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In Vivo Drug-Selectable Genes: A New Concept in Gene Therapy

^a Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland, USA;
^b University of Ulm, Department of Internal Medicine III, Ulm, Germany;
^c Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland, USA

Key Words. Chemotherapy • Hematopoietic stem cells • Multidrug resistance • Myelosuppression • P-glycoprotein • Dihydrofolate reductase • Transgenic animals • Retroviral vectors

Dr. M.M. Gottesman, Laboratory of Cell Biology, National Cancer Institute, NIH, Building 37, Room 1B22, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255, USA.

	Abstract

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Chemoresistance genes, initially considered to be a major impediment to the successful treatment of cancer, may become useful tools for gene therapy of cancer and of genetically determined disorders. Various target cells are rendered resistant to anticancer drugs by transfer of chemoresistance genes encoding P-glycoprotein, the multidrug resistance-associated protein-transporter, dihydrofolate reductase, glutathione-S-transferase, O⁶-alkylguanine DNA alkyltransferase, or aldehyde reductase. These genes can be used for selection in vivo because of the pharmacology and pharmacokinetics of their substrates. In contrast, several other selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture. Possible applications for chemoresistance genes include protection of bone marrow and other organs from adverse effects caused by the toxicity of chemotherapy. Strategies have also been developed to introduce and overexpress nonselectable genes in target cells by cotransduction with chemoresistance genes. Thereby expression of both transgenes can be increased following selection with drugs. Moreover, treatment with chemotherapeutic agents should restore transgene expression when or if expression levels decrease after several weeks or months. This approach may improve the efficacy of somatic gene therapy of hematopoietic disorders which is hampered by low or unstable gene expression in progenitor cells. In this article we review preclinical studies in tissue culture and animal models, and ongoing clinical trials on transfer of chemoresistance genes to hematopoietic precursor cells of cancer patients.

	Introduction

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Expression of drug resistance genes in tumor cells may indicate poor prognosis of patients suffering from various malignancies. For instance, detection of the multidrug resistance gene, MDR1, has been associated with failure of anticancer drug treatment of acute leukemias [1, 2], multiple myelomas [3, 4], sarcomas [5] and neuroblastomas [6]. Similarly, neuroblastomas in which the MRP transporter protein is overexpressed are likely to be resistant to chemotherapy [7]. Multiple chemoresistance genes conferring resistance to a broad spectrum of drugs can be overexpressed in malignant cells [8]. Several attempts have been undertaken to circumvent drug resistance by restoring chemosensitivity of cancer cells. Treatment modalities include high-dose chemotherapy with or without transplantation of progenitor cells, chemosensitization with inhibitors of proteins encoded by drug resistance genes [9-11], administration of monoclonal antibodies [12, 13] or immunotoxins [14], or by interference with transcription or translation of chemoresistance genes [15, 16].

A different strategy is aimed at overcoming chemoresistance by ameliorating the adverse effects of anticancer drugs to normal cells. For many drugs myelosuppression leading to leukocytopenia and thrombocytopenia is dose-limiting. Thus, protection of hematopoietic cells from the cytotoxicity of such drugs would allow safe dose intensification. We and others have suggested the transfer of chemoresistance genes to hematopoietic progenitor cells for this purpose [17-19]. Most preclinical studies have been performed with the DHFR (dihydrofolate reductase) and the MDR1 genes. We will therefore focus on these genes. Protection of bone marrow cells by other chemoresistance genes has been investigated more recently.

Chemoresistance genes reduce the sensitivity of cancer cells to a single class of drugs, or simultaneously to multiple compounds which are structurally unrelated. The DHFR gene which upon point mutation displays reduced affinity for methotrexate serves as an example for the first type of genes, since only antifolates like methotrexate or trimetrexate are affected [20-22]. In contrast, P-glycoprotein, the product of the MDR1 gene, and the multidrug resistance-associated protein, MRP, confer resistance to various natural toxic substrates including anthracyclines, Vinca alkaloids, epipodophyllotoxins and taxanes, and synthetic derivatives thereof [23-25]. Glutathione-S-transferases, O⁶-alkylguanine DNA alkyltransferase and aldehyde reductase confer different types of cross-resistance. These genes render cells resistant to alkylating agents such as nitrosoureas and derivatives of N-mustard, e.g., cyclophosphamide. In addition, resistance to anthracyclines and cis-platinum may be conveyed by glutathione-S-transferases.

	Transgenic Animal Models

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Overexpression of drug resistance genes in hematopoietic organs has first been studied in transgenic animal models. Mice transgenic for a human MDR1 cDNA under the control of a chicken ß-actin promoter expressed human P-glycoprotein in virtually all bone marrow cells. While the normal function of bone marrow remained unchanged, several-fold higher doses of taxol and daunomycin could be administered safely as compared with normal animals of the respective background strains [26, 27]. This protection of hematopoietic cells was specifically due to overexpression of the MDR1 transgene because it was reversed by coadministration of a chemosensitizing agent like verapamil [28]. In subsequent investigations bone marrow of MDR1-transgenic mice was transplanted into lethally irradiated normal mice. It was demonstrated that the recipients were also protected from myelosuppression following chemotherapy [29]. Similar experiments have been performed on mice transgenic for DHFR [30]. Transplantation of transgenic bone marrow protected recipient mice from lethal doses of methotrexate [31].

