Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the extracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy.

Transglutaminase 2 (TG2, a.k.a. tissue transglutaminase) belongs to a family of transglutaminase enzymes that stabilize proteins by affecting covalent crosslinking via formation of amide bonds. Cell surface TG2 is directly involved as an adhesive receptor in cell-extracellular matrix (ECM) interactions. Here, we show that TG2 activity is elevated in glioblastomas compared with non-neoplastic brain. Immunofluorescent studies showed increased staining of fibronectin colocalized with TG2 in the ECM in glioblastomas. In addition, small clusters of invading human glioblastoma cells present in non-neoplastic brain parenchyma secrete high levels of TG2 and fibronectin that distinguish them from normal brain stroma. Downregulation of TG2 in U87MG glioblastoma cells with RNAi demonstrated decreased assembly of fibronectin in the ECM. Treatment with KCC009 blocked the remodeling of fibronectin in the ECM in glioblastomas in both in vitro and in vivo studies. KCC009 treatment in mice harboring orthotopic glioblastomas (DBT-FG) sensitized the tumors to N,N'-bis(2-chloroethyl)-N-nitrosourea chemotherapy, as measured by reduced bioluminescence, increased apoptosis and prolonged survival. The ability of KCC009 to interfere with the permissive remodeling of fibronectin in the ECM in glioblastomas suggests a novel target to enhance sensitivity to chemotherapy directed not only at the tumor mass, but also invading glioblastoma cells.

MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase.

Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.

Overexpression of IRF9 confers resistance to antimicrotubule agents in breast cancer cells.

IRF9/p48/ISGF3gamma (IRF9) is an IFN regulatory factor that mediates signaling by type I IFNs (IFNalpha and IFNbeta). After single-step selection of breast adenocarcinoma cells in paclitaxel, differential display and single gene analysis demonstrated that transcriptional activation of IRF9 and other IFN-responsive genes, independent of IFN, corresponded with resistance to antimicrotubule agents. Transient overexpression of IRF9 reproduced the drug-resistance phenotype and induced expression of IFN-responsive genes. However, drug resistance was not induced by overexpression of Stat1 or Stat2, or treatment with IFNalpha per se. Using a donor-matched array of cDNA prepared from human tumor and normal tissue from a variety of organs, we observed overexpression of IRF9 in approximately one-half of breast and uterine tumors, which indicated that IRF9 may be important in signaling in these tumor types. These data identify a novel IFN-independent role for IRF9 in the development of resistance to antimicrotubule agents in breast tumor cells and may link downstream mediators of IFN signaling to drug resistance in human cancers.

Functional imaging of multidrug-resistant P-glycoprotein with an organotechnetium complex.

The multidrug-resistant P-glycoprotein (Pgp), a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR1), appears to function as an energy-dependent efflux pump. Many of the drugs that interact with Pgp are lipophilic and cationic at physiological pH. We tested the hypothesis that the synthetic gamma-emitting organotechnetium complex, hexakis(2-methoxyisobutylisonitrile)technetium(I) ([99mTc]SESTAMIBI), a lipophilic cationic radiopharmaceutical, could be a suitable Pgp transport substrate capable of functional imaging of the MDR phenotype. The cellular pharmacological profile of [99mTc]SESTAMIBI transport was examined in Chinese hamster V79 lung fibroblasts and the 77A and LZ derivative cell lines which express modestly low, intermediate, and very high levels of Pgp, respectively. Steady-state contents of [99mTc]SESTAMIBI in V79, 77A, and LZ cells were 10.0 +/- 0.5 (SEM) (n = 9), 3.6 +/- 0.5 (n = 8), and 0.4 +/- 0.02 (n = 9) fmol.(mg protein)-1 (nMo)-1, respectively, consistent with enhanced extrusion of the imaging agent by Pgp-enriched cells. Maximal doses (> 100 microM) of the multidrug-resistant reversal agents verapamil and cyclosporin A enhanced [99mTc]SESTAMIBI accumulation in V79, 77A, and LZ cells by approximately 10-, 25-, and 200-fold, respectively. The median effective concentration values for tracer accumulation in the presence of verapamil in V79, 77A, and LZ cells were 4, 100, and 200 microM, and those for cyclosporin A were 0.9, 3, and > 25 microM, respectively. Pgp-mediated [99mTc]SESTAMIBI transport occurred against its electrochemical gradient and was found to be ATP dependent displaying an apparent Km of 50 microM. Carrier-added [99Tc]SESTAMIBI was 11- to 13-fold less toxic in multidrug-resistant cells, and inhibited photolabeling of Pgp by [125I]iodoaryl azidoprazosin in a concentration-dependent manner; half-maximal displacement was observed at approximately 100- to 1000-fold molar excess [99Tc]SESTAMIBI. Exploiting the favorable gamma emission properties of 99mTc, functional expression of Pgp was successfully imaged in human tumor xenographs in nude mice with pharmacologically inert tracer quantities of [99mTc]SESTAMIBI. Functional imaging with these organotechnetium complexes may provide a novel mechanism to rapidly characterize Pgp expression in human tumors in vivo, target reversal agents in vivo, and ultimately provide a means to direct patients to specific cancer therapies.

