional guidelines for the care and use of laboratory animals.

A sample of mRNA was extracted from peripheral blood mononuclear cells.a Single-stranded cDNA reverse transcribed from the mRNA with a commercial kitb was used as a template for PCR procedures. Eight primers (Appendix) were designed on the basis of reported canine mdr1 sequences (GenBank accession No. AF045016) to amplify 4 overlapping segments covering its entire open reading frame. The PCR was initiated by a denaturation step of 98°C for 2 minutes and then followed by 35 cycles at 98°C for 20 seconds, 60°C for 2 minutes, and 72°C for 2 minutes, with a final extension step of 72°C for 10 minutes. Segments were sequenced with a capillary sequence analyzer.c The 4 segments were designed to use 3 restriction endonuclease sites (BglII, HindIII, XhoI) within the mdr1 sequence. BglII and BamHI restriction sites were added at the 5a and 3a ends of mdr1 cDNA, respectively. To construct the retroviral vector inserted with canine mdr1 cDNA (ie, pLNC-cMDR1), the 4 segments were successively inserted between the BglII and BamHI sites of a murine leukemia virus–based retroviral vector (ie, pLEGFP-N1),d by use of the BglII, HindIII, and XhoI sites of canine mdr1 cDNA fragments. The pLEGFP-N1 is a retroviral vector based on the Moloney murine leukemia virus genome and was designed to express the enhanced green fluorescent protein under the control of the cytomegalovirus promoter (Figure 1). Expression of P-gp from the obtained canine mdr1 cDNA was verified by transient transfection of COS-7 cells.e

Figure 1—
Figure 1—

Retroviral vector constructs and results of analysis of P-gp expression in COS-7 and MDCK cells. A—Schematic representation of pLEGFP-N1 and pLNCcMDR1 constructs. B—Immunoblot analysis of P-gp expression in COS-7 cells 48 hours after transfection with pLNC-cMDR1 as follows: lane 1, nontransfected COS-7 cells; lane 2, COS-7 cells transfected with the control plasmid (pLEGFP-N1); and lane 3, COS-7 cells transfected with pLNC-cMDR1 plasmid. C—Immunoblot analysis of P-gp expression in MDCK cells transduced with pLNC-cMDR1 as follows: lane 1, untreated MDCK cells; lane 2, MDCK cells transduced with the control retroviral vector (pLEGFP-N1); and lane 3, MDCK cells transfected with pLNC-cMDR1 plasmid. LTR = Long terminal repeat. MoMLV = Moloney murine leukemia virus. Neo = Neomycin resistance gene. CMV = Human cytomegalovirus immediate early promoter. EGFP = Enhanced green fluorescent protein gene.

Citation: American Journal of Veterinary Research 68, 1; 10.2460/ajvr.68.1.95

Production of RD114-pseudotyped vector viruses—To obtain vector viruses, the method described by Kelly et al11 was used. Briefly, vector genomes of pLNC-cMDR1 and pLEGFP-N1 were introduced into GP2-293T cells,d producing vector particles enveloped with the vesicular stomatitis virus G protein. The transfection was performed with a commercial reagent.f Vesicular stomatitis virus G-protein pseudotyped vectors were used to infect FLYRD-18g,12 cells to produce viral vectors pseudotyped with the RD114 envelope. Supernatants of the packaging cells were harvested every 24 hours after transfection and concentrated by ultracentrifugation at 80,000 × g at 4°C for 90 minutes. Viral pellets were resuspended in Dulbecco modified Eagle media and maintained at 80°C until use.13 Viral stocks were titered on HT-1080 cells.14,e

Retroviral transduction of MDCK cells—One day before inoculation with the vector viruses, MDCKh cells were seeded at the concentration of 104 cells/well on 24well plates. The MDCK cells were infected with the vector viruses at a multiplicity-of-infection value of 10 in the presence of hexadimethrine bromide (8 μg/mL)i for 16 hours and then cultured for 3 consecutive days. The pLNC-cMDR1–transduced cells (ie, MDCK/cMDR1 cells) were selected by increasing the concentrations of colchicinei in the medium up to 80 ng/mL. Colchicine is a substrate for P-gp and is useful for stringent selection of cells that express high amounts of P-gp.15 Cells transduced with the control vector do not survive colchicines selection because it causes mitotic arrest through interference with the mitotic spindle. For selection of cells transduced with pLEGFP-N1, MDCK cells were cultured in the presence of neomycin at a concentration of 1 mg/mL.

