[36]
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in more detail, how this epithelium transports electrolytes and, to some extent, how this epithelium controls electrolyte movements. We can now approach questions on intracellular mechanisms of electrolyte transporter regulation. This can be accomplished by combining tools from molecular biology, biochemistry, and pharmacology with electrolyte transport methodology to investigate the molecular nature of transporters and signal transduction at the plasma membrane and intracellular levels. Acknowledgments This research was supported in part by grants from the National Cystic Fibrosis Foundation and from the National Institutes of Health (DK-27651).
[36] Transformation of Airway Epithelial Cells with Persistence of Cystic Fibrosis or Normal
Ion Transport Phenotypes B y JAMES R. YANKASKAS a n d RICHARD C. BOUCHER
Introduction
Until 5 years ago, the pathophysiologic mechanisms unique to cystic fibrosis (CF) were difficult to study because of lack of in vitro specimens or animal models. The development of primary epithelial cell cultures from affected organs provided an important research tool. Cultured CF respiratory 1-6 and sweat duct7-9 epithelial cells demonstrate the abnormal regulation of an apical membrane chloride permeability that characterizes this disease. These tissue culture studies indicate that the abnormalities described in vivo are primary effects of the abnormal gene rather than I j. R. Y a n k a s ~ M. R. Knowles, J. T. Gatzy, and R. C. Boucher, Lancet 1, 954 (1985). 2 j. R. Yankaskas, C. U. Cotton, M. R. Knowles, J. T. Gatzy, and R. C. Boucher, Am. Rev. Respir. Dis. 132, 1281 (1985). 3 R. A. Frizzell, G. Rechkemmer, and R. L. Shoemaker, Science 233, 558 (1986). 4 M. J. Welsh and C. M. Liedtke, Nature (London) 322, 467 (1986). s j. H. Widdicombe, M. J. Welsh, and W. E. Finkbeiner, Proc. Natl. Acad. Sci. U.S.A. 82, 6167 (1985). 6 N. J. Willumsen and R. C. Boucher, Am. J. Physiol. 256, C226 (1989). 7 G. Collie, M. Buchwald, P. Harper, and J. R. Riordan, In Vitro 21, 597 (1985). 8 C. M. Lee, F. Carpenter, T. Coaker, and T. Kealey, J. Cell Sci. 83, 103 (1986). 9 p. S. Pedersen and E. H. Larsen, ICRS Med. Sci.: Libr. Compend. 14, 1159 (1986).
METHODSIN ENZYMOLOGY,VOL. 192
Copyright© 1990byAcademicPrim,Inc. Allrightsofleproductionin anyformreserved.
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secondary effects of infection or inflammation. Comprehensive studies of CF pathogenesis are still limited by the low availability of cystic fibrosis tissues and by the limited life of epithelial cells in primary culture. Cell lines derived from cystic fibrosis and normal epithelial cells have the potential to provide ample research material for genetic, physiologic, and biochemical studies. Such cell lines may be used to test the ability of normal counterparts of candidate genes to complement or reverse the physiologic abnormalities of the recessive CF genes and may be used to develop and test new therapeutic modalities. To be useful, a disease-specific cell line must have sufficient proliferation capability to substantially increase cell availability, express the abnormal gene, and exhibit phenotypic properties of interest. Developing such a cell line requires a supply of the affected tissues, a transforming agent that can increase cell proliferation without markedly changing differentiated cell properties, a means to introduce the transforming agent into the cells, selection criteria for cell lines that incorporate the transforming gene, and a means to assay the phenotypic characteristics. The following sections describe specific techniques for tissue procurement and cell culture, inserting a selected transforming gene with a retroviral shuttle vector, selecting for transforming gene expression and formation of intercellular tight junctions, and characterization of phenotypic properties. These procedures have been used to develop cystic fibrosis (CF/T43) and control (NL/T4) cell lines, which exhibit abnormal and normal regulation of an apical membrane chloride permeability, respectively.l° Tissue Procurement, Epithelial Cell Isolation; and P r i m a r y Culture Progressive airway obstruction from thick secretions, inflammation, and bacterial infection is currently the major cause of morbidity and death in cystic fibrosis. H I n vivo measurements ~2,13 and in vitro ~4.~5 studies of intact excised CF and normal airway tissues have identified abnormalities in regulation of a chloride permeability and sodium absorption in CF epithelia. These differences are manifest in nasal, tracheal, and bronchial tissue. Because of the high incidence of nasal polyposis in cystic fiio A. M. Jetten, J. R. Yankaskas,M. J. Stutts, N. J. Willumsen,and R. C. Boucher,Science 244, 1472(1989). ~ L. M. Taussig,L. I. Landau,and M. I. Marks,and L. M. Taussig,"CysticFibrosis,"p. 115. Thieme-Stratton,New York, 1984. ~2M. Knowles,J. Gatzy,and R. Boucher,N. Engl. J. Med. 305, 1489 ( 1981). ~3M. Knowles,J. Gatzy,and R. Boucher,J. Clin. Invest. 71, 1410(1983). 14M. R. Knowles,M. J. Stutts, A. Spook,N. Fischer,J. T. Gatzy,and R. C. Boucher,Science 221, 1067(1983). is R. C. Boucher,M. J. Stutts, M. R. Knowles,L. Canfley,and J. T. Gatzy,J. Clin. Invest. 78, 1245 (1986).
