Proc. Natl. Acad. Sci. USA Vol. 87, pp. 4012-4016, May 1990 Cell Biology

A cystic fibrosis pancreatic adenocarcinoma cell line (chloride channel/pancreas)

R. A. SCHOUMACHERa, J. RAMa, M. C. IANNUZZIc, N. A. BRADBURYad, R. W. WALLACEad, C. TOM HONc, D. R. KELLYawe, S. M. SCHMIDf, F. B. GELDERg, T. A. RADOah, AND R. A. FRIZZELLai aGregory Fleming James Cystic Fibrosis Research Center, and Departments of 'Physiology and Biophysics, bPediatrics, dPharmacology, ePathology, and hMedicine, University of Alabama at Birmingham, University Station, Birmingham, AL 35294; cDepartment of Internal Medicine, University of Michigan, Ann Arbor, MI 48103; bDepartment of Chemotherapy and Toxicology, Southern Research Institute, Birmingham, AL 35255; and gDepartment of Surgery, Louisiana State University, Shreveport, LA 71130

Communicated by I. S. Edelman, March 12, 1990 (received for review January 26, 1990)

We established a pancreatic adenocarcinoma ABSTRACT cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl--secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through >80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

tumors were established by using an explant technique (5). Ascites fluid was centrifuged and the pellet was resuspended in culture medium. All cultures were initially seeded and grown in RPMI 1640 medium (Sigma); after passage 2, we changed to the richer Iscove's modified Dulbecco's medium (Sigma), which has been used successfully for a variety of tumor cell lines (6). Media were supplemented with 10%1o fetal bovine serum and maintained at 370C in a 5% C02/95% air atmosphere. Cells were usually passaged at 1:10 dilution by exposure to 0.05% trypsin in a 1.5 mM EDTA buffer at 370C for 3 min. Xenograft culture in congenitally athymic mice was attempted with standard techniques (7). Morphology and Markers of Differentiation. Cells cultured on coverslips were fixed in 10%o neutral-buffered formalin and embedded in paraffin. Cytokeratin expression was evaluated by immunofluorescence (8), using pooled mouse monoclonal anti-keratin (Boehringer Mannheim). For transmission electron microscopy, cells were fixed with 3% glutaraldehyde in 0.1 M Sorenson's buffer for 1 hr and postfixed in 1% OS04 in Sorenson's buffer for 1 hr. After dehydration through an ethanol series, the cells were flat-embedded in Polybed 812 resin (Polysciences). The expression of several oncofetal antigens (CEA, POA, ACAA, CA 19-9) was evaluated by standard ELISA, performed on medium removed from nearconfluent cell cultures after 3 days of incubation. Unused medium served as control. Isotopic Anion Efflux. Cells were plated at a density of 105 cells per cm2 on 35-mm culture dishes that were used when 80-100% confluent. Effluxes were performed at 370C, using a phosphate-buffered Ringer's (PBR) solution containing 140 mM NaCl, 3.3. mM KH2PO4, 0.83 mM K2HPO4, 1 mM CaSO4, 1 mM MgSO4, 10 mM glucose, and 10 mM Hepes, buffered to pH 7.4. Cells were loaded with 1251 (1-2 ,uCi/ml; 1 Ci = 37 GBq) for 30 min, washed briefly with PBR to deplete extracellular 1251, and efflux was monitored by a samplereplace procedure. After five samples, the solutions contained 10 ,M forskolin, 2 ,uM ionomycin, or 10 ,uM A23187. Remaining monolayer 1"I was extracted in 0.1 M nitric acid. Sample and extract counts were summed and the efflux was expressed as a time course of the loaded 1251I remaining in the monolayer. This assay monitors conductive anion exit as evidenced by inhibition of efflux by raised extracellular [K+] to depolarize membrane voltage or by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine carboxylic acid (9), or indanyloxyacetic acid 94 (10) (data not shown), cAMP Determination. Cells were cultured as described in the efflux experiments. After a 30-min preincubation and wash with PBR, monolayers were exposed to control and experimental solutions for various times at 37°C. Intracellular cAMP was assayed according to published methods (11), using a radioimmunoassay kit (NEN); 0.1 pmol of [3H]cAMP

