Proc. Natl. Acad. Sci. USAVol. 93, pp. 3587-3591, April 1996Medical Sciences

Mutant cystic fibrosis transmembrane conductance regulatorinhibits acidification and apoptosis in C127 cells: Possiblerelevance to cystic fibrosisROBERTA A. GOTTLIEB*t AND AMRITA DOSANJHt

*Dcpartment of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037; tResearch Service, Department of Veterans AffairsMedical Center, San Diego, CA 92037; and tDepartment of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305

Connmmulnicated by Ernest Beitler, The Scripps Research Institute, La Jolla, CA, December 22, 1995 (received for review November 28, 1995)

ABSTRACT We have shown elsewhere that acidificationis an early event in apoptosis, preceding DNA cleavage. Cellsexpressing the most common mutation (deIF508) of the cysticfibrosis transmembrane regulator (CFTR) exhibit a higherresting intracellular pH and are unable to secrete chloride andbicarbonate in response to cAMP. We hypothesized thatdefective acidification in cells expressing delF508 CFTR wouldinterfere with the acidification that accompanies apoptosis,which, in turn, would prevent endonuclease activation andcleavage of DNA. We therefore determined whether the func-tion of the CFTR would affect the process of apoptosis inmouse mammary epithelial C127 cells stably transfected withthe wild-type CFTR (C127/wt) or the delF508 mutation of theCFTR (C127/508). C127 cells possessed an acid endonucleasecapable of DNA degradation at low pH. Sixteen hours aftertreatment with cycloheximide, C127/wt cells underwent cy-toplasmic acidification. In contrast, C127/508 cells failed todemonstrate acidification. Furthermore, the C127/508 cellsdid not show nuclear condensation or DNA fragmentationdetected by in situ nick-end labeling after treatment withcycloheximide or etoposide, in contrast to the characteristicfeatures of apoptosis demonstrated by the C127/wt cells.Measurement of cell viability indicated a preservation of cellviability in C127/508 cells but not in C127/wt cells. That thisresistance to the induction of apoptosis depended upon theloss of CFTR activity is shown by the finding that inhibitionof the CFTR with diphenylamine carboxylate in C127/wt cellsconferred similar protection. These findings suggest a role forthe CFTR in acidification during the initiation of apoptosis inepithelial cells and imply that a failure to undergo pro-grammed cell death could contribute to the pathogenesis ofcystic fibrosis.

Apoptosis, or programmed cell death, was first described byWyllie et al. (1) and is the physiologic process by whichorganisms eliminate unwanted cells. Apoptosis is essential formaintenance of homeostasis in continuously renewing tissues,for clearance of inflammatory cells during the resolution phaseof an infection, and for removing cells that have sustaineddamage that makes their continued survival undesirable (e.g.,cells with DNA damage). Apoptosis is a tightly regulatedprocess that represents a cellular response to stress, such asgrowth factor withdrawal, oxidative stress, or an external signal(e.g., Fas ligation). Agents such as cycloheximide and etopo-side have been shown to induce apoptosis in a variety of celltypes and were chosen for use in these studies. The stimulus toinduce apoptosis (death signal) initiates a complex, coordi-nated cellular response characterized by preservation of thecell membrane (preventing spillage of proinflammatory cellcontents), crosslinking of cellular proteins by a transglutami-nase to form a cornified envelope, DNA fragmentation,

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

condensation and fragmentation of the nucleus into smallapoptotic bodies, and expression of markers targeting thecells/apoptotic bodies for phagocytosis.The internucleosomal DNA cleavage characteristic of

apoptosis is often attributed to a calcium- and magnesium-dependent endonuclease active at neutral pH; both DNase Iand NUC18 have been implicated (2, 3). We and others haveidentified an acid endonuclease similar or identical to DNaseII (4, 5). This acid endonuclease is capable of digestingchromatin into oligonucleosomal fragments at pH valuesbelow 6.8.We and other investigators have shown that acidification

accompanies apoptosis (6-10), and we have also shown thatacidification is a prerequisite for DNA cleavage and possiblyother biochemical changes that accompany apoptosis (11).Control of intracellular pH depends upon activity of a varietyof ion channels and pumps, including the sodium/hydrogenexchanger, the anion exchanger, the vacuolar proton ATPase,and, in epithelial cells, the cystic fibrosis transmembraneregulator (CFTR).The CFTR is a transmembrane anion channel that opens in

