Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2Kb-tsA58 transgene

Marcia Takacs-Jarrett, William E. Sweeney, Ellis D. Avner, and Calvin U. Cotton

Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33°C with interferon-gamma ). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl- permeability and cAMP-stimulated Cl- secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl- secretory responses. Ca2+-activated Cl- secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.

cystic fibrosis; murine epithelial cell lines; ts-sv40 large T antigen; cystic fibrosis transmembrane conductance regulator; calcium-activated chloride secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MUTATIONS in the cystic fibrosis transmembrane conductance regulator (CFTR) are responsible for cystic fibrosis (CF), and the primary defect in CF is the loss of cAMP-regulated anion conductance in the apical plasma membrane of epithelial cells in affected tissues (29, 33). Identification, cloning (34, 35), and heterologous expression of CFTR and mutant forms of CFTR provide direct evidence that the protein functions as a cAMP-regulated anion channel (2, 3). It is not certain how loss of apical membrane Cl- conductance alone leads to the wide spectrum of phenotypic abnormalities associated with defective CFTR (37), including aberrant regulation of Na+ channels (9, 10, 41) and non-CFTR Cl- channels (38), altered regulation of exocytosis and endocytosis (13), abnormal composition of macromolecules, and increased adherence of certain bacteria to airway epithelial cells (12). A number of mechanisms have been proposed to explain CF pathophysiology (4, 38, 39); however, unifying hypotheses are few, and the relevance of certain observations to native epithelial cell function is unclear. Furthermore, most studies of CFTR function have been performed using airway and intestinal (T84) epithelial cells (7, 8, 43) or heterologous expression systems (5). Epithelial cells from other tissues affected by CF have received relatively little attention, primarily due to lack of access to appropriate human biological material (6). The development of the CF mouse has expanded the number of epithelial cell types available for study (16, 21, 40).

Primary culture of human epithelial cells (9) and immortalized epithelial cell lines (24, 25, 27, 28, 45, 46) have been widely used in biomedical research and have proven particularly useful for studies of CF and CFTR. Many of the immortalized CF and non-CF epithelial cell lines were generated by in vitro transfection of human airway epithelial cells with either SV40 large T antigen (24, 27, 28, 46) or human papilloma virus (45). The degree of differentiation appears to vary widely, as many of the cell lines do not form functional epithelial tight junctions (24, 46). However, the CF phenotype, namely loss of cAMP-stimulated Cl- permeability, is retained (24, 27, 28, 45, 46). Immortalized cell lines have also been developed from genetically modified mice that carry an SV40 large T antigen transgene (25). Recently, Jat and coworkers (26) produced a transgenic mouse line (H-2Kb-tsA58; ImmortoMouse) that carries a thermolabile mutant of SV40 large T antigen under the control of a ubiquitous interferon-gamma (IFN-gamma )-inducible promoter. This mouse line has been used to generate several conditionally immortalized cell lines to date (14, 26, 32, 42, 44). The major theoretical advantages of cell lines generated in this way are 1) the avoidance of unpredictable characteristics of in vitro transfection with oncogenes (e.g., variable copy number and multiple sites of integration), 2) the ability to immortalize a heterogeneous population of cells from an epithelium (e.g., ciliated, goblet, and basal cells from the airway) from which clones with specific properties may be selected at a later time, and 3) the opportunity to control large T antigen levels and thereby promote differentiation by switching the cells from permissive to nonpermissive culture conditions (36). The goal of this work was to cross the ImmortoMouse (26) with the University of North Carolina (UNC) CF knockout mouse (CFTR S489X) (40) and develop genetically well-matched, conditionally immortalized CF and non-CF epithelial cell lines.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Male mice, homozygous for a temperature-sensitive SV40 large T antigen transgene (ImmortoMouse; CBA/ca X C57B1/10 strain; Charles River Laboratories) (26), were bred with female mice that were heterozygous for the S489X CFTR mutation (UNC; CFTR +/-; C57BL/6J X F129 strain) (40). The offspring were genotyped by polymerase chain reaction (PCR) analysis of DNA extracts from tail sections. Sections of ~1 cm in length were digested overnight at 55°C with 700 µl proteinase K [Fisher; 10 mg/ml NTES buffer: 100 mM NaCl, 50 mM Tris (pH 8.0), 50 mM EDTA, and 1% SDS]. DNA was extracted from the digests by the phenol method, and spooled DNA was rinsed with 70% ethanol and dried. DNA was resuspended in 0.5 ml sterile water. Oligomer sequences (5'-3') used for PCR were as follows: primer 1, AGC GCT TGT GTC GCC ATT GTA TTA; primer 2, GTC ACA CCA CAG AAG TAA GGT TCC; primer 3, ATA CCG TCC ATC TTG GCA AAG GAG; primer 4, TTG CTT CAG TCT CTT GAG TAT TAG GAT TGC; primer 5, GGG AGA CTT GTG ATT GGA ATA ATT GGA CG; and primer 6, TCC TGC AGT TCA TTC AGG GCA CCG G. Primers 1 and 2 were used to identity the SV40 transgene (IM). Primers 3 and 4 were used to identify the CFTR wild-type allele (CFTR). Primers 5 and 6 were used to amplify the S489X neodisrupted allele of CFTR (NEO). The PCR reaction parameters were as follows: 30 cycles at 95°C for 1 min, 58°C for 2 min, and 70°C for 3 min. The first generation offspring were bred, and second generation pups were genotyped by PCR to identify animals (CFTR -/- and CFTR +/+ or +/- animals that carried at least 1 copy of the SV40 transgene) that were to be used for tissue isolation. All second generation offspring were maintained on a liquid diet to improve survival of CFTR -/- pups (18).

