Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1167-1172, February 1996 Medical Sciences

Uptake of fluorescent associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells (gene therapy) ROBERT P. WERSTO*t, EUGENE R. ROSENTHAL*, RONALD G. CRYSTAL*t, AND KENNETH R. SPRING§ *Pulmonary Branch and §Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Maurice B. Burg, National Institutes of Health, Bethesda, MD, October 12, 1995 (received for review January 3, 1995)

ABSTRACT Specific mutations in the cystic fibrosis ing the full length CFTR cDNA or mock virus, respectively transmembrane conductance regulator (CFTR), the most (10), were generously provided by R. Frizzell (University of common autosomal recessive fatal genetic disease of Cauca- Alabama, Birmingham). CFPAC cells were also cultured in sians, result in the loss of epithelial cell adenosine 3',5'-cyclic- IMDM. C127 cells (mouse mammary tumor cells) stably monophosphate (cAMP)-stimulated Cl- conductance. We overexpressing wild-type CFTR and a mock-transfected con- show that the influx of a fluorescent , dihydrorhodamine trol (11) were generously provided by S. Cheng (Genzyme) 6G (dR6G), is increased in cells expressing human CFTR after and were maintained in IMDM containing Geneticin sulfate retrovirus- and adenovirus-mediated gene transfer. dR6G (G418, GIBCO/BRL) at 200 gg/ml. influx is stimulated by cAMP and is inhibited by antagonists Recombinant, replication-deficient adenoviral vectors con- of cAMP action. Dye uptake is ATP-dependent and inhibited taining the full-length human CFTR cDNA (12) were used to by Cl- removal or the addition of 10 mM SCN-. Increased convey cAMP-dependent anion conductance to IB3-1 cells. staining is associated with functional activation of CFIR Cl- Adenoviral constructs expressing human catalase (AdCL; ref. permeability. dR6G staining enables both the fluorescent 13) and human al-antitrypsin (AdalAT; ref. 14) were used as assessment of C1ITR function and the identification of suc- viral controls. Recombinant virus was propagated, purified by cessfully corrected cells after gene therapy. cesium chloride density gradient centrifugation, and titered by plaque assay on human embryonic kidney 293 cells as de- From the time of the initial discovery of the gene coding for scribed (12). Recombinant virus was stored in vehicle buffer the human cystic fibrosis transmembrane conductance regu- [10% (vol/vol) glycerol/10 mM Tris HCl/1 mM MgCl2, pH lator (CFTR), questions were raised about its role in the 7.4]. Viral titers are expressed as the number of plaque- transport of solutes other than Cl- (1). CFTR belongs to the forming units (pfu) and infections are described as pfu per cell. "traffic ATPase" superfamily (2) which includes the MDR-1 CFPAC-PLJ, uninfected IB3-1 cells, or IB3-1 cells infected P-glycoprotein, responsible for the transport of diverse che- with AdCL or AdalAT adenoviral vectors lack both cAMP- motherapeutic drugs (3) and fluorescent dyes (4) out of cells. mediated anion permeability and CFTR protein expression as Although it is well established that CFTR is a Cl- channel judged by immunoprecipitation (9, 10). regulated by cAMP (5), it has been shown that it can regulate Cells for measurements were seeded at a the transport of other solutes (6) and may transport organic density of 2 x 104 cells per cm2 and grown for 72 hr in substrates (7, 8). In the present study, we identify two com- coverglass chambers (Nunc) coated with recombinant fi- pounds whose steady-state fluorescence and rate of uptake are bronectin (ProNectin; Biosource International, San Diego). preferentially associated with functional CFTR expression in IB3-1 cells were infected with adenoviral vectors at the time of epithelial cells and may serve as useful indicators of CFTR plating. function. Fluorescent Dyes. The following dyes were tested: Hoechst 33342; 123; ; fluo-3 acetoxymethyl ester (AM); 2',7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluo- MATERIALS AND METHODS rescein (BCECF) AM; acridine orange 10-nonyl bromide; Cell Culture and Viral Constructs. 1B3-1 cells, derived from Lucifer yellow; octadecyl acridine orange; 9-amino-6-chloro- the bronchial epithelium of an individual with cystic fibrosis 2-methoxyacridine; 7-amino-4-chloromethylcoumarin; hydro- (CF) having a compound heterozygote genotype (A508 mu- ethidium (HET); dihydrorhodamine 123; dihydrorhodamine tation on one allele) were a gift of P. Zeitlin (Johns Hopkins 6G (dR6G); and FluoroBora T. HET and FluoroBora T were University, Baltimore) and cultured on fibronectin-coated purchased from Polyscience. All other fluorescent dyes were substrates as described (9). NIH 3T3 cells (mouse fibroblast obtained from Molecular Probes. Cell membrane anion con- line) and 3T3 cells expressing the human MDR-1 P- ductance was assessed with 6-methoxy-N-(3-sulfopropyl) glycoprotein (3T3-MDR cells) were a gift of M. Gottesman quinolinium (SPQ; Molecular Probes). Once opened, HET (National Cancer Institute) and were grown in Iscove's mod- ified Dulbecco's medium (IMDM; GIBCO/BRL) containing Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane conduc- In tance regulator; CMFDA, 5-chloromethylfluorescein diacetate; 10% fetal bovine serum, 4 mM L-glutamine, and antibiotics. dR6G, dihydrorhodamine 6G; HET, hydroethidium; pfu, plaque- addition, the medium for the 3T3-MDR cells contained col- forming unit(s); SPQ, 6-methoxy-N-(3-sulfopropyl)quinolinium; cpt- chicine at 60 ng/ml. CFPAC-CFTR and CFPAC-PLJ cells cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic-monophosphate. (clone 10), pancreatic adenocarcinoma cells homozygous for tPresent address: Laboratory of Pathology, National Cancer Institute, the A508 mutation and transformed with a retrovirus contain- National Institutes of Health, Bethesda, MD 20892. tPresent address: Division of Pulmonary and Critical Care Medicine, Cornell University Medical College, New York, NY 10021. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Building 10, Room payment. This article must therefore be hereby marked "advertisement" in 6N307, National Heart, Lung, and Blood Institute, National Institutes accordance with 18 U.S.C. §1734 solely to indicate this fact. of Health, Bethesda, MD 20892.

