Xenobiotic transport across isolated brain microvessels studied by confocal microscopy.
AuthorsMiller DS,Nobmann SN,Gutmann H,Toeroek M,Drewe J,Fricker G
JournalMolecular pharmacology
PubMed ID11093774
Visualization of membrane-associated folate transport proteins.
AuthorsPope LE, Fan J, Chang CM, Minskoff SA, Vitols KS, Huennekens FM
JournalAdv Enzyme Regul
PubMed ID2516406
'Transport of Methotrexate (MTX) into cells, via the "reduced folate" transport system, is a critical factor in the effectiveness of the drug in cancer chemotherapy, and defective transport is one of the principal types of resistance to MTX. Probes capable of detecting membrane-associated folate transport proteins (ftp''s) in individual cells ... More
MRP2 and acquired tolerance to inorganic arsenic in the kidney of killifish (Fundulus heteroclitus).
'We used proximal tubules isolated from the killifish, Fundulus heteroclitus, to examine the effect of environmentally relevant, sublethal levels of arsenic on the function and expression of MRP2, an ABC transporter that transports xenobiotics into urine, including arsenic-glutathione conjugates. Exposure of fish to arsenic as sodium arsenite (4-14 days) increased ... More
Characterization of a lipophilic antifolate resistance provoked by treatment of mammalian cells with the antiparasitic agent pyrimethamine.
AuthorsAssaraf YG, Slotky JI
JournalJ Biol Chem
PubMed ID8440739
'We describe the characterization of an antitumor drug resistance following multiple step selection of hamster cells to the 2,4-diaminopyrimidines (DAP) metoprine, pyrimethamine (Pyr), and trimethoprim (Tmp). Pyr and Tmp are DAP lipophilic antifolates currently used as antiparasitic and antibacterial antibiotics, respectively. Dihydrofolate reductase (DHFR) from hamster cells bore a low ... More
Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia.
AuthorsTrippett T, Schlemmer S, Elisseyeff Y, Goker E, Wachter M, Steinherz P, Tan C, Berman E, Wright JE, Rosowsky A
JournalBlood
PubMed ID1381235
'Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4''-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of ... More
Flow cytometric analysis of mammalian glial cultures treated with methotrexate.
AuthorsSerrano EE, Schimke RT
JournalGlia
PubMed ID2148556
'Methotrexate (MTX) is an antineoplastic drug that acts by competitive inhibition of the enzyme dihydrofolate reductase (DHFR). MTX treatment of cultured cell lines leads to the emergence of resistant cell populations. Studies using stepwise selection procedures have demonstrated that MTX resistance conferred by overproduction of DHFR can be caused by ... More
Transport of fluorescein methotrexate by multidrug resistance-associated protein 3 in IEC-6 cells.
AuthorsLi T, Ito K, Horie T
JournalAm J Physiol Gastrointest Liver Physiol
PubMed ID12909565
'The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6. Enhanced accumulation of F-MTX at 4 degrees C suggests the existence of an active efflux system. MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the ... More
Fluorescent analogues of methotrexate: characterization and interaction with dihydrofolate reductase.
'The dansylated derivatives of lysine and ornithine analogues of methotrexate exhibit fluorescence properties characteristic of the dansyl moiety with an excitation at 328 nm and an emission maximum at 580 nm in aqueous media. As in the case of dansyl amino acids, the fluorescence emission is dependent upon the polarity ... More
Characterization by flow cytometry and fluorescein-methotrexate labeling of hydrophilic and lipophilic antifolate resistance in cultured mammalian cells.
AuthorsAssaraf YG
JournalAnticancer Drugs
PubMed ID8292810
'The aim of this review is to summarize currently available information on the rapid screening and initial characterization of the different mechanisms of resistance to hydrophilic [e.g. methotrexate (MTX)] and lipophilic antifolates [e.g. trimetrexate (TMTX)] in cultured mammalian cells using fluorescein-methotrexate (F-MTX) and flow cytometry. Toward this end an integrative ... More
A new fluorescent dihydrofolate reductase probe for studies of methotrexate resistance.
AuthorsRosowsky A, Wright JE, Shapiro H, Beardsley P, Lazarus H
JournalJ Biol Chem
PubMed ID7142200
'A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to ... More
Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy.
