Drug selection of MDR1-transduced hematopoietic cells ex vivo increases transgene expression and chemoresistance in reconstituted bone marrow in mice.

The MDR1 (multidrug resistance) gene, transferred to hematopoietic cells, is expected to protect them from anticancer chemotherapy and may serve as a selectable marker, restoring gene expression in vivo. Appropriate selection strategies, however, need to be established. To investigate whether preselection ex vivo affects chemoresistance, murine bone marrow cells were retrovirally transduced with high-titer or, as a model for suboptimal gene expression, low-titer retroviruses and exposed to daunomycin or colchicine for 48-96 h. Selection significantly increased chemoresistance of clonogenic progenitor cells. In tissue culture, the entire target population was rendered highly drug resistant after MDR1 transfer with high-titer viruses. If transduction was performed under suboptimal conditions, drug selection increased the frequency of chemoresistant colonies up to 40% over the number of unselected cells. Colchicine and daunomycin were equally efficient in increasing drug resistance ex vivo, but colchicine-preselected cells rescued lethally irradiated mice under conditions where daunomycin-selected bone marrow cells failed to do so. Hence, while hematopoietic cells can be protected by MDR1, the selection strategy is critical for repopulation of bone marrow with transduced cells. Preselection in culture before transplantation significantly increased P-gp expression and chemoresistance in vivo in mice reconstituted with transduced bone marrow cells. This study may help to facilitate the use of MDR1 as a selectable marker in gene therapy of the hematopoietic system. Gene Therapy (2000) 7, 348-358.

Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein.

Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro. To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.

Reversal of drug resistance in a human colon cancer xenograft expressing MDR1 complementary DNA by in vivo administration of MRK-16 monoclonal antibody.

One strategy to overcome multidrug resistance in neoplasia is to inhibit the gp170 glycoprotein (relative molecular mass, 170,000) that functions as a plasma membrane, energy-dependent, drug-efflux pump. The human colon cancer cell line HT-29, which grows as an ascitic tumor in athymic NCr-nu/nu nude mice, was made multidrug resistant by infection with an MDR1 (also known as PGY1) retrovirus. Referred to as HT-29mdr1, it was used to study reversal of drug resistance in vivo by the anti-P-glycoprotein monoclonal antibody MRK-16. Flow cytometry and radioimmunoassay demonstrated a marked increase in MRK-16 reactivity on HT-29mdr1 cells as compared with its reactivity on the parental, uninfected cell line (HT-29par). The 50% inhibitory concentrations (IC50) of vincristine on HT-29par and HT-29mdr1 cells were 2.5 and 15 ng/mL, respectively. The MRK-16 monoclonal antibody did not affect the vincristine sensitivity of the HT-29par cells. Pretreatment of HT-29mdr1 cells with 10 micrograms/mL MRK-16 in tissue culture partially restored the vincristine sensitivity (IC50 = 7 ng/mL). This modulation of vincristine sensitivity by MRK-16 was then tested in vivo. The median survival times of mice given intraperitoneal transplants of 5 x 10(6) HT-29par or HT-29mdr1 were 37 and 39 days, respectively. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, resulted in a significant increase in the median survival time of the HT-29par tumor-bearing mice (68 days, P less than .0001), but it had no effect on the HT-29mdr1 tumor-bearing mice. However, treatment of mice bearing the HT-29mdr1 tumor with MRK-16 before vincristine therapy reversed the resistance to the drug (median survival time = 64 days, P less than .0001). The MRK-16 monoclonal antibody alone had no effect on the median survival time of mice given an injection of either HT-29par or HT-29mdr1 cells. These results suggest that strategies employing monoclonal antibody against gp170 may be clinically useful to reverse multidrug resistance.