Downloaded by guest on October 2, 2021 1167 1168 Medical Sciences: Wersto et al. Proc. Natl. Acad. Sci. USA 93 (1996)

and dR6G were stored under vacuum at -70°C and were chlorophenylthio)adenosine 3',5'-cyclic-monophosphate (cpt- freshly dissolved in N,N-dimethylformamide and dimethyl cAMP) in NO--containing buffer. sulfoxide, respectively, for each experiment. Dye solutions that Statistics. Data are expressed as mean + SEM. Statistical were not colorless were discarded and a new batch of dye used. significance was calculated with Student's t test. dR6G and Cyclosporin A was generously provided by James Weaver SPQ rates were calculated by a linear least-squares method (Center for Drug Evaluation, Food and Drug Administration, (Curve-Fit 2D; Jandel Scientific, San Mateo, CA). Washington, DC). The protein kinase A inhibitor Rp-cpt- cAMPS was purchased from Biolog, La Jolla CA. Forskolin RESULTS was purchased from Calbiochem-Novabiochem. All other drugs were obtained from Sigma. Steady-State Fluorescence Differences. To screen a large Fluorescence Microscopy and Image Analysis. Coverglass number of diverse fluorescent dyes (see Materials and Meth- chambers containing stained cells were mounted on the stage ods) for staining differences between CFTR-expressing and of an inverted microscope (Diaphot, Nikon) and fluorescence -nonexpressing cells, CFPAC-CFTR and CFPAC-PLJ cells was visualized with a X40 (1.30 n.a.) oil-immersion lens. The were treated for 15 min with dyes (0.1-10 ,uM) and the microscope was equipped with a 75-W xenon lamp and mo- steady-state fluorescence was quantitated. Only two com- torized filterwheel (LEP, Hawthorne, NY) as the excitation pounds, HET and dR6G, showed statistically significant in- source. An environmental enclosure permitted experiments to creases in steady-state staining levels in CFTR-expressing be done at 37°C or room temperature (22°C). Digitized images cells. Likewise, increases in steady-state fluorescence were also were obtained with a CCD camera (CCD 200; Video Scope, observed in HET- or dR6G-stained IB3-1 cells infected with Sterling, VA) attached to a image intensifier (VS2525; Video an adenoviral vector conveying the full-length human CFTR Scope) and analyzed with the IMAGEl/FLUOR software pack- cDNA (Table 1). Experiments in other CFTR-expressing age (Universal Imaging, West Chester, PA). Excitation inten- systems [CFTR-transfected and mock-transfected C127 mouse sities were attenuated to 1% with neutral density filters to mammary tumor cells (10) and AdCFTR-infected mouse 3T3 minimize photobleaching. cells] showed similar increases in steady-state fluorescence in HET fluorescence was excited at 350 nm and measured at CFTR-expressing cells (data not shown) and further con- 620 nm; dR6G fluorescence was excited at 480 nm and firmed the association between increased HET and dR6G detected at 575 nm. SPQ fluorescence was excited at 350 nm staining and CFTR expression. and detected at 440 nm. All manipulations with HET and Both HET and dR6G are nonfluorescent and must be dR6G were carried out under darkroom conditions. dehyrogenated to their fluorescent parent compounds, Dye Staining. For the steady-state experiments, cells were ethidium and rhodamine 6G, respectively. Neither ethidium loaded with HET (14 ,ug/ml) by incubation for 15 min at 22°C nor rhodamine 6G exhibited increased levels of fluorescence in phosphate-buffered saline supplemented with 2 mM MgSO4 staining in CFPAC-CFTR cells or AdCFTR-infected IB3-1 and 2 mM CaCl2. After staining, cells were washed three times cells, suggesting that these forms were not associated with and incubated for 30 min at 37°C in bicarbonate- and phenol functional CFTR expression. Because the increase in HET or red-free minimal essential medium (MEM; GIBCO/BRL) dR6G dye accumulation by the CFPAC-CFTR or AdCFTR- supplemented with 25 mM Hepes. Cells were loaded with 1 infected IB3-1 cells could be due to accelerated