Proc. Natl. Acad. Sci. USA Vol. 88, pp. 7759-7762, September 1991 Cell Biology

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells [cystic fibrosis trausmembrane conductance regulator/CFTR/chloride aort/6-methoxy-N-(3-sulfopropryl)quinolinium/fiuorescent digital microscopy]

ERIC J. SORSCHER*t*§, KEVIN L. KIRK**, MARY L. WEAVER*t, TAMAS JILLING*t, J. EDWIN BLALOCK*, AND ROBERT D. LEBOEUF* *Department of Physiology and Biophysics and tDepartment of Medicine, Division of Hematology and Oncology, and the *Gregory Fleming James Cystic Fibrosis Research Center, The University of Alabama at Birmingham, Birmingham, AL 35294

Communicated by Robert W. Berliner, June 3, 1991

ABSTRACT We have tested the hypothesis that the cystic fibrosis (CF) gene product, cailed the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated ceols was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinoilnium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cels. Antisense oflgodeoxynucleotide treatment (3.9 or 1.3 FM) for 24 hr virtually abolished Cl transport in sweat duct cells compared with untreated cells or control ceols treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations ofantisense oligodeoxynucleotides were less effective at inhibiting CI transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl1 permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl permeability.

Cystic fibrosis (CF) is the most common lethal genetic disorder among Caucasians, occurring in approximately 1 in 2000 live births (1). Considerable progress has been made recently in two areas of CF research. First, the results of a number offunctional studies indicate that the "CF defect" is limited largely to epithelial tissues, such as lung and sweat gland, that exhibit abnormal regulation of plasma membrane Cl- permeability in this disease. The CF sweat duct exhibits a much reduced rate of Cl- reabsorption which accounts for the salty sweat that is a hallmark of the disease (2-8). Patch clamp studies of normal and CF epithelia have suggested that the compromised Cl- permeability of affected cells is due to a defective regulation of plasma membrane Cl- channels

(9-12).

chloride transport, and the CF gene has been renamed CFTR to reflect the function of its product.

The identification, cloning, and sequencing of CFTR now makes it possible to directly relate the expression of this gene to its presumed function-i.e., epithelial Cl- transport regulation. Two such experiments are required to confirm that CFTR functions to mediate or modulate epithelial Cl- permeability: (i) to express the wild-type gene in mutant CF cells and restore normal Cl- permeability regulation (i.e., a complementation or gain-of-function experiment) and (it) to block expression of the wild-type gene in normal cells and monitor subsequent decrements in Cl- transport (i.e., a loss-offunction experiment). Several laboratories have now reported successful complementation of the Cl- permeability defect by expressing the wild-type gene in cultured CF cells (16-18). The goal of the present study was to determine if blocking the expression of the wild-type gene in normal, untransfected cells inhibits epithelial Cl- transport. Toward that end we have used antisense oligodeoxynucleotides designed to block translation of the CFTR mRNA in normal sweat duct cells and examined these cells for subsequent decrements in Cl- transport using a previously described optical assay of Cl- flux. This antisense strategy has proven to be an effective means of specifically inhibiting the translation of a given gene product in a number of cell systems. Human sweat duct cells in primary culture were the experimental system of choice for the present study because these cells exhibit a maximal Cl- permeability in the absence of exogenous cyclic AMP- or Ca2+-mediated agonists, and this permeability is virtually abolished in CF (6). Our results document that treatment of normal sweat duct cells with antisense oligodeoxynucleotides, but not with control sense oligodeoxynucleotides, greatly inhibits Cl- permeability and thereby converts their "Cl- transport phenotype" to the CF phenotype. These results provide the needed evidence that inhibition of CFTR expression in a normal, untransfected cell inhibits epithelial Cl- permeability.

METHODS

The second area of CF research in which significant progress has been made involves the characterization of the gene responsible for CF. Using a combination of chromosomal walking and jumping, the gene responsible for CF has been identified (13), its cDNA characterized (14), and the most common mutation responsible for the disease elucidated (15). The gene product has been termed the CF transmembrane conductance regulator (CFTR) because ofits implicated role as a chloride channel or as a regulator of

Tissue Preparation and Cell Culture. Explants of sweat duct were isolated and cultured from normal adult surgical skin samples by described procedures (6). Individual sweat ducts were placed on coverslips and grown under serum-free conditions in 35-mm dishes containing supplemented MCDB 170 medium (6) and cultured for 10-20 days prior to experiments.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: CF, cystic fibrosis; SPQ, 6-methoxy-N-(3sulfopropryl)quinolinium; CFTR, cystic fibrosis transmembrane conductance regulator. §To whom reprint requests should be addressed. 7759

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Cell Biology: Sorscher et al.