Uptake of the cation hexakis(2-methoxyisobutylisonitrile)-technetium-99m by human carcinoma cell lines in vitro.

Uptake of the cationic compound hexakis(2-methoxyisobutylisonitrile)-technetium-99m ([99mTc]MIBI) was examined in nine human tumor cell lines. The concentration of [99mTc]MIBI after a 1-h incubation with the compound varies from 5 to 28% of the activity in the external medium. In contrast, normal V79 cells (Chinese hamster lung fibroblasts) and human peripheral blood mononuclear cells exhibit a minimal uptake of less than 2% of the activity in the medium. Kinetic experiments with SW-13 cells indicate a rapid uptake over time (t1/2 of 10 min) until a steady state is approached whose concentration appears directly correlated with the extracellular concentration of [99mTc]MIBI with no evidence of saturation over the range tested (10(-12)-10(-9) M). [99mTc]MIBI is taken up by a temperature dependent process that is restricted to living cells. Microautoradiography demonstrates that [99mTc]MIBI is clustered in the cytoplasm around the nucleus. Given that depolarizing the plasma membrane potential in high K+ buffer results in lowering the uptake of [99mTc]-MIBI and that alteration of the mitochondrial membrane potential with valinomycin or nigericin induces, respectively, a significant decrease or increase of [99mTc]MIBI uptake, we propose that the plasma and mitochondrial membrane potentials play a major role in the uptake. These data suggest that the gamma emitter [99mTc]MIBI exhibits interesting tumor cell interaction characteristics with promise for in vivo tumor imaging.

Membrane potential determination in large unilamellar vesicles with hexakis(2-methoxyisobutylisonitrile)technetium(I).

The accumulation of the lipophilic cation hexakis (2-methoxyisobutylisonitrile)technetium (99mTc-MIBI) within large unilamellar vesicles made from egg phosphatidylcholine was examined as a function of time and membrane potential (Em). Equilibrium distribution occurred within minutes at 30 degrees C. The transmembrane distribution of Tc-MIBI was measured at Em = 0 mV and at a series of negative membrane potentials. The distribution of Tc-MIBI was in close agreement with the Nernst equation for passive distribution of a permeant ion across a bilayer, permitting the membrane potential to be predicted from Tc-MIBI distribution. In this respect, Tc-MIBI behaves similarly to other radioprobes of membrane potential, but with unique properties including high specific activity (10(9) Ci/mol), rapid kinetics of distribution, low potential-independent binding, and short half-life (6.02 h). The results indicate a mechanism for tissue accumulation of Tc-MIBI in vivo that may in part account for its utility in clinical imaging of ischemic myocardium.

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange.

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.

Na/H exchange in cultured chick heart cells. pHi regulation.