Chemoresistance genes may play additional physiological roles in normal tissues. It has been proposed that they may be involved in the early steps of carcinogenesis by protecting cells from mutagenic compounds [32, 33]. It has been demonstrated that rapid repair of O⁶-methylguanine-DNA adducts protects transgenic mice from N-methyl-nitrosourea-induced thymic lymphomas [34]. This protection can be targeted to other organs like the liver by suitable promoter systems [35].

In conclusion, overexpression of drug resistance genes was found to protect transgenic animals from leukocytopenia due to the cytotoxicity of drugs and may exert additional benefits such as protection from carcinogenic events. No major disturbances of hematopoiesis were associated with the overexpression of chemoresistance genes in bone marrow. Based on these encouraging results, methods for retroviral transfer of chemoresistance genes to hematopoietic progenitor cells of normal animals were developed.

	Retroviral Transfer of Chemoresistance Genes

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Retroviral transfer of drug resistance genes was first investigated in cultured cells. Transfer of a full-length MDR1 cDNA to normal hematopoietic progenitor cells protects them from the cytotoxicity of antineoplastic agents. K562 erythroleukemia cells [36] and primary bone marrow cells [37] were transduced with the use of a vector containing a full-length MDR1 cDNA under control of Harvey sarcoma virus long terminal repeats [38]. Resistance to multiple drugs including taxol, colchicine and daunomycin was demonstrated in transduced cells.

A retroviral vector containing the MRP transporter sequence [39] may be useful for protection of hematopoietic cells, if MDR1-related endogenous chemoresistance has to be reversed in tumor cells. In contrast to P-glycoprotein, drug extrusion by the MRP transporter is not inhibited by chemosensitizing agents like verapamil or cyclosporine. One potential disadvantage of this approach is the relatively low-level resistance obtained with MRP and the requirement for intracellular derivitization of some drugs.

To confer resistance against alkylating agents, vectors containing glutathione-S-transferase or O⁶-alkylguanine DNA alkyltransferase have been engineered. NIH 3T3 fibroblasts transduced with rat glutathione-S-transferase Yc displayed resistance to chlorambucil and mechlorethamine, while their sensitivity to methotrexate, which is not a substrate of glutathione-S-transferase, remained unchanged [40]. A novel retroviral vector in which glutathione-S-transferase and MDR1 are driven from separate promoters conferred a broad range of chemoresistance to NIH 3T3 cells [41].

Taken together, development of novel vectors which contain chemoresistance genes conferring high levels of resistance are likely to improve transgene expression, thereby efficiently protecting target cells. Further improvements may be achieved by redesigned retroviral vector constructs. For instance, vectors based on Harvey murine sarcoma virus can be modified by removal of viral sequences 3' of the foreign sequence to be transduced. Containing a human full-length MDR1 cDNA, such a vector construct facilitated efficient virus production, gene transduction, and expression of MDR1 in target cells [42]. This approach may help augment the packaging capacity of retroviral vectors which is usually below 9 kb. Baum et al. [43] discovered that both the viral enhancer region in the U3 region of the long terminal repeat of Moloney leukemia viruses and a repressor element coincident with the primer binding site are limiting for expression in hematopoietic cells in a differentiation-dependent fashion. To overcome these limitations, vectors were constructed in which the U3 regions of either Friend mink cell focus-forming virus or myeloproliferative sarcoma virus were combined with the primer binding site of the murine embryonic stem cell virus. In comparison to conventional Moloney leukemia virus-derived vectors, the new vectors increased transduction efficiency and chemoresistance of hematopoietic cell lines when used to transduce MDR1.

	Animal Models for Transduction of Chemoresistance Genes to Hematopoietic Progenitor Cells

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Animal models for the use of the MDR1 gene in somatic gene therapy were subsequently developed: MDR1-transduced murine bone marrow cells were transplanted into anemic W/W^v mice [44] or lethally irradiated normal syngeneic mice [45]. Both investigators found increased levels of MDR1 expression after treatment of recipient mice with taxol. These experiments supported the idea of a selective advantage in vivo of hematopoietic cells expressing the MDR1 transgene. Further support was provided by experiments in which MDR1-transduced bone marrow was first transplanted into recipient mice. After taxol treatment of recipient animals the bone marrow was transplanted into a second generation of recipient mice. In several cycles of retransplantation and taxol treatment of recipient mice, increasingly high levels of chemoresistance were generated in vivo [46].

Likewise, vectors containing mutated DHFR genes have been engineered for transduction of hematopoietic progenitor cells [47-49]. Williams et al. [50] and Cline et al. [51] demonstrated protection of recipient animals from lethal doses of methotrexate. Retransplantation experiments performed with DHFR [52] gave results comparable to those obtained with MDR1: both MDR1 and DHFR may act as selectable marker genes in vivo.