[36]
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567
brosis, ~6,~7this tissue is more frequently available than lower respiratory tract tissues and provides more epithelial cells than can be isolated from the sweat glands in a typical skin biopsy. Nasal tissues are relatively free of bacterial and fungal infection compared to tracheal tissues obtained during autopsy. Consequently, surgically excised nasal and bronchial tissues were used to develop airway epithelial cell lines. Detailed studies of ion transport properties of cultured cystic fibrosis and normal airway epithelial cells2-4,~s-2° derived from excised specimens have defined the phenotypic ion transport properties of CF. Cystic fibrosis center directors and otolaryngologists identify patients undergoing elective nasal surgery. Excised cystic fibrosis or normal nasal polyps and turbinates 2,2~are rinsed in sterile saline solution in the operating room to remove blood and debris and transported to the laboratory at 4 ° in Joklik's minimum essential medium (JMEM) supplemented with antibiotics (penicillin, 50 U/ml; streptomycin, 50/~g/ml; and gentamicin, 40/tg/ml). Epithelial cells are prepared for culture by the following protocol: (1) place specimens in chilled (4*) modified Eagle's medium (MEM containing 0.1% protease XIV (Sigma, St. Louis, MO) and 100/~g/ml deoxyribonuclease (Sigma); (2) after 24-48 hr, add 10% (v/v) fetal bovine serum (FBS) to neutralize the protease; (3) concentrate the detached cells by centrifugation (800 g, 5 min) and wash in MEM plus 10% FBS; (4) determine cell number with a hemocytometer and cell viability by Trypan Blue exclusion; (5) concentrate cells by a final centfifugation (800 g, 5 min) and resuspend in a plating solution of supplemented F-12 medium 2~,22(F12-7X) that contains insulin (5/~g/ml), epidermal growth factor (20 ng/ml), triiodothyronine (3 X 10-s M), endothelial cell growth supplement (7.5/tg/ml), transferdn (5/~g/ml), hydrocortisone (10-~M, all from Collaborative Research, Lexington, MA) and cholera toxin (10 ng/ ml; Sigma); (6) plate cells on uncoated plastic tissue culture dishes or on culture substrates suitable to planned experiments; (7) after overnight incubation (37 °, 5% CO2, 95% air, 98% humidity), remove nonadherent cells bygentle washing. Refeed with supplemented medium three times per week. ~6H. Schwachman, L. L. Kulczycki, H. L. Mueller, and C. G. Flake, Pediatrics 30, 389 (1962). 17B. Taylor, J. N. G. Evans, and G. A. Hope, Arch. Dis. Child. 49, 133 (1974). ~sR. C. Boucher, C. U. Cotton, J. T. Gatzy, M. R. Knowles, and J. R. Yankaskas, J. Physiol. (London) 405, 77 (1988). ~9N. J. Willumsen, C. W. Davis, and R. C. Boueher, Am. J. Physiol. 256, C1045 (1989). 20M. Li, J. D. McCann, C. M. Liedke, A. C. Nairn, P. Greengard, and M. J. Welsh, Nature (London) 331, 358 (1988). 2z R. Wu, J. Yankaskas, E. Cheng, M. R. Knowles, and R. Boucher, Am. Rev. Respir. Dis. 132, 311 (1985). 22N. J. Willumsen, C. W. Davis, and R. C. Boucher, Am. J. Physiol. 256, C1033 (1989).