Oligodeoxynucleotide. A 23-nucleotide oligodeoxynucleotide complementary (antisense) to the human CFTR transcript and a 23-nucleotide control oligodeoxynucleotide homologous (sense) to this transcript (14) were synthesized. Both oligodeoxynucleotides encompassed the first 23 base pairs of the CFTR transcript beginning at the translation initiation AUG (14). Either the antisense oligodeoxynucleotide, 5'-TTTTCCAGAGGCGACCTCTGCAT-3', or the sense oligodeoxynucleotide, 5'-ATGCAGAGGTCGCCTCTGGAAAA-3', was added to experimental cultures in 20-50 ,ul of doubly distilled H20 for various periods of time (4 or 24 hr) prior to assaying anion permeability. Oligodeoxynucleotides were synthesized on a DuPont Coder 300 DNA synthesizer and were purified prior to addition to cultures by using Nensorb cartridges (DuPont) according to the manufacturer's protocol. Recovery of Labeled Oligodeoxynucleotides from Treated Sweat Duct Cells. CFTR gene transcript sense or antisense oligodeoxynucleotides were end-labeled with 32P by using T4 polynucleotide kinase and ['y-32P]ATP (3000 Ci/mmol, DuPont; 1 Ci = 37 GBq) (19). Free 32p was removed from labeled oligodeoxynucleotides by passage twice through Sephadex G-25 spin columns. The specific activities of the sense and antisense oligodeoxynucleotides were 2.18 x 106 cpm/,ug and 1.9 x 106 cpm/pxg, respectively. Normal sweat duct cells (1 x 103 cells per ml) were cultured for 24 hr in the presence of 5 FLM CFTR gene transcript sense or antisense oligodeoxynucleotides. After 24 hr, cells from each treatment were solubilized with Nonidet P-40 lysis buffer (19), nuclei were removed by centrifugation, and the supernatants were extracted three times with 24:1 (vol/vol) phenol/chloroform. The 32P radioactivity in the cytoplasmic extract from each treatment was determined. Approximately equivalent amounts of 32P radioactivity from cytoplasmic extracts from CFTR gene transcript sense- and antisense-treated cells (sense = 1.45 x 106 cpm; antisense = 1.26 x 106 cpm) were electrophoresed on 12% polyacrylamide sequencing gels, and the gel was exposed to Kodak AR film for 24 hr. Anion-Flux Measurements. Anion fluxes were assayed by using a fluorescent dye that is specifically quenched by halides (Br- > Cl-), 6-methoxy-N-(3-sulfopropryl)quinolinium (SPQ), in combination with fluorescence digital-imaging microscopy. This optical method provides a convenient assay of the macroscopic Cl- permeabilities of individual cells that has been applied to a variety of nonepithelial and epithelial cells, including sweat duct cells in primary culture (6, 17, 18). As described (6), sweat duct cells were hypotonically loaded with SPQ by exposing them to 5 mM dye in 1:1 (vol/vol) growth medium/sterile water for 10 min. Cells loaded with SPQ in this manner are viable on the basis of very low leak rates of SPQ and the ability to respond to regulators of Cl- permeability (6). After loading, individual coverslips of dye-loaded cells were placed in a perfusion chamber on the stage of an inverted microscope equipped for fluorescence digital imaging microscopy (6) and equilibrated in 150 mM NaCI/2.5 mM KCl/2.5 mM Na2HPO4/1.0 mM CaCl2/1.0 mM MgSO4/1.0 mM glucose, pH 7.4. Fluorescence images were collected at various times during the course of each experiment with a x40 planachromat objective lens (1.0 numerical aperture; Zeiss) and were electronically intensified by a microchannel plate intensifier (KS-1380; Videoscope Int., Herndon, VA) coupled to a Newvicon video camera (Dage MTI). Each intensified image was relayed to an image analysis system (GMR-270; Grinnell Systems) where it was digitized, signal-averaged, and stored on an optical memory disk. Individual cell fluorescence was estimated for four to eight cells in each field as the average intensity within a 2 x2 j.m window located over the cytoplasm of the cell as described (6). Each cell fluorescence measurement was corrected for background fluorescence, defined operationally as