Immunoblotting for P-gp—For extraction of the total cellular protein, cells grown on culture plates were treated with a lysis buffer (150mM NaCl, 20mM TrisHCl [pH, 7.4], and 1% Triton X) supplemented with a protease inhibitor cocktaili on ice for 30 minutes. The collected cellular extract was centrifuged at 15,000 × g for 10 minutes, and the supernatant was concentrated with centrifugal filter units.j Cellular protein samples (20 Mg) diluted in a sample buffer (62.5mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate, 25% glycerol, 0.01% bromophenol blue, and 5% 2-mercaptoethanol) were subjected to polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred onto polyvinylidene diflouride membranesk and incubated in PBS solution with 0.6% Tween 20 and 5% skim milk to prevent nonspecific binding. Membranes were incubated at 4°C for 6 hours with primary mouse monoclonal antibody C219 directed to P-gp,l which had been shown to react with human, murine, and canine P-gp.16,17 After washing, membranes were incubated with horseradish peroxidase–conjugated goat anti-mouse IgG for 1 hour. As an internal isotype control antibody, a mouse monoclonal antibody to β-actini was used. Chemiluminescence of the horseradish peroxidase in protein-antibody complexes was captured with a charge-coupled device camera.m

Measurement of Rh123 retention—The dynamic traffic of a fluorescent dye, Rh123, through the membrane of living cells is well characterized.18,19 Its intracellular accumulation occurs by passive diffusion, but efflux to the extracellular compartment is limited by the ATP-dependent transport through P-gp. For this reason, the efflux rate of Rh123 is used as a sensitive marker of P-gp activity in living cells.

Efflux rate of Rh123 through P-gp can be measured indirectly by determining the retention of the dye in cells. The more cells efflux the dye, less retention of Rh123 is expected.18 The MDCK and MDCK/cMDR1 cells (5 × 105) were incubated in serum-free RPMI 1640 containing Rh123 (150 ng/mL) at 37°C for 60 minutes (FRh123+ cells). After washing the cells twice, they were incubated in a medium without Rh123 for 15 minutes to measure the 15-minute dye efflux. The mean fluorescence intensity of each sample and the background fluorescence of cells in a medium without Rh123 (FRh123 cells) were measured with a flow cytometer. The Rh123 retention was calculated as the ratio of Rh123 present in cells before efflux to that present after 15 minutes of efflux by use of the following formula:20

article image

Drug sensitivity assay—One day before the examination of drug sensitivity, cells were plated at 3 × 103 cells/well of a 96-well microplate. After addition of serial dilutions of doxorubicin and vincristine into the culture medium, MDCK cells were cultured for 48 hours. Then, cells were washed and incubated in a solution containing 2-(2-methoxy-4-nitrophenyl)-3(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt,n which is cleaved by viable cells to result in a formazan dye, for 4 hours. Absorbance of the formazan dye was measured with a microplate ELISA reader with a 450-to-600 nm filter. Percentages of the viable cells were calculated from the ratios of the absorbances of cells cultured with a drug at a given concentration to those of the cells cultured without the drug (control). Percentages of viable cells at each drug concentration were plotted to generate a dose-response curve. The IC50 of drugs was obtained from the viable cell counts in the presence of several dilutions of the drugs.

Statistical analysis—Mean ± SD IC50 of 3 measurements was calculated for each cell line. Analysis was performed with the aid of a computer software package.o A 1way ANOVA was used for comparison of means, followed by the Student t test analysis between pairs, to identify groups significantly different from the control. Values of P < 0.05 were considered significant.

Results

Expression of P-gp in COS-7 cells after transient transfection with pLNCcMDR1—Molecular clones of canine mdr1 cDNA were obtained from the mRNA sample of the peripheral blood mononuclear cells from a Beagle. We compared the sequence of canine mdr1 mRNA obtained in this study (GenBank accession No. DQ068953) with the corresponding sequence of canine genome (GenBank accession No. NW_876258.1) and found 100% nucleotide sequence identity between the 2 sequences. However, when the canine mdr1 mRNA sequence obtained in this study was compared with 3 previously registered canine mdr1 mRNA sequences, GenBank accession Nos. AF045016, AF536758, and AY582533, 11-, 4-, and 2-nucleotide differences, respectively, were found.

To verify the expression of P-gp by the canine mdr1 cDNA obtained, pLNC-cMDR1 was transiently transfected into COS-7 cells and cells were examined by immunoblotting with an anti–P-gp antibody C219. At 48 hours, strong P-gp expression was detected in cells transfected with pLNC-cMDR1, but not in the parental cells or cells transfected with the control vector, pLEGFP-N1 (Figure 1).