Tissue and Cell Isolation

Collecting tubule cell lines. Ten mice that carried at least one copy of the H-2Kb-tsA58 transgene and that were heterozygous for the CFTR allele were killed, and the kidneys were removed, sliced, and digested with collagenase type IV (0.5% wt/vol) in Hanks' balanced salt solution (HBSS) for 30 min at 37°C. The digest was centrifuged (800 g for 5 min), and the cell pellet was resuspended in cold culture medium with 10% fetal bovine serum (FBS), centrifuged again, and resuspended in 5 mM glucose in phosphate-buffered saline (PBS). The cell suspension was plated onto tissue culture dishes (Falcon 1058; Falcon-Becton Dickinson, Lincoln Park, NJ) coated with Dolichos biflorus agglutinin (DBA; 4°C, 10 µg/ml in 0.1 M NaHCO3). DBA has been shown to specifically label the collecting duct (principal and intercalated cells) of the mouse kidney (30). Cells were allowed to adhere for 45 min at 4°C. Unbound cells were removed by being washed three times with PBS-glucose at 4°C. Lectin-adhered cells were eluted by being incubated in 10 ml of 150 mM galactose in PBS for 5 min. Cells were washed in PBS-glucose, centrifuged (800 g for 5 min), washed in culture medium, centrifuged (800 g for 5 min), resuspended, and plated in culture media at a density of 2 × 105 cells/ml. Cells were maintained as primary cultures at 37°C for 7 days in defined basal media for collecting tubule epithelial cells (CT media). Once colonies were established, recombinant mouse IFN-gamma (10 U/ml) was added to the basal CT media, and cultures were expanded at the 33°C permissive temperature. The resulting cell line is identified as mCT1 (42). An identical procedure was used to isolate CT cells from six mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mCT1-CF.

Pancreas cell lines. Four mice that carried at least one copy of the H-2Kb-tsA58 transgene and were heterozygous for the CFTR allele were killed, and the pancreati were removed. The tissues were placed in a HEPES-buffered Ringer solution (HR) that contained 0.25 mg/ml collagenase type I, 0.25 mg/ml collagenase type IV, and 0.1 mg/ml soy bean trypsin inhibitor. The tissues were minced with scissors and digested for 45 min at 37°C. At 15-min intervals, the tissue fragments were disrupted by repeat passage through a plastic pipette. The resulting tissue digest was passed through a nylon filter (149 × 149 µm), and the material trapped by the filter was retained for culture. The tissue fragments were resuspended in exocrine media and plated on tissue culture dishes. A combination of serum-free media and differential trypsinization was used to eliminate fibroblast contamination. The resulting cell line is identified as mPEC1. An identical procedure was used to isolate pancreatic epithelial cells from four mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mPEC1-CF.

Salivary gland cell lines. Three mice that carried at least one copy of the H-2Kb-tsA58 transgene and were homozygous for the wild-type CFTR allele were killed, and the submandibular salivary glands were removed. The tissues were placed in HR that contained 0.25 mg/ml collagenase type I, 0.25 mg/ml collagenase type IV, and 0.1 mg/ml soy bean trypsin inhibitor. The tissues were minced with scissors and digested for 60 min at 37°C. At 15-min intervals, the tissue fragments were disrupted by repeat passage through a plastic pipette. At the end of this period, the cell suspension was layered over a 4% bovine serum albumin (BSA in HR) solution and was allowed to settle for 10 min on ice. The supernatant was removed, and the resulting pellet was resuspended in exocrine media and plated in tissue culture dishes. After several passages, the contaminating fibroblasts were removed by differential trypsinization. The resulting cell line is identified as mSEC1. An identical procedure was used to isolate salivary gland epithelial cells from three mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mSEC1-CF.

Tracheal epithelial cell lines. Three mice that carried at least one copy of the H-2Kb-tsA58 transgene and were heterozygous for the CFTR allele were killed, and tracheas were removed. Isolated trachea were cleaned of connective tissue, opened along the posterior surface, and pinned to expose the epithelial surface. The tissues were exposed to 0.1% protease (type XIV, Sigma) and 0.1% collagenase (type IV, Sigma) in Ca2+- and Mg2+-free HBSS that contained 5 mM EDTA at 37°C for 20 min. The epithelium was freed from tracheal rings and lamina propria by pipetting a steady, forceful stream of fluid over the partially digested tracheal tissue. Tracheal epithelial cells were rinsed, centrifuged, resuspended in small airways growth media (SAGM; Clonetics), and plated on vitrogen gel-coated six-well culture plates. Cells were grown on vitrogen gels for three passages. Tracheal cell cultures were subsequently expanded and maintained on tissue culture dishes. The resulting cell line is identified as mTEC1. An identical procedure was used to isolate tracheal epithelial cells from two mice that carried at least one copy of the H-2Kb-tsA58 transgene with both CFTR alleles disrupted. The resulting cell line is identified as mTEC1-CF.