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[36]
Transforming Agent Selection and Retroviral Infection Viral oncogenes,23 hybrid viruses, ~ s and viral genesl°~s have been used to transform human airway epithelial cells. Chemical carcinogens26 have been effective transforming gents for rodent airway epithelial cells but have been less effective with human cells. The gene coding the simian virus 40 large T (SV40T) protein was selected for these studies because it can increase cell proliferation and has been shown to immortalize human keratinocytes while causing minimal changes in phenotypic characteristicsY An origin of replication-deficient (off-) SV40T mutant2s was used to minimize autologons viral replication. Different means are available to insert the selected gene into cells. Hybrid adeno/SV40 viruses can infect airway epithelial cells,2s but the adenoviral genes may complicate the transforming effects of SV40T or result in virus-producing cell fines. Genes incorporated into plasmids may be introduced into cells by a variety of transfection techniques, including DEAE dextran, 29 calcium phosphate~ or strontium phosphate31 coprecipiration, lipof~tion, 32 and electroporation)3 but these techniques have relatively low efficiency. DNA can be microinjected~ directly into cell nuclei, but relatively few cells can be injected in a reasonable time period. To increase transformation efficiency, an amphotrophic ret~oviral shuttle vector was used to transfer an ori- SV40T gene into primary cultures of human airway epithelial cells. The ~FAM packaging cell line harboring the recombinant retrovirus pZipneoSV(X)I/SV40T was obtained from P. S. Jar (MIT, Cambridge, MA)) 5,~s Rctroviral infections of l0 s cells/60-mm G. H. Yoakum, J. F. Lechner, E. W. Gabfieison, B. E. Korba, L. Malan-Shiblvy, J. C. WiUey, M. G. Valerio, A. M. Shamsuddin, B. F. Trump, and C. C. Harris, Science 227, 1174 (1985). 24R. R. Reddel, Y. Ke, B. I. Gerwin, M. G. McMenamin, J. F. Lechnvr, R. T. Su, D. E. Brash, J-B. Park, J. S. Rhim, and C. C. Harris, Cancer Res. 48, 1904 (1988). 25 B. J. Scholte, M. Kansen, A. T. Hoogeveen, R. Willems¢, J. S. Rhim, A. W. M. Van Der Kamp, and J. Bijman, Exp. CeURes. 182, 559 (1989). D. G. Thomassen, T. E. Gray, M. J. Mass, and J. (2. Barrett, Cancer Res. 43, 5956 (1983). 2~j. S. Rhim, G. Jay, P. Arnstein, F. M. Price, IC K. Sanford, and S. A. Aaronson, Science 227, 1250 (1985). M. B. Small Y. Gluzman, and H. L. Ozcr, Nature (London) 296, 671 (1982). 29j. H. McCutchan and J. S. ~ o . J. Natl. Cancer Inst. 41, 351 (1968). ~oF. L. Graham and A. J. Van der Eb, Virology 52, 456 (1973). a~ D. E. Brash, R. R. Reddel, M. Quanmd, K. Yang, M. P. Farrell, and C. C. Harris, Mol. Cell. Biol. 7, 2031 (1987). 32p. L F¢lgnvr, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 33M. C, Iannuzzi, J. L. Weber, J. Yankaskas, R. Boucher, and F. S. Collins, Am. Rev. Respir. Dis. 138, 965 (1988). A. Graessmann, M. Graessmann, and C. Muellvr, this series, Vol. 65, p. 816. 3~C. L. Cepko; B. E. Roberts, and R. C. Mulligan, Cell 37, 1053 (1984). P. S. Jar, C. L Cepko, R. C. Mulligan, and P. A. Sharp, Mol. Cell. Biol. 6, 1204 (1986).
[36]
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dish were carried out at 37 ° for 2 hr in 2 ml of a 1 : 1 mixture of keratinocyte growth medium (KGM, Clonetics Corp., San Diego, CA) and rctrovirus-containing (2 × 103 cfu/ml) RPMI 1640 with 5% FBS. Virus was then diluted by addition of 3 ml KGM. Two days later, the cells were exposed to the neomycin analog (3418 (50 gg/ml) for 10 to 14 days. After this time, (3418-resistant colonies were isolated with cloning cylinders and passaged to individual tissue culture flasks for further culture in KGM medium. One to six G418-resistant colonies/60-mm dish were obtained. Colonies were fed twice weekly and passaged during exponential growth. Colonies with continued proliferation at passage 3 were cyropreserved and aliquots screened for development of secondary selection features. Secondary Selection: Formation of Tight Junctions and Transepithelial Resistance Specialized epithelial functions such as barrier formation and vectorial transport of solutes depend on formation of tight junctions, which separate the plasma membrane into apical and basolateral rcgionsYSince the ion transport properties that characterize cystic fibrosis are dependent on such epithelial cell polarization, cell lines that form functional fight junctions (and hence develop a transepithelial resistance) are most likely to demonstrate vectorial transport of sodium and/or chloride ions. Such differentiation is partially dependent on the culture substrate. Permeable collagen matrix support (CMS) culture dishes2 support the best differentiated ion transport properties in primary cell cultures. These matrices are cast in a beveled orifice (4.5-ram diameter) in the bottom of a 2.5-cm-diameter polycarbonate cup by the following protocol. (1) Dissolve type III calfskin collagen (Sigma Cat. No. C-3511, 15 mg/ml) in 0.2% acetic acid at 37 °. (2) Add 0.5 vol of 2.5% giutaraldehyde and cool in an ice bath until jelling begins. (3) Apply a bead of collagen/glutaraldehyde around the orifice of the inverted polycarbonate cup, and drag mixture across the orifice with a Pasteur pipet, forming a thin sheet. (4) Dry in air (3-4 hr). (5) Apply a second coat of collagen (without giutaraldehyde) over the bottom surface of the matrix. Dry overnight. (6) Apply a third coat of collagen (without giutaraldehyde) to the inside surface of the matrix. Dry overnight. (7) Expose bottom side of CMS to two drips of 2.5% giutaraldehyde for 5 rain. (8) Rinse four times with bicarbonate containing saline solution. Dry overnight. (9) Sterilize unit in 70% ethanol for 30 rain. Rinse in sterile culture medium. Incubate overnight at 37 ° in culture medium to test sterility. (10) CMS dishes can be stored in physiologic saline at 4 ° for 3 - 4 weeks. 37 M. Cereijido, A. Ponce, and L. Gonzalez-Mariscal, J. Membr. Biol. 110, 1 (1989).