response to cAMP. Under physiologic conditions, the direc-tion of flow for chloride and bicarbonate is out of the cell; theloss of bicarbonate would tend to lower the intracellular pH(12). It has been shown that cells expressing the mutant formof the CFTR do not secrete chloride or bicarbonate, have abaseline pH that is higher than in comparable normal cells, andfail to fully acidify cytoplasmic organelles (13-15). We hypoth-esized that the cytoplasmic alkalinization (or inability toacidify) caused by the mutant CFTR might interfere with theprocess of apoptosis that depends upon acidification for DNAfragmentation. We considered it possible that in cystic fibrosisdefective apoptosis could lead to the release of undigestedDNA from senescent epithelial cells and leukocytes. Thiscould explain a cardinal feature of cystic fibrosis: the accu-mulation of thick, viscous mucus containing high molecularweight DNA. We sought to test this hypothesis in cell cultureby using cycloheximide and etoposide to induce apoptosis inepithelial cells transfected with the normal and delF508 mu-tant CFTR gene.

MATERIALS AND METHODSCell Culture. The mouse mammary C127 epithelial cell line

(which does not express the CFTR) was used to establish stablytransfected sublines expressing the wild-type human CFTR(C127/wt) or the delF508 mutation (which is the most com-mon mutation in patients with cystic fibrosis) (C127/508);these cell liens have been described elsewhere (16). TheCFTR-transfected cell lines were grown in Dulbecco's modi-fied Eagle's medium with 10% fetal calf serum and 2 mML-glutamine, and passaged by trypsinization twice weekly.

Abbreviations: CFTR, cystic fibrosis transmembrane regulator; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

3587

Dow

nloa

ded

by g

uest

on

July

17,

202

1

3588 Medical Sciences: Gottlieb and Dosanjh

Preparation of Nuclear Extracts. Epithelial cells werewashed in phosphate-buffered saline (PBS) then lysed in 10mM Tris (pH 7.5), 1.5 mM MgCl2, and 0.5% Nonidet P-40 for15 min on ice. The nuclear pellet (typically representing 30 t1l)obtained after sedimentation (2000 x g) was resuspended in100 t1l nuclear extract buffer (20 mM Tris, pH 7.5/0.4 MNaCl/1.5 mM MgCl2/1 mM dithiothreitol/25% glycerol) andincubated for 30 min at 4°C with gentle shaking. Aftersedimentation at 17,000 x g, the supernatant (nuclear extract)was stored at -70°C until use.

Plasmid DNA Digestion Assay. Nuclear extract (1 gul) wasincubated at 37°C for 1 hr with 100 ng supercoiled plasmidDNA in APT buffer (10 mM sodium acetate/10 mM potas-sium phosphate/10 mM Tris) at pH 5.5 or cation buffer (30mM Tris, pH 7.4/5 mM CaCl2/5 mM MgCl2) as indicated ina volume of 50 tal (4). At the end of the 1-hr reaction, SDS(0.2%) and EDTA (10 mM) were added, followed by glycerolsample buffer. Samples were resolved electrophoretically on a1% agarose gel containing 0.5 jug of ethidium bromide per ml.Determination of Intracellular pH. C127/wt and C127/508

cell lines were treated overnight with dimethyl sulfoxide(vehicle control), etoposide (160 ,tg/ml), or cycloheximide(100 ,tg/ml), then harvested by trypsinization with EDTA.Cells in suspension were labeled for 30 min with 10 ,LMcarboxy SNARF-1 AM acetate (Molecular Probes), washedand resuspended in Hanks' balanced salt solution with 0.1 mMEDTA (to prevent aggregation), and analyzed by flow cytom-etry. A Coulter model Elite flow cytometer was used withexcitation at 488 nm and emission analyses at 575 and 620 nm(17). Ten thousand events are collected and data plotted as cellnumber vs. fluorescence ratio. Intracellular pH is estimated bycomparison with the fluorescence intensity ratio of cells thatare pH adjusted by incubation with nigericin (10 jtM) in ahigh-potassium buffer as described (18).Morphologic Assessment of Apoptosis. Cultured cells were