The purpose of this study was to establish the existence of Na/H exchange in cardiac muscle and to evaluate the contribution of Na/H exchange to pHi regulation. The kinetics of pHi changes in cultured chick heart cells were monitored microfluorometrically with 6-carboxyfluorescein and correlated with Nai content changes analyzed by atomic absorption spectrophotometry; transmembrane H+ movements were evaluated under pH stat conditions. After induction of an intracellular acid load by pretreatment with NH4Cl, a regulatory cytoplasmic alkalinization occurred with a t1/2 of 2.9 min. pHi regulation required external Na+ and was concomitant with transmembrane H+ extrusion as well as a rapid rise in Nai content in an Na/H ratio of 1:1. Microelectrode recordings of membrane potential demonstrated directly the electroneutral character of pHi regulation. Acid-induced net Na+ uptake could be either stimulated by further decreasing pHi or inhibited by decreasing pHo; Na+ uptake was unaffected by tetrodotoxin (10 micrograms/ml), quinidine (10(-3) M), DIDS (10(-4) M), Clo-free solution, or HCO3-free solution. Amiloride (10(-3) M) maximally inhibited both pHi regulation and Na+ uptake; the ID50 for amiloride inhibition of Na+ uptake was 3 microM. Nao-dependent H+ extrusion showed half-maximal activation at 15 mM Nao; Li+, but not K+ or choline+, could substitute for Na+ to support H+ extrusion. Cao-free solution also stimulated acid-induced Na+ uptake. We conclude that pHi regulation following an acid load in cardiac muscle cells is by an amiloride-sensitive, electroneutral Na/H exchange. Stimulation of Na/H exchange up to 54 pmol/cm2 X s indicates the rapidity of this exchange across cardiac cell membranes. Na/H exchange may also participate in steady state maintenance of pHi.

Theoretical effects of transmembrane electroneutral exchange on membrane potential.

Transmembrane electroneutral transport mechanisms [e.g., Na/H exchange, Cl/HCO3 exchange, (K + Cl) cotransport] have recently been identified in a wide variety of cell types. If these exchanges sum to give a net electroneutral Na/K exchange, they may hyperpolarize the membrane potential beyond the value calculated from the Mullins-Noda equation, provided the cell maintains steady state intracellular ionic concentrations. In extreme circumstances, the membrane potential could hyperpolarize beyond the potassium reversal potential. This effect is mediated by the electrogenic Na/K pump. If either Na or K exchanges electroneutrally against a third ion (e.g., Na/Ca exchange), then the exchange may depolarize the membrane potential.

Transmembrane chloride flux in tissue-cultured chick heart cells.

To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)-1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min-1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent.

Novel gallium(III) complexes transported by MDR1 P-glycoprotein: potential PET imaging agents for probing P-glycoprotein-mediated transport activity in vivo.

BACKGROUND: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients. Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy. RESULTS: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate. Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry. Radiolabeled (67)Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp(-)) and MDR (Pgp(+)) tumor cells. Compared with control, lead compound 6. demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells. Futhermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6. in brain and liver tissues of mdr1a/b((-/-)) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood-brain barrier and hepatocellular biliary cannalicular surface in vivo. CONCLUSIONS: These results indicate that gallium(III) complex 6. is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate (68)Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET.

Characterization of functional assays of multidrug resistance P-glycoprotein transport activity.

P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy. The P-glycoprotein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of chemotherapeutic agents. Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials. Characterization of P-glycoprotein functional activity is critical in determining if these multidrug resistance reversal agents improve therapeutic responses of tumors expressing P-glycoprotein. In this report, we directly compare and characterize assays using rhodamine 123, dimethyloxadicarbocyanine iodide (DiOC2), [3H]daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ([(99m)Tc]Sestamibi) as P-glycoprotein transport probes to quantitate functional activity. The accumulation of certain substrates is concentration dependent and the parameters which determine probe accumulation are impacted by the level of P-glycoprotein expression. In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expressing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein transport activity is higher than generally recognized. Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentrations of potent reversal agents. These results highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein function.

Tumor receptor imaging: proceedings of the National Cancer Institute workshop, review of current work, and prospective for further investigations.