Cystic fibrosis (CF) is a chronic, inherited disease that impairs the electrolyte transport properties of epithelial cells in the airways, sweat glands, pancreas, and other organs (1). The primary culture of human epithelial cells from some of these tissues has permitted studies of the functional basis of the CF defect. Single-channel patch-clamp analysis has revealed apical membrane Cl- channels in CF cells that are defective in their activation by cAMP-mediated agonists (2, 3) but are stimulated by agents that raise intracellular Ca2+ (2). While patch-clamp studies are feasible using a limited supply of human epithelial cells, an understanding of the molecular and biochemical basis of the CF defect in epithelial cells is likely to require access to permanent cell lines. A patient at the University of Alabama at Birmingham Cystic Fibrosis Care Center developed pancreatic adenocarcinoma; the clinical presentation is described in detail elsewhere (4). The patient was a 26-year-old white male with CF, pancreatic insufficiency, moderate pulmonary involvement, and insulin-dependent diabetes mellitus. Exploratory laparotomy revealed a large tumor in the head of the pancreas and multiple smaller lesions in the liver. Histology showed a well-differentiated pancreatic ductal adenocarcinoma. Ascites fluid and tumor fragments were obtained at autopsy, 6 weeks after larparatomy. This report details the development and characterization of a continuous cell line from this material, including analysis of the regulated anion transport properties and the CF genotype.

MATERIALS AND METHODS Culture Techniques. Primary cultures of epithelial cells from fragments of pancreatic (primary) and liver (metastatic) 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.

Abbreviation: PKA, protein kinase A.

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was added to each sample to assess recovery. Data are reported as pmol of cAMP per mg of protein (12). Protein Kinase A (PKA) Activity. Cytosolic fractions (100,000 x g; 60 min supernatant) were prepared from cells grown to confluence in 100-mm dishes. cAMP-dependent PKA activity was determined by the ability of cAMP to stimulate incorporation of 32P04 from [y-32P]ATP (Amersham) into the synthetic substrate Kemptide (13). Protein kinase activities are reported as pmol of P04 incorporated per min per mg of cytosolic protein. Single-Channel Recording. Techniques for single-channel recording using the cell-attached and excised, inside-out configurations of the patch-clamp method have been described in detail (2, 14). The recording pipette contained 150 mM NaCI, 2 mM CaCl2, 5 mM MgCl2, and 5 mM Hepes (pH 7.2) or a similar solution in which NaCl was replaced by 135 mM N-methyl-D-glucamine chloride plus 15 mM NaCl. Bath solutions contained 150 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 1 mM EGTA, and 5 mM Hepes (pH 7.2). Estimated free Ca2W of the bath was 10 ,uM. Relative ion permeabilities were calculated as described (14). To determine the relative permeabilities to sodium and chloride, bath NaCl was changed to 300 or 50 mM. For the halide selectivity determinations, bath NaCl was replaced by NaBr or Nal. DNA and RNA Analyses. Cells were harvested and resuspended in PBS to a concentration of 5 x 106 cells per ml. DNA and RNA were prepared as described (15, 16). DNA was amplified by using Thermus aquaticus DNA polymerase (Taq polymerase) (17). The amplification sequences for genomic DNA were 5'-CAGTTlTCCTGGATTATGCCTGGCAC-3' and 5'-GTTGGCATGClT-GATGACGCTTC-3'. RNA was amplified via cDNA (16). Reactions for reverse transcriptase were performed in a 50-,ul volume. Two reaction mixtures each contained 1 ,ug of total RNA, 21 units of Moloney murine leukemia virus reverse transcriptase (Boehringer Mannheim), and 3 ug of oligo(dT) as primer. One reaction mixture without reverse transcriptase served as a control for DNA contamination of the RNA preparation. All three were incubated for 1 hr at 37°C. For RNA amplification, the reaction mixtures were the same as for DNA except that 10 ,ul of the RNA reaction mixture was used in place of genomic DNA and the primer sequences were from exon 7 (5'-CCACCATCTCATTCTGCATTGTTCTGC-3') and exon 13 (5'-TCCAGGAGACAGGAGCATCTCCTTCTAATG-3'). After amplification, 50 ,ul of chloroform was added and each tube was centrifuged to separate the aqueous phase from the mineral oil. ..a

