Multidrug Resistance Proteins

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P-glycoprotein (P-gp) is encoded by the multidrug-resistance-1 gene (mdrl) and is the prototypic member of the ATP-binding cassette family of drug resistance proteins. P-gp was identified as the cellular protein responsible for the pleiotropic cross-resistance certain cell lines acquired to unrelated chemotherapy drugs, such as anthra-cyclins, epipodophyllotoxins, taxanes, and vinca-alkaloids (59,60). Drug resistance is established by the ATP-dependent efflux of these compounds from the cell, preventing intracellular concentrations from becoming cytotoxic (see Fig. 3).

The potential for using P-gp to protect bone marrow from myelosuppression was first demonstrated in transgenic mice (61,62). High level P-gp expression in transgenic bone marrow protected animals from daunomycin and taxol treatments that caused myelosuppression in normal mice. Transplantation of mdrl-transgenic bone marrow

Fig. 3. Drug efflux pumps. The ABC-family of drug efflux proteins are localized in the plasma membrane where they actively export cytotoxic drugs from the cytosol to the extracellular space. Cells that overexpress these proteins are protected from higher drug concentrations.

into lethally-irradiated control animals was sufficient to transfer long-term drug resistance to recipient animals (63). These results set the framework for retroviral gene transfer experiments. In 1992 two groups demonstrated that retroviral delivery of mdrl to bone marrow progenitor cells resulted in increased drug tolerance that correlated with enrichment for the transduced population (64,65). Transduction of long-term repopulat-ing cells with mdrl was subsequently shown to protect murine transplant recipients from repetitive administration of normally myelotoxic chemotherapy treatments (66,67), while simultaneously sensitizing tumor cells (68). More recent in vivo selection studies using mdrl have been carried out in other animal models. Schiedlmeier et al. demonstrated the first significant evidence of mdr 1-mediated selection of human hematopoietic progenitors in vivo, using NOD/SCID recipients treated with paclitaxel (69).

Clinical trials have also been carried out to evaluate the use of mdrl gene transfer for cancer patients receiving autologous transplants to reduce treatment-induced myelo-suppression (70-75). Early trials were limited by poor transduction efficiencies and resulted in transient marking levels indicative of short-term repopulating cell contributions. Higher marking rates (up to 52%) have been obtained using plates coated with the recombinant fibronectin fragment CH-296 (75). A maximum of 15% mdrl-marked bone marrow CFU were detected a year after transplant, but evidence of mdrl-mediated selection was limiting.

More than 50 other ATP-binding cassette family members have been identified, some of which have been associated with antineoplastic drug resistance in cancer cells (76). In experiments aimed at resolving the cycling status of HSCs, Goodell et al. discovered a population of cells resistant to labeling with the fluorescent DNA stain, Hoechst 33342 (77). Interestingly, lymphomyeloid repopulating activity was enriched over 1000-fold in the population of cells with the highest degree of Hoescht exclusion (designated SP cells). The verapamil-sensitivity of this phenomenon indicated that P-gp, or a similar drug efflux pump, was responsible and expressed at higher levels in bone marrow stem cells. Subsequent studies have identified SP stem cells in a variety of tissues. ABCG2/BCRP1 has been identified as the transporter responsible for Hoechst 33342 efflux in SP cells. However, enforced ABCG2 expression in murine bone marrow reduced progenitor cell differentiation in vitro (78,79). Similar results were previously reported for murine bone marrow cells transduced with mdrl (80). Overexpression of mdrl in the absence of drug selection resulted in SP cell expansion ex vivo and the onset of a myeloproliferative syndrome when these cells were transplanted into lethally irradiated recipients. Another group has recently reported an increased frequency of leukemogenesis associated with retroviral delivery of mdrl to murine BM cells at a high-copy number (81). However, in this study insertional mutagenesis was also seen using fluorescent reporter vectors, but at a reduced frequency. The high expression levels of these proteins in primitive hematopoietic cells suggest that these transporters may have a role in stem cell biology beyond protection from drug exposure (82,83). However, both mdrl and ABCG2 knockout animals exhibit normal hematopoiesis (79,84). No myeloproliferative disorders were detected during the mdrl clinical trials. Further, no aberrant expansion was detected in rhesus macaques after transplanting cells transduced with conditions similar to those that caused the disorder in mouse models (85).

Drug selection of stem cells transduced with mdrl or ABCG2 may be limited by the high endogenous levels of these proteins in primitive hematopoietic cell populations. Specific point mutations of mdrl (86) or ABCG2 (87) that are resistant to inhibition, or have altered substrate recognition, may be more potent agents for differentially selecting transduced stem cells in vivo.

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