Human MDR1 Research Articles

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.

Identification of two nuclear protein binding sites and their role in the regulation of the murine multidrug resistance mdr1a promoter.

Multidrug resistance genes (mdr) that encode P-glycoproteins (P-gp) are transcriptionally regulated in normal tissues and in some multidrug-resistant (MDR) cells. Several lines of evidence suggest that regulation of P-gp overexpression at the transcriptional level is also important in human tumors. In murine MDR cells, mdr1a and/or mdr1b genes are overexpressed and P-gp isoforms are overproduced. To identify the mdr1a promoter regions that are required for transcription, the promoter has been linked to the chloramphenicol acetyltransferase (CAT) gene in transient expression vectors. 5'-Deletions of the promoter sequences have demonstrated that the region between -155 to +89 bp is crucial for basal activity of the mdr1a gene. DNase I footprinting, methylation interference, and gel retardation assays identified two nuclear protein binding sites within these sequences. One of the nuclear protein binding sites contains an 11-bp DNA sequence that interacts with nuclear protein(s) and is conserved in the promoters of the murine mdr1a and mdr1b, hamster pgp1, and human MDR1 genes. The conserved SP1 site (5'-GGGCGGG-3') that is present further downstream was shown to interact with its nuclear factor. These observations suggest that at least part of mdr gene transcriptional regulation is mediated by conserved mdr cis-regulatory elements and common nuclear factors.

From laboratory expertise to clinical practice: multidrug-resistance-based gene therapy becomes available for urologists.

Many human tumors such as bladder carcinoma that are initially responsive to chemotherapy eventually fail to respond to treatment. For most drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow limit cytotoxic therapy. Approaches to prevent or circumvent myelosuppression are therefore a high priority of research on dose intensification protocols. One such strategy is to protect bone marrow cells by virtue of expression of the multidrug-resistance (MDR1) gene encoding for P-glycoprotein. In our first set of experiments, we transplanted bone marrow cells derived from transgenic mice that constitutively express MDR1 to lethally irradiated recipients (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and bone marrow specimens as indicated by Southern-blot analysis and expressed MDR1 RNA in bone marrow samples as detected by slot-blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal bone marrow reacted with a significant decrease in their white blood counts. In a second set of experiments, we retrovirally transfected a construct consisting of a murine long-terminal repeat (LTR) promoter and the human MDR1 gene into CD34-positive bone marrow stem cells from rhesus monkeys using the same technique as in the ongoing clinical ADA gene-therapy protocol. Upon transplantation, high-level and long-lasting expression of the human MDR1 gene was observed in recipient monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between rhodamine 123 transport, cell volume, and ion-channel function of P-glycoprotein.

The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl- transport blockers suggests a possible role for Cl- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl- (and also Na+ and HCO3-/CO2) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a approximately 60-min removal of Cl- (or by exposure to Na(+)-free, or nominally HCO3-/CO2-free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl- current by hyposmotic solution. The lack of detectable effect of removal of Cl-, Na+, or HCO3- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl- current denotes that Pgp-mediated transport of organic substrates and Pgp-associated Cl- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl- transport.

Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene.

It has been shown recently that heterologous expression of human MDR-1 gene, which is responsible for multidrug resistance during cancer therapy, causes appearance of volume-sensitive Cl- currents, thus suggesting that the product of the MDR-1 gene (the P-glycoprotein) has a Cl- channel activity (Valverde, M. A., Diaz, M., Sepulveda, M. A., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833). In the present work, we have tested four epithelial cell lines both for the expression of MDR-1 gene and for the presence of volume-sensitive Cl- currents. LoVo/H and LoVo/Dx cells derive from a human colon adenocarcinoma, the latter cell line being resistant to high concentrations of the antitumoral drug doxorubicin. 9HTEo- cells were obtained by transformation of human tracheal epithelium. The 9HTEo-/Dx cell line was established from these cells by selection in doxorubicin. As expected, higher levels of P-glycoprotein expression were detected in LoVo/Dx and 9HTEo-/Dx by means of reverse transcriptase polymerase chain reaction technique, indirect immunofluorescence, and Western immunoblot assays. In contrast with these data, the size of swelling-induced Cl- current was the same in the sensitive cell line and in its drug-resistant counterpart. Actually, the Cl- conductance of 9HTEo- and 9HTEo-/Dx was 4-fold higher than that of either LoVo/H or LoVo/Dx cells. This indicates that the amplitude of this conductance is not directly related to the expression of the MDR-1 gene.

Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.

Multidrug Resistance in the Laboratory and Clinic

Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases. Studies have demonstrated that this form of drug resistance occurs in cultured tumor cell lines as well as in human cancers. P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural-product drugs from the cell before they have a chance to exert their cytotoxic effects. Using the tools of molecular biology, studies are beginning to reveal the true incidence of multidrug resistance, as mediated by the MDR1 gene, in the clinical setting. It has been demonstrated, at least in the laboratory, that resistance mediated by P-glycoprotein may be modulated by a wide variety of compounds, including verapamil and cyclosporine A. These are compounds which, by themselves, generally have little or no effect on the tumor cells, but when used in conjunction with antineoplastic agents act to decrease, and in some instances eliminate, drug resistance. The mechanism(s) by which these agents act to reverse resistance is not fully understood. Clinical trials to modulate P-glycoprotein activity are now under way to determine whether such strategies will be feasible. The detection of the P-glycoprotein in patient samples is very important in the design of these studies, as it appears that drug-resistant cells lacking P-glycoprotein will be unaffected by agents such as verapamil. Clinical studies are needed in which patients are stratified into chemotherapy protocols based on levels of MDR1 mRNA or P-glycoprotein expression in the primary tumors. Several research areas have been identified that are important to the transfer of the discovery of the MDR1 gene and its protein product from the research laboratory to the clinical environment. There is an immediate need for comprehensive information on the prevalence and levels of expression of the human MDR genes and their protein products in human organs and tissues. Data are needed on P-glycoprotein levels in specific subpopulations (e.g., according to age, sex, race, and diet), and the study of the heterogeneity and variability of expression of P-glycoprotein in normal human tissues should be given high priority. Since early studies have indicated some successes in identifying patients with classic multidrug resistance who might be responsive to chemosensitization, it can be anticipated that clinical research will accelerate in this area. The next wave of clinical studies will provide clinical investigators with opportunities to develop and evaluate P-glycoprotein tests and correlate test results with clinical outcomes.

Reversal of the human and murine multidrug-resistance phenotype with megestrol acetate.

MA is an orally active PG derivative with an excellent safety profile that is used primarily for the treatment of carcinomas of the breast and endometrium. We investigated the potential application of MA as an MDR-reversal agent using cell culture and human tumor xenograft models. The reversing activity of MA in vitro was compared with that of PG and VER in two human MDR cell lines, the colon carcinoma HCT-116/VM46 and the breast carcinoma MCF-7/ADR, and in a murine cell line, J774.2. At concentrations as low as 3 microM, MA was capable of partially restoring sensitivity to Act D in the HCT-116/VM46 cells and sensitivity to DOX in the MCF-7/ADR cells. Although less effective than VER, MA was about 2.5 times more potent than PG in reversing MDR at equimolar concentrations. Increased accumulation of DOX in drug-resistant cells that were treated simultaneously with MA was observed by flow cytometry. In vivo, using established human colon and breast carcinoma xenografts implanted s.c. in athymic mice, the combined therapy with MA and DOX resulted in enhanced antitumor activity relative to that of DOX alone in the MDR sublines. These results suggest that MA may be a promising clinical MDR-reversing agent.

Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P‐glycoprotein

Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance. Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4. The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs. In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected. None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein. The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5–30 times the IC 50 (30 μg mg ml −1) of parental cells. The vinblastine-resistant parasites were also resistant to promycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug effiux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance ( mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdrl, and demonstrate that this gene is amplified on an extrachromosomal circle of 35–40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr 1 and 83% identity with the L. donovani 1mdr 1 gene. The lemdr 1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.

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.