Cell culture. Renal cell lines were maintained in culture media (CT media) that contained a 1:1 mix of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium supplemented with 1.3 µg/l sodium selenite, 1.3 µg/l 3,5,3'-triiodo-L-thyronine, 5 mg/l insulin, 5 mg/l transferrin, 25 µg/l prostaglandin E1, 2.5 mM glutamine, 5 nM dexamethasone, 50,000 U/l nystatin, 50 mg/l streptomycin, 30 mg/l penicillin G, and 10,000 U/l recombinant mouse IFN-gamma . mCT1 and mCT1-CF cells were maintained on plastic tissue culture dishes in CT media in a humidified 33°C incubator with 5% CO2. Media was changed every other day, and cells were passaged weekly. Cells used for experiments reported here were between passages 15 and 25. Pancreatic and salivary gland epithelial cell lines were maintained in culture media (exocrine media) that contained 1:1 mix of DMEM and Ham's F-12 medium supplemented with 0.5 mM isobutyl methyl xanthine, 2 mM glutamine, 10 µg/l epidermal growth factor, 100 mg/l streptomycin sulfate, 60 mg/l penicillin G, 50,000 U/l nystatin, 2.5% FBS, and 10,000 U/l IFN-gamma . Salivary and pancreatic epithelial cells were maintained on plastic tissue culture dishes in exocrine media in a humidified 33°C incubator with 5% CO2. Media was changed every other day, and cells were passaged weekly. Cells used for experiments reported here were between passages 10 and 25. Tracheal epithelial cells were maintained in SAGM (Clonetics) supplemented with 500 mg/l BSA, 0.5 µg/l human epidermal growth factor, 5 mg/l insulin, 6.5 µg/l 3,5,3'-triiodo-L-thyronine, 10 mg/l transferrin, 30 mg/l bovine pituitary extract, 0.5 mg/l epinephrine, 0.5 mg/l hydrocortisone, 0.1 µg/l retinoic acid, 50 mg/l gentamycin sulfate, 50 µg/l amphotericin B, and 10,000 U/l IFN-gamma . Tracheal epithelial cells were maintained on plastic tissue culture dishes in SAGM in a humidified 33°C incubator with 5% CO2. Media was changed every other day, and cells were passaged weekly. Cells used for experiments reported here were between passages 15 and 30.

Immunolocalization of ZO-1. Cells were seeded onto Costar Transwell clear polyester filters and maintained under permissive conditions until the cultures became confluent. The epithelial monolayers were rinsed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. The monolayers were then washed three times with PBS, permeabilized by exposure to 0.1% Triton X-100 in PBS for 5 min, and then washed three times with PBS. The monolayers were blocked with 10% FBS in PBS and then incubated with primary antibody (diluted 1:10; ZO-1; R26.4C; obtained from the Developmental Studies Hybridoma Bank at the Univ. of Iowa) for 60 min at room temperature. The cells were washed three times with PBS and then exposed to secondary antibody (diluted 1:100; FITC-conjugated Affinipure goat anti-rat IgG; Jackson Immunochemicals) for 60 min at room temperature. The monolayers were washed three times with PBS, and a section of the filter was cut, mounted on a glass slide with a drop of Slow Fade (Molecular Probes), covered with a coverslip, and sealed with clear nail polish. The samples were examined using a confocal microscope.

Transepithelial electrical measurements. Cells were seeded (1-3 × 105 cells/filter) on collagen-coated, permeable supports (Millicell-CM 12 filters, Millipore) cut to a height of 4 mm, with the "feet" removed (17). The filter surface was coated with 125 µl/cm2 calf skin collagen (Sigma) dissolved in acetic acid (7.5 mg/ml 0.2% glacial acetic acid) and allowed to dry. The collagen coating was cross-linked by exposure to ammonium hydroxide vapors (3.5% solution) for 10 min followed by immersion in glutaraldehyde (2.5%) for 10 min. This procedure was followed by a thorough rinsing in distilled water, 70% ethanol, distilled water, and finally, culture media. Filter-grown cells were cultured with IFN-gamma at 33°C for 7-14 days. Media was changed every 48 h. Cell monolayers grown on modified supports were clamped between Lucite flux chambers and bathed on both sides by equal volumes (usually 6-10 ml) of Krebs-Ringer bicarbonate (KRB) solution. The solutions were circulated through the water-jacketed glass reservoir by gas lifts (95% O2-5% CO2) to maintain solution temperature at 37°C and pH at 7.4. Transepithelial voltage difference (VT) was measured between two Ringer-agar bridges, each positioned within 3 mm of the monolayer surface. Calomel half-cells connected the bridges to a high-impedance voltmeter. Current from an external direct current source was passed by silver-silver chloride electrodes and Ringer-agar bridges to clamp the spontaneous VT to 0. The current required (short-circuit current, Isc) was corrected for solution and filter series resistance. Monolayers were maintained under short-circuit conditions except for brief 3- to 5-s intervals when the current necessary to clamp the voltage to a nonzero value (usually +2 mV) was measured to calculate transepithelial resistance (RT).