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OTHER EPITHELIA
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Prior to seeding, CMS dishes are incubated (37 °, overnight) in 35-mmdiameter culture dishes with culture medium in both the inner cup and the outer dish. Medium in the inner cup is removed by aspiration and epithelial cells (removed from plastic tissue culture dishes with trypsin/EDTA) in KGM medium are seeded at 2 X 106 cells/cm2 in 10 to 30/zl of medium. Additional medium is added to the inner cup after 24 hr, and the cells are washed vigorously at 48 hr. Cell attachment and confluency are monitored by phase-contrast microscopy. Transepithelial electric potential and resistance are measured by established techniques.22 Characterization
Expression of the transforming SV40T antigen and epithelial cell-specific keratins by standard Western blot and hybridization techniques~°tests transforming gene expression and the epithelial nature of the cells. The persistence of the cystic fibrosis gene3s in the CF/T43 cell line was confirmed by amplifying the exon 10 region of the cystic fibrosis transmembrahe regulator (CFTR) gene by polymerase chain reaction (PCR) 39 and hybridizing with oligonucleotide probes specific to presence of deletion of the Phe5°8 codon. CF/T43 cells are homozygous for Phe5°8 deletion. Similarly, expression of the CFTR messenger RNA was confirmed by Northern hybridization of messenger RNA extracted from CF/T43 cells by Chirgwin's cesium chloride methOd4° and an oligonucleotide probe. Phenotypic characterization may be performed with any technique that differentiates CF and normal airway epithelial cells. For the CF/T43 and NL/T4 cell lines, chloride conductance of the apical membrane (Go-) was assessed with transepithelial and chloride-selective intracellular microelectrodes, z2 Compared to NL/T4 cells, CF/T43 cells have a reduced Go- in the basal state. This conductance is not activated by cAMP-dependent agonists, but is increased by Ca2+-mediated agonists. These findings were confirmed with single-channel patch-clamp techniques.l° Summary These studies demonstrate the feasibility of transforming human airway epithelial cells while inducing only modest changes in function. Feaas j. R. Riordan, J. M. Rommens, B-S. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J-L. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L-C. Tsui, Science 245, 1066 (1989). 39 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985). 4oj. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979).
[37]
BOVINE CORNEAL ENDOTHELIAL CELL CULTURE
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tures central to the pathophysiology of cystic fibrosis, i.e., abnormal regulation of a chloride permeability in the apical cell membrane, appear to be preserved in the CF/T43 transformed cell line. This work indicates that additional cystic fibrosis and normal cell lines may be developed, as well as epithelial cell lines from other diseases of interest. In addition to SV40T gene, temperature-sensitive viral genes, 41 or genes driven by inducible promoters (e.g., glucocorticoids, heavy metals42) may produce cell lines in which proliferation or differentiation can be controlled. For example, the temperature-selective SV40A gene is expressed in cells cultured at the permissive (33 °) temperature but is degraded at the nonpermissive (40 °) temperature. 41 Thus, the transfected gene may induce proliferation to increase cell number, and then be suppressed to permit expression of a differentiated phenotype. Out strategy of initially selecting clones by G418 resistance and then selecting clones that develop functional tight junctions (and a transepithelial resistance) was useful in identifying a cell line with highly differentiated phenotypic properties. Cell lines that do not form transepithelial resistances may be valuable for studies that do not depend on cell polarization. Initial evidence suggests that some of the differentiated properties of CFfr43, i.e., formation of functional fight junctions and a transepithelial resistance, are lost at late passages. Although these properties may be a function of the culture medium constituents and the nature of the culture substmte, it is possible that the transforming gene caused further mutations that resulted in subclones that have increased growth rate but different phenotypic properties. The continuing expression of the CFTR messenger RNA in these late passage cells indicates that they will be an important tool for future CF research. 4~ j. y . Chou, Proc. Natl. Acad. Sci. U.S.A. 75, 1409 (1978). 42 O. Hurko, L. McKee, and J. G. E. M. Zuurveld, Ann. Neurol. 20, 573 (1986).