More recently, investigations have attempted to transfer chemoresistance genes to isolated subpopulations of progenitor cells, namely to hematopoietic stem cells from various origins. Hematopoietic stem cells are thought to be ideal target cells for gene therapy. Their life span is virtually indefinite because they have the capacity of self-renewal, whereas more mature progenitor cells become more mature with each step of cell replication and eventually undergo terminal differentiation followed by apoptosis. Targeting of more differentiated, lineage-committed progenitor cells may therefore result in merely transient gene expression [53]. Incidental transduction of stem cells has been confirmed in numerous investigations by gene transfer to unfractionated bone marrow. Transduction of true pluripotent stem cells, though feasible, has usually been found to be elusive [54]. A major obstacle to better transduction is the quiescence of the majority of stem cells [55], while cell proliferation is required for stable integration of retroviral sequences into the genome [56]. The efficiency of gene transfer has been improved with the use of growth factors and cytokines, or by chemotherapeutic pretreatment of bone marrow donors.

We have transferred an MDR1 cDNA to a small proportion of murine bone marrow cells that expressed neither lineage-specific antigens nor the MHC class II-associated I-A antigen, but had high levels of Sca-1, also referred to as Ly6A/E (Lin^–MHC II^–Sca-1⁺ cells). Stem cell characteristics of this cell fraction were confirmed in vivo by transplantation in sublethally irradiated severe combined immunodeficiency (SCID) mice. These experiments revealed sustained presence of an MDR1 marker cDNA in recipient mice, multilineage engraftment, and the presence of the marker gene after retransplantation into a second generation of recipient mice. The isolated cells were expanded ex vivo with the use of growth factors while gene transfer was performed by coculturing with retrovirus producing GP + E86 fibroblasts. Functional human P-glycoprotein was detected in more than 60% of expanded cells. Following transplantation of the ex vivo-transduced cell population into SCID mice, P-glycoprotein was expressed in a high proportion of bone marrow cells of recipient animals at levels comparable to those observed in comparison to multidrug-resistant cancers in the clinic [57].

Stem cells mobilized into peripheral blood of splenectomized mice by administration of G-CSF and stem cell factor have been found to be useful targets for MDR1 gene transfer [58]. Additional sources for activated stem cells, many of which are in cell cycle, are cord blood and fetal liver. It should be mentioned that fetal liver stem cells lack receptors for amphotropic retroviruses as reported by Richardson et al. [59]

In a recent article, retroviral MDR1-transfer to MO7e cells was reported. As compared with nontransduced cells of this factor-dependent human leukemia cell line, they became 20-fold more resistant to taxol following MDR1-gene transfer. After transplantation of MO7e cells into immunodeficient non-obese diabetic (NOD) SCID-mice, cells expressing the MDR1 gene were able to survive taxol treatment which was not observed with nontransduced cells [60].

	Transfer of Chemoresistance Genes to Human Hematopoietic Cells and Ongoing Clinical Trials

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Gene transfer to human hematopoietic cells is usually less efficient than to their murine counterparts. Nevertheless, transfer of the MDR1 gene to human CD34⁺ precursor cells has been demonstrated [61-64]. Several clinical trials on transfer of the MDR1 gene to bone marrow cells have been initiated in the United States [65-67]. Bone marrow from patients suffering from advanced-stage breast or ovarian cancers, or non-Hodgkin's lymphomas will be transduced with the use of retroviruses. Main goals of these studies are to prove feasibility, efficiency and safety of the gene therapy procedure. In addition, in some trials patients will be treated with taxol following reinfusion of transduced cells. Thereby enrichment of transduced cells, i.e., selection of transduced cell, should be demonstrated.

There may be a risk of transferring chemoresistance genes to previously undetected cancer cells. Micrometastases are not uncommon in advanced breast cancer. In addition, integration of viral sequences into the genome bears a small risk of insertional mutagenesis if oncogenes become activated or anti-oncogenes are disrupted. If this should happen, it is necessary to remove multidrug-resistant cells. First, transduced cells should display unaltered sensitivity to treatment with drugs that are not inactivated by the respective mechanism of drug resistance, i.e., alkylating agents or antimetabolites, if MDR1 is transduced to bone marrow cells. Second, chemosensitization with inhibitors of P-glycoprotein such as calcium channel blockers permits killing of multidrug-resistant cancer cells. Third, MDR1 mRNA or P-glycoprotein can be targeted with antibodies, immunotoxins or ribozymes as mentioned above.

Nevertheless, it would be advantageous to remove malignant cells from samples of hematopoietic progenitors prior to transduction with chemoresistance genes. Thus, future trials should address whether this procedure can be improved with the use of immunologically separated or purged preparations. Preparations of pure hematopoietic stem cells which have been depleted from contaminating cells should substantially enhance the safety of this method.