dye uptake, ,tM dR6G at 22°C in bicarbonate- and phenol red-free MEM decreased dye extrusion, or the increased activity of cellular containing 20 mM Hepes and 0.1% glucose. After 15 min, dye dehydrogenases (16), we reasoned that HET would be inap- was aspirated and replaced with fresh medium and the cells propriate for dye influx or efflux studies because of its toxicity were incubated for an additional 30 min at 37°C. For the dR6G as a potent mutagen and its high affinity for nucleic acids (17). uptake studies, cellular fluorescence was measured at various Since dR6G does not share these properties, subsequent influx times following the addition of 1 ,uM dR6G to the medium. experiments used dR6G exclusively. Cells were isotonically loaded with SPQ by inclusion of 10 Increased Staining Is Not Due to MDR-1. Increased steady- mM dye in the culture medium for 12-16 hr. Cells were state staining of CFPAC-CFTR cells by dR6G, above the levels incubated in Nal buffer for 10 min prior to measurements. In observed in control CFPAC-PLJ cells, could be due to dye this assay, SPQ fluorescence is quenched by 1- and not by extrusion in the CFPAC-PLJ cells mediated by the MDR-1 NO-. Thus, an increase in SPQ fluorescence is a measure of P-glycoprotein. To exclude this possibility, CFPAC-CFTR and NO- entry and I- efflux (15). An increase in cAMP- CFPAC-PLJ cells were treated with agents which interfere dependent anion permeability due to CFTR was evaluated by with MDR-1 P-glycoprotein drug transport and then stained exposure of cells to a mixture of 20 ,uM forskolin, 500 ,uM with HET or dR6G. Neither verapamil (16 ,uM, 15 min), a 3-isobutyl-1-methylxanthine (IBMX), and 200 ,uM 8-(4- Ca2+ channel blocker (18), nor cyclosporin A (1 ,ug/ml, 5-15 Table 1. Relative steady-state fluorescence of stained cells CPFAC-CFTR IB3-1 AdCFTR IB3-1 AdCL NIH 3T3 MDR-1 Dye vs. CFPAC-PLJ vs. IB3-1 vs. IB3-1 vs. NIH 3T3 dR6G 1.95 + 0.14* (n = 360) 1.48 ± 0.08* (n = 810) 1.19 ± 0.07 (n = 622) 0.01 ± 0.00 (n = 90) HET 3.87 + 0.43* (n = 180) 2.50 ± 0.39* (n = 240) 0.90 ± 0.14 (n = 180) 0.27 ± 0.05 (n = 150) R123 0.85 ± 0.05 (n = 237) 0.91 ± 0.06 (n = 180) 0.90 ± 0.06 (n = 180) 0.01 ± 0.00 (n = 87) dR123 0.58 ± 0.031 (n = 240) 0.77 ± 0.07 (n = 90) 0.84 ± 0.04 (n = 90) 0.02 ± 0.00 (n = 90) Fluo-3 0.66 ± 0.11 (n = 180) 1.09 ± 0.05 (n = 90) 0.82 ± 0.05 (n = 90) 0.08 ± 0.01 (n = 90) Steady-state fluorescence of dyes loaded into transfected cells is shown divided by that of control cells (CFPAC-CFTR vs. CFPAC-PLJ; AdCFTR-infected IB3-1 vs. uninfected; AdCL-infected IB3-1 vs. uninfected; NIH 3T3 MDR-1 vs. NIH 3T3). The total number of cells is denoted by n; typically 30 cells were measured in each chamber. A ratio > 1 represents dye accumulation; a ratio < 1 indicates dye extrusion. dR6G staining was lacking in NIH 3T3 MDR cells because rhodamine 6G is an efficient substrate for MDR-1 and was presumably extruded as rapidly as the dR6G was dehydrogenated. Statistically significant differences (P < 0.025) in staining between CFPAC-CFTR and CFPAC-PL cells, AdCFTR-infected IB3-1 and uninfected IB3-1 cells, and AdCFTR-infected IB3-1 and AdCL-infected IB3-1 cells are indicated (*). Differences between uninfected IB3-1 and AdCL-infected IB3-1 cells were not statistically significant (P > 0.68) for HET and dR6G staining. R123, ; dR123, dihydrorhodamine 123. Downloaded by guest on October 2, 2021 Medical Sciences: Wersto et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1169 min; ref. 19) had any effect on dR6G steady-state staining surement of dR6G staining and cAMP-stimulated anion per- differences or on cAMP-mediated anion permeability (data meability could not be accomplished because rhodamine 6G, not shown). MDR-1 P-glycoprotein expression, assessed by the reduced form of dR6G in the cell, is quenched by I- and , was absent in both the CFPAC-CFTR and NO- used in the SPQ assay (22). Increased staining of CFPAC-PLJ cells (data not shown). By