[37] C e l l C u l t u r e o f B o v i n e C o r n e a l E n d o t h e l i a l C e l l s a n d Its A p p l i c a t i o n t o T r a n s p o r t S t u d i e s By
M I C H A E L WIEDERHOLT a n d THOMAS J. JENTSCH
Introduction Ion transport studies using cultured epithelial cells have become increasingly popular in the past few years. Advantages of studies of cultured cells as opposed to investigations of the tissue in situ include the availabilMETHODS IN ENZYMOLOGY,VOL. 192
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
TRANSFORMATION OF AIRWAY EPITHELIAL CELLS
565
in more detail, how this epithelium transports electrolytes and, to some extent, how this epithelium controls electrolyte movements. We can now approach questions on intracellular mechanisms of electrolyte transporter regulation. This can be accomplished by combining tools from molecular biology, biochemistry, and pharmacology with electrolyte transport methodology to investigate the molecular nature of transporters and signal transduction at the plasma membrane and intracellular levels. Acknowledgments This research was supported in part by grants from the National Cystic Fibrosis Foundation and from the National Institutes of Health (DK-27651).
[36] Transformation of Airway Epithelial Cells with Persistence of Cystic Fibrosis or Normal
Ion Transport Phenotypes B y JAMES R. YANKASKAS a n d RICHARD C. BOUCHER
Introduction
Until 5 years ago, the pathophysiologic mechanisms unique to cystic fibrosis (CF) were difficult to study because of lack of in vitro specimens or animal models. The development of primary epithelial cell cultures from affected organs provided an important research tool. Cultured CF respiratory 1-6 and sweat duct7-9 epithelial cells demonstrate the abnormal regulation of an apical membrane chloride permeability that characterizes this disease. These tissue culture studies indicate that the abnormalities described in vivo are primary effects of the abnormal gene rather than I j. R. Y a n k a s ~ M. R. Knowles, J. T. Gatzy, and R. C. Boucher, Lancet 1, 954 (1985). 2 j. R. Yankaskas, C. U. Cotton, M. R. Knowles, J. T. Gatzy, and R. C. Boucher, Am. Rev. Respir. Dis. 132, 1281 (1985). 3 R. A. Frizzell, G. Rechkemmer, and R. L. Shoemaker, Science 233, 558 (1986). 4 M. J. Welsh and C. M. Liedtke, Nature (London) 322, 467 (1986). s j. H. Widdicombe, M. J. Welsh, and W. E. Finkbeiner, Proc. Natl. Acad. Sci. U.S.A. 82, 6167 (1985). 6 N. J. Willumsen and R. C. Boucher, Am. J. Physiol. 256, C226 (1989). 7 G. Collie, M. Buchwald, P. Harper, and J. R. Riordan, In Vitro 21, 597 (1985). 8 C. M. Lee, F. Carpenter, T. Coaker, and T. Kealey, J. Cell Sci. 83, 103 (1986). 9 p. S. Pedersen and E. H. Larsen, ICRS Med. Sci.: Libr. Compend. 14, 1159 (1986).
METHODSIN ENZYMOLOGY,VOL. 192
Copyright© 1990byAcademicPrim,Inc. Allrightsofleproductionin anyformreserved.
566
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[36]
secondary effects of infection or inflammation. Comprehensive studies of CF pathogenesis are still limited by the low availability of cystic fibrosis tissues and by the limited life of epithelial cells in primary culture. Cell lines derived from cystic fibrosis and normal epithelial cells have the potential to provide ample research material for genetic, physiologic, and biochemical studies. Such cell lines may be used to test the ability of normal counterparts of candidate genes to complement or reverse the physiologic abnormalities of the recessive CF genes and may be used to develop and test new therapeutic modalities. To be useful, a disease-specific cell line must have sufficient proliferation capability to substantially increase cell availability, express the abnormal gene, and exhibit phenotypic properties of interest. Developing such a cell line requires a supply of the affected tissues, a transforming agent that can increase cell proliferation without markedly changing differentiated cell properties, a means to introduce the transforming agent into the cells, selection criteria for cell lines that incorporate the transforming gene, and a means to assay the phenotypic characteristics. The following sections describe specific techniques for tissue procurement and cell culture, inserting a selected transforming gene with a retroviral shuttle vector, selecting for transforming gene expression and formation of intercellular tight junctions, and characterization of phenotypic properties. These procedures have been used to develop cystic fibrosis (CF/T43) and control (NL/T4) cell lines, which exhibit abnormal and normal regulation of an apical membrane chloride permeability, respectively.l° Tissue Procurement, Epithelial Cell Isolation; and P r i m a r y Culture Progressive airway obstruction from thick secretions, inflammation, and bacterial infection is currently the major cause of morbidity and death in cystic fibrosis. H I n vivo measurements ~2,13 and in vitro ~4.~5 studies of intact excised CF and normal airway tissues have identified abnormalities in regulation of a chloride permeability and sodium absorption in CF epithelia. These differences are manifest in nasal, tracheal, and bronchial tissue. Because of the high incidence of nasal polyposis in cystic fiio A. M. Jetten, J. R. Yankaskas,M. J. Stutts, N. J. Willumsen,and R. C. Boucher,Science 244, 1472(1989). ~ L. M. Taussig,L. I. Landau,and M. I. Marks,and L. M. Taussig,"CysticFibrosis,"p. 115. Thieme-Stratton,New York, 1984. ~2M. Knowles,J. Gatzy,and R. Boucher,N. Engl. J. Med. 305, 1489 ( 1981). ~3M. Knowles,J. Gatzy,and R. Boucher,J. Clin. Invest. 71, 1410(1983). 14M. R. Knowles,M. J. Stutts, A. Spook,N. Fischer,J. T. Gatzy,and R. C. Boucher,Science 221, 1067(1983). is R. C. Boucher,M. J. Stutts, M. R. Knowles,L. Canfley,and J. T. Gatzy,J. Clin. Invest. 78, 1245 (1986).
