Human Multiple Drug Resistance Gene Research Articles

Hetero-oligomerization does not compromise 'gain of function' of tumor-derived p53 mutants.

Tumor-derived p53 mutants activate transcription from promoters of various growth-related genes. We tested whether this transactivation function of the mutant protein is sufficient to induce tumorigenesis ('gain of function'). Tumor-derived mutant p53-281G transactivates the promoters of human epidermal growth factor receptor (EGFR) and human multiple drug resistance gene (MDR-1). To determine whether the C-terminal domain functions only as an oligomerization domain in mutant p53-mediated transactivation, we have replaced the tetramerization domain of p53 by a heterologous tetramerization domain; although this mutant protein formed tetramers in solution, it failed to transactivate significantly. Therefore, for successful mutant p53-mediated transactivation, sequences near the C-terminus of mutant p53 are required to perform functions in addition to tetramerization. We also demonstrate that co-expression of a deletion mutant of p53 (p53 del 1-293), which retains the p53 oligomerization domain, inhibits this transactivation. p53 del 1-293 co-immunoprecipitates with p53-281G suggesting that hetero-oligomers of p53-281G and p53 del 1-293 are defective in transactivation. We also show that a cell line stably transfected with p53-281G expresses higher levels of endogenous NF-kappaB and proliferating cell nuclear antigen (PCNA) compared to that transfected with vector alone. On co-expression, p53 del 1-293 lowered the levels of NF-kappaB and PCNA in p53-281G-expressing cells. However, on co-expression, p53 del 1-293 did not inhibit the tumorigenicity and colony forming ability of p53-281G expressing cells. Our earlier work showed that a deletion of the C-terminal sequences of p53-281G overlapping the oligomerization domain obliterates 'gain of function'. Taken together, the above information suggests that the C-terminal sequences have some critical role in 'gain of function' in addition to transactivation.

Gene therapy of cancer

Gene therapy of cancer

Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.

We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells. Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner. Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells. In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells. In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus. On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus. When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

Preselection of transduced murine hematopoietic stem cell populations leads to increased long-term stability and expression of the human multiple drug resistance gene

We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte-macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL-HSC subpopulations show the greatest number of circulating granulocyte-macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.

Preselection of Transduced Murine Hematopoietic Stem Cell Populations Leads to Increased Long-Term Stability and Expression of the Human Multiple Drug Resistance Gene

We have been using the human multiple drug resistance (MDR) gene to transduce murine hematopoietic cells via retroviruses as a model system for potential human gene therapy. In this paper, we show that transplantation of MDR-transduced midgestational fetal liver cells (FLCs) into lethally irradiated mice leads to the continued presence and expression of the human MDR gene in the short-lived granulocyte-macrophages of recipients' peripheral blood (PB) for up to 12 months. We have also shown the ability of this retroviral system to efficiently transduce several murine FLC subpopulations enriched for hematopoietic stem cells (FL-HSCs) both (1) short-term by MDR-polymerase chain reaction analysis of individual day 12 colony-forming unit-spleen and (2) long-term by in vivo maintenance of MDR and expression of its product, p-glycoprotein, up to 1 year in PB. More highly enriched FL-HSC subpopulations show the greatest number of circulating granulocyte-macrophage cells expressing MDR long-term. These studies also show that preselection by fluorescence-activated cell sorting of MDR-transduced and -expressing cells before transplant significantly increases the percentage of circulating granulocyte-macrophage cells that express MDR at all time points analyzed posttransplant as compared with unsorted cells transduced in the same manner (P < .01). These results have potentially significant implications for future human gene therapy trials.

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34(+) cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34(+) cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.

Transfer and expression of the human multiple drug resistance gene in human CD34+ cells

The human multiple-drug resistance (MDR1) gene has been transferred into human hematopoietic progenitors using retroviral gene transfer. Human bone marrow cells and isolated CD34+ cells isolated from marrow were exposed to growth factors interleukin-3 (IL-3), IL-6, and stem cell factor for 48 hours and then to two changes of MDR retroviral supernatants over the next 24 hours. Progenitor assays in methylcellulose at this time showed that 18% to 70% of BFU-E and 30% to 60% of CFU-GM contain the transferred MDR gene by polymerase chain reaction analysis. Up to 11.2% of the progeny of these cells express increased amounts of MDR glycoprotein on their surface by fluorescence- activated cell sorter (FACS) analysis. In addition, transduced cells are enriched in high MDR-expressing cells after exposure to taxol as assessed by FACS analysis, and by resistance of BFU-E to taxol (Bristol- Myers Squibb, Princeton, NJ). These studies indicate the feasibility of using MDR gene transfer as a means of enriching marrow for MDR- transduced cells. They also provide the basis of a phase 1 clinical protocol in patients with advanced cancers not involving the bone marrow for the use of MDR gene transfer as a means of protecting marrow cells, which normally express low levels of MDR, from the myelosuppressive effects of drugs like taxol.

Transfer and Expression of the Human Multiple Drug Resistance Gene in Human CD34+ Cells

The human multiple-drug resistance (MDR1) gene has been transferred into human hematopoietic progenitors using retroviral gene transfer. Human bone marrow cells and isolated CD34+ cells isolated from marrow were exposed to growth factors interleukin-3 (IL-3), IL-6, and stem cell factor for 48 hours and then to two changes of MDR retroviral supernatants over the next 24 hours. Progenitor assays in methylcellulose at this time showed that 18% to 70% of BFU-E and 30% to 60% of CFU-GM contain the transferred MDR gene by polymerase chain reaction analysis. Up to 11.2% of the progeny of these cells express increased amounts of MDR glycoprotein on their surface by fluorescence-activated cell sorter (FACS) analysis. In addition, transduced cells are enriched in high MDR-expressing cells after exposure to taxol as assessed by FACS analysis, and by resistance of BFU-E to taxol (Bristol-Myers Squibb, Princeton, NJ). These studies indicate the feasibility of using MDR gene transfer as a means of enriching marrow for MDR-transduced cells. They also provide the basis of a phase 1 clinical protocol in patients with advanced cancers not involving the bone marrow for the use of MDR gene transfer as a means of protecting marrow cells, which normally express low levels of MDR, from the myelosuppressive effects of drugs like taxol.

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.

CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.

Localization of the human multiple drug resistance gene, MDR1, to 7q21.1.

Multiple drug resistance has been shown to be associated with amplification/increased expression of a gene designated MDR. The localization of one member of the MDR gene family, MDR1, to the long arm of chromosome 7 by in situ hybridization is reported.