contrast, stably trans- AdCFTR-infected IB3-1 cells by dR6G was not a consequence fected NIH 3T3 cells over-expressing MDR-1 P-glycoprotein of adenoviral infection. Cells treated with AdCL or AdalAT did not stain with rhodamine 123, Fluo-3 AM, dihydrorho- adenoviral constructs, used at the highest equivalent AdCFTR damine 123, HET, or dR6G whereas control, nontransfected dose, did not exhibit increased rates of dR6G fluorescence NIH 3T3 cells readily stained with all dyes tested (Table 1). (Fig. 1) or differences in steady-state staining intensities The lack of HET and dR6G fluorescence in 3T3-MDR-1 cells (Table 1). was presumably due to the efflux of the dehyrogenated dyes by Cl- Dependence and Ionic Sensitivity of dR6G Staining. As MDR-1 P-glycoprotein. shown in Fig. 2D, increased dR6G uptake by AdCFTR- Increased dR6G Staining Is Not Due to Endocytosis or infected IB3-1 cells required Cl-. Replacement of Cl- by Organic Ion Transport. Increased fluid-phase endocytosis was gluconate abolished any staining differences between Ad- ruled out as a source of the increased dR6G staining by CFTR-infected and noninfected IB3-1 cells. SCN- (10 mM), experiments with Lucifer yellow, an impermeant dye used as which has been shown to inhibit competitively the Cl- con- a volume marker for endocytosis (20). No differences were ductance of CFTR (23), also abolished the increased dR6G observed in the staining patterns or in the fluorescent inten- staining of AdCFTR-infected cells. Increased dR6G staining sities of CFPAC-CFTR and CFPAC-PLJ cells exposed to the was still observed in CFPAC-CFTR cells (compared with dye at 500 ,ug/ml (data not shown). Furthermore, the addition CFPAC-PLJ) when cells were incubated and stained under of 2.5 mM probenecid, a competitive inhibitor of the organic depolarizing conditions (125 mM K+; data not shown), ruling ion transporter (21), did not diminish the increased dR6G out a role for differences in cell membrane potential in dR6G staining of CFPAC-CFTR cells (data not shown). accumulation. Correlation of dR6G Staining with Anion Permeability. To test directly for effects of dR6G and HET on Cl- Besides increased steady-state staining in CFTR-expressing permeability, CFPAC-CFTR and CFPAC-PLJ cells were cells, the rate of increase of dR6G fluorescence was signifi- loaded with 36C1- and efflux was measured (24) in the cantly higher in CFPAC-CFTR cells and AdCFTR-infected presence of one of these dyes, before and after stimulation with IB3-1 cells that in control cells (Fig. 1). Intracellular quenching cAMP agonists. Pretreatment with either of the dyes (10, 15, of the fluorescence of the dye SPQ by halides is an accepted and 30 min) did not alter the rate of 36C1- efflux from method for the evaluation of cell membrane anion conduc- CFTR-expressing cells. Exposure of CFPAC-CFTR cells to 10 tance. To address the association between dR6G staining and ,uM diphenylamine 2-carboxylate, a nonspecific inhibitor of cAMP-mediated anion permeability, IB3-1 cells were infected Cl- channels (25), did not alter dR6G uptake or cAMP- with increasing amounts of AdCFTR. Both SPQ and dR6G mediated anion permeability as assessed by SPQ (data not fluorescence showed similar AdCFTR dose-dependent in- shown). creases (Fig. 2 A and B, respectively). Likewise, the rates of cAMP Dependence and dR6G Staining. If dR6G uptake is increase of SPQ and dR6G fluorescence were linearly related due to uptake mediated or regulated by CFTR, the rate of as a function of CFTR protein expression (indicated by increase of fluorescence should be increased by exogenous increasing AdCFTR viral dose; Fig. 2C). Simultaneous mea- cAMP and reduced by cAMP antagonists. To test this hypoth- esis, a membrane-permeant form of cAMP (cpt-cAMP) was added to the dR6G staining buffer and the fluorescence of AdCFTR-infected IB3-1 cells was measured. As shown in Fig. 1A, raising intracellular cAMP increased the rate of dR6G staining of the