36Cl- efflux. Efflux assays were performed as described previously (1, 31). Briefly, cells were grown to confluence in 35-mm tissue culture dishes. The monolayers were then washed three times with HR to remove media, and monolayers were incubated with 36Cl- (NaCl, 5 µCi/ml; Amersham, Arlington Heights, IL) in 1 ml HR for 1 h. After the cells were loaded with 36Cl-, they were rapidly washed (3 times with warmed isotope-free HR) to remove extracellular 36Cl-. Efflux of 36Cl- was measured at 30-s intervals for 8 min. The effect of elevation of cAMP on 36Cl- efflux was determined by adding forskolin (10 µM) and isobutyl methyl xanthine (100 µM) during time intervals of 2 to 8 min. After the last sample was removed, the cells were lysed by the addition of 0.5 ml of 1 N HCl for 20 min. The sample was neutralized with NaOH. All samples were mixed with liquid scintillation fluid (Ecolume, ICN) and assayed for 36Cl- activity (LS 5801; Beckman Instruments, Fullerton, CA). The apparent rate constant (r, in min-1) was calculated for each efflux interval from the following equation: r = [ln(C1- ln(C2)]/(t2 - t1), where ln(C1) and ln(C2) are the natural logs of the percentage of counts remaining in the cell layer, at times t1 and t2, respectively.

Solutions and chemicals. HR was composed of (in mM) 10 HEPES, 138 NaCl, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose. KRB was composed of (in mM) 115 NaCl, 25 NaHCO3, 5 KCl, 2.5 Na2HPO4, 1.8 CaCl2, 1 MgSO4, and 10 glucose.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Epithelial cell lines were successfully derived from trachea, pancreas, salivary gland, and renal CT from CF and non-CF mice that carried the temperature-sensitive SV40 transgene. Each of the eight cell lines had been maintained in culture under permissive conditions (33°C plus INF-gamma ) for >1 yr with >50 passages. Multiple attempts to generate cell lines from the small intestine and colon were not successful. The cell lines grow as epithelial monolayers, and each cell line expresses the epithelial tight junction protein ZO-1 (Fig. 1), consistent with the formation of functional tight junctions (as revealed by measurements of transepithelial electrical resistance).


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Fig. 1.   Immunolocalization of ZO-1 in confluent monolayers of cystic fibrosis (CF) and non-CF epithelial cells. Cells were grown to confluence on uncoated Transwell filters under permissive conditions. The monolayers were fixed, permeabilized, stained, and examined for ZO-1 expression. In each of the 8 cell lines, the ZO-1 immunoreactivity is restricted to the lateral surface of the epithelial cells near the apical membrane. A: mTEC1 cells; B: mTEC1-CF cells; C: mCT1 cells; D: mCT1-CF cells; E: mSEC1 cells; F: mSEC1-CF cells; G: mPEC1 cells; H: mPEC1-CF cells. Magnification, ×270.

Cell Line Genotypes

The genotypes of the cell lines were determined by PCR analysis of genomic DNA, and the results are shown in Fig. 2. The CF cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) were negative for the wild-type CFTR allele (faint 200-bp bands are non-CFTR PCR products) and positive for the S489X neodisrupted allele of CFTR. In contrast, three of the non-CF cell lines (mTEC1, mCT1, and mPEC1) carried both the wild-type CFTR allele and the S489X neodisrupted allele, whereas one of the non-CF cell lines (mSEC1) was positive for the wild-type CFTR allele and negative for the S489X neodisrupted allele of CFTR. All eight cell lines were positive for the SV40 transgene. The results were as expected based on the genotypes of the animals used for cell isolation.


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Fig. 2.   Genotypes of conditionally immortalized epithelial cell lines. Each of the 8 cell lines were grown to confluence under permissive conditions, the cells were harvested, and DNA was extracted. Wild-type cystic fibrosis transmembrane conductance regulator (CFTR), neomycin disrupted S489X CFTR (NEO), and Immorto (IM) alleles were identified from PCR products separated by gel electrophoresis. Specific primers were used that yielded products of 500, 650, and 1,000 bp for the CFTR, NEO, and IM, respectively (see METHODS). All 8 cell lines were positive for the Immorto gene. The 4 CF cell lines were negative for the wild-type CFTR allele (CFTR) and positive for the disrupted CFTR allele (NEO). Three of the non-CF cell lines (mTEC1, mCT1, and mPEC1) were heterozygous for the CFTR allele (CFTR, +/-), and 1 cell line (mSEC1) carried 2 copies of wild-type CFTR (CFTR +/+). pos con and neg con, Positive and negative control samples, respectively.

cAMP-Regulated Cl- Permeability

The CF phenotype of the resulting cell lines was determined by measuring cAMP-stimulated 36Cl- efflux from the cells. As illustrated in Fig. 3, plasma membrane Cl- permeability of each of the four non-CF epithelial cell lines was increased by exposure to forskolin/isobutyl methyl xanthine. The time course for the response was the same in each of the four cell lines (i.e., 30- to 60-s delay) and was similar to what we have observed previously in bovine pancreatic duct cells (1, 31). Cl- efflux from the CF pancreatic and CF salivary epithelial cells (mPEC1-CF and mSEC1-CF) was increased slightly after an ~180-s delay. The increase in 36Cl- efflux observed in these two cell lines was prevented by addition of bumetanide (10 µM) to the efflux media, suggesting that the small increase in Cl- permeability was due to stimulation of Na+-K+-2Cl- cotransport-mediated efflux (data not shown). In contrast, there was no response to elevation of cAMP in the CF renal CT and CF tracheal cells (mCT1-CF and mTEC1-CF).


