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Uptake of Fluorescent Dyes Associated with the Functional Expression of The Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1167-1172, February 1996 Medical Sciences Uptake of fluorescent dyes 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 dye, 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 fluorescence 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; rhodamine 123; rhodamine 6G; 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
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