BBB transport and P-glycoprotein functionality using MDR1A (-/-) and wild-type mice. Total brain versus microdialysis concentration profiles of rhodamine-123

PURPOSE: The effect of P-glycoprotein (Pgp) on brain distribution using mdr1a (-/-) mice was investigated. METHODS: Fluorescein (Flu) and FD-4 were used to check whether blood-brain barrier (BBB) integrity was maintained in mdr1a (-/-) mice. The Pgp substrate rhodamine-123 (R123) was infused and total brain, blood and brain microdialysate concentrations in mdr1a (-/-) mice and wild-type mice were compared. RESULTS: Maintenance of BBB integrity was indicated by equal total brain/blood ratios of Flu and FD-4 in both mice types. R123 concentrations in brain after i.v. infusion were about 4-fold higher in mdr1a (-/-) than in wild-type mice (P < 0.05), without changes in blood levels. After microdialysis experiments the same results were found, excluding artifacts in the interpretation of Pgp functionality by the use of this technique. However the 4-fold ratio in brain was not reflected in corresponding microdialysates. No local differences of R123 in the brain were found. By the no-net-flux method in vivo recovery appeared to 4.6-fold lower in mdrla (-/-) mice compared with wild-type mice. CONCLUSIONS: Pgp plays an important role in R123 distribution into the brain. Using intracerebral microdialysis, changes in in vivo recovery by the absence or inhibition of Pgp (or active efflux in general) need to be considered carefully.     

Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene

The promoter of the rat pgp2/mdr1b gene has a GC-rich region (pgp2GC) that is highly conserved in mdr genes and contains an consensus Sp1 site. Sp1's role in transactivation of the pgp2/mdr1b promoter was tested in Drosophila Schneider cells. The pgp2/mdr1b promoter was strongly activated by co-transfected wild type Sp1 but not mutant Sp1 and mutation of the Sp1 site abrogated Sp1-dependent transactivation. In gel shift assays, the same mutations abolished Sp1-DNA complex formation. Moreover, basal activity of the pgp2/mdr1b Sp1 mutant promoter was dramatically lower. Enforced ectopic overexpression of Sp1 in H35 rat hepatoma cells revealed that cell lines overexpressing Sp1 had increased endogenous pgp2/mdr1b mRNA, demonstrating that Sp1 activates the endogenous pgp2/mdr1b gene. Pgp2GC oligonucleotide also bound Egr-1 in gel shift assays and Egr-1 competitively displaced bound Sp1. In transient transfections of H35 cells (and human LS180 and HepG2 cells) Egr-1 potently and specifically suppressed pgp2/mdr1b promoter activity and mutations in the Egr-1 site decreased Egr-1 binding and correlated with pgp2/mdr1b up-regulation. Ectopic overexpression of Egr-1 in H35 cells decreased Pgp expression and selectively increased vinblastine sensitivity. In conclusion, Sp1 positively regulates while Egr-1 negatively regulates the rat pgp2/mdr1b gene. Moreover, competitive interactions between Sp1 and Egr-1 in all likelihood determine the constitutive expression of the pgp2/mdr1b gene in H35 cells.     

Decreased uptake of cyclosporin A by P-glycoprotein (Pgp) expressing CEM leukemic cells and restoration of normal retention by Pgp blockers

The P-glycoprotein (Pgp) molecules which are expressed on multidrug resistant (MDR) tumor cells can efflux a variety of cytostatics. In both normal and tumoral epitheliums, Pgp molecules are selectively expressed on the apical surface of the epithelial cells. Such a distribution seems to be responsible for the transcellular transport of Pgp substrates, including cyclosporin A (CsA), from the basal to the apical side. Some normal lymphoid cells also express small amounts of Pgp molecules, for as yet unknown functions. Nevertheless, the sensitivity of their mitogen-induced proliferation to cytostatics, including doxorubicin and CsA, could be increased by the Pgp blockers. Using isotopically-labeled CsA and tumoral lymphoid cell lines, we now show a higher CsA retention in Pgp-lacking parental ('Par') cells than in Pgp-expressing MDR cells. The Pgp blockers can restore the CsA retention in the MDR cells to its level in the Par cells.     