plated in Chamber Slides (Nunc) and treated with drugsovernight as indicated. The following day, the slides wererinsed with PBS, then fixed for 5 min with 4% bufferedformaldehyde and rinsed with methanol before air drying.Nuclear morphology was assessed after staining with a 4jtg/mL solution of acridine orange (19). Cells were photo-graphed at x400 magnification through a fluorescence micro-scope. To control for loss of nonadherent cells during fixationand staining, in some experiments cells in chamber slides werestained in acridine orange and scored without media exchangeor fixation.

Detection of Apoptosis by in Situ Nick-End Labeling. Cellsplated in chamber slides were treated with cycloheximide oretoposide overnight and labeled by a modification of themethod of Wijsman et al. (20), in which cells were fixed for 5min with 4% formaldehyde in PBS and rinsed with methanol.Samples were incubated with a reaction mix containing 0.05,LM biotin-14-dATP and 5 LtM each dTTP, dCTP, and dGTP,in 50 mM Tris-HCl (pH 7.5), 5 mM MgC12, 0.1 mg/ml bovineserum albumin, and 10 units/ml Klenow fragment of DNApolymerase I, for 30 min at room temperature. Slides were rinsedwith PBS, incubated with 0.28% periodic acid, rinsed, incubatedwith PBS containing 2% bovine serum albumin for 5 min, thenincubated with streptavidin/peroxidase (1:1000) (Kirkegaard &Perry Laboratories) for 30 min and rinsed twice with PBS. Colorwas developed with Enhance Black according to the manufac-turer's instructions (Kirkegaard & Perry Laboratories).

Cell Viability Assay. Cells from the wild-type and delF508CFTR-transfected lines were plated (5000 cells/well in 96-wellplates) and allowed to adhere for 1 hr. Test drugs were addedto triplicate wells and the cells were cultured overnight. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide] in a 5 mg/ml solution in PBS was added to each well (10jul per 100 til well) and the cells were incubated for anadditional 4 hr. The assay was terminated by the addition of

5.5 7.5 Ca" Mg+ Both

oc

sc

FIG. 1. Endonuclease activity of C127 nuclear extracts. Nuclearextracts were incubated with 100 ng of supercoiled plasmid DNA for1 hr at 37°C, then the products were resolved on a 1% agarose gelcontaining ethidium bromide. SC, supercoiled DNA; OC, open circle.Lanes: 1, reaction mix at pH 5.5; 2, pH 7.5; 3, pH 7.5 with 5 mM Ca2+;4, pH 7.5 with 5 mM Mg2+; 5, pH 7.5 with both Ca2 and Mg2+.Endonuclease activity is indicated by the conversion of supercoiledplasmid DNA to open circle form, followed by linearization andprogressive digestion. Plasmid DNA is completely digested by thenuclear extract at pH 5.5. Partial digestion of the plasmid DNA isapparent at neutral pH in the presence of divalent cations.

20% SDS in dimethylformamide. The plates were red in anELISA plate reader at 550 nm. The use of this assay fordetermination of cell viability is well established (21).

RESULTSSince we hypothesized that DNA degradation was dependentupon acidification and activity of an acid endonuclease, it wasimportant to determine if the C127 cell line possessed acidendonuclease activity. Nuclear extracts were prepared andendonuclease activity was measured under a variety of pH andionic conditions. As shown in Fig. 1, these cells contained acidendonuclease activity (indicated by complete degradation ofplasmid DNA) as well as neutral endonuclease activity in thepresence of divalent cations, indicated by degradation ofsupercoiled DNA to open circle and linear forms as well as

degradation to smaller fragments. Thus, the C127 cells pos-sessed the necessary machinery for DNA cleavage underconditions of low pH.We have shown acidification to accompany apoptosis in

several other cell types. To determine if this occurred in thesecells, we treated them with cycloheximide for 20 hr, thenloaded them with the pH-sensitive fluor, carboxy SNARF-1AM acetate, and analyzed them by flow cytometry. The resultscan be seen in Fig. 2. Cycloheximide induced acidification inthe C127/wt cell line, but had no effect on intracellular pH inthe C127/508 cells, consistent with the absence of apoptosis inthese cells.To determine if cytotoxic treatment of the cells was inducing

nuclear condensation suggestive of apoptosis, we examinedfresh or formalin-fixed cells stained with acridine orange. As

C127/WT .!X C127/508

.4*A.. - .