Alternatively, a transcriptional gene fusion may increase the safety of drug resistance genes in gene therapy of cancer. Sugimoto et al. constructed a vector in which MDR1 is coexpressed with a thymidine kinase cDNA from Herpes simplex virus [68]. The latter gene sensitizes transduced cells for an antiviral agent, ganciclovir. Treatment with ganciclovir would therefore selectively kill transduced cells while nontransduced cells would not be affected.

	Increased Coexpression of the Multidrug Resistance Gene and Nonselectable Therapeutic Genes following Retroviral Transduction and Drug Selection

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Fibroblasts and hematopoietic cells display increased expression of MDR1 following selection with substrates of P-glycoprotein. Because it was questionable whether drug-treated hematopoietic progenitor cells would retain the capacity of repopulation of bone marrow, we performed transplantation experiments on lethally irradiated mice. We found that MDR1-transduced progenitor cells, selected in daunomycin or colchicine at high concentrations (30 ng/ml or 50 ng/ml, respectively), rescued the recipient animals, provided transduction of MDR1 was performed at sufficient retroviral titers. In addition, these experiments revealed increased P-glycoprotein expression in vivo if cells were selected with drugs prior to transplantation [69].

An alternative strategy for enrichment of transduced cells was reported by Richardson et al. [70]. If chemoresistance genes are expressed at the outer surface of cells, e.g., membrane-bound transporter molecules such as P-glycoprotein or the MRP-transporter, they can also be sorted with the use of monoclonal antibodies. In this investigation, preselection by flow cytometry of MDR1-transduced hematopoietic stem cell populations led to increased long-term stability and expression of human P-glycoprotein in murine bone marrow.

Based on animal studies that showed increased expression of the transduced gene after drug treatment, it has been suggested that drug resistance genes may be useful not only by virtue of rendering hematopoietic cells chemoresistant but also by introducing overexpression of a second ‘passenger' gene. This approach may improve the efficiency of somatic gene therapy which has the potential to cure genetically determined diseases, but is still hampered by low gene expression in target organs. In particular, this is true for gene therapy of hematopoietic disorders because the efficiency of gene transfer is limited, and stable expression of transgenes in bone marrow has been found difficult to accomplish. In preclinical primate investigations and in clinical studies the percentage of hematopoietic cells with long-term expression of transgenes has been found to be disappointingly low [71, 72]. To employ drug resistance genes for gene therapy, they can be coexpressed with nonselectable genes using different strategies. The simplest approach is cotransduction of separate vectors, but this does not necessarily ensure stable expression of both genes. Alternatively, two genes can be integrated into one common vector. They can be driven from separate promoters (two-gene vectors) but, with this strategy, expression of the unselected gene may be extinguished. Negative interactions between promoters may lead to loss of expression of the unselected genes [73-75]. A different approach is to construct gene fusions encoding for a bifunctional chimeric protein. A fusion between MDR1 and the adenosine deaminase gene which encodes a chimeric protein has been used to stably transduce fibroblasts [76, 77]. However, this approach cannot be used for proteins that are physiologically expressed in different cell compartments. Interestingly, a fusion gene in which MDR1 was linked to the glucocerebrosidase gene, failed to be translated into a bifunctional protein. Instead, two separate functional proteins, P-glycoprotein and glucocerebrosidase, were synthesized suggesting either alternative splicing of transcripts or rapid processing of a fusion protein into two proteins [78].

A preferred approach is the use of polycistronic mRNAs that facilitate transcription of multiple genes by internal ribosomal entry sites. Such transcriptional fusion vectors have been developed containing the MDR1 cDNA and cDNAs that correct inherited metabolic disorders [79]. In these cassettes, a single mRNA containing both genes is transcribed from one retroviral promoter. This transcript is translated from two open reading frames into two separate proteins. Although the size of these was close to the presumed maximal packaging capacity of retroviral vectors, in PA317 packaging cells titers of 1 x 10⁵ cfu/ml are achievable. Metz et al. [80] compared the efficacy of two-gene vectors and bicistronic vectors. In this study expression from two-gene vectors was subject to host-specific limitations on internal promoter activity which was not observed with bicistronic vectors.

Bicistronic vectors were engineered to correct different monogenic disorders of the hematopoietic system. For instance, Aran et al. [81] described a transcriptional fusion between MDR1 and the glucocerebrosidase gene which is defective in Gaucher disease, an inherited metabolic disease characterized by accumulation of a glucosylceramide in glucocerebrosidase-deficient hematopoietic cells, particularly in macrophages. Patients suffer from severe hepatosplenomegaly, skeletal lesions and hemolytic anemia. The bicistronic vector allowed expression of functional glucocerebrosidase in NIH 3T3 fibroblasts which was increased by selection with cytotoxic substrates of P-glycoprotein. Furthermore, a new strategy of selection allowed for complete restoration of the enzymatic deficiency in fibroblasts of Gaucher patients at very low dose levels of anticancer drugs. This was accomplished by partial inhibition of the function P-glycoprotein by simultaneous exposure to chemosensitizing agents [82].