Bryostatin 1 affects P-glycoprotein phosphorylation but not function in multidrug-resistant human breast cancer cells

The function of P-glycoprotein (Pgp), which confers multidrug resistance by active efflux of drug, is thought to be dependent on phosphorylation. Previous studies have suggested that protein kinase C (PKC) plays an important role in Pgp phosphorylation. We report here the effects of bryostatin 1, a unique PKC activator and inhibitor, on Pgp function in a multidrug-resistant MCF-7 human breast cancer subline which overexpresses PKC-alpha. Bryostatin 1 (100 nM) decreased Pgp phosphorylation after 24 h of treatment. In contrast, it did not affect Pgp function as demonstrated by the accumulation of [3H]vinblastine and rhodamine 123. We compared the effect of bryostatin 1 treatment on PKC-alpha with that of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM). 12-O-tetradecanoylphorbol-13-acetate caused translocation of PKC-alpha from the cytosol to the cell membrane after a 10-min treatment and its down-regulation after 24 h of treatment. Likewise, bryostatin 1 (100 nM) caused translocation, but only after longer treatment (1 h), and it caused down-regulation of PKC-alpha at 24 h of treatment. Thus, while the MCF-7TH cells overexpress the PKC-alpha isoform, and its down-regulation by bryostatin 1 is associated with decreased Pgp phosphorylation, these alterations do not modulate drug transport. We conclude that, while bryostatin 1 may be useful clinically because of its ability to inhibit PKC, it is not able to reverse Pgp-mediated multidrug resistance.     

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

Wall effects observed in tissue-equivalent proportional counters from 1.05 GeV/nucleon iron-56 particles

A strong overlap between P-glycoprotein (Pgp) and cytochrome P450 3A (CYP3A) substrates and modulators has been reported. To test the hypothesis that CYP3A and Pgp are coordinately regulated, we examined the effects of known inducers of CYP3A (triacetyloleandomycin, rifampicin, dexamethasone, pregnenolone 16alpha-carbonitrile) on Pgp expression in rat liver. We also investigated the gender-specific expression of Pgp and compared its response to dexamethasone between male and female rats. In male rats, western blot analyses showed that rifampicin and dexamethasone caused 50% and 5-fold increases in Pgp levels, respectively. RNase protection assays using gene-specific probes for the three Pgp isoforms revealed a 3-fold increase in mdr2 mRNA levels after dexamethasone administration and a 2-fold increase following rifampicin treatment. Triacetyloleandomycin and pregnenolone 16alpha-carbonitrile had no effect on Pgp expression and mRNA levels. We also observed that the basal level of Pgp was 40% lower in male rats than in females and that mdr2 mRNA levels in male rats were one-half those in females. As opposed to the results in male rats, dexamethasone reduced Pgp expression by approximately 60% and caused a 30% decrease in mdr2 mRNA levels in female rats. Mdr1a was not affected and mdr1b was not detected in female or male rats. We conclude that, at the dosage regimen used, CYP3A and Pgp responses to CYP3A inducers are regulated independently in rat liver. In addition, this study shows that Pgp expression and regulation are gender specific.     

Functional multidrug resistance phenotype associated with combined overexpression of Pgp/MDR1 and MRP together with 1-beta-D-arabinofuranosylcytosine sensitivity may predict clinical response in acute myeloid leukemia