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Fig. 3.   Effect of elevation of cAMP on the rate (r) of 36Cl- efflux. Cells were seeded onto tissue culture dishes and maintained under permissive growth conditions until the cultures were confluent. The cells were loaded with 36Cl- for 1 h, and the rate of efflux of isotope was measured as described in METHODS. During the period indicated by hatched bars, cells were exposed to vehicle (open circle ) or forskolin (10 µM) and IBMX (100 µM) (). Values are means +/- SE; n = 3-4 for each condition.

Electrogenic Ion Transport

Transepithelial electrical measurements were made on epithelial monolayers to determine whether the cell lines formed polarized epithelial barriers and expressed the appropriate Cl- transport phenotype. Cells were cultured on permeable supports and mounted in Ussing chambers for measurements of electrogenic ion transport (Isc). As summarized in Table 1, each of the eight cell lines formed polarized epithelial barriers and exhibited electrogenic ion transport. Amiloride-sensitive Isc was observed in renal, tracheal, and pancreatic cell lines but not in salivary cell lines. As illustrated in Fig. 4 and summarized in Table 1, each of the four non-CF cell lines exhibited cAMP-stimulated ion transport. In contrast, all four of the CF cell lines failed to respond to elevation of cAMP. However, subsequent exposure to extracellular ATP (100 µM added to the luminal bath to elevate intracellular Ca2+) elicited a large, transient increase in Isc in both non-CF and CF cell lines. Thus cAMP-stimulated Cl- secretion (CFTR-mediated) is expressed in each of the non-CF cell lines and is missing in the CF cell lines, whereas Ca2+-stimulated Cl- secretion is common to all eight cell lines.

                              
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Table 1.   Summary of bioelectric properties of epithelial monolayers



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Fig. 4.   Effect of forskolin and ATP on short-circuit current (Isc) across confluent epithelial monolayers. Cells were seeded onto collagen-coated permeable supports and maintained under permissive conditions until confluent monolayers were formed. The filters were mounted in Ussing chambers, bathed on both sides with Krebs-Ringer bicarbonate, and maintained under short-circuit conditions except during 3-s pulses when transepithelial voltage was clamped to +2 mV (vertical deflections) to measure transepithelial resistance. The monolayers were treated with amiloride (10 µM added to the mucosal bathing solution) to inhibit transepithelial Na+ absorption. At the time indicated by the first bar, forskolin (10 µM) was added to the serosal bathing solution. At the second bar, ATP (100 µM) was added to the mucosal bathing solution.

Effects of Nonpermissive Culture Conditions on Ion Transport Properties of mCT1 Cells

The cell lines described in this report were derived from animals that carry a temperature-sensitive form of large T antigen. We have previously shown that the amount of large T antigen and the rate of cell proliferation are reduced by placing the cultures at 39°C and removing the INF-gamma from the culture media (42). To determine the effects of the switch from permissive to nonpermissive conditions on electrogenic ion transport, confluent monolayers of mCT1 cells were maintained under permissive conditions for 12 days or under permissive conditions for 8 days followed by an additional 4 days under nonpermissive conditions. Amiloride-sensitive Na+ absorption and cAMP-stimulated Cl- secretion were measured in paired monolayers. As shown in Fig. 5, cultures maintained under nonpermissive conditions for 4 days (a time when large T antigen levels fell by >95%; see Ref. 42) exhibited an increase in amiloride-sensitive Na+ absorption and a decrease cAMP-stimulated Cl- secretion.


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Fig. 5.   Effect of culture conditions on amiloride-sensitive Na+ absorption and cAMP-stimulated Cl- secretion in mCT1 cells. mCT1 cells were seeded onto permeable supports and maintained under permissive conditions for 12 days (filled bars; n = 6) or under permissive conditions for 8 days followed by nonpermissive conditions for 4 days (open bars; n = 6). Amiloride-sensitive Isc (10 µM amiloride) and cAMP-stimulated Isc (10 µM forskolin) were measured on all monolayers on day 12. *Drug-induced change in Isc is significantly different between monolayers maintained under permissive and nonpermissive conditions (P < 0.05, unpaired t-test).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this work was to generate genetically well-matched, immortalized epithelial cell lines from CF and non-CF mice. The results of the 36Cl- efflux studies demonstrate that the non-CF cell lines each respond to elevation of cAMP with a characteristic increase in Cl- permeability. The small increase in 36Cl- efflux seen in mSEC1-CF and mPEC1-CF cells (after a 3-min delay) suggests that in these cell types, elevation of cAMP stimulates bumetanide-sensitive Na+-K+-2Cl- cotransport. Without concurrent stimulation of apical Cl- conductance, activation of basolateral Na+-K+-2Cl- cotransport would not be expected to increase Isc (see Fig. 4). The transepithelial electrical measurements provide direct evidence for cAMP-activated Cl- secretion in non-CF cell lines but not in the corresponding CF cell lines. Therefore, as expected, the cAMP-dependent Cl- secretory phenotype of the cell lines accurately reflects the genotypes of the animals from which the cells were derived (9, 11, 28).