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To test for the presence of either the common CF mutation or the normal sequence, the samples were divided and run in duplicate agarose gels, and the DNA was transferred to Nytran (Schleicher & Schuell) by Southern blotting using lOx SSC (lx SSC = 0.15 M NaCl/0.015 M sodium citrate). Hybridization with allele-specific oligonucleotides was performed as described (17).

RESULTS In Vitro Cultre and Morphology. Growth was observed from >70% of adherent tumor explants; the first outgrowths were noted 2-3 weeks after plating. Three morphologies were observed in the initial cultures: (i) epithelial cells, which grew in a cobblestone pattern and had a high nucleus/cytoplasm ratio; (ii) fibroblastoid cells; and (iii) round cells, both of which had lower nucleus/cytoplasm ratios, grew in a disorganized pattern and occasionally contained multiple nuclei. Differential trypsinization (16) was used to purify cells with epithelial morphology from the mixed primary cultures of metastatic liver tumor; this cell line was designated CFPAC1, and its properties are described in this report. CFPAC-1 has been in continuous culture for >24 months and 80 passages, without evidence of altered morphology (by light microscopy) or anion transport (by isotopic flux; see below). The cells lack both contact inhibition and dilution limitation of their proliferation. Their doubling times were 30 and 32 hr at passages 17 and 74, respectively. They have been cloned by the Noble agar and limiting-dilution techniques. Both tumor and ascites fluid samples were tumorigenic in athymic mice, suggesting a potential for virtually unlimited cell volume expansion at low in vitro passage number. Karyotype analyses of CFPAC-1 cells at passage 25 (n = 7) revealed hyperdiploidy with 65-75 chromosomes (median, 73) per nucleus, including several translocations. Two or three normal-appearing copies of chromosome 7, the locus of the CF gene, were observed in every metaphase photographed. Epithelial Markers and Ultrastructure. Transmission electron microscopy of CFPAC-1 grown on plastic coverslips reveals epithelial polarization with apical microvilli, tight junctions, and gap junctions (Fig. la). CFPAC-1 cells stain strongly positive for keratin by immunofluorescence (Fig. lb), confirming their epithelial origin. They express oncofetal antigens that are associated with pancreatic ductal adenocarcinoma (18, 19). ELISA of culture medium supernatants revealed 9 ng of carcinoembryonic antigen per ml, 28 ng of

-I.

I

FIG. 1. (a) Transmission electron micrograph from confluent in vitro culture of CFPAC-1, passage 10, showing apical microvilli and glycocalyx, subapical cytoplasmic vacuoles containing mucin granules, and tightjunctions between adjacent cells. (x 10,100.) (b) Keratin staining by immunofluorescence of CFPAC-1 cells, passage 10. (x550.)