Similar bicistronic vectors have been engineered which facilitate coexpression of MDR1 with -galactosidase or the gp91phox gene, respectively [83, 84]. Defects of -galactosidase are the cause of Fabry disease, a storage disease. gp91phox mutations result in X-linked chronic granulomatous disease, a disorder of immune response due to failure of superoxide production by phagocytic cell oxidase.

Following introduction of bicistronic vectors containing MDR1 into hematopoietic cells of patients, drug treatment will eliminate untransduced cells whose genetic defect has not been corrected. All surviving cells should express both MDR1 and the passenger gene. Because chemotherapy should eliminate unprotected cells, surviving progenitor cells should expand and repopulate the marrow in response to compensate for the lack of mature leukocytes in peripheral blood. Thereby high levels of transgene expression should be achievable in hematopoietic cells. This approach should restore the expression of therapeutic genes which tend to decrease after several weeks or months.

To investigate this concept in gene therapy, a bicistronic marking vector in which MDR1 is coexpressed with lacZ has been constructed [85]. Cells expressing ß-galactosidase, the product of the nonselectable lacZ gene, can easily be detected by histochemical staining. This vector should be useful for analyzing expression in individual hematopoietic progenitor cells of different lineages after chemotherapy.

In these bicistronic vectors the ‘passenger' gene was cloned downstream from the internal ribosomal entry sites. It was found that cDNAs in the downstream position were translated at three- to fivefold lower levels than the upstream MDR1 gene. If the positions were reversed, i.e., if MDR1 was positioned downstream, fewer chemoresistant clones were obtained, but almost all of them expressed the nonselectable gene at high levels. Vectors can thus be designed to either confer high levels of drug resistance and a good yield of drug-selectable cells, or to facilitate very high levels of expression of a nonselectable therapeutic gene. Whether or not the latter type of vectors will prove suitable for gene therapy of hematopoietic cells depends on the achievable levels of gene expression in target cells. Sufficient transgene expression requires good retroviral titers and suitable flanking regions from which transgenes are promoted. Development of vector systems for high tissue-specific transgene expression and increased packaging capacity may substantially improve the efficacy of clinical gene therapy.

	Conclusion

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 
Overexpression of chemoresistance genes can protect hematopoietic tissues from the toxicity of antineoplastic drugs as first shown in transgenic animal models. Retroviral transfer of chemoresistance genes to normal hematopoietic progenitor cells revealed that these cells have a selective advantage over nontransduced cells when exposed to drugs. Following transplantation of transduced progenitor cells into recipient mice, expression of transgenes can be increased in bone marrow by administration of chemotherapeutic drugs. Thus, these genes may act as selectable markers in vivo. In ongoing clinical studies safety and efficiency of retroviral transfer to hematopoietic cells are being investigated.

Drug resistance genes may be useful in gene therapy by protecting cancer patients from the hematological side effects of chemotherapy. In addition, introduction and stable expression of nonselectable genes in target organs may be facilitated by cotransduction with chemoresistance genes. For this purpose, chimeric fusion vectors and polycistronic vectors have been constructed in which the multidrug resistance gene is combined with therapeutic genes that are capable of correcting inherited disorders of the hematopoietic system. Further investigations are needed to determine the efficacy of this new approach in vivo.

	Acknowledgments

 
T. Licht and F. Herrmann are grant recipients of Dr. Mildred Scheel Stiftung (Deutsche Krebshilfe).

	References

Top Abstract Introduction Transgenic Animal Models Retroviral Transfer of... Animal Models for Transduction... Transfer of Chemoresistance... Increased Coexpression of the... Conclusion References

 

1. Pirker R, Wallner J, Gotzl M et al. MDR1 RNA expression is an independent prognostic factor in acute myeloid leukemia. Blood 1992;80:557-559.[Free Full Text]

2. Zhou DC, Marie JP, Suberville AM et al. Relevance of mdr1 gene expression in acute myeloid leukemia and comparison of different diagnostic methods. Leukemia 1992;6:879-885.[Medline]

3. Salmon SE, Grogan TM, Miller T et al. Prediction of doxorubicin resistance in vitro in melanoma, lymphoma, and breast cancer by P-glycoprotein staining. J Natl Cancer Inst 1989;81:696-701.[Abstract/Free Full Text]

4. Musto P, Lombardi G, Matera R et al. The expression of the multidrug transporter P-170 glycoprotein in remission phase is associated with early and resistant relapse in multiple myeloma. Haematologica 1991;76:513-516.[Medline]

5. Chan HSL, Thorner PS, Haddad G et al. Immunohistochemical detection of P-glycoprotein: prognostic correlation in soft tissue sarcoma of childhood. J Clin Oncol 1990;8:689-704.[Abstract]

6. Goldstein L, Fojo A, Ueda K et al. Expression of the multidrug resistance gene, MDR1, gene in neuroblastomas. J Clin Oncol 1990;8:128-136.[Abstract/Free Full Text]

7. Norris MD, Bordow SB, Marshall GM et al. Expression of the gene for multidrug-resistance associated-protein and outcome in patients with neuroblastoma. N Engl J Med 1996;334:231-238.[Abstract/Free Full Text]

