Human Multidrug Resistance Gene Research Articles

MULTIDRUG-RESISTANCE GENE (MDR1) EXPRESSION IN HUMAN TUMOR XENOGRAFTS

We examined the expression of human multidrug resistance gene (MDR1); 13 of 34 xenografts showed various MDR1 gene transcript levels by reverse transcriptase-polymerase chain reaction. MDR1 was detected in 3 of 10 lung, 5 of 8 gastric, 3 of 3 colon and 2 of 3 pancreatic cancers, but not in 4 breast cancers, or 6 gliomas. We evaluated drug-sensitivity of xenografts in vivo; 28 of 34 xenografts showed drug resistance to vincristine and/or doxorubicin, whereas 13 of 28 drug-resistant xenografts expressed MDR1. The expression of MDR1 may explain the intrinsic drug resistance in certain xenografts.

Effect of unmodified triple helix-forming oligodeoxyribonucleotide targeted to human multidrug-resistance gene mdr1 in MDR cancer cells

The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 μM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB 100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.

Retrovirus‐mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells

We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer. Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, efficiency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 micrograms/ml taxol. In uninfected control, 1-2% of CFU-GM and CFU-GEMM were found to be drug-resistant, while 14-31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB-derived compared to BM-derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometric analysis of the expression of CD34 and P-glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P-glycoprotein was found on 17-25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P-glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB-cells expressed P-glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB- and BM-derived progenitor cells seems a feasible and attractive approach to generate a drug-resistant haemopoiesis.

Ultrastructural localization of P-glycoprotein on capillary endothelial cells in human gliomas.

The P-glycoprotein (P-Gp) encoded by the human multidrug-resistance gene MDR1 has been suggested to play certain roles in the blood-brain barrier (BBB). However, the detailed mechanism of the activity of P-Gp in multidrug-resistance (MDR) remains unclear in human glioma. We examined the localization of P-Gp in human glioma by immunohistochemical (IHC) and immunoelectron microscopic (IEM) methods with anti P-Gp monoclonal antibodies (C219, MRK16). We also examined MDR1 expression in primary glioma and xenografts by reverse transcription-polymerase chain reaction (RT-PCR) with human MDR1-specific primers. The IHC study showed no P-Gp expression on tumour cells but it was present on capillary endothelial cells and IEM analysis showed definitive localization on their luminal surface. MDR1 gene expression was detected in eight primary glioma and three normal brain specimens by RT-PCR, but not in glioma xenografts. The lack of MDR1 expression in these cells appears to be a consequence of the replacement of the original human stroma, including blood vessels, by murine stroma in glioma xenografts. The unique distribution of P-Gp on the capillary blood vessels was confirmed in human glioma by the results of immunohistochemical and molecular biological studies.

Detection of Soluble P-Glycoprotein in Culture Media and Extracellular Fluids

Multidrug resistance (MDR) is a unique phenomenon in cancer patients and is commonly associated with an overexpression of the human MDR gene mdr1, which encodes an energy-dependent Mr 180 kDa membrane bound protein, known as P-glycoprotein. P-glycoprotein serves as a membrane efflux to pump the drugs out of the cancer cells. Western blot analysis, using a newly generated monoclonal antibody F4 which recognizes specifically an extracellular epitope of human MDR1 P-glycoprotein, reveals that soluble P-glycoprotein is detected in the cultured media of viable adriamycin-resistant human ovarian carcinoma 2780AD cells, whereas those of the drug-sensitive parent A2780 cells contain no detectable level of soluble P-glycoprotein. Soluble P-glycoprotein also is detected in extracellular fluids of cancer patients, such as malignant ascites and serum, and is not detectable in serum samples of normal healthy individuals. The Mr of soluble P-glycoprotein is the same as that of membrane bound P-glycoprotein. The presence of soluble P-glycoprotein in extracellular fluids may provide the basis for its use as a quantitative parameter of MDR and as a means to lessen or reverse MDR.

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.