the fluorescence intensity measured off the cell monolayer. The location of the measuring window for each cell remained invariant throughout the protocol. Two protocols were used to assess Cl- transport in oligodeoxynucleotide-treated cells. First, in most experiments the extracellular Cl- was replaced with an impermeant anion [gluconate (150 mM)] to provide a driving force for net Clefflux from the cells. This procedure resulted in an increase in intracellular SPQ fluorescence that reflected a decrease in the intracellular concentration of the quenching anion-i.e., Cl- (Fig. 1). Second, in additional experiments the cells were preequilibrated in the CL-free, gluconate-containing medium, and then the extracellular gluconate was replaced with Br-, an effective quencher of SPQ fluorescence and permeater of epithelial Cl- channels. This maneuver resulted in a decrease in SPQ fluorescence as Br- entered the cells and quenched the fluorescence. Each of these protocols has the advantage that the rate of net halide influx or efflux is measured under conditions where anion exchange should be minimal. (Note that the solutions were bicarbonate free.)

RESULTS Uptake of Antisense and Sense Oftodeoxynudeotides by Sweat Duct Cells. Antisense inhibition of gene expression involves cellular uptake of the antisense oligodeoxynucleotide followed by hybridization of the exogenous nucleotide

B

a

FIG. 1. Increases in intracellular SPQ fluorescence after removal of extracellular Cl-. Cells loaded with SPQ were equilibrated in Cl--containing medium (A) and then placed in a medium that was Clfree-i.e., containing gluconate (B). During incubation in Cl--free medium, efflux of intracellular Cl- leads to an increase in intracellular fluorescence as the SPQ dye is dequenched. (x850.)

Cell Biology: Sorscher et A

Proc. Natl. Acad. Sci. USA 88 (1991)

sequence to the target transcript and subsequent inhibition of translation (20-24). In a series of control experiments we examined the toxicity, uptake, and stability of antisense and sense oligodeoxynucleotides after their incubation with sweat duct cells. Treatment of sweat duct cells with either the sense or antisense oligodeoxynucleotide for 24 hr at concentrations up to 3.9 MAM was not cytotoxic as determined by trypan blue exclusion (95% viability). Cells treated with 32P-labeled sense or antisense oligonucleotides did not differ in their cell-associated 32P radioactivity after 24 hr of treatment (sense = 39,722 cpm per 100 cells; antisense = 37,791 cpm per 100 cells), suggesting an equivalent uptake ofthe two sequences. We also recovered 32P-labeled sense and antisense oligodeoxynucleotides from cells that had been treated for 24 hr, and we obtained sufficient quantities of intact, undegraded oligodeoxynucleotide to be visualized by polyacrylamide gel electrophoresis. Dose-Dependent Inhibition of Cl- Efflux by the Antisense Oligodeoxynucleotide. Fig. 2 summarizes the results obtained for sweat duct cells that were exposed to various concentra''-

1.40

control 3.9 M (sense) .33z M -o °1.3 M - *3.9MM

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tions of sense or antisense oligodeoxynucleotides for 24 hr and then assayed for Cl- efflux by the "gluconate" protocol. The replacement ofbath Cl- with gluconate results in net Clefflux from the cells that is reported as a time-dependent increase in SPQ fluorescence. Previously we showed that this fluorescence increase can be blocked by the Cl- channel blocker diphenylamine carboxylate (6, 26) at concentrations that are specific for Cl- conductive pathways. Sense-treated cells showed no statistically significant difference from untreated control cells regarding the rate of Cl- efflux at any of the three concentrations examined (3.9, 1.3, and 0.33 MM). However, cells treated with antisense oligodeoxynucleotides at 1.3 AM and 3.9 MM, but not at 0.33 MM, exhibited significant inhibition of the rate of efflux. In fact, the highest concentration of antisense oligodeoxynucleotide nearly completely inhibited Cl- efflux (Fig. 2). Time-Dependent Inhibition of Anion Transport by Antisense Oligodeoxynucleotide. The experiments depicted in Fig. 3 were conducted for two reasons: (i) to establish that antisense inhibition of sweat duct anion permeability is a time-dependent phenomenon, as expected if antisense treatment decreases Cl- transport because of an inhibition of gene translation, and (it) to document that the observed inhibition of anion transport is protocol independent. Sweat duct cells were treated with 3.9 MM antisense oligodeoxynucleotide for 4 or 24 hr and then assayed for anion transport by the "bromide" protocol outlined in Methods. Cells treated for 24 hr showed an inhibition of Br- uptake compared with untreated cells, whereas cells treated for 4 hr showed no significant inhibition. Thus, antisense inhibition of anion transport was observed at 24 hr of treatment irrespective of the specific flux protocol employed. The lack of significant inhibition after 4 hr of treatment is consistent with the notion that inhibition of Cl- transport occurs subsequent to an inhibition of gene translation. antisense, 24hours antisense, 4hours control