Overexpression of P-glycoprotein (Pgp) or MDR1 mRNA has been shown to be a negative prognostic factor for clinical outcome in acute myeloid leukemia (AML). However, resistance to chemotherapy also occurs in the absence of Pgp overexpression. Therefore, besides Pgp expression, we have assessed the expression of MRP, a novel drug transporter gene, along with the functional multidrug-resistant (MDR) phenotype of leukemic cells. These MDR parameters are correlated with clinical outcome in individual patients. We found functional changes in fresh leukemic cells from de novo or relapsed patients similar to those reported for tumor cell lines with the MDR phenotype. These changes were reduced drug accumulation as assessed with radiolabeled doxorubicin (factor 1.6), daunomycin (factor 1.13), and vincristine (factor 1.6) in patients who were refractory to the combination treatment of 1-beta-D-arabinofuranosylcytosine (ara-C) and daunomycin or mitoxantrone as opposed to patients who had complete responses. Also, the intracellular distribution of doxorubicin fluorescence (nuclear/cytoplasmic ratio), as assessed with laser scan microscopy, was reduced 1.4-fold in blasts from refractory patients. Based on historically known clinical response to single-agent daunomycin or ara-C in the group of responding de novo AML patients, we have set a threshold level such that a defined part of the samples that had the highest drug accumulation or nuclear to cytoplasmic ratios were above this threshold value. This allowed discrimination between patients responding to daunomycin from those who were refractory to this drug. By using this threshold level, in the refractory group clinical resistance corresponded with high sensitivity with a resistant phenotype. A similar threshold was set for the data of the in vitro ara-C sensitivity test. By combining both assays for all individual patients, clinical refractoriness as well as sensitivity could be predicted with high accuracy. There appeared to be no stringent relationship between the functional MDR phenotype with expression of either Pgp (fluorescence-activated cell sorting analysis) or MRP mRNA (RNase protection). However, by combining both parameters the functional MDR phenotype correlated with the overexpression of either one or both of the parameters in 94% of the samples studied. It is concluded that this combined overexpression in conjunction with functional changes for MDR drugs and ara-C reveal a correlation of MDR phenotype with clinical resistance to combination chemotherapy in AML patients and hereby may adequately predict clinical MDR in individual AML patients.     

In vitro and in vivo reversal of cancer cell multidrug resistance by the semi-synthetic antibiotic tiamulin

A large number of multidrug resistance (MDR) modulators, termed chemosensitizers, have been identified from a variety of chemicals, but most have been proven to be clinically toxic. Low concentrations of the pleuromutilin-derived semi-synthetic antibiotic tiamulin (0.1 to 10 microM) sensitized the three highly resistant P-glycoprotein (Pgp)-overexpressing tumor cell lines P388 (murine lymphoid leukemia), AS30-D (rat hepatoma), CEM (human lymphoblastic leukemia), and the barely resistant AS30-D/S cell lines to several MDR-related anticancer drugs. Flow cytometric analysis showed that tiamulin significantly increased the intracellular accumulation of daunomycin. When compared to reference modulating agents such as verapamil and cyclosporin A, tiamulin proved to be 1.1 to 8.3 times more efficient in sensitizing the resistant cell lines. Moreover, when given i.p. (1.6 microg/mg body weight), tiamulin increased the survival rate of adriamycin-treated mice bearing the P388/ADR25 tumor line by 29%. In the presence of an anticancer drug, tiamulin inhibited both ATPase and drug transport activities of Pgp in plasma membranes from tumor cells. Tiamulin is thus a potent chemosensitizer that antagonizes the Pgp-mediated chemoresistance in many tumor cell lines expressing the MDR phenotype at different levels and displays no toxic effects on contractile tissues at active doses, therefore providing the promise for potential clinical applications.     