[36]
TRANSFORMATION OF AIRWAY EPITHELIAL CELLS
567
brosis, ~6,~7this tissue is more frequently available than lower respiratory tract tissues and provides more epithelial cells than can be isolated from the sweat glands in a typical skin biopsy. Nasal tissues are relatively free of bacterial and fungal infection compared to tracheal tissues obtained during autopsy. Consequently, surgically excised nasal and bronchial tissues were used to develop airway epithelial cell lines. Detailed studies of ion transport properties of cultured cystic fibrosis and normal airway epithelial cells2-4,~s-2° derived from excised specimens have defined the phenotypic ion transport properties of CF. Cystic fibrosis center directors and otolaryngologists identify patients undergoing elective nasal surgery. Excised cystic fibrosis or normal nasal polyps and turbinates 2,2~are rinsed in sterile saline solution in the operating room to remove blood and debris and transported to the laboratory at 4 ° in Joklik's minimum essential medium (JMEM) supplemented with antibiotics (penicillin, 50 U/ml; streptomycin, 50/~g/ml; and gentamicin, 40/tg/ml). Epithelial cells are prepared for culture by the following protocol: (1) place specimens in chilled (4*) modified Eagle's medium (MEM containing 0.1% protease XIV (Sigma, St. Louis, MO) and 100/~g/ml deoxyribonuclease (Sigma); (2) after 24-48 hr, add 10% (v/v) fetal bovine serum (FBS) to neutralize the protease; (3) concentrate the detached cells by centrifugation (800 g, 5 min) and wash in MEM plus 10% FBS; (4) determine cell number with a hemocytometer and cell viability by Trypan Blue exclusion; (5) concentrate cells by a final centfifugation (800 g, 5 min) and resuspend in a plating solution of supplemented F-12 medium 2~,22(F12-7X) that contains insulin (5/~g/ml), epidermal growth factor (20 ng/ml), triiodothyronine (3 X 10-s M), endothelial cell growth supplement (7.5/tg/ml), transferdn (5/~g/ml), hydrocortisone (10-~M, all from Collaborative Research, Lexington, MA) and cholera toxin (10 ng/ ml; Sigma); (6) plate cells on uncoated plastic tissue culture dishes or on culture substrates suitable to planned experiments; (7) after overnight incubation (37 °, 5% CO2, 95% air, 98% humidity), remove nonadherent cells bygentle washing. Refeed with supplemented medium three times per week. ~6H. Schwachman, L. L. Kulczycki, H. L. Mueller, and C. G. Flake, Pediatrics 30, 389 (1962). 17B. Taylor, J. N. G. Evans, and G. A. Hope, Arch. Dis. Child. 49, 133 (1974). ~sR. C. Boucher, C. U. Cotton, J. T. Gatzy, M. R. Knowles, and J. R. Yankaskas, J. Physiol. (London) 405, 77 (1988). ~9N. J. Willumsen, C. W. Davis, and R. C. Boueher, Am. J. Physiol. 256, C1045 (1989). 20M. Li, J. D. McCann, C. M. Liedke, A. C. Nairn, P. Greengard, and M. J. Welsh, Nature (London) 331, 358 (1988). 2z R. Wu, J. Yankaskas, E. Cheng, M. R. Knowles, and R. Boucher, Am. Rev. Respir. Dis. 132, 311 (1985). 22N. J. Willumsen, C. W. Davis, and R. C. Boucher, Am. J. Physiol. 256, C1033 (1989).