Patients with metastatic breast cancer will receive 4-5 cycles of induction chemotherapy on one of the ongoing Medicine Branch protocols. Patients achieving at least a partial response, and who do not have evidence of bone marrow involvement and who do not have metastatic bone disease, will undergo PBSC and bone marrow harvest when hematologic recovery has occurred. Patients who have not achieved a PR, but who are responding to therapy, may be treated with additional cycles of therapy in an attempt to achieve a PR. Such patients will be eligible for transplant if a PR is obtained. 70% of the bone marrow and PBSC will be cryopreserved. The CD34+ subpopulation from the remaining 30% of the bone marrow and PBSC harvest will be obtained using an anti-CD34+ antibody and immunoabsorption column. The bone marrow and peripheral blood CD34 cells will be transduced with a retroviral vector expressing the human MDR-1 cDNA. Patients with positive bone scans or histologic evidence of bone marrow involvement will be excluded from the gene transfer component of the protocol. The MDR-1 transduced CD34 cells will be reinfused along with the non-transduced bone marrow and PBSC into patients following high dose ICE chemotherapy. Serial peripheral blood and bone marrow samples will be obtained to study hematopoietic reconstitution with MDR-1 transduced cells. Patients with residual or progressive disease after ABMT will be treated with taxol or vinblastine. In these relapsed patients, peripheral blood and bone marrow samples will be obtained to study whether chemotherapy amplifies the proportion of hematopoietic cells containing the MDR-1 provirus. We will monitor the nadir blood counts of each patient receiving salvage chemotherapy for evidence of myeloprotection and correlate this data with changes in the mean proviral copy number. Sites of relapsed tumor will be biopsied to test for the presence of the MDR-1 provirus.

Stage-specific distribution of P-glycoprotein in first-trimester and full-term human placenta

The distribution of P-glycoprotein in human placenta has been examined by immunohistochemistry using a battery of monoclonal antibodies (MRK-16, C219 and JSB-1). P-glycoprotein was located on the syncytiotrophoblast microvillus border in first-trimester placentas and some of the placental macrophages (Hofbauer cells) showed weak cytoplasmic staining. In term placentas, however, staining was not observed in the trophoblast but most of the Hofbauer cells displayed strong cytoplasmic staining. In situ hybridization with specific gene probes suggested that both human multidrug resistance genes were expressed in the placenta, although only the multidrug resistance-1 gene product would have been detected by the MRK and JSB-1 antibodies. These results point to distinct functions for P-glycoprotein during the different stages of placental development and indicate that its expression may be under developmental control.

Expression of the Multidrug Resistance Gene (MDR1) in Non‐small Cell Lung Cancer

To examine the clinical relevance of P‐glycoprotein, encoded by the human multidrug resistance gene (MDR1), to multidrug resistance in lung cancer, we examined the expression of MDR1 in 107 non‐small cell lung cancer (NSCLC) specimens and 20 corresponding specimens of normal lung tissues. We also evaluated the relationship between MDR1 expression and the histopathology and pathological staging of NSCLC. The tumors consisted of 60 adenocarcinomas, 38 squamous cell carcinomas, 8 large cell carcinomas, and 1 adenosquamous carcinoma. MDR1 expression was semi‐quantified by use of the reverse transcriptase‐polymerase chain reaction method. We subclassified the NSCLC into 3 grades according to the MDR1 expression level (‐, +, ++). Sixty‐one of the 107 tumor specimens (57%) and 18 of the normal lung tissue specimens (90%) expressed various levels of the MDR1 gene. Only one tumor specimen showed higher MDR1 expression than the corresponding normal lung tissue. The relationship between pathological stage and MDR1 expression levels was not significant. These results suggest that the level of MDR1 expression in lung cells is decreased as cells progress from the normal to the transformed state.

Gene transfer of drug resistance genes. Implications for cancer therapy.

Two general approaches to the gene therapy of cancer have been proposed: (1) strategies that use exogenous genes to modify cancer cells so that they are less malignant or more susceptible to host defenses or to killing by exogenous agents; and (2) approaches that modify host cells so that they are more effective in eliminating cancer cells or more resistant to agents that are used to treat cancer. In both cases, the development of vectors that encode in vivo selectable phenotypes, such as drug resistance, would be extremely valuable because of the inherent inefficiency of gene transfer and the potential of such vectors to protect normal tissues against toxic agents. To allow the selection of cells in vivo that have been transduced with vectors for gene therapy, we have utilized the human multidrug resistance (MDR1) gene. The product of this gene is a 170,000-dalton glycoprotein known as P-glycoprotein, which acts as an energy-dependent efflux pump for a great many cytotoxic anticancer drugs, including doxorubicin, daunorubicin, etoposide, teniposide, actinomycin D, and taxol. Vectors encoding an MDR1 cDNA are able to transduce many cell types, including bone marrow cells, with high efficiency to allow selection of drug resistance in vitro and in vivo in mouse models. Thus, it should be possible to protect the bone marrow of patients undergoing intensive chemotherapy by transduction of their bone marrow with MDR1 vectors. Furthermore, the ability to select for the presence of the MDR1 cDNA in vivo means that it can be used to introduce otherwise nonselectable genes into the bone marrow for therapy of cancer and other diseases.