AuthorsMasereeuw R, Russel FG, Miller DS
JournalAm J Physiol
PubMed ID8997391
'Previous studies with p-aminohippurate (PAH) and fluorescein (FL) have shown that cellular uptake and tubular secretion of organic anions is driven by indirect coupling to sodium. Here we used killifish proximal tubules and laser-scanning confocal microscopy to study the transport of a larger organic anion, fluorescein-methotrexate (FL-MTX, mol mass 923 ... More
Enhancement of the frequency of methotrexate resistance by gamma-radiation in Chinese hamster ovary and mouse 3T6 cells.
AuthorsSharma RC, Schimke RT
JournalCancer Res
PubMed ID2736529
'We have studied the effects of gamma-radiation on the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. gamma-Irradiation of Chinese hamster ovary cells resulted in enhancement of the frequency of methotrexate resistant colonies (maximum enhancement: 2000-fold after 1000 rads). The enhancement of methotrexate resistance was dependent on the dose ... More
Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays.
AuthorsRemy I, Michnick SW
JournalProc Natl Acad Sci U S A
PubMed ID10318894
'Two strategies are described for detecting constitutive or induced protein-protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP ... More
Characterization by flow cytometry of fluorescein-methotrexate transport in Chinese hamster ovary cells.
AuthorsAssaraf YG, Seamer LC, Schimke RT
JournalCytometry
PubMed ID2917475
'We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ovary cells. Fluorescein-methotrexate appears to enter cells via a mechanism different from the carrier-mediated system for methotrexate. This conclusion is supported by the following observations: 1) Fluorescein-methotrexate is transported equally well into normal and mutant cells defective ... More
Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy.
AuthorsBreen CM, Sykes DB, Baehr C, Fricker G, Miller DS
JournalAm J Physiol Renal Physiol
PubMed ID15126245
'One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath ... More
Cytological, flow cytometric, and molecular analysis of the rapid evolution of mammalian chromosomes containing highly amplified DNA sequences.
AuthorsFendrock B, Destrempes M, Kaufman RJ, Latt SA
JournalHistochemistry
PubMed ID3086259
'Transfection of a mouse dihydrofolate reductase (DHFR) cDNA contained in a plasmid "expression vector" into DHFR deficient Chinese hamster cells, followed by progressive selection of cells in increasing concentrations of methotrexate (MTX), leads to marked amplification of the exogenous DHFR sequences in the recipient hamster cells. This gene amplification is ... More
Quantitation of dihydrofolate reductase in individual parental and methotrexate-resistant murine cells. Use of a fluorescence activated cell sorter.
AuthorsKaufman RJ, Bertino JR, Schimke RT
JournalJ Biol Chem
PubMed ID670236
Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells.
AuthorsFan J, Vitols KS, Huennekens FM
JournalJ Biol Chem
PubMed ID1869524
'Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3''-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step ... More
Xenobiotic efflux pumps in isolated fish brain capillaries.
AuthorsMiller DS, Graeff C, Droulle L, Fricker S, Fricker G
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID11742838
'To identify specific transporters that drive xenobiotics from the central nervous system to blood, the accumulation of fluorescent drugs was studied in isolated capillaries from killifish and dogfish shark brain using confocal microscopy and quantitative image analysis. In killifish brain capillaries, luminal accumulation of fluorescent derivatives of cyclosporin A and ... More
Cross-resistance to the lipid-soluble antifolate trimetrexate in human carcinoma cells with the multidrug-resistant phenotype.
AuthorsAssaraf YG, Molina A, Schimke RT
JournalJ Natl Cancer Inst
PubMed ID2521520
'We are studying mechanisms of resistance to hydrophilic and lipophilic antifolates in cultured mammalian cells. We determined the cytotoxicity of methotrexate and the lipid-soluble antifolate trimetrexate to various human carcinoma cells and their doxorubicin-resistant sublines. These multidrug-resistant cells were 17-fold to 26-fold more resistant to trimetrexate but as sensitive to ... More
A fluorescein-methotrexate-based flow cytometric bioassay for measurement of plasma methotrexate and trimetrexate levels.
AuthorsAssaraf YG, Molina A, Schimke RT
JournalAnal Biochem
PubMed ID2526605
'We describe a bioassay for the quantitation of plasma methotrexate and trimetrexate levels employing intact cells. This assay is based on the intracellular saturation of dihydrofolate reductase with fluorescein-methotrexate (F-MTX) and its dose-dependent displacement by methotrexate or trimetrexate as monitored by flow cytometry. Serially diluted methotrexate-containing plasma, representing a wide ... More
Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated methotrexate and daunorubicin.
AuthorsSherwood SW, Assaraf YG, Molina A, Schimke RT
JournalCancer Res
PubMed ID2143099
'We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry. Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining ... More
Fluorescent methotrexate labeling and flow cytometric analysis of cells containing low levels of dihydrofolate reductase.