AdCFTR-infected cells. When AdCFTR- infected IB3-1 cells were incubated with Rp-cpt-cAMPS, an inhibitor of protein kinase A (26), the cAMP-dependent C increase in dR6G staining was eliminated. Rp-cpt-cAMPS also E prevented cpt-cAMP stimulation of anion permeability as- 0 C: sessed by SPQ (Fig. 3B). cpt-cAMP had no effect on dR6G u0 staining or cAMP-mediated anion permeability in uninfected 0 -D or AdCL-infected IB3-1 cells. cc 0 ATP Dependence of dR6G Uptake. CFTR function requires a) ._- ATP (2) and should be blocked by agents which decrease co intracellular ATP. Fig. 4A shows that the cAMP-dependent anion conductance of AdCFTR-infected IB3-1 cells, assessed by SPQ fluorescence, was completely eliminated by treatment with 50 mM 2-deoxyglucose and 0.1 ,uM antimycin A for 10 min. Likewise, the rate of dR6G staining in CFTR-expressing cells was reduced by this treatment (Fig. 4B). CFPAC CFPAC IB3-1 IB3-1 IB3-1 Locating Cells with Functional CFTR. Since gene therapy PLJ CFTR UNIF AdCL AdCFTR using adenoviral vectors, or protein replacement therapy, for CF is likely to result in transfer of CFTR to only a fraction of FIG. 1. The rate of increase of dR6G fluorescence in CFTR- the cells in the target tissue, we evaluated the utility of dR6G expressing (CFPAC-CFTR and AdCFTR-infected IB3-1) and CFTR- staining for the identification of cells expressing functional nonexpressing [CFPAC-PLJ and AdCl-infected and uninfected CFTR. AdCFTR-infected IB3-1 cells were labeled with the (UNIF) IB3-1] cells is shown as the mean ± SEM for a field of 30 cells cell tracking dye 5-chloromethylfluorescein diacetate per experiment (n = 5 experiments for CFPAC-CFTR and CFPAC- (CMFDA) and mixed in various ratios with either uninfected PLJ; n = 6 for uninfected IB3-1 and AdCL-infected IB3-1 cells; n = or AdCL-infected IB3-1 cells. Fig. 5 Upper shows images of 10 for AdCFTR-infected IB3-1 cells). IB3-1 cells were infected with cells mixed with adenoviral vectors at 200 pfu per cell for 48-72 hr. P < 0.001 for CMFDA-labeled, AdCFTR-infected IB3-1 differences in HET or dR6G staining between CFTR-expressing and unlabeled, uninfected IB3-1 cells. Fig. 5A shows subsequent -nonexpressing cells. Hoechst 33258 staining used to delineate nuclear boundaries Downloaded by guest on October 2, 2021 1170 Medical Sciences: Wersto et al. Proc. Natl. Acad. Sci. USA 93 (1996) A B 250 FIG. 2. cAMP-stimulated anion per- SPQ I 200 meability and dR6G staining in AdCFTR- ' 200- 50 infected IB3-1 cells. (A) Fluorescence of C 0 400 cells loaded with the dye SPQ and exposed 0 co 150- to cocktail containing forskolin, 3-isobu- 0 200 o tyl-l-methylxanthine, and cpt-cAMP as o described in Materials and Methods. Cells a 100oo 50 0 5 0- VHC were infected with AdCFTR at 50, 200, or 400 per cell as pfu indicated at right. a: 50- VHC a) :r Uninfected cells were exposed to vehicle cc buffer (VHC). (B) dR6G staining of IB3-1 cells infected with AdCFTR at 50 or 200 pfu per cell, showing the dependence of 0 5 10 15 20 dR6G staining on CFTR expression. Time, min Time, min dR6G was added at time zero. Experi- ments were done at 22°C to slow dye uptake. Data are the mean ± SEM from 30 Chloride a field of 30 cells for each observation. (C) Correlation between the rate of increase 25 of SPQ fluorescence following stimulation E c with cAMP agonists and the rate of in- (3 (D 0 0-cJ 20 crease of dR6G fluorescence staining with a) a) 0Co increased AdCFTR dose. 0, Uninfected c 0 0 15 :3 Glu t cells; *, AdCL at 200 pfu per cell; *, a1) AdCFTR at pfu *, AdCFTR at uconate 50 cell; 200 cell = U) 10 CU1 pfu per AdCFTR. n 6 + cocktail experi- a) cc ments for uninfected IB3-1 and AdCL- 5 infected IB3-1 cells and n = 10 experi- ments for AdCFTR-infected IB3-1 cells. (D) Dependence of dR6G fluorescence in 0 5 10 15 20 25 0 5 10 IB3-1 cells on extracellular Cl-. Data are SPQ rate, Time, min the mean ± SEM from a field of 30 cells relative fluorescence/min for each observation.