A variety of secondary defects have been identified in CF cells, including hyperabsorption of Na+ (9, 20, 23) and altered cAMP-dependent regulation of epithelial Na+ channels (10, 37, 41) and outward-rectifying Cl- channels (38). Six of the eight cell lines that we generated exhibited small, amiloride-sensitive currents; however, Na+ hyperabsorption was not observed in the CF cell lines. This is not unexpected, since freshly isolated CF mouse trachea does not exhibit Na+ hyperabsorption compared with non-CF trachea (22). The reason for tissue- and species-specific Na+ hyperabsorption (9, 20, 23) in CF is not known but may depend on the relative and absolute levels of expression of CFTR and epithelial Na+ channel. CFTR is also known to regulate cAMP-activated, DIDS-sensitive Cl- channels, perhaps via release of ATP, although this hypothesis remains controversial (37, 38). CFTR-dependent activation of DIDS-sensitive Cl- secretion is observed in mPEC1 monolayers but not in mPEC1-CF cells (data not shown). Thus murine pancreatic cell lines appear to retain abnormal regulation of DIDS-sensitive Cl- channels and may be useful for studies of the interaction of CFTR and non-CFTR Cl- channels.

It is difficult to make comparisons between the cell lines that we generated and native murine epithelia due to the paucity of ion transport data from freshly isolated tissues. Transepithelial ion transport data are not available from native murine pancreatic ducts; however, Githens et al. (19) reported that primary cultures of mouse pancreatic ducts expressed amiloride-sensitive Na+ absorption and cAMP- and Ca2+-stimulated Cl- secretory responses. The responses of the mPEC1 cell line resembled those reported by Githens et al. (19), but quantitative comparisons cannot be made since they showed only single traces with no summary data. We are unaware of transepithelial ion transport data from murine salivary ductal epithelial cells. A number of renal epithelial cell lines have been generated from various nephron segments, including CT (36, 42). Nearly all of the CT cell lines express amiloride-sensitive Na+ absorption and cAMP-stimulated Cl- secretion, similar to the results obtained with the mCT1 cell line. The relatively small amiloride-sensitive Isc of mCT1-CF cells was unexpected but is probably unrelated to the lack of CFTR expression, since amiloride-sensitive Na+ absorption appears to be poorly retained in epithelial cell lines. We have previously shown that mCT1 cells express aquaporin-2 and vasopressin receptors (42), properties characteristic of CT principal cells. The ion transport properties of freshly isolated tissues and primary cultures of murine tracheal epithelium have been established. Grubb and coworkers (22) reported cAMP-activated Cl- secretion of ~10 µA/cm2 in both non-CF and CF mouse trachea, whereas primary cultures of non-CF and CF tracheal epithelium responded to elevation of cAMP with ~5 and 0 µA/cm2, respectively (15). The anomalous secretory response of CF trachea to cAMP is thought to be mediated by cAMP-dependent release of Ca2+ and activation of Ca2+-stimulated Cl- secretion (22). The response is absent in primary cultures of CF tracheal cells (15) and in the immortalized mTEC1-CF cell line (this report). The cAMP-activated secretory response of mTEC1 cells might be enhanced by modifications in culture conditions such as addition of cholera toxin to the media and/or growth at an air-liquid interface. The Cl- secretory response elicited by elevation of intracellular Ca2+ in CF and non-CF trachea (~25-30 µA/cm2) (15) and primary cultures of non-CF and CF tracheal cells (~20-40 µA/cm2) (20) is similar to that observed in our immortalized tracheal cell lines (~17-27 µA/cm2) (this report).

The strategy used to generate the cell lines (CF knockout mice crossed with the ImmortoMouse) avoided the problems (variable integration site and transgene copy number) associated with in vitro immortalization of primary cell cultures. Furthermore, the use of a temperature-sensitive SV40 large T antigen provides an opportunity to control large T antigen levels and cell proliferation. The results presented above were obtained from cells maintained under permissive growth conditions (33°C with IFN-gamma ); however, several cell lines derived from the ImmortoMouse show tissue-specific differentiation when the cells are placed under nonpermissive growth conditions (14, 26, 42, 44). The mCT1 cells are derived from principal cells of the CT and are expected to exhibit amiloride-sensitive Na+ absorption. Thus the increase in amiloride-sensitive Na+ absorption coupled with the decrease in cAMP-stimulated Cl- secretion (Fig. 5) when the cells are maintained under nonpermissive conditions is consistent with acquisition of a more differentiated transport phenotype. Additional studies will be required to examine the effect of nonpermissive growth conditions on each of the cell lines.