8. Cheng A-L, Su I-J, Chen Y-C et al. Expression of P-glycoprotein and glutathione-S-transferase in recurrent lymphomas: the possible role of Epstein-Barr virus, immunophenotypes, and other predisposing factors. J Clin Oncol 1993;11:109-115.[Abstract]

9. Lum BL, Fisher GA, Brophy NA et al. Clinical trials of modulation of multidrug resistance. Pharmacokinetic and pharmacodynamic considerations. Cancer 1993;72(suppl 11):3502-3514.[Medline]

10. Ford JM, Hait WN. Pharmacologic circumvention of multidrug resistance. Cytotechnology 1993;12:171-212.[Medline]

11. Lehnert M. Reversal of P-glycoprotein associated multidrug resistance: from bench to bedside. Onkologie 1994;17:8-15.

12. Pearson JW, Fogler WE, Volker K et al. Reversal of drug resistance in a human colon cancer xenograft expressing MDR1 complementary DNA by in vivo administration of MRK-16 monoclonal antibody. J Natl Cancer Inst 1991;83:1386-1391.[Abstract/Free Full Text]

13. Tsuruo T, Hamada H, Sato S et al. Inhibition of multidrug-resistant human tumor cell growth in athymic mice by anti-P-glycoprotein monoclonal antibodies. Jpn J Cancer Res 1989;80:627-631.[Medline]

14. Mickisch GH, Pai LH, Siegsmund M et al. Pseudomonas exotoxin conjugated to monoclonal antibody MRK16 specifically kills multidrug resistant cells in cultured renal cell carcinomas and in MDR-transgenic mice. J Urol 1993;149:174-178.[Medline]

15. Kiehntopf K, Brach MA, Licht T et al. Ribozyme mediated cleavage of MDR-1 transcript restores chemosensitivity in previously resistant cancer cells. EMBO J 1994;13:4645-4652.[Medline]

16. Scanlon KJ, Ishida H, Kashani-Sabet M. Ribozyme-mediated reversal of the multidrug-resistant phenotype. Proc Natl Acad Sci USA 1994;91:11123-11127.[Abstract/Free Full Text]

17. Gottesman MM, Germann UA, Aksentijevitch I et al. Gene transfer of drug resistance genes. NY Acad Sci 1994;716:126-139.

18. Banerjee D, Zhao SC, Li MX et al. Gene therapy utilizing drug resistance genes: a review. STEM CELLS 1994;12:378-385.[Abstract]

19. Licht T, Pastan I, Gottesman MM et al. The multidrug-resistance gene in gene therapy of cancer and hematopoietic disorders. Ann Hematol 1996;72:184-193.[Medline]

20. Haber DA, Beverly SM, Kiely ML et al. Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts. J Biol Chem 1981;256:9501-9510.[Abstract/Free Full Text]

21. Simonsen CC, Levinson AD. Isolation and expression of an altered mouse dihydrofolate reductase cDNA. Proc Natl Acad Sci USA 1983;80:2495-2499.[Abstract/Free Full Text]

22. Dicker AP, Volkenandt M, Schweitzer BI et al. Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate resistant CHO cell line Pro^–3 MTX^RIII. J Biol Chem 1990;265:8317-8321.[Abstract/Free Full Text]

23. Endicott JA, Ling V. The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 1989;58:137-171.[Medline]

24. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 1993;62:385-427.[Medline]

25. Grant CE, Valdimarsson G, Hipfner DR et al. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res 1994;54:357-361.[Abstract/Free Full Text]

26. Galski H, Sullivan M, Willingham MC et al. Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin induced leukopenia. Mol Cell Biol 1989;9:4357-4363.[Abstract/Free Full Text]

27. Mickisch GH, Licht T, Merlino GT et al. Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency and toxicity. Cancer Res 1991;51:5417-5424.[Abstract/Free Full Text]

28. Mickisch GH, Merlino GT, Galski H et al. Transgenic mice which express the human multidrug resistance gene in bone marrow enable a rapid identification of agents which reverse drug resistance. Proc Natl Acad Sci USA 1991;88:547-551.[Abstract/Free Full Text]

29. Mickisch GH, Aksentijevich I, Schoenlein PV et al. Transplantation of bone marrow cells from transgenic mice expressing the human MDR1 gene results in long-term protection against the myelosuppressive effect of chemotherapy in mice. Blood 1992;79:1087-1093.[Abstract/Free Full Text]

30. Isola LM, Gordon JW. Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene. Proc Natl Acad Sci USA 1986;83:9621-9625.[Abstract/Free Full Text]

31. May C, Gunther R, McIvor RS. Protection of mice from lethal doses of methotrexate by transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase activity. Blood 1995;86:2439-2448.[Abstract/Free Full Text]

32. Ferguson L, Baguley BC. Multidrug resistance and mutagenesis. Mutat Res 1993;285:79-90.[Medline]

33. Gottesman MM. Multidrug-resistance during chemical carcinogenesis: a mechanism revealed? J Natl Cancer Inst 1988;80:1352-1353.[Free Full Text]