Involvement of a DNA Binding Protein, MDR-NF1/YB-1, in Human MDR1 Gene Expression by Actinomycin D

The human multidrug resistance 1 (MDR1) gene is an SOS gene that responds to environmental stress including various anticancer agents. The chloramphenicol acetyltransferase (CAT) gene was linked to various lengths of MDR1 promoter, and these constructs were integrated into the genome of human cancer KB cells. Using these cell lines, we previously demonstrated that various environmental stimuli lead to an increased abundance of both CAT enzymatic activity and CAT mRNA in a sequence dependent manner. We examined the molecular mechanism of this stress response using actinomycin D, a potent RNA synthesis inhibitor. We found that CAT activity was significantly increased more than 10 fold by actinomycin D itself without comparable elevation of CAT mRNA. CAT induction was, however, lost in the presence of a deletion from position −136 to −76. Gel mobility shift assays showed that the specific DNA binding activity of the transacting protein, MDR-NF1/YB-1, which binds to the inverted CCAAT box, was augmented in nuclear extracts from the cells treated with actinomycin D. We also found that actinomycin D increased the steady state levels of MDR-NF1/YB-1 mRNA, which encodes the inverted CCAAT box binding protein. These results indicate that MDR-NF1/YB-1 mediates the response of the MDR1 gene to environmental stress.

Effect of colchicine and heat shock on multidrug resistance gene and P-glycoprotein expression in rat liver

The multidrug resistance genes encode plasma membrane glycoproteins named P-glycoproteins, that act as an ATP-dependent drug efflux pump and decrease the cytosolic concentration of chemotherapeutic agents. It has been hypothesized that in rat liver, this protein may have a physiological role as a biliary transporter of xenobiotics and endobiotics. Some human tumor cell lines turn on the human multidrug resistance gene in response to high temperature and after exposure to toxic chemicals. Accordingly, it has been proposed that the human multidrug resistance gene is a heat shock gene. We have assessed whether two environmental stresses, heat shock or acute exposure to cytotoxic drugs (colchicine, vincristine, vinblastine and daunomycin), induce changes in the expression of multidrug resistance genes in the rat. Total cellular RNA extracted from rat liver was hybridized to a labeled human multidrug resistance gene cDNA probe. Temperature upshift did not increase the steady-state of mdr mRNA levels in the tissues studied, suggesting that the mdr genes are not activated as part of a heat shock response. The mdr mRNA levels increased in rat liver as early as 3 h after a single injection of colchicine, reached a peak (500%; p < 0.05) after around 24 h and returned to constitutive levels after 48 h. Changes in the relative content of mdr mRNA were not detected in kidney, adrenal gland and small bowel, suggesting that the in vivo induction of the mdr gene in the liver is a tissue-specific response. The other cytotoxic drugs that were tested did not increase the steady-state of mdr mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Circumvention of chemotherapy-induced myelosuppression by transfer of the mdr1 gene.

Drug-induced myelosuppression is a frequent reason for curtailing chemotherapy in cancer patients. 'Rescue' of myelosuppressed patients with autologous marrow transplants is reasonably advanced and permits an increase in the dose of anticancer drugs. Despite this improvement, patients often relapse with drug resistance disease. The human multidrug resistance (mdr1) gene might make it possible to render hemopoietic stem cells resistant to anticancer drugs after transfer of this gene. By introducing resistant stem cells into patients it might be possible to treat these patients repeatedly with otherwise ablative therapy. This review explores the feasibility of mdr1 gene therapy.

Activation of the human MDR1 gene promoter in differentiated neuroblasts.

When treated in vitro with retinoic acid, many neuroblastoma cell lines undergo neuronal differentiation and show a significant increase in human multi-drug resistance gene (MDRI) transcript levels. To determine whether over-expression of the gene may result from activation of its promoter by differentiating agents, the activity of the human MDRI proximal promoter (MDRI pp) transfected to 2 neuroblastoma lines treated with retinoic acid and forskolin was first measured using transient expression assays. The MDRI gene transcript levels of these 2 lines were then measured by Northern blots. The results indicate that retinoic acid and forskolin were able to activate the human MDRI pp in a dose-dependent manner after transfection of the MDRI pp-CAT constructs in the 2 cell models tested, i.e., SK-N-SH and IGR-N-91, a new human neuroblastoma cell line. A significant increase in MDRI gene transcript levels was observed upon treatment with differentiation inducers in SK-N-SH but not in IGR-N-91 neuroblasts. These results suggest that the induction of the MDRI gene promoter is necessary but not sufficient to lead to an increase in MDRI gene transcript levels, according to the neuroblast cell line considered.