The cell lines that we have generated are unique in several regards: 1) they represent the first cell lines obtained from CF knockout mice, 2) the lines are derived from several tissues relevant to CF, 3) the lines are genetically well matched, and 4) the use of a temperature-sensitive SV40 large T antigen oncogene should provide a means to regulate growth and differentiation in culture. The cell lines should prove useful for studies of the role of CFTR in normal epithelial cell physiology. Since the CF cells are derived from CFTR -/- mice, they represent a null background suitable for studies of mutant forms of CFTR and should help to define alterations in cell function associated with loss of CFTR and/or specific CFTR mutations in the context of an epithelial cell. Finally, the non-CF cell lines will be useful as model systems for studies of regulation of epithelial function.


    ACKNOWLEDGEMENTS

We thank Mike Haley for expert technical assistance.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-53318, the Cystic Fibrosis Foundation, and the Polycystic Kidney Research Foundation.

Address for reprint requests and other correspondence: C. U. Cotton, 2109 Adelbert Rd., Biomedical Research Bldg., Cleveland, OH 44106-4948 (E-mail: cuc{at}po.cwru.edu).

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

Received 22 May 2000; accepted in final form 29 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Al-Nakkash, L, and Cotton CU. Bovine pancreatic duct cells express cAMP-and calcium-activated apical membrane Cl- conductances. Am J Physiol Gastrointest Liver Physiol 273: G204-G216, 1997[Abstract/Free Full Text].

2.   Anderson, MP, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, Smith AE, and Welsh MJ. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253: 202-205, 1991[ISI][Medline].

3.   Anderson, MP, Rich DP, Gregory RJ, Smith AE, and Welsh MJ. Generation of cAMP-activated chloride currents by expression of CFTR. Science 251: 679-682, 1991[ISI][Medline].

4.   Barasch, J, Kiss B, Prince A, Saiman L, Gruenert D, and Al-Awqati Q. Defective acidification of intracellular organelles in cystic fibrosis. Nature 352: 70-73, 1991[ISI][Medline].

5.   Berger, HA, Travis SM, and Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. J Biol Chem 268: 2037-2047, 1993[Abstract/Free Full Text].

6.   Bhaskar, KR, Turner BS, Grubman SA, Jefferson DM, and LaMont JT. Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-f508) cystic fibrosis transmembrane conductance regulator. Hepatology 27: 7-14, 1998[ISI][Medline].

7.   Boucher, RC. Human airway ion transport. II. Am J Respir Crit Care Med 150: 581-593, 1994[ISI][Medline].

8.   Boucher, RC. Human airway ion transport. I. Am J Respir Crit Care Med 150: 271-281, 1994[ISI][Medline].

9.   Boucher, RC, Cotton CU, Gatzy JT, Knowles MR, and Yankaskas JR. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures. J Physiol (Lond) 405: 77-103, 1988[Abstract].

10.   Boucher, RC, Stutts MJ, Knowles MR, Cantley L, and Gatzy JT. Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 78: 1245-1252, 1986[ISI][Medline].

11.   Boucher, RC, Yankaskas JR, Cotton CU, Knowles MR, and Stutts MJ. Cell culture approaches to the investigation of human airway ion transport. Eur J Respir Dis 153: 59-67, 1987.

12.   Bradbury, NA. Intracellular CFTR: localization and function. Physiol Rev 79, Suppl 1: S175-S191, 1999[Medline].

13.   Bradbury, NA, Jilling T, Berta G, Sorscher EJ, Bridges RJ, and Kirk KL. Regulation of plasma membrane recycling by CFTR. Science 256: 530-532, 1992[ISI][Medline].

14.   Chambers, T, Owens J, Hattersley G, Jat P, and Noble M. Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse. Proc Natl Acad Sci USA 90: 5578-5582, 1993[Abstract].

15.   Clarke, LL, Grubb BR, Gabriel SE, Smithies O, Koller BH, and Boucher RC. Defective epithelial chloride transport in a gene-targeted mouse model of cystic fibrosis. Science 257: 1125-1128, 1992[ISI][Medline].

16.   Clarke, LL, Grubb BR, Yankaskas JR, Cotton CU, McKenzie A, and Boucher RC. Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in CFTR (-/-) mice. Proc Natl Acad Sci USA 91: 479-483, 1994[Abstract].

17.   Cotton, CU, and Al-Nakkash L. Isolation and culture of bovine pancreatic duct epithelial cells. Am J Physiol Gastrointest Liver Physiol 272: G1328-G1337, 1997[Abstract/Free Full Text].

18.   Eckman, EA, Cotton CU, Kube DM, and Davis PB. Dietary changes improve survival of the CFTR S489X homozygous mutant mouse. Am J Physiol Lung Cell Mol Physiol 269: L625-L630, 1995[Abstract/Free Full Text].

19.   Githens, S, Schexnayder JA, Moses RL, Denning GM, Smith JJ, and Frazier ML. Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. In Vitro Cell Dev Biol 30A: 622-635, 1994.

20.   Grubb, BR, and Boucher RC. Enhanced colonic Na+ absorption in cystic fibrosis mice vs. normal mice. Am J Physiol Gastrointest Liver Physiol 272: G393-G400, 1997[Abstract/Free Full Text].

21.   Grubb, BR, and Boucher RC. Pathophysiology of gene-targeted mouse models for cystic fibrosis. Physiol Rev 79, Suppl 1: S193-S214, 1999[Medline].