34. Liu L, Allay E, Dumenco LL et al. Rapid repair of O⁶-methylguanine-DNA adducts protects transgenic mice from N-methylnitrosourea-induced thymic lymphomas. Cancer Res 1994;54:4648-4652.[Abstract/Free Full Text]

35. Nakatsuru Y, Matsukuma S, Nemoto N et al. O⁶-methylguanine DNA methyltransferase protects against nitrosamine-induced hepatocarcinogenesis. Proc Natl Acad Sci USA 1993;90:6468-6472.[Abstract/Free Full Text]

36. DelaFlor-Weiss E, Richardson C, Ward M et al. Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells. Blood 1992;80:3106-3111.[Abstract/Free Full Text]

37. McLachlin JR, Eglitis MA, Ueda K et al. Expression of a human complementary DNA for the human multidrug resistance gene in murine hematopoietic precursor cells with the use of retroviral gene transfer. J Natl Cancer Inst 1990;82:1260-1263.[Abstract/Free Full Text]

38. Pastan I, Gottesman MM, Ueda K et al. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci USA 1988;85:4486-4490.[Abstract/Free Full Text]

39. D'Hondt V, Caruso M, Ward M et al. Expression of the multidrug resistance gene using retroviral vectors. Blood 1995;86:466a.

40. Greenbaum M, Letourneau S, Assar H et al. Retrovirus-mediated gene transfer of rat glutathione S-transferase Yc confers alkylating drug resistance in NIH 3T3 mouse fibroblasts. Cancer Res 1994;54:4442-4447.[Abstract/Free Full Text]

41. Doroshow JH, Metz MZ, Matsumoto L et al. Transduction of NIH 3T3 cells with a retrovirus carrying both human MDR1 and glutathione S-transferase pi produces broad-range multidrug resistance. Cancer Res 1995;55:4073-4078.[Abstract/Free Full Text]

42. Metz MZ, Best DM, Kane SE. Harvey murine sarcoma virus/MDR1 retroviral vectors: efficient virus production and foreign gene transduction using MDR1 as a selectable marker. Virology 1995;208:634-643.[Medline]

43. Baum C, Hegewisch-Becker S, Eckert HG et al. Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells. J Virol 1995;69:7541-7547.[Abstract]

44. Sorrentino BP, Brandt SJ, Bodine D et al. Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1. Science 1992;257:99-103.[Abstract/Free Full Text]

45. Podda S, Ward M, Himelstein A et al. Transfer and expression of the human multiple drug resistance gene into live mice. Proc Natl Acad Sci USA 1992;89:9676-9680.[Abstract/Free Full Text]

46. Hanania EG, Deisseroth AB. Serial transplantation shows that early hematopoietic precursor cells are transduced by MDR-1 retroviral vector in a mouse gene therapy model. Cancer Gene Ther 1994;1:21-25.[Medline]

47. Miller AD, Law M-F, Verma IM. Generation of a helper free amphotropic retrovirus that transduces a dominant acting methotrexate resistant dihydrofolate reductase gene. Mol Cell Biol 1985;5:431-437.[Abstract/Free Full Text]

48. Li M-X, Banarjee D, Zhao S-C et al. Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction. Blood 1994;83:3403-3408.[Abstract/Free Full Text]

49. Zhao SC, Li M-X, Banerjee D et al. Long term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double copy vector retrovirus containing a mutant dihydrofolate reductase. Cancer Gene Ther 1994;1:27-33.[Medline]

50. Williams DA, Hsieh K, DeSilva A et al. Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate resistant bone marrow. J Exp Med 1987;166:210-218.[Abstract/Free Full Text]

51. Cline MJ, Stang H, Mercola K et al. Gene transfer in intact animals. Nature 1980;284:422-425.[Medline]

52. Corey CA, DeSilva AD, Holland CA et al. Serial transplantation of methotrexate resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells. Blood 1990;76:337-343.

53. Lemischka IR. Clonal, in vivo behavior of the totipotent hematopoietic stem cell. Semin Immunol 1991;3:349-355.[Medline]

54. Nolta JA, Dao MA, Wells S et al. Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice. Proc Natl Acad Sci USA 1996;93:2414-2419.[Abstract/Free Full Text]

55. Ogawa M. Differentiation and proliferation of stem cells. Blood 1993;81:2844-2853.[Abstract/Free Full Text]

56. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1992;10:4239-4242.