In February 1994, the National Cancer Institute held a workshop to evaluate the current and future role of emission tomographic imaging methods, positron emission tomography and single-photon emission computed tomography, in improving the accuracy of cancer diagnosis and the effectiveness of treatment and in elucidating basic aspects of human cancer biology. Reviews covered many of the receptor and transport systems for hormones and growth factors, as well as metabolic changes important in human cancer, and topical presentations reviewed the current status of receptor-based imaging in the most well-characterized systems: somatostatin receptor imaging of neuroendocrine tumors, estrogen receptor imaging of breast cancer, and epidermal growth factor receptor and tumor metabolic imaging. A critical analysis was made of the current research and of new directions for the future development and use of receptor-imaging methods in oncology. In each area, recommendations were made for further investigation, where emerging understanding of tumor cell biology and defined molecular targets might be combined with the methods of radiopharmaceutical design and evaluation, to develop new approaches to critical issues in the diagnosis, staging, and treatment of cancer through tumor receptor imaging.

Effects of multidrug resistance (MDR1) P-glycoprotein expression levels and coordination metal on the cytotoxic potency of multidentate (N4O2) (ethylenediamine)bis[propyl(R-benzylimino)]metal(III) cations.

Enhanced mitochondrial transmembrane potentials in tumor cells have been proposed to confer tumor-selective-targeting properties to modestly lipophilic monocationic compounds. To explore the potential cytotoxic activity of lipophilic cationic metallopharmaceuticals containing a highly flexible hexadentate N4O2 Schiff-base phenolic ligand, we first synthesized precursors H3Mabi (1) and H3DMabi (2) by condensation of an appropriate linear tetraamine with substituted salicylaldehydes. The desired N4O2 ligands, (ethylenediamine)-N,N'-bis[propyl[(2-hydroxy-3-methoxybenzyl)imino]] and (ethylenediamine)-N,N'-bis[propyl[2-hydroxy-4,6-dimethoxybenzyl)-imino]] (R-ENBPI), were obtained by cleavage of the imidazolidine ring, and their corresponding monocationic complexes were produced by reaction with appropriate hydrated salts or acetylacetonates of Al(III), Fe(III), Ga(III), and In(III). All complexes were stable to neutral hydrolysis. In human epidermal carcinoma KB-3-1 cells, cytotoxic potencies of racemic mixtures of these complexes were in the low micromolar range and, for a given ligand, depended on the identity of the coordinating central metal. The active 4,6-dimethoxy-ENBPI complexes were more potent than their 3-methoxy analogs, while the free ligands and metal(III) ions showed little or no cytotoxic activity. Furthermore, in colchicine-selected KB-8-5 multidrug resistant (MDR) cells, modest cellular expression of human MDR1 P-glycoprotein conferred protection from the cytotoxic activities of Al(III), Fe(III), and Ga(III) R-ENBPI complexes indicating that these complexes were recognized as transport substrates by the P-glycoprotein efflux transporter. However, the cytotoxic activities of the corresponding In(III) complexes, while among the lowest in potencies, were also not altered by expression of MDR1 P-glycoprotein. Thus, for the Group III elements, human cells were capable of distinguishing R-ENBPI complexes formed of the same ligands with different metals. Furthermore, selected R-ENBPI metal(III) complexes may be useful as novel anticancer metallopharmaceuticals.

Novel hexakis(areneisonitrile)technetium(I) complexes as radioligands targeted to the multidrug resistance P-glycoprotein.

Transport substrates and modulators of the human multidrug resistance (MDR1) P-glycoprotein (Pgp) are generally lipophilic cationic compounds, many with substituted aryl moieties. We sought to synthesize aromatic technetium-isonitrile complexes to enable functional detection in vivo of Pgp expression in tissues. A series of substituted aromatic isonitrile analogs were synthesized from their corresponding amines by reaction with dichlorocarbene under phase transfer-catalyzed conditions, and the non-carrier-added hexakis(areneisonitrile)Tc-99m(I) complexes were produced by reaction with pertechnetate in the presence of sodium dithionite. Cellular accumulation in vitro, whole body biodistribution, and the imaging properties of these lipophilic, monocationic organometallic complexes were determined in Chinese hamster lung fibroblasts expressing MDR Pgp, in normal rats, and in rabbits, respectively. For this initial series, verapamil (50 microM), the classical Pgp modulator, significantly enhanced cellular accumulation or displaced binding of Tc complexes of 1b, 1d, 1h, 2a, 2d, 3a, and 3b, indicative of targeted interactions with Pgp. Most complexes, despite their modestly high lipophilicity, were excluded by the blood/brain barrier, and several complexes displayed simultaneously high hepatobiliary and renal excretion in vivo, consistent with the physiological expression pattern of Pgp in these tissues. Selected Tc- and Re-areneisonitrile complexes of this class have potential applicability to the functional imaging and modulation, respectively, of MDR Pgp in human tissues.