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FIG. 2. CFTR gene transcript antisense inhibition of Cl- transport. The vertical axis represents the fluorescence normalized to the baseline fluorescence (F.) determined just prior to the change to the Cl[-free, gluconate-containing bathing solution at: = 0. Each data point represents the mean relative fluorescence (± SEM) from several control or CFTR transcript antisense experiments (control, n = 18; 0.33 AM, n = 3; 1.3 ILM, n = 5; 3.9 1LM, n = 6) and from six sense experiments. In each experiment and at each time point, the fluorescence for four to eight cells was measured as described. Rates of change in fluorescence were analyzed by regression analysis, and rates (regression slopes) were compared by an F test for comparing the equality of two regression slopes. Nine-minute time points were compared by Student's t test after determining that the variances for these samples were homogeneous (F.,. test). All statistical analyses were performed as described (25). The rate of change of intracellular fluorescence in experiments in which cells were treated with 1.3 JIM or 3.9 tuM antisense oligodeoxynucleotide was significantly less than the rate of change of intracellular fluorescence in control cells (P < 0.025). Additionally, the mean fluorescence measured 9 min after the removal of extracellular Cl- (i.e., after initiating Cl- efflux) for 1.3 1AM and 3.9 MM antisense oligodeoxynucleotide-treated cells was significantly less than corresponding measurements in control cells (P < 0.05). Cells treated with 0.33 AM antisense oligodeoxynucleotide or with sense oligodeoxynucleotide at 0.33, 1.3, and 3.9 FM did not differ from untreated control cells.

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FIG. 3. CFTR gene transcript antisense inhibition of Br- uptake. Cells were equilibrated in Cl--free, gluconate-containing buffer, and a stable baseline fluorescence (FO) was obtained just prior to the change to Br--containing buffer. The vertical axis represents the relative fluorescence expressed as fluorescence normalized to the baseline fluorescence (F.) just prior to the change to Br--containing solution. Each data point represents the mean relative fluorescence (± SEM) from untreated cells (n = 47) or cells treated for either 4 hr (n = 36) or 24 hr (n = 34) with antisense oligodeoxynucleotide. The mean relative fluorescence at 1, 2, and 3 min was compared by Student's t test (25). Br- entry into normal sweat duct cells measured in this way was diminished after 24 hr of antisense CFTR oligodeoxynucleotide treatment (3.9 MM) compared with untreated control cells (P < 0.05 at 1, 2, and 3 min). No statistical difference was observed between untreated control cells and normal sweat duct cells that were treated with the CFTR antisense oligodeoxynucleotide for 4 hr prior to the assay (P > 0.05).

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Cell Biology: Sorscher et al.

DISCUSSION Using antisense oligodeoxynucleotides designed to inhibit the translation of the CFTR product, we have been able to produce an anion permeability defect in normal sweat duct cells that is similar to the Cl- transport abnormality seen in CF. Antisense oligodeoxynucleotides targeted to a diversity of other genes have been shown to block translation and lead to an absence or decrease of the gene product (20-24). In this report, we have shown that normal sweat duct cells treated with the antisense oligodeoxynucleotide, but not the sense oligodeoxynucleotide, exhibit a markedly reduced Cl- permeability similar to that of sweat duct cells cultured from CF patients. These results indicate a role for the normal CFTR product in maintenance of Cl- transport in reabsorptive sweat duct cells. Our studies complement those that recently have shown correction of the anion transport defect in CF cells after transfection with the full-length CFTR gene (16, 17). However, unlike those complementation experiments in which CF cells were engineered to overexpress the wild-type gene, the present study was performed on untransfected cells that were presumably expressing CFTR at physiologic levels. Thus, our results provide direct evidence for a role of CFlR in modulating or mediating epithelial Cl permeability in a normal, untransfected cell. The results of our study also suggest that the CFTR has a relatively rapid turnover rate (

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion tra...
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