Expression of P-gp in MDCK cells transduced with pLNC-cMDR1—Titers of retroviral vectors produced by transduction of pLNC-cMDR1 into FLYRD18 packaging cells ranged from 6.5 to 2.0 × 107 CFU/ mL. The MDCK cells infected with the resultant vector virus were subjected to selection by increasing the concentration of colchicine in the culture medium. The IC50 of colchicine for the nontransduced MDCK cells was 15.3 ± 1.8 ng/mL. The MDCK/cMDR1 cells were able to survive at increasing doses of colchicine. Because a delay in doubling time (data not shown) was observed at higher colchicine concentrations (> 80 ng/ mL), MDCK/cMDR1 cells were maintained with colchicine at 80 ng/mL for further experiments.

Expression of P-gp protein was examined by immunoblotting with antibody C219 (Figure 1). Expression of P-gp was not observed in parental MDCK cells or cells transduced with pLEGFP-N1. However, in MDCK/cMDR1 cells, strong expression of P-gp was detected.

Rh123 efflux activity in MDCK/cMDR1 cells—Flow cytometric analysis was performed to depict the retention and efflux of Rh123 (Figure 2). A shift to the left of the fluorescence intensity peaks indicated enhanced Rh123 efflux activity and, therefore, enhanced P-gp pumping activity in MDCK/ cMDR1 cells, compared with the nontransduced MDCK cells. The degree of Rh123 efflux was quantified by measuring the percentage of Rh123 retention, which was less in MDCK/cMDR1 cells than in nontransduced MDCK cells. The MDCK cells transduced with the control vector, pLEGFP-N1, could not be used in the Rh123 efflux assay because the fluorescence wavelength of green fluorescent protein overlaps with that of Rh123.

Figure 2—
Figure 2—

Results of analysis of Rh123 efflux and retention in MDCK and MDCK/ cMDCR1 cells. A—Representative histogram of flow cytometric analysis of Rh123 efflux in MDCK and MDCK/cMDR1 cells. Fluorescence of MDCK and MDCK/ cMDR1 cells after 15 minutes of efflux of Rh123 are shown in comparison to the fluorescence of MDCK cells incubated with (Rh123+) and without Rh123 (Rh123+) as references. B—Percentage of Rh123 retention in MDCK and MDCK/cMDR1 cells calculated from the fluorescence intensity values of 3 independent experiments. Values are mean ± SD of 3 independent experiments.

Citation: American Journal of Veterinary Research 68, 1; 10.2460/ajvr.68.1.95

Chemoresistance of MDCK/cMDR1 cells—To evaluate the degree of drug resistance conferred by the transduction of pLNC-cMDR1, a drug sensitivity or cytotoxicity assay was performed. Nontransduced MDCK cells, MDCK cells transduced with pLEGFP-N1 (control vector), and MDCK/ cMDR1 cells were cultured in the presence of various concentrations of vincristine and doxorubicin (Figure 3), and the percentages of viable cells cultured at each drug concentration were calculated from control cells cultured without drug. Plotting the resulting percentages at each drug concentration, a sigmoid dose-response curve was obtained for each cell line. Transduction of pLEGFPN1 did not confer any survival advantage on the parental MDCK cells, as observed by the proximity of the 2 sigmoid curves. On the other hand, MDCK/cMDR1 cells had a remarkable increase in the chemoresistance to vincristine and doxorubicin, as observed by the shift to the right of the sigmoid curve. From results of the cytotoxic assays, it was possible to calculate the IC50. Vincristine IC50 in MDCK/cMDR1 cells was 59-fold higher than that in the nontransduced MDCK cells. Similarly, the transduction of mdr1 conferred remarkable resistance to doxorubicin in MDCK cells, with a 25-fold higher IC50 (Table 1).

Table 1—

Mean ± SD IC50 of vincristine and doxorubicin for MDCK cells, MDCK cells transduced with the control vector, and MDCK/cMDR1 cells.

DrugIC50 (μmol)
MDCKControl vectorMDCK/cMDR1
Vincristine0.066 ± 0.007l0.073 ± 0.0393.893 ± 2.328*
Doxorubicin0.576 ± 0.0581.295 ± 0.10514.889 ± 0.986

Results reported as mean values of 3 independent experiments.

Significantly (P < 0.05) different from MDCK cells.

Significantly (P < 0.01) different from MDCK cells.

Figure 3—
Figure 3—

Dose-response curves of the cytotoxic effects of doxorubicin (A) and vincristine (B) on nontransduced MDCK cells, MDCK cells transduced with the control retroviral vector (pLEGFP-N1), and MDCK/cMDR1 cells. Values represent the mean of 3 independent experiments.