Multidrug resistance P-glycoprotein monoclonal antibody JSB-1 crossreacts with pyruvate carboxylase.

Multidrug resistance (MDR) is associated with overexpression of a 170 KD plasma membrane P-glycoprotein (P-gp), a putative energy-dependent efflux transporter that reduces intracellular accumulation of chemotherapeutic agents. For detection of P-gp expression in normal and malignant tissues, an MDR1-specific monoclonal antibody (MAb) JSB-1 has been used extensively. In this report we show that MAb JSB-1 crossreacts with a protein of M(r) approximately 130,000 present in rat liver mitochondrial inner membrane/matrix fractions. Peptide mapping and microsequencing identify this protein as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. MAb JSB-1 also crossreacts with purified PC from bovine liver. Under immunoblotting conditions, this crossreactivity is partially abolished by pre-incubation of MAb JSB-1 with a 1000-fold molar excess of MAb C494 epitope-specific peptide (PNTLEGN), indicating that the epitope of MAb JSB-1 may either overlap with or be in close proximity to that of MAb C494. Immunohistochemical cross-reactivity was also demonstrated in cryosections of human skeletal muscle, a tissue known not to express P-gp. MAb JSB-1 strongly immunostained Type 1 fibers, the subtype known to contain abundant mitochondria. Use of MAb JSB-1 for detection of MDR1 P-gp expression should be approached with caution.

Effect of mitochondrial and plasma membrane potentials on accumulation of hexakis (2-methoxyisobutylisonitrile) technetium(I) in cultured mouse fibroblasts.

Hexakis(2-methoxyisobutylisonitrile) technetium(I) (Tc-MIBI) is representative of a class of 99mTc-based lipophilic cationic myocardial perfusion imaging agents. To test the hypothesis that the mechanism of cellular uptake may involve distribution across biologic membranes in response to membrane potential, Tc-MIBI net uptake and retention were determined in cultured mouse BALB/c 3T3, NIH 3T3, and v-src transformed NIH 3T3 fibroblasts as well as in cultured chick embryo heart cells. Isovolumic depolarization of plasma membrane potentials with 130 mM K 20 mM Cl buffer decreased Tc-MIBI net cell uptake in all preparations. In BALB/c 3T3 cells, depolarizing mitochondrial membrane potential with valinomycin in high K buffer or with the protonophore CCCP inhibited net uptake and retention of Tc-MIBI while hyperpolarizing mitochondrial and plasma membrane potentials with the K+/H+ exchanger nigericin increased Tc-MIBI net uptake. These results indicated that net cellular uptake and retention of Tc-MIBI in fibroblasts were determined by both mitochondrial and plasma membrane potentials; the gamma-emitting properties of Tc-MIBI may therefore raise the possibility of monitoring membrane potential in vivo.

Modulation of the multidrug resistance P-glycoprotein: detection with technetium-99m-sestamibi in vivo.