Development and characterisation of novel human multidrug resistant mammary carcinoma lines in vitro and in vivo

Clinical chemotherapy of breast carcinomas must be considered insufficient, mainly due to the appearance of drug resistance. The multidrug resistance (MDR) phenotype, either intrinsically occurring or acquired, e.g., against a panel of different antineoplastic drugs, is discussed in relation to several MDR-associated genes such as the MDR-gene mdr1 encoding the P-glycoprotein (PGP), the MRP gene (multidrug resistance protein) encoding an MDR-related protein or the LRP gene encoding the lung resistance protein. Numerous experimental and clinical approaches aiming at reversing resistance require well-characterised in vitro and in vivo models. The aim of our work was to develop multidrug resistant sublines from human xenotransplanted breast carcinomas, in addition to the broadly used line MCF-7 and its multidrug resistant subline MCF-7/AdrR. MDR was induced in vitro with increasing concentrations of Adriablastin (ADR) for several weeks, resulting in a 3.5- to 35-fold increase in IC50 values using the MTT-test. Cell lines were cross-resistant toward another MDR-related drug, vincristine, but remained sensitive to non-MDR-related compounds such as cisplatin and methotrexate. The resistance toward Adriamycin and vincristine was confirmed in vivo by a lack of tumour growth inhibition in the nude mouse system. Gene expression data for the mdr1/PGP, MRP/MRP and LRP/LRP on both the mRNA (RT-PCR) and the protein levels (immunoflow cytometry) demonstrated that induction of mdr1 gene expression was responsible for the acquired MDR phenotype. Rhodamine efflux data, indicated by PGP overexpression, underlined the development of this MDR mechanism in the newly established breast carcinoma lines MT-1/ADR, MT-3/ADR and MaTu/ADR.     

A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.     

Functional characterization of the rat mdr1b encoded P-glycoprotein: not all inducing agents are substrates

The multidrug resistance (mdr) genes encode P-glycoproteins, integral membrane proteins which function as drug efflux transporters. Exposure of animals in vivo and cells in vitro to a variety of xenobiotics leads to increased mdr1 gene expression and higher levels of P-glycoprotein. This response may protect cells from the cytotoxic effects of these compounds. In this investigation we functionally expressed the rat mdr1b gene in NIH 3T3 cells and assessed the ability of the encoded P-glycoprotein to protect these cells from the cytotoxicity of xenobiotics known to induce mdr1b expression. In long-term colony survival assays, stably expressed mdr1b conferred resistance to cytotoxic drugs such as colchicine, vinblastine and doxorubicin, but not to 5-fluorouracil nor to the carcinogens aflatoxin B1 and N-hydroxy-acetylaminofluorene. The mdr reversal agent verapamil restored cytotoxicity of colchicine, doxorubicin, actinomycin D, vinblastine and taxol, but had no effect on the sensitivity of these cells to 5-fluorouracil, aflatoxin B1 or N-hydroxy-acetylaminofluorene. In a competitive transport assay, verapamil and, to a lesser extent, colchicine blocked the increased efflux of the fluorescent dye rhodamine 123 from mdr1b-transfected cells, whereas aflatoxin B1 did not compete for this export. These data demonstrate that expression of the rat mdr1b encoded P-glycoprotein can protect cells from a diverse group of compounds previously identified to be mdr substrates, however, other effective inducers of mdr expression, such as aflatoxin B1 and N-hydroxy-acetylaminofluorene, remain potent cytotoxins despite high levels of P-glycoprotein. The fact that compounds which are not themselves substrates can induce P-glycoprotein expression may have implications for pharmacokinetic interactions and chemotherapy.     