FIG. 2. Flow cytometric analysis of intracellular pH of C127/wtand C127/508 cells treated with cycloheximide for 24 hr. Cells weretreated with buffer (shaded region, solid line) or with 100 ,tg/mlcycloheximide (clear region, dotted line) for 24 hr, detached bytrypsinization, loaded with carboxy SNARF-1 AM, and analyzed bydual emission ratio analysis. Ten thousand events were analyzed.Vertical axis denotes cell number, and horizontal axis denotes fluo-rescence ratio, with lower pH to the left. (Left) Acidification ofC127/wt cells treated with cycloheximide (compared to control cells).(Right) No change in pH in C127/508 cells treated with cycloheximide.

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

July

17,

202

1

Proc. Natl. Acad. Sci. USA 93 (1996) 3589

shown in Fig. 3, the characteristic nuclear condensation inapoptosis is observed with high frequency in C127/wt cellstreated with cycloheximide or etoposide, but only rarely seenin C127/508 cells. Quantitation of these results is shown inTable 1 and indicates that changes of nuclear condensationconsistent with apoptosis were at least three times morefrequent in the C127/wt cells treated with cycloheximide oretoposide compared with the C127/508 cells. Results weresimilar in three separate experiments.We next examined the ability of these cells to degrade their

DNA upon exposure to cycloheximide. Cells treated for 20 hrand fixed as above were subjected to in situ nick-end labeling.Nuclei with DNA fragmentation were strongly labeled by thismethod (data not shown). Cycloheximide treatment inducedthe fragmentation of DNA in the C127/wt cells but not theC127/508 cells.

Cell viability was also assessed using the MTT assay, whichdepends upon metabolic activity for reduction of the tetrazo-lium salt. As shown in Fig. 4, cells expressing the delF508mutation of the CFTR were more resistant to the cytotoxicagents cycloheximide or etoposide than cells expressing thewild-type form of the CFTR. Inhibition of chloride channelfunction with the inhibitor diphenylamine carboxylate (1 mM)protected the C127/wt cell line against the induction ofapoptosis, again directly implicating chloride channel activityin the process leading to cell death (Fig. 4).To determine if the resistance to apoptosis in the C127/508

line could be overcome by promoting acidification, cells were

Table 1. Quantitation of apoptotic nuclei after treatment withcycloheximide or etoposide

% apoptoticTreatment C127/wt C127/508

Control 3.0 3.4Cycloheximide 39.6 1.9Etoposide 40.0 13.4

Apoptotic nuclei were scored afterminimum of 300 cells per conditionrepresentative of three experiments.

acridine orange staining. Awere counted. Results are

cultured with cycloheximide in the presence or absence of theweak organic acid, propionic acid (10 mM, adjusted to pH 7.4).After overnight culture, cell viability was measured using theMTT assay. As shown in Fig. 5, propionic acid made theC127/508 cells more susceptible to the induction of apoptosisby cycloheximide, but did not affect viability of C127/wt cellstreated with cycloheximide.

DISCUSSIONThis work demonstrates the essential role of a functionalCFTR in permitting apoptosis in epithelial cells, and providessupport for the hypothesis that DNA processing during apo-ptosis is abnormal in cystic fibrosis. Cystic fibrosis is anautosomal recessive genetic disease due to mutation of theCFTR; the most common mutation is a 3-bp deletion at codon