Citation: American Journal of Veterinary Research 68, 1; 10.2460/ajvr.68.1.95

Discussion

As a retroviral vector, pLNC-cMDR1 was shown to express the canine P-gp in the transduced cells and was demonstrated to be an effective vehicle for inducing chemoresistance in canine cells. The canine mdr1 cDNA sequence obtained in our laboratory from a clinically normal Beagle had a complete match with the sequence of a canine genome. However, compared with the mRNA sequence previously reported21 and those previously registered in GenBank, some nucleotide differences were found among these sequences. Many of these differences are conceivably caused by single nucleotide polymorphisms as found in human mdr122; however, some might have derived from errors during the PCR, step which was generally used to obtain cDNA clones. To verify the genetic heterogeneity of mdr1 in dogs, accumulation of larger numbers of the sequence data will be required.

When assessing P-gp expression in cells transfected with mdr1, it is always mandatory to differentiate expression of the transfected vector from the expression of intrinsic P-gp because P-gp represents one of the natural defenses of most cells to toxic compounds. P-glycoprotein was strongly expressed 48 hours after transfection of the pLNC-cMDR1 clone into COS-7 cells. Expression was not observed in parental COS-7 cells or in COS-7 cells transfected with pLEGFP-N1. This indicates that P-gp expression is derived from the vector mdr1 and not from the cellular mdr1.

The MDCK cells were not as permissive as COS-7 cells for the expression of mdr1. For this reason, the P-gp substrate colchicine was used for selection of cells with higher expression of P-gp. It was supposed that the presence of the P-gp substrate could induce the expression of endogenous P-gp rather than that of the retroviral vector-derived P-gp in MDCK cells. To verify this possibility, nontransduced and pLEGFP-N1–transduced MDCK cells were cultured in the presence of colchicine at the same dose as used for the pLNC-cMDR1– transduced cells. None of these cells could survive the colchicine selection, leading to a conclusion that the drug-resistant phenotype observed in MDCK/cMDR1 cells was conferred by the expression of mdr1 transduced by the retroviral vector.

The increased Rh123 efflux activity observed in cells transduced with pLNC-cMDR1 is evidence of existence of pumping activity in P-gp expressed from the transduced mdr1. Unfortunately, Rh123 efflux activity could not be evaluated in cells transduced with the control vector because of technical limitations. However, because P-gp was not detected by immunoblotting in these cells, Rh123 efflux activity is expected to be lower in these cells as well.

The IC50 of doxorubicin in control vector (pLEGFP-N1)–transduced MDCK cells was significantly higher than that in nontransduced MDCK cells. The low drug resistance acquired in the pLEGFP-N1–transduced cells might be the result of the vector itself or the selection procedure. The MDCK cells transduced with pLEGFP-N1 as a control vector were cultured in the presence of neomycin to select cells with higher expression of green fluorescent protein. Although neomycin is not known as a substrate for P-gp and P-gp expression was not detected by immunoblotting of the cells, the selection with neomycin could have stimulated a crossresistance to doxorubicin.

The final goal of our laboratory is to construct a gene transfer system applicable to clinical use. Our focus is to develop more effective chemotherapy for canine lymphoma; novel therapeutic strategies that are aimed at further prolongation of survival time are required.23 The rationale of the advance for the treatment of canine lymphoma may lie on the use of more aggressive chemotherapy to kill the maximum number of neoplastic cells. But, unfortunately, an increase in the dose of the chemotherapeutic agents or the use of more aggressive drugs is invariably followed by cytotoxicity to normal hematopoietic cells.

The transfer of mdr1 into bone marrow cells could provide a benefit to overcome the myelosuppression after aggressive chemotherapy. For efficient gene transfer into hematopoietic cells, hematopoietic stem cells expressing the CD34 marker are conceivably the best target.24 CD34+ hematopoietic cells have a capacity to self renew, to proliferate and propagate, and, finally, to differentiate into the cells of multiple hematopoietic lineages. Because retroviral vectors can integrate into the host genome, cells originating from the transduced cells are expected to provide long-term chemoresistance to the various substrates of P-gp.

One disadvantage of the onco-retroviral vectors based on Moloney murine leukemia virus is the need of cell divisions for its integration into the host genome. In the case of CD34+ hematopoietic stem cells, cell division could result in the differentiation and loss of the characteristics of primitive cells. To overcome this problem, lentiviral vectors, which can integrate into the host genome of nonmitotic cells, have been developed as promising viral vectors for transduction of CD34+ hematopoietic stem cells.25 For the development of a gene transfer system into CD34+ stem cells, lentiviral vectors are conceivably more suitable than the oncoretroviral vectors used in our study.