Involvement of protein kinase in environmental stress-induced activation of human multidrug resistance 1 (MDR1) gene promoter

The human MDR1 gene can be induced in response to various environmental stimuli. To examine whether such stress-induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter. H-7, a protein kinase C inhibitor, at more than 40 μM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5-fluorouracil or UV irradiation. DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H-7. Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences. Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter. The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.

A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene.

Basal transcription of the human multidrug resistance (mdr1) promoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines. Deletion of mdr1 DNA sequences to -89 relative to the start of transcription (at +1) had little effect on expression. Deletion of nucleotide sequences from -89 to -70, however, resulted in a 5-10-fold reduction in mdrCAT expression. DNase I footprint analysis demonstrated that the region from -85 to -70 was protected from nuclease digestion using nuclear extracts from these cell lines. The sequence between -82 and -73 is perfectly homologous with the 10-base pair Y-box consensus sequence found in the promoters of all major histocompatibility complex class-II (MHC II) genes. The Y-box sequence in MHC II genes is required for accurate and efficient transcription and contains the sequence CCAAT in the reverse orientation (Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C., and Mathis, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6249-6253). Mutations in the reverse CCAAT sequence of the Y-box consensus substantially reduced expression of an mdrCAT vector and eliminated nucleoprotein binding in an electrophoretic mobility shift assay. These results suggest that proteins which bind to the putative Y-box consensus sequence are critical for basal transcriptional regulation of the human mdr1 gene.

Purification and characterization of NF-R2 that regulates the expression of the human multidrug resistance (MDR1) gene.

NF‐R2 is a DNA‐binding protein that interacts with the MDR1 gene proximal promoter sequence. We previously reported that NF‐R2 binds within the promoter's — 126 and — 102 regions, which contain the ATTCAGTCA motif. In the present study, we have purified NF‐R2 from the nuclear extract of K562/ADM cells, a multidrug‐resistant cell line derived from human myelogenous leukemia K562 cells, using sequential chromatography on Sephacryl S‐300, DEAE‐Sepharose, heparin‐Sepharose and a DNA affinity column consisting of a repetitive synthetic ATTCAGTCA motif coupled to Sepharose. NF‐R2 runs as a single protein of 75 kDa on SDS‐PAGE (sodium dodecyl sulfate‐polyacrylamide gel electrophoresis). CAT (chloramphenicol acetyltransferase) expression assay and gel mobility shift competition assay with mutated promoters revealed that the ATTCAGTCA motif is a positive regulatory element of MDR1 gene and that the motif is important for NF‐R2 binding. These results suggest that NF‐R2 may be involved in the positive regulation of the MDR1 gene transcription.

4] Use of multidrug resistance gene in mammalian expression vectors

Publisher Summary This chapter discusses the use of multidrug resistance in mammalian expression vectors. Virus-based vector systems—such as retroviruses, vaccinia virus, and bovine papillomavirus—have proved useful for both stable and transient gene expression. Gene transfer can be efficient and reproducible with such viruses and infection of a variety of cell types is often possible. Many plasmid vectors make use of strong promoter elements for high-level gene expression in transfected cells, and tissue-specific or inducible promoters allow expression under more controlled conditions. Amplifiable selection systems have been developed for stable overexpression of foreign sequences, the most common of these being based on dihydrofolate reductase, which confers resistance to folate analogs. This chapter explains the theory and applications of another such amplifiable selection system, based on the human multidrug resistance gene (MDR1), and resistance to a variety of clinically relevant drugs, such as colchicine, adriamycin (doxorubicin), and vinblastine.

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

Expression of a human multidrug resistance gene in human ovarian carcinoma cell lines.

To investigate the possible role of the multidrug resistance phenotype to chemoresistance in human ovarian carcinoma, we have analyzed human multidrug resistance gene (mdr 1) expression in 8 human ovarian adenocarcinoma cell lines. An increase in P-glycoprotein level specific to multidrug-resistant tumor cells was not apparently associated with the increase in resistance to vincristine (VCR) or doxorubicin (Adriamycin). Mdr 1 transcripts (4.5 kilobases) were observed in the RNA preparation obtained from only one cell line (SHIN-3) that showed the highest resistance to both drugs in vitro and in vivo. No cell lines showed mdr 1 DNA amplification. These results suggest that the insensitivity of human ovarian carcinoma to chemotherapy could be partly explained by the expression of mdr 1.

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.