UNLABELLED: Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) has been documented in nearly all forms of human cancers and increased levels of Pgp in some tumors correlate with poor response to treatment. Technetium-99m-sestamibi has recently been validated as a Pgp transport substrate. Pgp is also normally expressed along the biliary canalicular surface of hepatocytes and the luminal side of proximal tubule cells in the kidney, while not expressed in heart. METHODS: Focused on these organs with known Pgp status, we present the findings on 99mTc-sestamibi scintigraphy of three patients with refractory cancer who were imaged before and after administration of SDZ PSC 833, a second-generation, high-potency modulator of Pgp. RESULTS: Before treatment with SDZ PSC 833, scintigraphy using 99mTc-sestamibi showed normal, prompt clearance of the radiotracer from the liver and kidneys relative to the heart. After administration of the Pgp modulator, 99mTc-sestamibi was selectively retained in the liver and kidneys. CONCLUSION: Hepatobiliary and renal clearance of 99mTc-sestamibi are Pgp-mediated, and inhibition of Pgp transport in these organs can be successfully imaged using 99mTc-sestamibi in patients. Similar results might be expected with this and related radiopharmaceuticals for functional imaging of Pgp transport and modulation in tumors.

Enhancement by tetraphenylborate of technetium-99m-MIBI uptake kinetics and accumulation in cultured chick myocardial cells.

Myocellular uptake and retention of technetium-99m-hexakis (2-methoxyisobutylisonitrile) (Tc-MIBI), a lipophilic cationic myocardial perfusion and viability imaging agent, is dependent on both mitochondrial and plasma-membrane potentials. To test for enhancement of uptake kinetics by lipophilic anions, cultured chick heart cells were exposed to tetraphenylborate (TPB), which produced a concentration-dependent maximal 15-fold increase in Tc-MIBI uptake kinetics (at 3 x 10(-5) M) and enhanced peak accumulation of Tc-MIBI from 165.4 +/- 26.3 to 705.6 +/- 61.3 fmoles/mg protein.nMo (P less than 0.001). Carbonyl cyanide-m-chloro phenylhydrazone (CCCP; 10(-5) M), a mitochondrial uncoupler, rapidly depleted cellular content of Tc-MIBI in the presence of TPB (10(-5) M) from 300.0 +/- 30.0 to 42.5 +/- 1.9 fmole/mg protein.nMo (p less than 0.001). TPB enhanced both uptake rates and net accumulation of Tc-MIBI at all buffer Ko concentrations between 130 mM and 0.54 mM. Tc-MIBI influx rates allowed estimation of plasma-membrane potential as a function of Ko in the presence of valinomycin with a slope of -67 mV/decade (r = -0.99). The results further support a potential-dependent mechanism for cell uptake of Tc-MIBI and suggest a rational approach for increasing tissue extraction fraction in vivo.

Expression of recombinant human multidrug resistance P-glycoprotein in insect cells confers decreased accumulation of technetium-99m-sestamibi.

UNLABELLED: The multidrug-resistant P-glycoprotein is a M(r) 170,000 plasma membrane protein encoded by the mammalian multidrug resistance gene (MDR) which appears to function as an efflux transporter of a variety of potent chemotherapeutic agents. METHODS: To directly demonstrate that 99mTc-sestamibi is recognized by the human P-glycoprotein, we overexpressed recombinant human MDR1 P-glycoprotein in host Sf9 insect cells using a baculoviral vector and correlated expression of the gene product with 99mTc-sestamibi accumulation. RESULTS: In parental Sf9 cells and in wild-type baculoviral infected (control) cells, 99mTc-sestamibi accumulation asymptotically approached a plateau of 650 fmoles (mg protein)-1 (nMo)-1 and 337 fmoles (mg protein)-1 (nMo)-1, respectively. In MDR1 baculoviral infected cells, P-glycoprotein expression was maximal at 72 hr postinfection, while 99mTc-sestamibi accumulation was reduced to 12 fmole (mg protein)-1 (nMo)-1. Verapamil (500 microM), the classical MDR modulator, produced an approximately 300% enhancement of 99mTc-sestamibi accumulation in Sf9 cells expressing MDR1 P-glycoprotein, but only a 50% enhancement in parental Sf9 cells, consistent with verapamil-induced inhibition of P-glycoprotein-mediated 99mTc-sestamibi efflux. CONCLUSIONS: These data demonstrate that the recombinant protein is transiently expressed in a functional state capable of drug transport in Sf9 cell membranes and that 99mTc-sestamibi is a transport substrate recognized by the human MDR1 P-glycoprotein. Technetium-99m-sestamibi may prove useful for functionally characterizing P-glycoprotein expression in human tumors in vivo.