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a

pancreatic oncofetal antigen per ml, 5 Ag of adenocarcinomaassociated antigen per ml, and 12,000 units of CA 19-9 per ml (n = 2). Virgin medium contained no detectable amounts of these antigens. These findings suggest that CFPAC-1 is derived from a tumor of the pancreatic duct (20). Radioiodide Efflux. The macroscopic anion transport properties of CFPAC-1 cells were assessed by using an isotopic anion (1251) efflux assay (Fig. 2). The Cl--secreting human colonic adenocarcinoma cell line, T84, served as a positive control for the effects of cAMP- and Ca2+-mediated secretory agonists. 1251 efflux from T84 monolayers increased 7-fold in response to the adenylate cyclase activator, forskolin, or the Ca2+ ionophores, A23187 or ionomycin (Fig. 2a). In contrast, CFPAC-1 responded to the Ca2+ ionophores with 7- and 27-fold increases in anion efflux but did not respond to forskolin (Fig. 2b). In other experiments, 125j efflux from CFPAC-1 also was not affected by the cAMP analogues 8-Br-cAMP and chlorophenylthio-cAMP, or the phosphodiesterase inhibitor isobutylmethylxanthine (100 AM each). cAMP and PKA Levels. Levels of the intracellular mediators of forskolin's effects, cAMP and the cAMP-dependent PKA, were determined in CFPAC-1 and T84 cells. Basal cAMP levels in T84 and CFPAC-1 were 11 ± 2 (SEM) (n = 16) and 10 ± 1 pmol per mg of protein (n = 15), respectively. After 15 min of exposure to 10 ,uM forskolin, cAMP in T84 and CFPAC-1 increased to 1570 ± 225 (n = 14) and 415 ± 78 pmol per mg of protein (n = 13), respectively. Fig. 3a shows that the time courses of cellular cAMP accumulation in T84 and CFPAC-1 during the initial 2 min offorskolin stimulation, a time that correlates with the initiation of the anion transport response (Fig. 2), do not differ in T84 and CFPAC-1.

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FIG. 2. Time course of 1251 efflux from T84 (a) and CFPAC-1 (b) cells. Stimulation of efflux by agonist is expressed as the ratio of the maximal slope observed after agonist addition/slope at 1.00-1.25 min. The rapid initial efflux component (0-0.5 min) is due to washout of extracellular 1251 (C. J. Venglarik and R.A.F., unpublished data).

Cytosolic extracts of T84 and CFPAC-1 contained similar levels of PKA activity. A 5-fold increase in enzyme activity was elicited by 5 ,uM cAMP in both cell lines (Fig. 3b). PKA inhibitor (21) caused a slight reduction in basal enzyme activity and abolished the stimulation by cAMP. The concentration-response relation between protein kinase activity and cAMP (Fig. 3c) did not differ in CFPAC-1 and T84; no significant differences in Vmax or Kd (the apparent dissociation constant for cAMP) were observed (Kd = 0.20 jM for T84 vs. 0.23 uM for CFPAC-1). These Kd values are similar also to those found in control (0.13 ,uM) and CF (0.14 ,uM) airway cells in primary culture (22). The findings indicate that the absence of an anion transport response to forskolin in CFPAC-1 cannot be related to deficient cAMP production or to an alteration in the total activity of PKA or its sensitivity to cAMP.

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Electrophysiology. Patch-clamp techniques were used tto identify Cl- channels in CFPAC-1 cells. Channels weire activated after patch excision by a sustained depolarizinIg voltage, usually +80 mV, cytoplasmic side positive. In 26 cof 51 excised, inside-out membrane patches, depolarizatio n induced the activity of an outwardly rectifying anion channtel (Fig. 4a). Two to four levels of single-channel current wer present in 11 of the 26 channel-containing patches. Voltagere induced activation of outwardly rectifying Cl- channels i observed in normal and CF airway cell membranes antId serves as a positive control for the presence of channels ilin kinase-activation studies (23, 24). The slope conductance oAf this channel was 39 ± 2 pS at 0 mV in symmetric 150 mNA NaCl bath and pipette solutions, and chord conductance s were 68 ± 3 pS at +80 mV and 24 ± 1 pS at -80 mV (Fig 4a). With asymmetric NaCI solutions, PNa/PC1 was 0.05 (n = 2); substitution of pipette Na+ with N-methyl-D-glucamin4 e did not alter the current-voltage relationship. The halidee anion selectivity, assessed from biionic substitution experi iments (Fig. 4a) was 1 (1.7) > Br (1.4) > Cl (1.0) (n = 3). Thesee properties are identical to those reported for the outwardl)Y rectifying Cl- channel in the apical membranes of colonicc a