Characterisation of a vindesine-resistant human small-cell lung cancer cell line

We established a vindesine-resistant (x 11.6) human small-cell lung cancer cell line (H69/VDS) by stepwise exposure of parent line H69 to vindesine. H69/VDS showed cross-resistance to taxol (x 10.1), vincristine (x 6.9) and colchicine (x 3.4) but not to doxorubicin, cisplatin or etoposide. There was no significant difference in intracellular [3H]-vincristine and doxorubicin accumulation between H69 and H69/VDS cells. The human mdr1 mRNA was not detected in either of the cell lines. These results indicated that H69/VDS did not express a typical multidrug resistant phenotype. Addition of 20 microM verapamil enhanced the growth inhibitory effect of vindesine on both H69/VDS (x 12.0) and H69 cells (x 3.8). The amount of total tubulin in H69/VDS cells was lower than that in the H69 parental cells. No significant increase was observed in the amount of total and polymerised tubulins of H69 cells. In H69/VDS cells, however, verapamil increased the amount of total tubulin to the level of parental cells, but decreased the amount of polymerised tubulin. Modulation of tubulin may play a role in the resistance to vindesine.

A signal transduction pathway for activation of the mdr1 promoter involves the proto-oncogene c-raf kinase

To investigate the regulation of expression of the human mdr1 gene, the response of the mdr1 promoter to signals involved in cell proliferation was examined. The activity of the mdr1 promoter was measured in transiently transfected NIH 3T3 cells, which were stimulated to enter the cell cycle by addition of serum, platelet-derived growth factor, or transforming growth factor alpha. Addition of serum to quiescent NIH 3T3 cells resulted in a 6-8-fold activation of mdr1 promoter activity, which peaked at 10 h, just prior to S-phase. Treatment of serum-starved cells with the serum mitogens platelet-derived growth factor and transforming growth factor alpha also activated the mdr1 promoter. To define components of the signal cascade resulting in mdr1 promoter activation, the role of c-Raf kinase, a serine/threonine kinase important in mitogen-activated signal transduction, was examined. We measured the effects of a constitutively activated Raf kinase, v-raf, and a dominant negative Raf mutant, c-Raf-C4, on mdr1 promoter activity in NIH 3T3 and HepG2 cells. In serum-starved NIH 3T3 cells, v-raf increased mdr1 promoter activity approximately 10-fold compared to a v-raf frame-shift control. Raf responsiveness of the mdr1 promoter was localized to sequences between -69 and -41, relative to the initiation site. Serum stimulation of the mdr1 promoter was blocked by co-transfection of NIH 3T3 cells with the dominant negative Raf mutant c-Raf-C4. In the human hepatoma cell line HepG2, which has high endogenous Raf kinase activity (Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Dev. 6, 545-556), co-transfection with c-Raf-C4 decreased mdr1 promoter activity 20-fold. Taken together, these data suggest that the mdr1 gene is transcriptionally regulated by normal cellular signaling events involving activation of c-Raf kinase.

MDR1 GENE-EXPRESSION IN A MULTIDRUG-RESISTANT HUMAN NON-HODGKINS-LYMPHOMA XENOGRAFT MODEL

In order to investigate the biology of tumor cells which express MDR1 gene and to test the activity of different P-glycoprotein blocking agents in vivo, we established a nude mice model. Five Non Hodgkin's Lymphoma (NHL) tumor specimens were xenografted to nude mice. One of them, obtained from a chemotherapy-refractory patient gave rise to a mice transplantable model. This tumor xenograft model, IGR-NHL-90, showed overexpression of the human MDR1 gene. In this tumor model, histology, mitotic index, phenotypic and karyotypic traits remained stable at subsequent passages. The in vitro resistance of vincristine was reversed by verapamil for these NHL tumor cells, suggesting that the MDR1 resistance is a relevant mechanism in this model. In the absence of chemotherapy a higher biological aggressivity of the heterotransplanted NHL was noted in subsequent nude mice passages. This was associated with decreased passage time and higher MDR1 m-RNA transcript levels. Thus IGR-NHL-90 may represent a suitable material to study regulation of MDR1 gene transcription in vivo and also to test the activity of various P-glycoprotein reversing agents with concurrent chemotherapy.