568
OTHER EPITHELIA
[36]
Transforming Agent Selection and Retroviral Infection Viral oncogenes,23 hybrid viruses, ~ s and viral genesl°~s have been used to transform human airway epithelial cells. Chemical carcinogens26 have been effective transforming gents for rodent airway epithelial cells but have been less effective with human cells. The gene coding the simian virus 40 large T (SV40T) protein was selected for these studies because it can increase cell proliferation and has been shown to immortalize human keratinocytes while causing minimal changes in phenotypic characteristicsY An origin of replication-deficient (off-) SV40T mutant2s was used to minimize autologons viral replication. Different means are available to insert the selected gene into cells. Hybrid adeno/SV40 viruses can infect airway epithelial cells,2s but the adenoviral genes may complicate the transforming effects of SV40T or result in virus-producing cell fines. Genes incorporated into plasmids may be introduced into cells by a variety of transfection techniques, including DEAE dextran, 29 calcium phosphate~ or strontium phosphate31 coprecipiration, lipof~tion, 32 and electroporation)3 but these techniques have relatively low efficiency. DNA can be microinjected~ directly into cell nuclei, but relatively few cells can be injected in a reasonable time period. To increase transformation efficiency, an amphotrophic ret~oviral shuttle vector was used to transfer an ori- SV40T gene into primary cultures of human airway epithelial cells. The ~FAM packaging cell line harboring the recombinant retrovirus pZipneoSV(X)I/SV40T was obtained from P. S. Jar (MIT, Cambridge, MA)) 5,~s Rctroviral infections of l0 s cells/60-mm G. H. Yoakum, J. F. Lechner, E. W. Gabfieison, B. E. Korba, L. Malan-Shiblvy, J. C. WiUey, M. G. Valerio, A. M. Shamsuddin, B. F. Trump, and C. C. Harris, Science 227, 1174 (1985). 24R. R. Reddel, Y. Ke, B. I. Gerwin, M. G. McMenamin, J. F. Lechnvr, R. T. Su, D. E. Brash, J-B. Park, J. S. Rhim, and C. C. Harris, Cancer Res. 48, 1904 (1988). 25 B. J. Scholte, M. Kansen, A. T. Hoogeveen, R. Willems¢, J. S. Rhim, A. W. M. Van Der Kamp, and J. Bijman, Exp. CeURes. 182, 559 (1989). D. G. Thomassen, T. E. Gray, M. J. Mass, and J. (2. Barrett, Cancer Res. 43, 5956 (1983). 2~j. S. Rhim, G. Jay, P. Arnstein, F. M. Price, IC K. Sanford, and S. A. Aaronson, Science 227, 1250 (1985). M. B. Small Y. Gluzman, and H. L. Ozcr, Nature (London) 296, 671 (1982). 29j. H. McCutchan and J. S. ~ o . J. Natl. Cancer Inst. 41, 351 (1968). ~oF. L. Graham and A. J. Van der Eb, Virology 52, 456 (1973). a~ D. E. Brash, R. R. Reddel, M. Quanmd, K. Yang, M. P. Farrell, and C. C. Harris, Mol. Cell. Biol. 7, 2031 (1987). 32p. L F¢lgnvr, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 33M. C, Iannuzzi, J. L. Weber, J. Yankaskas, R. Boucher, and F. S. Collins, Am. Rev. Respir. Dis. 138, 965 (1988). A. Graessmann, M. Graessmann, and C. Muellvr, this series, Vol. 65, p. 816. 3~C. L. Cepko; B. E. Roberts, and R. C. Mulligan, Cell 37, 1053 (1984). P. S. Jar, C. L Cepko, R. C. Mulligan, and P. A. Sharp, Mol. Cell. Biol. 6, 1204 (1986).