FIG. 3. Nuclear morphology of C127/wt and C127/508 cells after exposure to cycloheximide. Cells were plated on chamber slides and treatedwith cycloheximide (100 ,tg/ml) or etoposide (160 jug/ml) overnight, then fixed with 2% formaldehyde, stained with acridine orange, andphotographed under fluorescence. (A, C, and E) C127/wt cell line. (B, D, and F) C127/508 cell line. (A and B) Control. (C and D) Cycloheximide.(E and F) Etoposide. Typical apoptotic nuclei show brightly fluorescent chromatin (arrows). The results shown are representative of threeexperiments.

~~~~~~~~~~~~~~~~WW.........._~_-~__

Medical Sciences: Gottlieb and Dosanjh

Dow

nloa

ded

by g

uest

on

July

17,

202

1

3590 Medical Sciences: Gottlieb and Dosanjh

-I wt

100 [

COc)

>-

>

80

60 [

40 [

20

II^n1^IIIIIII

E,¢i 508

CON ETO CHX D/CHX

FIG. 4. Viability of C127/wt and C127/508 cells after overnightexposure to cycloheximide or etoposide. C127 cells expressing thewild-type CFTR (clear bars) or the delF508 CFTR (hatched bars) wereplated in 96-well plates and treated with 100 /ug/ml cycloheximide(CHX), 160 jig/ml etoposide (ETO), or 1 mM diphenylamine car-

boxylate plus cycloheximide (D/CHX) for 24 hr, then assayed forMTT reduction. Viability is expressed as percent of control activity (+1 SD) (cells treated with vehicle alone: water for cycloheximide and0.1% dimethyl sulfoxide for etoposide). CON, control.

508 (delF508) (22). In exocrine glands, epithelia do notrespond with chloride secretion to agents that elevate intra-cellular cAMP, and it has been suggested that this is respon-sible for the thick, viscous mucus secretions that are the mosttroublesome aspect of the disease. The introduction of nebu-lized recombinant DNase I into the airways has been shown toreduce the viscosity of the mucus and to provide clinicalimprovement (12, 23). This suggests that the presence of theviscous DNA is a major pathogenetic feature of the disease.We suggest that in cystic fibrosis, defective apoptosis-arising

-*- wto -- 508

I(D.0

CO3'>

80

o060 l

T-0

-L

,oI0--------

20

Ctrl PropFIG. 5. Effect of propionic acid on viability of cells after overnight

exposure to cycloheximide. C127/wt cells (e) and C127/508 cells (0)were treated with cycloheximide in the absence (Ctrl) or presence(Prop) of propionic acid (10 mM, pH 7.4). Results are presented as

percent viability relative to the untreated control (+ 1 SD). Threeexperiments are shown. For the C127/508 cell line, the differencebetween the absence and presence of propionic acid is significant at P< 0.01.

from an inability to achieve sufficient cytoplasmic acidifica-tion-contributes to the presence of undigested DNA in themucus of airways and other exocrine organs.

Apoptosis is a tremendously important process in tissuehomeostasis. The rate of cell death must equal that of renewal,and apoptotic cells must be neatly disposed of to avoid aninflammatory response to cellular contents. This is accom-plished by packaging the digested and condensed chromatin inmembrane-bound organelles termed apoptotic bodies, whichcan then be phagocytosed. Apoptosis is a coordinated processof cellular self-destruction that involves (among other things)cytoplasmic acidification and DNA degradation (4-6, 8, 10, 11).We show that the C127 mammary epithelial cells transfected