Sensitivity of K562 human chronic myelogenous leukemia blast cells transfected with a human multidrug resistance cDNA to cytotoxic drugs and differentiating agents.

The blast crisis of chronic myelogenous leukemia (CML) is refractory to most forms of cancer chemotherapy, but may be amenable to drugs that differentiate rather than kill leukemic cells. One mechanism implicated in resistance to cytodestructive drugs is overexpression of P-glycoprotein, the MDR1 gene product. While several classes of drugs sensitize multidrug-resistant (MDR) cells by interfering with the function of P-glycoprotein in vitro, few sensitizers have been effective in vivo. We have developed a preclinical model of MDR/CML uncomplicated by other mechanisms of drug resistance to evaluate the effects of MDR1 overexpression on cytodestructive and differentiation therapy and the ability of sensitizers to restore chemosensitivity in this disease. The CML-derived cell line K562 was transfected with a human MDR1 cDNA from the pHaMDR1/A expression vector and selected with vinblastine. Resistant K562 clones were 20-30-fold resistant to vinblastine, were cross-resistant to doxorubicin and etoposide, and remained sensitive to cytosine arabinoside, 6-thioguanine, hydroxyurea, and mechlorethamine. Resistance was associated with decreased cellular accumulation of cytotoxic drug and was reversed by cyclosporin A and trans-flupenthixol. The MDR phenotype did not adversely affect the ability of K562 cells to produce fetal hemoglobin in response to hemin, and was associated with increased responsiveness of cells to differentiate with cytosine arabinoside. Upon differentiation, the resistant clones increased MDR1 mRNA and P-glycoprotein. These studies suggest that the overexpression of the MDR1 gene in CML may not adversely affect the ability to undergo erythroid differentiation and that these resistant K562 cell lines are good models for studying drug resistance mediated by P-glycoprotein in CML.

Induction of Multidrug Resistance in Human Cells by Transient Exposure to Different Chemotherapeutic Drugs

The MDR1 (multidrug resistance) gene (also known as PGY1), which encodes the transmembrane efflux pump P-glycoprotein (Pgp), confers resistance to Pgp-transported cytotoxic drugs in tissue culture. The increase in MDR1 expression in tumors after chemotherapy is usually attributed to selection of pre-existing multidrug-resistant cells by Pgp-transported drugs. MDR1 expression in tissue culture can be increased by several types of stress-inducing treatment, including agents that activate protein kinase C (PKC). Previous studies, however, failed to demonstrate that short-term exposure to any chemotherapeutic drug can induce the expression of the resident MDR1 gene in drug-sensitive human cells. This study was designed to utilize highly sensitive assays to determine if transient exposure to chemotherapeutic drugs would have an effect on MDR1 expression in human cells and to assess if PKC inhibitors would influence such an effect. We analyzed the MDR1 gene expression in several human cell lines derived from leukemias or solid tumors, after treatment with different cytotoxic drugs, in the presence or absence of PKC inhibitors. Pgp function and expression were studied by flow cytometric assays, and MDR1 messenger RNA (mRNA) was assayed by polymerase chain reaction. Transient exposure to chemotherapeutic drugs, including agents that are not transported by Pgp, induced Pgp and MDR1 mRNA expression in subpopulations of treated cells in most of the tested cell lines. This induction was observed along with microscopically detectable cell damage. The drug-induced MDR1 expression and the associated twofold to threefold increase in resistance to vinblastine were sustained in K562 leukemia cells for at least several weeks after the removal of the drug. Drug-mediated MDR1 induction was blocked by nonspecific protein kinase inhibitors that are active against PKC, but not by a protein kinase inhibitor ineffective against PKC. Expression of the human MDR1 mRNA and Pgp can be induced in response to cellular damage by cytotoxic drugs regardless of whether the drugs are transported by Pgp. This induction can be prevented by protein kinase inhibitors. Induction of MDR1 expression in response to cellular damage may account for increased MDR1 expression in treated human tumors. Protein kinase inhibitors may be useful in preventing the emergence of multidrug resistance during cancer chemotherapy.