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Table 1. Cl- channel activation in CFPAC-1 Condition Forskolin A23187 0 8 C/A, activated by agonist 4 1 I/O, activated by depolarization 1/O, not activated by either 1 10 agonist or depolarization Total 5 19 Results are expressed as number of membrane patches examined under cell-attached (C/A) or inside-out (I/O) recording conditions. Six minutes was allowed for activation. See legend to Fig. 4 for other details.

tumor (T84) cells (14) and human airway (2, 3) and sweat gland (25) cells in primary culture. Cell-attached patches (Fig. 4b) were electrically quiet in the absence of agonists, and five cell-attached patches remained quiet in the presence of 10 AtM forskolin (Table 1). After patch excision to form the inside-out recording configuration, Cl- channels were subsequently activated by depolarizing voltage (+80 mV) in four of these patches (Fig. 4b); one remained quiet. In contrast, 10 AM A23187 elicited Clchannel currents in 8 of 19 cell-attached patches (Table 1). Of the 11 nonresponding patches, 1 was activated by depolarization after excision (inside-out). The cell-attached currents elicited by A23187 were typically less well defined than those recorded in the same patch after excision (Fig. 4c). However, these currents were never observed in the absence of ionophore, and both inward and outward currents were observed in cell-attached recordings with N-methyl-D-glucamine chloride in the pipette, a condition in which the inward currents must be due to Cl- flow from cell to pipette. This conclusion is supported by the 125J efflux studies (Fig. 2) and by the reversible, Ca2+-dependent stimulation of Cl- currents by A23187 and ionomycin during whole-cell recording from CFPAC-1 (26). These results indicate that CFPAC-1 expresses the outwardly rectifying Cl--selective channel found in other Cl--secreting epithelial cells and that it can be activated by Ca2+ ionophores and depolarizing voltages but not by cAMP-dependent agonists. DNA and RNA Analyses of the CF Genotype. The CF genotype was assessed by amplification of genomic DNA and RNA via cDNA. The amplified products were transferred to Nytran membranes for Southern blotting and were hybridized to allele-specific oligonucleotides; the results are shown in Fig. 5. DNA samples homozygous for the normal sequence and homozygous for the common CF mutation, a 3-base-pair deletion, served as controls. CFPAC-1 cells are homozygous for the common CF mutation, a deletion of three nucleotides that results in the absence of phenylalanine at position 508 (AF5N) of the deduced amino acid sequence. In addition, they

express this mutant CF gene mRNA. The possibility of DNA contamination of the RNA was ruled out since no signal was 1

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FIG. 4. (a) Current-voltage relations of the outwardly rectifying Cl- channel in CFPAC-1 recorded in the inside-out (I/O) excised patch configuration with NaCl (o), NaBr (A), or Nal (v) in the bath. (b) Failure of forskolin to activate Cl- channels during cell-attached (C/A) recording from CFPAC-1 (top trace), and records from an inside-out (I/O) patch after voltage activation. (c) Activation of currents during C/A recording by 10 FM A23187 at ±60 mV (top two traces), and currents recorded subsequently I/O at +80 mV (bottom two traces). Holding voltages are given on the left. Horizontal lines denote the closed state.

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B FIG. 5. Allele-specific oligonucleotide hybridization. Autoradiographs show the hybridization results with two specific oligonucleotide probes. (A) The mutant (AF508) sequence is detected. (B) The normal DNA sequence is detected. Samples were amplified by PCR. Lanes: 1, genomic DNA control, homozygous AFSO8 mutant; 2, genomic DNA control, homozygous normal sequence; 3, CFPAC-1 RNA, one round of amplification; 4, CFPAC-1 RNA, two rounds of amplification; 5, CFPAC-1 DNA.

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detected after two rounds of amplification of the RNA reaction without reverse transcriptase (data not shown). These results show that CFPAC-1 cells express the CF gene product and are homozygous for the common CF mutation.