with the CFTR possess endonuclease activity capable of DNAdigestion and that cells expressing the wild-type CFTR un-dergo acidification following cycloheximide treatment to in-duce apoptosis. In contrast, cells expressing the delF508 mu-tation of CFTR do not acidify. In addition, we show by severalcriteria (nuclear condensation, DNA fragmentation, loss ofmetabolic activity) that the cells expressing the delF508 CFTRare relatively resistant to the induction of apoptosis by cyclo-heximide or etoposide. Since the only difference between thetwo cell lines is expression of a functional or mutant (andnonfunctional) CFTR, this suggests that the CFTR may par-ticipate in the induction of apoptosis. The absence of acidifi-cation and diminished apoptosis in the C127/508 cells are inagreement with our previous studies in the Jurkat T-lymphoblastline, where we showed that preventing acidification could protectcells from apoptosis mediated by Fas ligation, cycloheximide, orultraviolet radiation (8). The finding that diphenylamine carbox-ylate protects the wild-type cells (C127/wt) from induction ofapoptosis is of therapeutic interest, because it may be possible touse this agent to protect intestinal epithelial cells from apoptosisinduced by chemotherapy or abdominal irradiation.The CFTR may participate directly in cytoplasmic acidifi-

cation, through secretion of bicarbonate (24), or indirectly,through its effect on other ion channels, including the out-wardly rectifying chloride channel (25-27). One additionalobservation suggesting a requirement for CFTR activity in theinduction of apoptosis is the finding that inhibition of theCFTR with diphenylamine carboxylate protects C127/wt cellsagainst apoptosis. Diphenylamine carboxylate does not pro-vide additional protection to C127/508 cells, indicating that itsprotective effect is related to inhibition of the CFTR ratherthan an unrelated mechanism. Because acidification appearsto depend upon activity of the CFTR, which is known to beactivated by cAMP, it suggests that cAMP-dependent phos-phorylation may occur during the induction of apoptosis (28).However, the CFTR may be regulated by other protein kinases(e.g., protein kinase C) (29). In addition, treatment of cellswith forskolin or cholera toxin does not induce apoptosis(R.A.G., unpublished results), suggesting that other or addi-tional signals may be required.One consequence of cytoplasmic acidification is activation

of an acidic endonuclease, which is known to be present inthese cells. The absence of DNA cleavage measured by in situnick-end labeling in the C127/508 cells treated with cyclohex-imide suggests that activation of an endonuclease (either pHdependent or divalent cation dependent) did not occur. Acid-ification may bring about a new metabolic state in whichchromatin is cleaved, condensed, and packaged into apoptoticbodies that would be disposed of by neighboring phagocyticcells in tissue. If this process fails to occur, one can envisionrelease of high-molecular-weight undigested DNA and otherinflammatory cellular contents from senescent epithelial cellssloughed into the lumen of airways and other organs. Thiscould explain the presence of viscous DNA in the mucus ofpatients of cystic fibrosis (30-32). That the DNA contributesto the viscosity of the mucus and to the clinical features of thedisease is evidenced by the beneficial effects of inhaled DNase

D . . F r r f a a r r x s Proc. Natl. Acad. Sci. U/SA 93 (1996)

T TI /kI,

/r//R/;//.MI

Dow

nloa

ded

by g

uest

on

July

17,

202

1

Proc. Natl. Acad. Sci. USA 93 (1996) 3591

I therapy (23, 24). It has been suggested that the DNA inairways derives from leukocytes; this is possible becauseneutrophils and lymphocytes also express the CFTR, althoughat lower levels (33, 34), and we have shown acidification tooccur in neutrophils and lymphoblasts as well (8, 11). Theobservation that propionic acid is sufficient to restore sensi-tivity of the C127/508 line to the induction of apoptosissuggests that it may be possible to modulate apoptosis throughalteration of intracellular pH. If undigested DNA contributesto the high viscosity of mucus in patients with cystic fibrosis,then exogenous acidification may have therapeutic potential.