AuthorsGaudray P, Trotter J, Wahl GM
JournalJ Biol Chem
PubMed ID3700393
'Previous studies have demonstrated the usefulness of flow cytometry in the analysis of dihydrofolate reductase (EC 1.5.1.3) gene amplification. However, this powerful and potentially sensitive method for analyzing gene expression in individual cells has not seen widespread use. This is due in part to the difficulty of producing fluorescent methotrexate ... More
Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate.
AuthorsAssaraf YG, Molina A, Schimke RT
JournalJ Biol Chem
PubMed ID2572592
'We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic ... More
Gene amplification, drug resistance, and cancer.
AuthorsSchimke RT
JournalCancer Res
PubMed ID6713376
Flow cytometric studies of methotrexate resistance in human squamous carcinoma cell cultures.
AuthorsWright JE, Rosowsky A, Boeheim K, Cucchi CA, Frei E
JournalBiochem Pharmacol
PubMed ID3579991
Transport of a fluorescent antifolate by methotrexate-sensitive and methotrexate-resistant human leukemic lymphoblasts.
AuthorsRosowsky A, Wright JE, Cucchi CA, Boeheim K, Frei E
JournalBiochem Pharmacol
PubMed ID3942605
A fluorescent derivative of methotrexate as an intracellular marker for dihydrofolate reductase in L1210 cells.
AuthorsHenderson GB, Russell A, Whiteley JM
JournalArch Biochem Biophys
PubMed ID6772112
Erythropoietin receptor activation by a ligand-induced conformation change.
AuthorsRemy I, Wilson IA, Michnick SW
JournalScience
PubMed ID9974393
Erythropoietin and other cytokine receptors are thought to be activated through hormone-induced dimerization and autophosphorylation of JAK kinases associated with the receptor intracellular domains. An in vivo protein fragment complementation assay was used to obtain evidence for an alternative mechanism in which unliganded erythropoietin receptor dimers exist in a conformation ... More
Use of fluorescence-activated cell sorter to isolate mutant mammalian cells deficient in an internal protein, dihydrofolate reductase.
AuthorsUrlaub G, McDowell J, Chasin LA
JournalSomat Cell Mol Genet
PubMed ID3856331
Dihydrofolate reductase-deficient mutants of Chinese hamster ovary cells have been selected based on their inability to bind a fluorescent derivative of methotrexate, a substrate analog. Nonfluorescent mutant cells were isolated from mutagenized populations using a fluorescence-activated cell sorter. After multiple rounds of sorting plus regrowth, the mutant cell frequency was ... More
Fluorescein-methotrexate transport in brush border membrane vesicles from rat small intestine.
AuthorsLi T, Tomimatsu T, Ito K, Horie T
JournalLife Sci
PubMed ID12967687
The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed ... More
Synthesis of a fluorescent derivative of amethopterin,.
AuthorsGapski GR, Whiteley JM, Rader JI, Cramer PL, Henderson GB, Neef V, Huennekens FM
JournalJ Med Chem
PubMed ID807734
Fluorescein isothiocyanate was treated with excess diaminopentane and the remaining unsubstituted amino group of the product was condensed, via a carbodiimide-promoted reaction, with a carboxyl group of amethopterin. The final product, a fluorescent derivative of amethopterin, was isolated by chromatography on AE-cellulose and preparative electrophoresis on polyacrylamide. It was shown ... More
The immunoassay of methotrexate by capillary electrophoresis with laser-induced fluorescence detection.
AuthorsSuzuki Y, Arakawa H, Maeda M
JournalAnal Sci
PubMed ID12558033
Immunoassay is widely employed as a highly sensitive, specific analytical method for hormones and drugs in biological samples. A technique utilizing capillary electrophoresis with laser-induced fluorescence detection was examined based on the reaction process of these immunoassays in order to develop a protocol characterized by high sensitivity and high speed. ... More
Nucleoside phosphonate interactions with multiple organic anion transporters in renal proximal tubule.
AuthorsMiller DS
JournalJ Pharmacol Exp Ther
PubMed ID11602668
The interactions of two antiviral, acyclic nucleoside phosphonates, adefovir and cidofovir, with xenobiotic transporters was studied in intact killifish (Fundulus heteroclitus) renal proximal tubules by using fluorescent substrates, confocal microscopy, and quantitative image analysis. Both drugs reduced in a concentration-dependent manner the transport of fluorescein on the classical organic anion ... More
Visualization of biochemical networks in living cells.