An artificial intelligence system for computer-assisted menu planning

BACKGROUND: The role of P-glycoprotein (Pgp) associated multidrug resistance for neuroblastoma patients is controversial. Therefore we asked whether at all the typical functional features of the multidrug resistance phenotype could be found in neuroblastoma cells and studied the prognostic relevance of Pgp expression. PATIENTS AND METHODS: Tumor touch preparations and tumor cell infiltrated bone marrow smears of 62 neuroblastoma patients were investigated. The expression of Pgp was determined by a highly sensitive immunosandwich technique. Drug resistance studies were performed by exposing cells to Pgp-dependent cytostatic drugs in tissue cultures. Intracellular drug accumulation was examined by rhodamine-123 fluorescence microscopy. RESULTS: Pgp expression was demonstrable for the SK-N-SH cell line, but not detectable in CHP-100 and ten other neuroblastoma cell lines by immunocytochemistry. In tissue cultures, SK-N-SH cells showed a relative resistance to vincristine and adriamycin (45.1 and 12.7-fold resp.) and reduced intracellular accumulation of rhodamine-123 which could be normalized by the Pgp blocker verapamil. Pgp expression was detected by immunocytochemistry in 14 out of 62 tumors (22.6%). No correlation was found to the stage of the disease (P = 0.33), histopathological grading (P = 0.82), N-myc oncoprotein expression (P = 0.76) or N-myc oncogene amplification (P = 0.20). Kaplan-Meier analysis of event free survival for stage 4 tumors revealed a weak trend of inferior survival for patients with Pgp positive tumors (log-rank analysis: P = 0.069). CONCLUSIONS: Though Pgp expression is detectable and functional in neuroblastoma cells, but its presence does not provide much information to the complex phenomenon of chemotherapy resistance in patients.     

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (-/-) mice]. In spite of the host of functions speculatively attributed to the mdrl-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal in mdr1a/1b (-/-) mice. The high level of mdrlb P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (-/-) mice behaved similarly to the previously analyzed mdr1a (-/-) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (-/-) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (-/-) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.     

Low-flow ischemia leads to translocation of canine heart GLUT-4 and GLUT-1 glucose transporters to the sarcolemma in vivo

BACKGROUND: Myocardial ischemia increases heart glucose utilization in vivo. However, whether low-flow ischemia leads to the translocation of glucose transporter (GLUT)-4 and/or GLUT-1 to the sarcolemma in vivo is unknown. METHODS AND RESULTS: In a canine model, we evaluated myocardial glucose metabolism in vivo and the distribution of GLUT-4 and GLUT-1 by use of immunoblotting of sarcolemma and intracellular membranes and immunofluorescence localization with confocal microscopy. In vivo glucose extraction increased fivefold (P < .001) and was associated with net lactate release in the ischemic region. Ischemia led to an increase in the sarcolemma content of both GLUT-4 (15 +/- 2% to 30 +/- 3%, P < .02) and GLUT-1 (41 +/- 4% to 58 +/- 3%, P < .03) compared with the nonischemic region and to a parallel decrease in their intracellular contents. Immunofluorescence demonstrated the presence of both GLUT-4 and GLUT-1 on cardiac myocytes. GLUT-1 had a more prominent cell surface pattern than GLUT-4, which was primarily intracellular in the nonischemic region. However, significant GLUT-4 surface labeling was found in the ischemic region. CONCLUSIONS: Translocation of the insulin-responsive GLUT-4 transporter from an intracellular storage pool to the sarcolemma occurs in vivo during acute low-flow ischemia. GLUT-1 is also present in an intracellular storage pool from which it undergoes translocation to the sarcolemma in response to ischemia. These results indicate that both GLUT-1 and GLUT-4 are important in ischemia-mediated myocardial glucose uptake in vivo.     

Alpha-fetoprotein-mediated targeting--a new strategy to overcome multidrug resistance of tumour cells in vitro

The possibility of overcoming the multidrug resistance of human malignant cells by using doxorubicin conjugated to alpha-fetoprotein (AFP) was studied. It was shown that this type of antitumour drugs, penetrating the cell by receptor-mediated endocytosis with AFP as a vehicle, raises the sensitivity of the tumour cells that are resistant due to the expression of the multidrug resistance gene mdr1. The sensitivity of antibiotic-resistant cell lines SKVLB (a human ovarian carcinoma) and MCF-7 AdrR (a human breast carcinoma) increased by 10- and 4-fold, respectively, when AFP-conjugated doxorubicin was used. The rationale of using human AFP-antitumour drug conjugates for the development of new chemotherapeutic approaches to cancer treatment is discussed.     