of cells in the mixed populations. The CMFDA-labeled cells stained with 2 ,tM 3,3'-dioctadecylcarbocyanine, a cationic are readily identified in Fig. 5B by their green fluorescence, membrane tracking dye (data not shown). which correlates with dR6G staining shown in Fig. 5C. Fig. 5 Lower quantitates the fluorescence of the CMFDA-labeled, DISCUSSION dR6G-stained cells and indicates that dR6G staining was largely restricted to the CMFDA-labeled cells. A few cells in Our results are consistent with the conclusion that the increased each field of view exhibited dR6G staining without any dR6G staining of epithelial cells is associated with CFTR func- CMFDA label. We attribute this pattern to diffusion of dR6G tion. Several lines of evidence support this conclusion: dR6G through gap junctional contacts between infected and unin- staining is stimulated and inhibited by agents which either stim- fected cells. Spread of dye was even more pronounced in ulate or inhibit cAMP-dependent anion conductance; dR6G mixtures of CFPAC-CFTR and CFPAC-PLJ cells, possibly due staining is ATP-dependent, requires the presence of extracellular to the same phenomenon. The correlation between CMFDA Cl-, is inhibited by SCN- (which competes with Cl- for transport and dR6G staining was not a unique property of the CMFDA through CFTR), and increases in a dose-dependent manner with dye itself, as similar results were obtained when cells were increasing CFTR levels of cAMP-mediated anion permeability