We acknowledge Judy Nordberg for expert flow cytofluorimetryand Grant Meisenholder and Joe DeLaCruz for technical assistance.We thank Bernard M. Babior and Robert L. Engler for support. Thiswork was supported by a Minority Scientist Award (to R.A.G.) fromthe American Heart Association and by U.S. Public Health ServiceGrant K08 AI01345-01.

1. Wyllie, A. H., Kerr, J. F. R. & Currie, A. R. (1980) Int. Rev. Cytol.68, 251-306.

2. Gaido, M. L. & Cidlowski, J. A. (1991) J. Biol. Chem. 266,18580-18585.

3. Peitsch, M. C., Polzar, B., Stephan, H., Crompton, T., Mac-Donald, H. R., Mannherz, H. G. & Tschopp, J. (1993) EMBO J.12, 371-377.

4. Barry, M. A. & Eastman, A. (1993)Arch. Biochem. Biophys. 300,440-450.

5. Gottlieb, R. A., Giesing, H., Engler, R. L. & Babior, B. M. (1995)Blood 86, 2414-2418.

6. Barry, M. A., Reynolds, J. E. & Eastman, E. (1993) Cancer Res.53, 2349-2357.

7. Caceres-Cortes, J., Rajotte, D., Dumouchel, J., Haddad, P. &Hoang, T. (1994) J. Biol. Chem. 269, 12084-12091.

8. Gottlieb, R. A., Giesing, H., Zhu, J., Engler, R. L. & Babior,B. M. (1995) Proc. Natl. Acad. Sci. USA 92, 5965-5968.

9. Li, J. & Eastman, A. (1995) J. Biol. Chem. 270, 3203-3211.10. Perez-Sala, D., Collado-Escobar, D. & Mollinedo, F. (1995) J.

Biol. Chem. 270, 6235-6242.11. Gottlieb, R. A., Nordberg, J., Skowronski, E. & Babior, B. M.

(1995) Proc. Natl. Acad. Sci. USA 93, 654-658.12. Poulsen, J. H., Fischer, H., Illek, B. & Machen, T. E. (1994) Proc.

Natl. Acad. Sci. USA 91, 5340-5344.13. Smith, J. J. & Welsh, M. J. (1992) J. Clin. Invest. 89, 1148-1153.

14. Barasch, J., Kiss, B., Prince, A., Saiman, L., Gruenert, D. &Al-Awqati, Q. (1991) Nature (London) 352, 70-73.

15. Elgavish, A. & Meezan, E. (1992) Am. J. Physiol. 263, C176-C186.

16. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J.,Smith, A. E. & Welsh, M. J. (1992) Nature (London) 358, 761-764.

17. Bassnett, S., Reinisch, L. & Beebe, D. C. (1990) Am. J. Physiol.258, C171-C178.

18. Musgrove, E. A. & Hedley, D. W. (1990) Methods Cell Biol. 33,59-69.

19. Duke, R. C. & Cohen, J. J. (1992) Curr. Protein Immunol. 1,3.17.1-3.17.19.

20. Wijsman, J. H., Jonker, R. R., Keijzer, R., Van De Velde,C. J. H., Cornelisse, C. J. & Van Dierendonck, J. H. (1993) J.Histochem. Cytochem. 41, 7-12.

21. Mosmann, T. (1983) J. Immunol. Methods 65, 55-63.22. Kerem, B., Rommens, J. M., Buchanan, J. A., Markiewicz, D.,

Cox, T. K., Chakravarti, A., Buchwald, M. & Tsui, L.-C. (1989)Science 245, 1073-1080.

23. Ranasinha, C., Assoufi, B., Shak, S., Christiansen, D., Fuchs, H.,Empey, D., Geddes, D. & Hodson, M. (1993) Lancet 342,199-202.

24. Shak, S., Capon, D. J., Hellmiss, R., Marsters, S. A. & Baker,C. L. (1990) Proc. Natl. Acad. Sci. USA 87, 9188-9192.

25. Egan, M., Flotte, T., Afione, S., Solow, R., Zeitlin, P. L., Carter,B. J. & Guggino, W. B. (1992) Nature (London) 358, 581-584.

26. Fulmer, S. B., Schweibert, E. M., Morales, M. M., Guggino,W. B. & Cutting, G. R. (1995) Proc. Natl. Acad. Sci. USA 92,6832-6836.

27. McPherson, M. A. & Dormer, R. L. (1992) Br. Med. Bull. 48,766-784.

28. Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh,M. J. & Smith, A. E. (1991) Cell 66, 1027-1036.

29. Berger, H. A., Travis, S. M. & Welsh, M. J. (1993) J. Biol. Chem.268, 2037-2047.

30. Hubbard, R. C., McElvaney, N. G., Birrer, P., Shak, S., Robinson,W. W., Jolley, C., Wu, M., Chernick, M. M. & Crystal, R. G.(1992) N. Engl. J. Med. 325, 812-815.

31. Lethem, M. I., James, S. L., Marriott, C. & Burke, J. F. (1990)Eur. Respir. J. 3, 19-23.

32. Picot, R., Das, I. & Reid, L. (1978) Thorax 33, 235-242.33. Graff, I., Schram-Doumont, A. & Szpirer, C. (1991) Cell. Sig-

nalling 3, 259-266.34. McDonald, T. V., Nghiem, P. T., Gardner, P. & Martens, C. L.

(1992) J. Biol. Chem. 267, 3242-3248.

Medical Sciences: Gottlieb and Dosanjh

Dow

nloa

ded

by g

uest

on

July

17,

202

1