Growth factor stimulation of hematopoietic cells leads to membrane translocation of AKT1 protein kinase

AKT1 is the human homolog of the v-akt oncogene. AKT1 has two distinct protein domains, one serine/threonine kinase domain and one pleckstrin homology (PH) domain. We studied the expression and activity of AKT1 in hematopoietic cell lines. The expression of AKT1 was constitutive in hematopoietic cells of various stages of development. In the growth factor dependent MO7e cells, serum and growth factor starvation resulted in an early 50% fall in activity which was maintained over 24 h. Treatment of cells which growth factors or agents which induce differentiation activated AKT1. The subcellular localization of AKT1 in MO7e cells was altered as it was activated. High AKT1 kinase activity was associated with membrane fractions in stimulated cells, in contrast to the much lower AKT1 activity in membranes of cells starved of serum and growth factor for 1 h. These results demonstrate AKT1 kinase activity and its regulation by extracellular signaling factors in vivo in hematopoietic cells, and suggest that the activation of AKT1 involves intracellular translocation of the kinase from cytosol to membrane.     

Influence of catheter materials and tissue composition on low dose rate iridium-192 seed implant dosimetry

Drug sensitivity was studied for the tubulin inhibitors taxol, taxotere, rhizoxin and for doxorubucin and cisplatin, in human lung and breast cancer cell lines, including drug-selected cell lines, overexpressing the membrane transporter P-glycoprotein (Pgp) or the multidrug resistance protein (MRP). All tubulin-inhibiting agents were more potent than doxorubicin and cisplatin in all cell lines. In the drug resistance-selected cell lines (doxorubicin or mitoxantrone resistant) there was cross-resistance between the tubulin inhibitors and the selecting agent; however, MRP overexpressing cells were relatively less resistant to taxanes than the Pgp overexpressing cells. Polymerization of microtubules after exposure to taxol was observed in drug sensitive cell lines, but not in resistant cell lines, even at high taxol concentrations and after long exposure times. In the Pgp overexpressing cell lines, steady accumulation of 14C-taxol was defective and could be reverted by verapamil. MRP overexpressing cells did not have a significant accumulation defect of taxol, compared to the parental cell lines, and verapamil did not have any effect. These data confirm that the Pgp overexpression is an important mechanism of resistance to taxanes and rhizoxin in human lung and breast tumor cells. However, the presence of mechanisms other than transport defects may play an important role in non-Pgp expressing cells, and these may include an altered function of tubulins.     

Identification of the in vivo phosphorylation sites for acidic-directed kinases in murine mdr1b P-glycoprotein

P-glycoprotein, the multidrug resistance transporter, is phosphorylated in vivo and the major phosphorylation domain has been identified as the linker region (amino acids 629-686). The linker region is a highly charged segment of the transporter in which the negative and positive amino acid side chains are spatially segregated. Both of these charged domains contain several consensus phosphorylation sites for protein kinases. Three of the consensus phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein are utilized in vivo and have been identified as serines 665, 669, and 681. Mutagenesis of all the consensus basic-directed kinase phosphorylation sites in the linker region of human MDR1 P-glycoprotein did not alter the ability of the mutated transporter to confer the multidrug resistance phenotype in stably transfected cell lines. These studies would suggest that phosphorylation/dephosphorylation within the basic domain of the linker region is not directly involved in regulation of drug transporter activity. We now report that the linker region of mdr1b P-glycoprotein is also phosphorylated in vivo within the acidic domain (amino acids 631-658). These sites have been mapped using casein kinase II, a prototypic acidic-directed kinase, and a recombinant mdr1b linker region peptide (amino acids 621-687). Electrospray mass spectrometry demonstrated that casein kinase II could introduce up to five phosphates into the recombinant peptide. Two-dimensional phosphopeptide mapping indicated that all the phosphates were contained in a tryptic peptide consisting of amino acids 631-658. Phosphopeptide mapping of in vivo labeled P-glycoprotein, isolated from either J7.V1-1, a murine vinblastine-resistant cell line, or HeLa cells stably transfected with mdr1b P-glycoprotein cDNA, revealed that this tryptic peptide was phosphorylated in both proteins.