DISCUSSION Pancreatic function is markedly compromised in =80% of CF patients (1). When compared to non-CF subjects with similar trypsin secretion rates, CF patients have reduced HCO- and Cl- secretion, suggesting a primary defect in anion secretion by pancreatic duct cells (27). Recent studies of microperfused rat pancreatic ducts suggest that anion secretion across the apical membrane involves an apical Cl- conductance in parallel with Cl-/HCO- exchange (28). This combination of transport processes exploits the inwardly directed chemical gradient for Cl- to drive HCO- secretion via electroneutral anion exchange and regulates HCO- secretion rate by recycling Cl- to the lumen via a cAMP-regulated Cl- channel. A defect in cAMP-mediated activation of apical Cl- channels would impair pancreatic HCO- and Cl- secretion and functionally link the defect in CF pancreatic duct cells to the Clchannel regulatory defect identified in airway and sweat gland cells, where transepithelial Cl- secretion is impaired.j Studies of CF have proceeded along two parallel tracks aimed at defining the CF mutation: a molecular approach to identify the CF gene, and a functional approach to identify the defective gene product. Now that the gene has been identified (30), the CFPAC-1 cell line should be useful in providing virtually limitless quantities of cells for further biochemical and physiologic investigations of the CF defect. In addition, these cells should be useful in genetic complementation experiments (31) designed to correct the CF defect in Cl- channel regulation by stable expression of the normal counterpart of the CFgene. This would provide well-matched CF and normal cell lines for detailed functional evaluation of the gene product and for manipulation of the Ca2+-mediated Cl- channel regulatory pathways that are not impaired by CF. The results of patch-clamp studies (24, 25) indicate that CF Cl- channels have normal ion conduction properties and Ca2+-dependent activation. This has given rise to the idea that the CF defect may not lie in the channel itself but in a membrane-associated regulatory protein that mediates channel activation by PKA (24). The likely candidate for this role is the CF gene product, the CF transmembrane regulator (CFTR). If this is true, then the structural features necessary for genetic complementation of defective cAMP-dependent channel regulation may be in place in CFPAC-1 cells since they express the outwardly rectifying Cl- channel and Ca2' but not cAMP-dependent regulation of channel activity. JRecently, single-channel studies identified an outwardly rectified Cl- channel in primary cultures of human fetal pancreatic duct (29). It was activated in excised patches by depolarizing voltages. A 5 pS Cl- channel was observed frequently and its basal activity (open time) was enhanced -3-fold by the cAMP-mediated agonist secretin. The single-channel response to Ca2l-dependent agonists was not evaluated. Inasmuch as there is no anion transport response to cAMP in CFPAC-1, we cannot compare our findings to those obtained from primary cultures of pancreatic duct. We thank A. Tousson and B. R. Brinkley (Department of Cell Biology and Anatomy, University of Alabama at Birmingham) for the cytokeratin localization studies, Dr. A. Carroll (Laboratory of Medical Genetics, University of Alabama at Birmingham) for the karyotype analyses, Dr. R. Greger (Albert-Ludwigs University, Freiburg, F.R.G.) for 5-nitro-2-(3-phenylpropylamino)benzoic acid,