22.   Grubb, BR, Paradiso AM, and Boucher RC. Anomalies in ion transport in CF mouse tracheal epithelium. Am J Physiol Cell Physiol 267: C293-C300, 1994[Abstract/Free Full Text].

23.   Grubb, BR, Vick RN, and Boucher RC. Hyperabsorption of Na+ and raised Ca2+-mediated Cl- secretion in nasal epithelia of CF mice. Am J Physiol Cell Physiol 266: C1478-C1483, 1994[Abstract/Free Full Text].

24.   Gruenert, DC, Basbaum CB, Welsh MJ, Li M, Finkbeiner WE, and Nadel JA. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc Natl Acad Sci USA 85: 5951-5955, 1988[Abstract].

25.   Hopfer, U, Jacobberger JW, Gruenert DC, Eckert RL, Jat PS, and Whitsett JA. Immortalization of epithelial cells. Am J Physiol Cell Physiol 270: C1-C11, 1996[Abstract/Free Full Text].

26.   Jat, PS, Noble MD, Ataliotis P, Tanaka Y, Yannoutsos N, Larsen L, and Kioussis D. Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 trangenic mouse. Proc Natl Acad Sci USA 88: 5096-5100, 1991[Abstract].

27.   Jefferson, DM, Valentich JD, Marini FC, Grubman SA, Iannuzzi MC, Dorkin HL, Li M, Klinger KW, and Welsh MJ. Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 259: L496-L505, 1990[Abstract/Free Full Text].

28.   Jetten, AM, Yankaskas JR, Stutts MJ, Willumsen NJ, and Boucher RC. Persistence of abnormal chloride conductance regulation in transformed cystic fibrosis epithelium. Science 244: 1472-1475, 1989[ISI][Medline].

29.   Knowles, MR, Stutts MJ, Spock A, Fischer N, Gatzy JT, and Boucher RC. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science 221: 1067-1070, 1983[ISI][Medline].

30.   Laitinen, L, Virtanen I, and Saxen L. Changes in the glycosylation pattern during embryonic development of mouse kidney as revealed with lectin conjugates. J Histochem Cytochem 35: 55-65, 1987[Abstract].

31.   Marino, LR, and Cotton CU. Immortalization of bovine pancreatic duct epithelial cells. Am J Physiol Gastrointest Liver Physiol 270: G676-G683, 1996[Abstract/Free Full Text].

32.   Morgan, JE, Beauchamp JR, Pagel CN, Peckham M, Ataliotis P, Jat PS, Noble MD, Farmer K, and Partridge TA. Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines. Dev Biol 162: 486-498, 1994[ISI][Medline].

33.   Quinton, P, and Bijman J. Higher bioelectric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis. N Engl J Med 108: 1185-1189, 1983.

34.   Riordan, JR, Rommens JM, Kerem BS, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, Drumm ML, Iannuzzi MC, Collins FS, and Tsui LC. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073, 1989[ISI][Medline].

35.   Rommens, JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, Zsiga M, Buchwald M, Riordan JR, Tsui LC, and Collins FS. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065, 1989[ISI][Medline].

36.   Ronco, PM, Prie D, Piedagnel R, and Lelong B. Oncogene-transformed renal cell lines: physiological and oncogenetic studies. NIPS 9: 208-214, 1994[Abstract/Free Full Text].

37.   Schwiebert, EM, Benos DJ, Egan ME, Stutts MJ, and Guggino WB. CFTR is a conductance regulator as well as a chloride channel. Physiol Rev 79, Suppl 1: S145-S166, 1999[Medline].

38.   Schwiebert, EM, Egan ME, Hwang TH, Fulmer SB, Allen SS, Cutting GR, and Guggino WB. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063-1073, 1995[ISI][Medline].

39.   Smith, JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[ISI][Medline].

40.   Snouwaert, JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088, 1992[ISI][Medline].

41.   Stutts, MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[ISI][Medline].

42.   Takacs-Jarrett, M, Sweeney WE, Avner ED, and Cotton CU. Morphological and functional characterization of a conditionally immortalized collecting tubule cell line. Am J Physiol Renal Physiol 275: F802-F811, 1998[Abstract/Free Full Text].

43.   Wagner, JA, McDonald TV, Nghiem PT, Lowe AW, Schulman H, Gruenert DC, Stryer L, and Gardner P. Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents. Proc Natl Acad Sci USA 89: 6785-6789, 1992[Abstract].

44.   Whitehead, RH, VanEeden PE, Noble MD, Ataliotis P, and Jat PS. Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. Proc Natl Acad Sci USA 90: 587-591, 1993[Abstract].

45.   Yankaskas, JR, Haizlip JE, Conrad M, Koval D, Lazarowski E, Paradiso AM, Rinehart CA, Jr, Sarkadi B, Schlegel R, and Boucher RC. Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype. Am J Physiol Cell Physiol 264: C1219-C1230, 1993[Abstract/Free Full Text].

46.   Zeitlin, PL, Lu L, Rhim J, Cutting G, Stetten G, Kieffer KA, Craig R, and Guggino WB. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection. Am J Respir Cell Mol Biol 4: 313-319, 1991[ISI][Medline].


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