AuthorsRemy I, Michnick SW
JournalProc Natl Acad Sci U S A
PubMed ID11438723
Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a ... More
BacMam recombinant baculovirus in transporter expression: a study of BCRP and OATP1B1.
AuthorsHassan NJ, Pountney DJ, Ellis C, Mossakowska DE
JournalProtein Expr Purif
PubMed ID16481201
Human BCRP and OATP1B1 have recently been identified as important transporters in the absorption, distribution, and elimination of clinically significant drugs. In this report, we illustrate the use of modified baculoviruses, termed BacMam viruses for the expression of functional BCRP and OATP1B1 in mammalian cells. We show a variety of ... More
Direct visualization of protein interactions in plant cells.
AuthorsSubramaniam R, Desveaux D, Spickler C, Michnick SW, Brisson N
JournalNat Biotechnol
PubMed ID11479572
The protein NPR1/NIM1 is required for the induction of systemic acquired resistance (SAR) in plants and has been shown to interact with members of the TGA/OBF family of basic leucine zipper (bZIP) transcription factors. However, to date, there is no method available to monitor such interactions in plant cells. We ... More
We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell ... More
Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.
AuthorsYoshikawa T, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, Kishimoto M, Suga KI
JournalBiotechnol Bioeng
PubMed ID11427945
In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed ... More
Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein.
AuthorsIsrael DI, Kaufman RJ
JournalProc Natl Acad Sci U S A
PubMed ID8483945
A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to ... More
Dual pathways for organic anion secretion in renal proximal tubule.
AuthorsMiller DS, Pritchard JB
JournalJ Exp Zool
PubMed ID9392867
Transport on the "classical" organic anion system in renal proximal tubule is specific, active, Na-dependent, and ouabain sensitive. Here we review recent studies using intact teleost proximal tubules and laser scanning confocal microscopy which show that the secretion of large organic anions, such as, fluorescein-methotrexate (FL-MTX, Mw 923 Da) is ... More
Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule.
AuthorsNotenboom S, Miller DS, Smits P, Russel FG, Masereeuw R
JournalAm J Physiol Renal Physiol
PubMed ID14970002
In killifish renal proximal tubules, endothelin-1 (ET-1), acting through a basolateral ET(B) receptor, nitric oxide synthase (NOS), and PKC, decreases cell-to-lumen organic anion transport mediated by the multidrug resistance protein isoform 2 (Mrp2). In the present study, we examined the roles of guanylyl cyclase and cGMP in ET signaling to ... More
Effect of amplification of dhfr and lac Z genes on growth and beta-galactosidase expression in suspension cultures of recombinant CHO cells.
AuthorsGu MB, Kern JA, Todd P, Kompala DS
JournalCytotechnology
PubMed ID1369176
Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human beta-interferon (beta-IFN) or the lac Z gene which ... More
P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule.
AuthorsGutmann H, Miller DS, Droulle A, Drewe J, Fahr A, Fricker G
JournalBr J Pharmacol
PubMed ID10694230
1. Transepithelial transport of a fluorescent derivative of octreotide (NBD-octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus). 2. Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD-octreotide into the tubular ... More
Interactions of HIV protease inhibitors with ATP-dependent drug export proteins.
AuthorsGutmann H, Fricker G, Drewe J, Toeroek M, Miller DS
JournalMol Pharmacol
PubMed ID10419558
We used renal proximal tubules from a teleost fish (killifish; Fundulus heteroclitus), fluorescent substrates and confocal microscopy to study the interactions between human immunodeficiency virus protease inhibitors and drug-transporting ATPases. Both saquinavir and ritonavir inhibited luminal accumulation of a fluorescent cyclosporin A derivative (a substrate for P-glycoprotein) and of fluorescein ... More
Characteristics of transport of fluoresceinated methotrexate in rat small intestine.
AuthorsNagakubo J, Tomimatsu T, Kitajima M, Takayama H, Aimi N, Horie T
JournalLife Sci
PubMed ID11487087
The small intestinal damage induced by the methotrexate (MTX) treatment results in malabsorption and diarrhea. The fluoresceinated methotrexate (F-MTX) may possibly be useful to study such effects of MTX on the small intestine. The purpose of this study is to characterize the transport of F-MTX in the small intestine in ... More
Excretory transport of xenobiotics by dogfish shark rectal gland tubules.