Three distinct nuclear protein binding sites in the promoter of the murine multidrug resistance mdr1b gene.

Multidrug resistance in mammalian cells is often associated with the overproduction of a membrane glycoprotein, P-glycoprotein, that is encoded by mdr genes. Multidrug resistance cell lines selected with either vinblastine, colchicine, or taxol from the drug-sensitive murine macrophage-like cell line J774.2 overexpress the mdr1a and/or mdr1b genes, and overproduce P-glycoprotein. To elucidate the mechanisms of mdr1b gene expression, the mdr1b 5'-flanking sequences have been isolated from a normal mouse liver genomic library and analyzed by gel shift and DNase I footprinting assays. These analyses have demonstrated three nuclear protein binding sites, from -82 to -59 (site 1), from -123 to -101 (site 2), and from -272 to -249 (site 3), which interact with proteins present in nuclear extracts from both sensitive and resistant cells. Although site 1 contains a partially conserved AP-2 consensus sequence, our results indicate that the nuclear protein binding to site 1 is not AP-2 protein. The sequence of site 2 is conserved in the murine mdr1a, human mdr1, and hamster pgp1 promoters. Such conservation suggests that this sequence may have an important role in mdr gene expression. The use of a transient chloramphenicol acetyltransferase expression vector containing the basal promoter for herpes simplex virus thymidine kinase (tkCAT) and either site 1 or site 2 or both revealed that the sequences of sites 1 and 2 enhanced tkCAT activity. DNase I footprinting analyses demonstrated that site 3 is recognized by human AP-1 protein, indicating that the nuclear protein binding to this site is an AP-1-like protein. These observations suggest that mdr1b gene expression is mediated by preexisting transcription factors present in sensitive and resistant cells.

Identification of 5‘ and 3‘ sequences involved in the regulation of transcription of the human mdr1 gene in vivo.

The human mdr1 gene encodes a putative drug efflux pump (P-glycoprotein) whose overexpression is associated with the development of multidrug resistance (MDR). The promoter and 5'-flanking DNA of this gene were isolated from a human genomic DNA library and used to prepare a series of chloramphenicol acetyltransferase (CAT) fusion vectors under the transcriptional control of the mdr1 promoter (mdrCAT vectors). Transient transfection of these mdrCAT vectors produced CAT activities similar to those produced by transfection of CAT vectors containing viral promoters. The regulation of mdr1 expression was examined in two MDR tumor cell lines selected for resistance to doxorubicin and their corresponding parental cell lines. Although nuclear run-on analysis indicates that the expression of the mdr1 gene in these two MDR cell lines is regulated by transcriptional mechanisms, mdrCAT expression was not significantly increased in either of these lines relative to parental cells. Thus, the sequences involved in the transcriptional regulation in these cells are apparently not included in the constructs studied (-4741 to +286). Analyses of a series of deletion constructs show that the basal mdr1 promoter activity is encoded by sequences that span a region adjacent to the transcription start site (-134 to +286) and that sequences 3' to the start of mdr1 transcription are necessary for proper initiation of transcription in vivo. Structural and functional studies indicate that an initiator (Inr) sequence surrounding the major transcription start site governs accurate initiation of mdr1 transcription.

Detection of P-glycoprotein in squamous cell carcinomas of the head and neck.

One established mechanism of multidrug resistance is elevated expression of P-glycoprotein. The expression of P-glycoprotein by immunohistochemistry was examined in squamous cell carcinomas of the head and neck using a newly developed monoclonal antibody, UIC2, which is specific to the human MDR1 gene product and recognizes an external epitope of the protein. P-glycoprotein was detected in 60% of the samples. MDR1 expression was compared with clinical response to chemotherapy in eight patients who received MDR1-dependent drugs and response was accurately predicted in seven (89%) of eight patients. Positive P-glycoprotein staining correlated with the degree of tumor differentiation, but not with other clinical factors. Therefore, analyzing the expression of P-glycoprotein may play a role when planning chemotherapeutic regimens for patients with head and neck cancer and may be an additional prognostic and diagnostic tool in these patients.