When retroviral vector-mediated gene transfer into hematopoietic stem cells is applied to treatment in the clinical setting, its safety issues should be carefully considered. Bunting et al26 reported the occurrence of myeloproliferative syndrome in mice following retroviral vectormediated transduction of mdr1. Recently, Modlich et al27 demonstrated that combinational insertional mutagenesis was associated with induction of leukemias in mice that received retroviral vector-mediated mdr1 transfer.

Although the frequency of developing neoplastic change following retroviral integration is conceivably not high, we should mention that the system itself harbors a risk of insertional mutagenesis and that the riskto-benefit ratio of gene therapy should be considered at the moment of patient selection.28 Moreover, when target cells for mdr1 transfer are widespread in the body after successful gene therapy, it is conceivable that they become resistant to P-gp substrate drugs including some corticosteroids, antimicrobial drugs, immunosuppressive drugs, and antineoplastic drugs. To avoid this, strategies to use tissue-specific or inducible promoters would be recommended to improve the safety for treatment by use of mdr1 transfer.

Findings in our study indicate that use of a gene transfer system to induce chemoresistance in cultured canine cell lines is feasible. Transduction of retroviral vectors harboring canine mdr1 may provide a useful tool for stable integration and long-term expression of P-gp in canine cells.

ABBREVIATIONS

mdr1

Multidrug resistance 1 gene

P-gp

P-glycoprotein

pLNC-cMDR1

Retroviral vector with canine mdr1 cDNA insertion

pLEGFP-N1

Retroviral vector with enhanced green fluorescent protein

MDCK

Madin-Darby canine kidney

MDCK-cMDR1

pLNC-cMDR1– transduced MDCK

Rh123

Rhodamine 123

FRh123

Rh123 fluorescence

IC50

50% inhibitory concentration

a.

Micro-Fast Track 2.0 Kit, Invitrogen, Carlsbad, Calif

b.

cDNA Cycle Kit, Invitrogen, Carlsbad, Calif

c.

Applid Biosystems/Hitachi, Foster City, Calif

d.

BD Biosciences Clontech, Palo Alto, Calif

e.

Health Science Research Resources Bank, Osaka, Japan

f.

Lipofectamine 2000, Invitrogen, Carlsbad, Calif

g.

European Collection of Cell Cultures (ECACC), Porton Down, Wiltshire, UK

h.

Riken Bioresource Center, Ibaraki, Japan

i.

Sigma-Aldrich Inc, St Louis, Mo

j.

Microcon YM-100 filters, Millipore, Bedford, Mass

k.

Hybond-P, Amersham Biosciences, Little Chalfont, Bucks, UK

l.

Calbiochem, San Diego, Calif

m.

ATTO Corp, Tokyo, Japan

n.

WST-8 cell counting kit-8, Dojindo, Kumamoto, Japan

o.

Jump, version 5.0.1J, SAS Institute Inc, Cary, NC.

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Appendix

Primers for PCR amplification of the open reading frame of canine mdr1.

Canine mdr1 fragmentsForwardReverse
Nucleotide position*Primer sequenceNucleotide position*Primer sequence
Fragment I61–765′-GAGAGATCTATGGATCCTGAAGGAGG-3′1332–13055′-CCCACTCTGAACCTTCAGGTTGAGACCC-3′
Fragment II1057–10775′-GGACAGGTACTCACTGTCTTC-3′2198–21775′-GGCCATTCAGTTGAGTTCAGC-3′
Fragment III2075–20985′-CTCGCAGGAGTATACATGCACCAC-3′3350–33275′-GAACCAGCCAAGGGGTCATAGAAG-3′
Fragment IV3201–32225′-CTATCCCACTCGACCAGACATC-3′3924–38975′-GGCCACGGGATCCTAGCGCTTTGCTCC-3′

Nucleotide positions in canine mdr1 sequence (GenBank accession No. AF045016)

Contributor Notes

Dr. Matsuura's present address is Department of Molecular and Experimental Medicine, Division of Oncovirology, The Scripps Research Institute, 10550 N Torrey Pines Rd, La Jolla, CA 92037.

Supported by grants from Japan Society for the Promotion of Science.

This work was presented in part at the 24th Annual Conference of the Veterinary Cancer Society, Kansas City, MO, November 2004.

Address correspondence to Dr. Tsujimoto.