Proc. Natl. Acad Sci. USA 87 (1990) and Dr. D. W. Landry (Columbia University) for indanyloxyacetic acid 94. This work was supported by the National Institutes of Health (DK38518 and DK41330), the Cystic Fibrosis Foundation, and a Parker B. Francis Foundation fellowship to R.A.S. 1. Taussig, L. M. (1984) Cystic Fibrosis (Thieme-Stratton, New York). 2. Frizzell, R. A., Rechkemmer, G. R. & Shoemaker, R. L. (1986) Science 233, 558-560. 3. Welsh, M. J. & Liedtke, C. M. (1986) Nature (London) 322, 467-470. 4. McIntosh, J. C., Schoumacher, R. A. & Tiller, R. E. (1988) Am. J. Med. 85, 592. 5. Wallace, D. H. & Hegre, 0. D. (1979) In Vitro 15, 270-277. 6. de St. Groth, S. F. (1983) J. Immunol. Methods 57, 121-136. 7. Fogh, J. M. & Orfoe, T. (1977) J. Natl. Cancer Inst. 59, 221-225. 8. Brinkley, B. R., Fistel, S. H., Marcum, J. M. & Pardue, R. L. (1980) Int. Rev. Cytol. 63, 59-95. 9. Wangemann, P., Wittner, M., Di Stefano, A., Englert, H. C., Lang, H. J., Schlatter, E. & Greger, R. (1986) Pflugers Arch. 407, S128-S141. 10. Landry, D. W., Akabas, M. H., Redhead, C., Edelman, A., Cragoe, E. J., Jr., & Al-Awqati, Q. (1989) Science 244, 14691472. 11. Smith, P. L., Welsh, M. J., Stoff, J. S. & Frizzell, R. A. (1982) J. Membr. Biol. 70, 217-226. 12. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 13. Clegg, C. H., Correll, L. A., Cadd, G. G. & McKnight, S. (1987) J. Biol. Chem. 262, 13111-13119. 14. Halm, D. R., Rechkemmer, G. R., Schoumacher, R. A. & Frizzell, R. A. (1988) Am. J. Physiol. 254, C505-C511. 15. lannuzzi, M. C., Dean, M., Drumm, M. L., Hidaka, N., Cole, J. L., Perry, A., Stewart, C., Gerrard, B. & Collins, F. S. (1989) Am. J. Hum. Genet. 44, 695-703. 16. Rappolee, D. A., Mark, D., Banda, J. & Werb, Z. (1988) Science 241, 708-712. 17. Lemna, W. K., Feldman, G. L., Kerem, B.-S., Fernbach, S. D., Zevkovich, E. P., O'Brien, W. E., Riordan, J. R., Collins, F. S., Tsui, L.-C. & Beaudet, A. L. (1990) N. Engl. J. Med. 322, 291-2%. 18. Hayakawa, T., Kondo, T., Shibata, T., Hamano, H., Kitagawa, M., Sakai, Y. & Ono, H. (1988) Cancer 61, 1827-1831. 19. Pinto, V. B., Gelder, F. B. & Morris, D. M. (1986) Cancer Res. 46, 6520-6524. 20. Cubilla, A. L. & Fitzgerald, P. J. (1984) Tumors ofthe Exocrine Pancreas (Armed Forces Inst. Pathol., Washington, DC). 21. Cheng, H. C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M. & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992. 22. Barthelson, R. A. & Widdicombe, J. H. (1987) J. Clin. Invest. 80, 1799-1802. 23. Schoumacher, R. A., Shoemaker, R. L., Halm, D. R., Tallant, E. A., Wallace, R. W. & Frizzell, R. A. (1987) Nature (London) 330, 752-754. 24. Li, M., McCann, J. D., Liedtke, C. M., Nairn, A. C., Greengard, P. & Welsh, M. J. (1988) Nature (London) 331, 358-360. 25. Krouse, M. E., Hagiwara, G., Chen, J., Lewiston, N. J. & Wine, J. J. (1989) Am. J. Physiol. 257, C129-C140. 26. Cliff, W. H. & Frizzell, R. A. (1989) Pediatr. Pulmonol. Suppl. 4, 123 (abstr.). 27. Forstner, G. G., Kopelman, H. R., Durie, P. R. & Corey, M. L. (1987) in Genetics and Epithelial Cell Dysfunction in Cystic Fibrosis, eds. Riordan, J. & Buchwald, M. (Liss, New York), pp. 101-104. 28. Novak, I. & Greger, R. (1988) Pflugers Arch. 411, 546-553. 29. Gray, M. A., Harris, A., Coleman, L., Greenwell, J. R. & Argent, B. E. (1989) Am. J. Physiol. 257, C240-C251. 30. Rommens, J. M., Tannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J. R., Tsui, L.-C. & Collins, F. S. (1989) Science 245, 1059-1065. 31. lannuzzi, M. C., Weber, J. L., Yankaskas, J. R., Boucher, R. C. & Collins, F. S. (1988) Am. Rev. Respir. Dis. 138, %5-968.