57. Licht T, Aksentijevich I, Gottesman MM et al. Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells. Blood 1995;86:111-121.[Abstract/Free Full Text]

58. Bodine DM, Seidel NE, Gale MS et al. Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor. Blood 1994;84:1482-1491.[Abstract/Free Full Text]

59. Richardson C, Ward M, Podda S et al. Mouse fetal liver cells lack amphotropic retroviral receptors. Blood 1994;84:433-439.[Abstract/Free Full Text]

60. Schwarzenberger P, Spence S, Lohrey N et al. Gene transfer of multidrug resistance into a factor-dependent human progenitor cell line: in vivo model for genetically transferred chemoprotection. Blood 1996;87:2723-2731.[Abstract/Free Full Text]

61. Ward M, Richardson C, Pioli P et al. Transfer and expression of the human multiple drug resistance gene in human CD34⁺ cells. Blood 1994;84:1408-1414.[Abstract/Free Full Text]

62. Boesen JJ, Nooter K, Valerio D. Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr1 gene. Biotherapy 1993;6:291-302.[Medline]

63. Bertolini F, de Monte L, Corsini C et al. Retrovirus-mediated transfer of the multidrug resistance gene into human haematopoietic progenitor cells. Br J Haematol 1994;88:318-324.[Medline]

64. Ward M, Pioli P, Ayello J et al. Retroviral transfer and expression of the human multiple drug resistance (MDR) gene in peripheral blood progenitor cells. Clin Cancer Res 1996;2:873-876.[Abstract]

65. Deisseroth AB, Kavanagh J, Champlin R. Use of safety-modified retroviruses to introduce chemotherapy resistance sequences into normal hematopoietic cells for chemoprotection during the therapy of ovarian cancer: a pilot trial. Hum Gene Ther 1994;5:1507-1522.[Medline]

66. O'Shaughnessy JA, Cowan KH, Nienhuis AW et al. Retroviral mediated transfer of the human multidrug resistance gene (MDR-1) into hematopoietic stem cells during autologous transplantation after intensive chemotherapy for metastatic breast cancer. Hum Gene Ther 1994;5:891-911.[Medline]

67. Hesdorffer C, Antman K, Bank A et al. Human MDR1 gene transfer in patients with advanced cancer. Hum Gene Ther 1994;5:1151-1160.[Medline]

68. Sugimoto Y, Hrycyna CA, Aksentijevich I et al. Co-expression of a multidrug resistance gene (MDR1) and Herpes Simplex Virus thymidine kinase gene as a part of a bicistronic mRNA in a retrovirus vector allows selective killing of MDR1-transduced cells. Clin Cancer Res 1995;2:447-457.[Abstract]

69. Licht T, Aran JM, Goldenberg SK et al. Cytotoxic drug selection of murine bone marrow cells following transfer of the MDR1 (multidrug resistance) gene increases gene expression and chemoresistance in vivo. Blood 1995;86:243a.

70. Richardson C, Bank A. Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene. Blood 1995;86:2579-2589.[Abstract/Free Full Text]

71. Dunbar CE, Bodine DM, Sorrentino B et al. Gene transfer into hematopoietic cells: implications for cancer therapy. Ann NY Acad Sci 1994;716:216-224.[Medline]

72. Bodine DM, Moritz T, Donahue RE et al. Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34⁺ bone marrow cells. Blood 1993;82:1975-1980.[Abstract/Free Full Text]

73. Emerman M, Temin HH. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 1984;39:459-467.

74. Emerman M, Temin HH. Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol Cell Biol 1986;6:792-800.[Abstract/Free Full Text]

75. Olsen JC, Johnson LG, Wong-Sun ML et al. Retrovirus-mediated gene transfer to cystic fibrosis airway epithelial cells: effect of selectable marked sequences on long-term expression. Nucleic Acids Res 1993;21:663-669.[Abstract/Free Full Text]

76. Germann UA, Gottesman MM, Pastan I. Expression of a multidrug resistance-adenosine deaminase fusion gene. J Biol Chem 1989;264:7418-7424.[Abstract/Free Full Text]

77. Germann UA, Chin K-V, Pastan I et al. Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene. FASEB J 1990;4:1501-1507.[Abstract]

78. Aran JM, Germann UA, Gottesman MM et al. Construction and characterization of a selectable multidrug resistance-glucocerebrosidase fusion gene. Cytokines Mol Ther 1996;2:47-57.[Medline]

79. Sugimoto Y, Aksentijevich I, Gottesman MM et al. Efficient expression of drug-selectable genes under control of an internal ribosome entry site. Biotechnology 1994;12:694-698.[Medline]

80. Metz MZ, Matsumoto L, Winters KA et al. Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene. Virology 1996;217:230-241.[Medline]

81. Aran JM, Gottesman MM, Pastan I. Drug-selected coexpression of human glucocerebrosidase and P-glycoprotein using a bicistronic vector. Proc Natl Acad Sci USA 1994;91:3176-3180.[Abstract/Free Full Text]

82. Aran JM, Licht T, Gottesman MM et al. Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy. Hum Gene Ther 1996;7:2165-2175.[Medline]

83. Sugimoto Y, Aksentijevich I, Murray G et al. Retroviral co-expression of a multidrug-resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease. Hum Gene Ther 1995;6:905-915.[Medline]

84. Sokolic RA, Sekhsaria S, Sugimoto Y et al. A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression. Blood 1996;87:42-50.[Abstract/Free Full Text]

85. Aran JM, Licht T, Gottesman MM et al. Design of a multidrug-resistance-marking vector. Proc Am Assoc Cancer Res 1995;36:336.

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