A B

250 -I1 cpt-cAMP

0 100- ._. 0) (U

cc

1 0 5 10 15 20 Time, min Time, min

FIG. 3. Activators and inhibitors of CFTR function affect dR6G staining and cAMP-dependent anion permeability. (A) dR6G fluorescence of AdCFTR-infected IB3-1 cells (200 pfu per cell, 48 hr) exposed to 200 ,uM cpt-cAMP at time zero (top curve) or incubated for 30 min with 200 ,LM Rp-cpt-cAMPS prior to addition of cpt-cAMP at time zero (middle curve). The lower curve, labeled control, shows dR6G uptake by AdCFTR-infected IB3-1 cells not treated with either drug. dR6G was added at time zero. (B) SPQ fluorescence in response to stimulation by cpt-cAMP (control) in AdCFTR-infected IB3-1 cells. The lower curve shows that treatment with Rp-cpt-cAMPS inhibited cAMP-dependent anion permeability. Each curve represents data from 30 cells, and data are expressed as the mean ± SEM. Downloaded by guest on October 2, 2021 Medical Sciences: Wersto et aL Proc. Natl. Acad. Sci. USA 93 (1996) 1171

A B zu 200 -SPQ dR6G a 200- Control v Control a) a 150 c 0 0 0) co - a) a 150 0 0 DOG

DOG a 100- 0-- : 50 cc50 - Iodide Nitrate Cocktail 0- 0- ... u fE to 4101 154r0 20 0 5 10 15 20 Time, min Time, min