Marine elasmobranch rectal gland is a specialized, osmoregulatory organ composed of numerous blind-ended, branched tubules emptying into a central duct. To date, NaCl excretion has been its only described function. Here we use isolated rectal gland tubule fragments from dogfish shark (Squalus acanthias), fluorescent xenobiotics, and confocal microscopy to describe ... More
Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon.
AuthorsPublicover NG, Horowitz NN, Sanders KM
JournalAm J Physiol
PubMed ID1550205
Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and ... More
Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule.
AuthorsMasereeuw R, Terlouw SA, van Aubel RA, Russel FG, Miller DS
JournalMol Pharmacol
PubMed ID10617679
In the kidney, endothelins (ETs) are important regulators of blood flow, glomerular hemodynamics, and sodium and water homeostasis. They have been implicated in the pathophysiology of acute ischemic renal failure, nephrotoxicity by cyclosporine, cisplatin and radiocontrast agents, and vascular rejection of kidney transplants. Here, we used intact killifish renal proximal ... More
Characterization of the coexisting multiple mechanisms of methotrexate resistance in mouse 3T6 R50 fibroblasts.
AuthorsAssaraf YG, Feder JN, Sharma RC, Wright JE, Rosowsky A, Shane B, Schimke RT
JournalJ Biol Chem
PubMed ID1372892
We have studied the discrepancy in the degree of methotrexate (MTX) resistance that exists between two clonal cell lines, mouse 3T6 R50 cells and Chinese hamster ovary B11 0.5 cells that overexpress comparable levels of dihydrofolate reductase, yet exhibit a 100-fold difference in MTX resistance while maintaining similar sensitivity to ... More
Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.
AuthorsFan JG, Pope LE, Vitols KS, Huennekens FM
JournalBiochemistry
PubMed ID1902381
Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the ... More
Nephrotoxicants induce endothelin release and signaling in renal proximal tubules: effect on drug efflux.
AuthorsTerlouw SA, Masereeuw R, Russel FG, Miller DS
JournalMol Pharmacol
PubMed ID11353803
We previously used killifish proximal tubules, fluorescent substrates, and confocal microscopy to demonstrate that transport mediated by the multidrug resistance protein (Mrp2) and by P-glycoprotein was reduced by nanomolar concentrations of endothelin-1 (ET), acting through a basolateral B-type ET receptor and protein kinase C (PKC). Here we show that representatives ... More
Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule.
AuthorsTerlouw SA, Graeff C, Smeets PH, Fricker G, Russel FG, Masereeuw R, Miller DS
JournalJ Pharmacol Exp Ther
PubMed ID11961059
We recently demonstrated in isolated killifish renal proximal tubules that two classes of nephrotoxicants, aminoglycoside antibiotics and radiocontrast agents, rapidly decrease transport mediated by multidrug resistance protein 2 (Mrp2) by causing endothelin (ET) release and signaling through an ET(B) receptor and protein kinase C (PKC). In the present study, we ... More
Synthesis and biological evaluation of a fluorescent analogue of folic acid.
A fluorescein derivative of the lysine analogue of folic acid, N alpha-pteroyl-N epilson-(4'-fluoresceinthiocarbamoyl)-L-lysine (PLF), was synthesized as a probe for dihydrofolate reductase (DHFR) and a membrane folate binding protein (m-FBP). Excitation of PLF at 282 nm and at 497 nm gave a fluorescence emission maximum at 518 nm. Binding of ... More
A phenotype conferring selective resistance to lipophilic antifolates in Chinese hamster ovary cells.
AuthorsSharma RC, Assaraf YG, Schimke RT
JournalCancer Res
PubMed ID1674447
Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them ... More
Confocal microscopy visualization of antifolate uptake by the reduced folate carrier in human leukaemic cells.
AuthorsJolivet J, Faure MP, Wong SC, Taub JW, Matherly LH
JournalBr J Cancer
PubMed ID9310238
Confocal microscopy was used to visualize the intracellular uptake of the fluorescent methotrexate analogue, fluorescein-MTX (F-MTX), in human leukaemic cell lines and leukaemic blasts. Cytosolic labelling of wild-type K562 human erythroleukaemia cells was detected during 3-60 min incubations with F-MTX (1 microM) and was completely inhibited by co-exposure to either ... More
Role of NO in endothelin-regulated drug transport in the renal proximal tubule.
AuthorsNotenboom S, Miller DS, Smits P, Russel FG, Masereeuw R
JournalAm J Physiol Renal Physiol
PubMed ID11832426
We previously demonstrated in intact killifish renal proximal tubules that endothelin (ET), acting through an ET(B) receptor and protein kinase C (PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, and Miller DS. ... More