FIG. 4. dR6G staining and cAMP-dependent increase in anion permeability cells are dependent on ATP levels. (A) SPQ fluorescence of AdCFTR-infected IB3-1 cells (200 pfu per cell) in response to forskolin, 3-isobutyl-1-methylxanthine and cpt-cAMP cocktail (upper curve, control). Pretreatment of cells in a glucose-free buffer containing 50 mM 2-deoxyglucose (DOG) and 0.1 ,uM antimycin A for 10 min was used to reduce intracellular ATP levels. (B) dR6G staining in AdCFTR-infected IB3-1 cells under control or ATP-depleted (DOG) conditions. Influx was reduced in 2-deoxyglucose/antimycin A-treated cells compared with untreated controls. P = 0.03 for differences between control and ATP-depleted cells in dR6G staining during the 12- to 16-min portions of the curves. Each curve represents data from 30 cells, and data are expressed as the mean ± SEM. conveyed by increasing levels of AdCFTR infection. Control modulate cAMP-stimulated anion permeability has fueled experiments have ruled out fluid-phase endocytosis, organic ion speculation that CFTR can interact with unidentified cytoskel- transport, the effects of adenoviral infection, and transport by etal elements or modulate the activities of other membrane MDR-1 P-glycoprotein as potential sources for the increased rate channels and transporters (27). Differences in redox potential of dye uptake and steady-state fluorescence in cells expressing as a consequence of CFTR expression could be responsible for functional CFT1R. the more rapid dehydrogenation of dR6G in cells having The dyes HET and dR6G are nonfluorescent and must be functional CFTR. However, it is not clear how differences in converted by. intracellular dehydrogenases to ethidium and redox potential could be responsible for the inhibition by rhodamine 6G, respectively. Neither of these nonhydroge- SCN- or dependence of Cl- on dR6G staining. nated dyes was preferentially accumulated by cells with func- dR6G staining was increased in cells having functional tional CFTR. The finding that NADP+ redox potential can CFTR and absent in cells expressing the MDR-1 P-

0a)en CO

: ax a:

FIG. 5. dR6G staining offunctional CFTR expression correlates with cell tracking ofAdCFTR-infected cells. (Upper) (A) Hoechst 33258 staining to locate cells. (B) Fluorescence of CMFDA-labeled AdCFTR-infected (200 pfu per cell) IB3-1 cells admixed with uninfected cells. (C). dR6G staining of the cell mixture. (Lower) Quantitation of CMFDA and dR6G fluorescence confirms the lack of dR6G staining of uninfected cells. Each bar represents the data from 30 cells and is expressed as the mean ± SEM. P < 0.001 for differences in dR6G and CMFDA staining between the AdCFTR-infected and uninfected IB3-1 cells. Downloaded by guest on October 2, 2021 1172 Medical Sciences: Wersto et al. Proc. Natl. Acad. Sci. USA 93 (1996) glycoprotein. It has been reported that CFTR and MDR-1 11. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., have complementary patterns of expression in the colonic Smith, A. E. & Welsh, M. J. (1992) Nature (London) 358, 761- epithelium during cell development (28) and in liver regener- 764. ation (29). It has been shown that the induction of the MDR-1 12. Rosenfeld, M. A., Yoshimura, K., Trapnell, B. C., Yoneyama, K., phenotype can downregulate CFTR expression (30). The Rosenthal, E. R., Dalemans, W., Fukayama, M., Bargon, M. M., uptake of fluorescent dyes by CFTR, shown in the present Strier, L. E., Stratford-Perricaudet, L., Perricaudet, M., Guggino, study, may be a useful model to improve chemotherapeutic W. B., Pavirani, A., Leqcocq, J.-P. & Crystal, R. G. (1992) Cell drug uptake by tumor cells. 68, 143-155. Previous investigators have speculated that CFTR is in- 13. Erzurum, S. C., Lemarchand, P., Rosenfeld, M. A., Yoo, J.-H. & volved in the uptake of Crystal, R. G. (1993) Nucleic Acids Res. 21, 1607-1612. leukotrienes (1), the extrusion of 14. Rosenfeld, M. 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