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569
dish were carried out at 37 ° for 2 hr in 2 ml of a 1 : 1 mixture of keratinocyte growth medium (KGM, Clonetics Corp., San Diego, CA) and rctrovirus-containing (2 × 103 cfu/ml) RPMI 1640 with 5% FBS. Virus was then diluted by addition of 3 ml KGM. Two days later, the cells were exposed to the neomycin analog (3418 (50 gg/ml) for 10 to 14 days. After this time, (3418-resistant colonies were isolated with cloning cylinders and passaged to individual tissue culture flasks for further culture in KGM medium. One to six G418-resistant colonies/60-mm dish were obtained. Colonies were fed twice weekly and passaged during exponential growth. Colonies with continued proliferation at passage 3 were cyropreserved and aliquots screened for development of secondary selection features. Secondary Selection: Formation of Tight Junctions and Transepithelial Resistance Specialized epithelial functions such as barrier formation and vectorial transport of solutes depend on formation of tight junctions, which separate the plasma membrane into apical and basolateral rcgionsYSince the ion transport properties that characterize cystic fibrosis are dependent on such epithelial cell polarization, cell lines that form functional fight junctions (and hence develop a transepithelial resistance) are most likely to demonstrate vectorial transport of sodium and/or chloride ions. Such differentiation is partially dependent on the culture substrate. Permeable collagen matrix support (CMS) culture dishes2 support the best differentiated ion transport properties in primary cell cultures. These matrices are cast in a beveled orifice (4.5-ram diameter) in the bottom of a 2.5-cm-diameter polycarbonate cup by the following protocol. (1) Dissolve type III calfskin collagen (Sigma Cat. No. C-3511, 15 mg/ml) in 0.2% acetic acid at 37 °. (2) Add 0.5 vol of 2.5% giutaraldehyde and cool in an ice bath until jelling begins. (3) Apply a bead of collagen/glutaraldehyde around the orifice of the inverted polycarbonate cup, and drag mixture across the orifice with a Pasteur pipet, forming a thin sheet. (4) Dry in air (3-4 hr). (5) Apply a second coat of collagen (without giutaraldehyde) over the bottom surface of the matrix. Dry overnight. (6) Apply a third coat of collagen (without giutaraldehyde) to the inside surface of the matrix. Dry overnight. (7) Expose bottom side of CMS to two drips of 2.5% giutaraldehyde for 5 rain. (8) Rinse four times with bicarbonate containing saline solution. Dry overnight. (9) Sterilize unit in 70% ethanol for 30 rain. Rinse in sterile culture medium. Incubate overnight at 37 ° in culture medium to test sterility. (10) CMS dishes can be stored in physiologic saline at 4 ° for 3 - 4 weeks. 37 M. Cereijido, A. Ponce, and L. Gonzalez-Mariscal, J. Membr. Biol. 110, 1 (1989).
570
OTHER EPITHELIA
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Prior to seeding, CMS dishes are incubated (37 °, overnight) in 35-mmdiameter culture dishes with culture medium in both the inner cup and the outer dish. Medium in the inner cup is removed by aspiration and epithelial cells (removed from plastic tissue culture dishes with trypsin/EDTA) in KGM medium are seeded at 2 X 106 cells/cm2 in 10 to 30/zl of medium. Additional medium is added to the inner cup after 24 hr, and the cells are washed vigorously at 48 hr. Cell attachment and confluency are monitored by phase-contrast microscopy. Transepithelial electric potential and resistance are measured by established techniques.22 Characterization
Expression of the transforming SV40T antigen and epithelial cell-specific keratins by standard Western blot and hybridization techniques~°tests transforming gene expression and the epithelial nature of the cells. The persistence of the cystic fibrosis gene3s in the CF/T43 cell line was confirmed by amplifying the exon 10 region of the cystic fibrosis transmembrahe regulator (CFTR) gene by polymerase chain reaction (PCR) 39 and hybridizing with oligonucleotide probes specific to presence of deletion of the Phe5°8 codon. CF/T43 cells are homozygous for Phe5°8 deletion. Similarly, expression of the CFTR messenger RNA was confirmed by Northern hybridization of messenger RNA extracted from CF/T43 cells by Chirgwin's cesium chloride methOd4° and an oligonucleotide probe. Phenotypic characterization may be performed with any technique that differentiates CF and normal airway epithelial cells. For the CF/T43 and NL/T4 cell lines, chloride conductance of the apical membrane (Go-) was assessed with transepithelial and chloride-selective intracellular microelectrodes, z2 Compared to NL/T4 cells, CF/T43 cells have a reduced Go- in the basal state. This conductance is not activated by cAMP-dependent agonists, but is increased by Ca2+-mediated agonists. These findings were confirmed with single-channel patch-clamp techniques.l° Summary These studies demonstrate the feasibility of transforming human airway epithelial cells while inducing only modest changes in function. Feaas j. R. Riordan, J. M. Rommens, B-S. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J-L. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L-C. Tsui, Science 245, 1066 (1989). 39 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985). 4oj. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18, 5294 (1979).
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BOVINE CORNEAL ENDOTHELIAL CELL CULTURE
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tures central to the pathophysiology of cystic fibrosis, i.e., abnormal regulation of a chloride permeability in the apical cell membrane, appear to be preserved in the CF/T43 transformed cell line. This work indicates that additional cystic fibrosis and normal cell lines may be developed, as well as epithelial cell lines from other diseases of interest. In addition to SV40T gene, temperature-sensitive viral genes, 41 or genes driven by inducible promoters (e.g., glucocorticoids, heavy metals42) may produce cell lines in which proliferation or differentiation can be controlled. For example, the temperature-selective SV40A gene is expressed in cells cultured at the permissive (33 °) temperature but is degraded at the nonpermissive (40 °) temperature. 41 Thus, the transfected gene may induce proliferation to increase cell number, and then be suppressed to permit expression of a differentiated phenotype. Out strategy of initially selecting clones by G418 resistance and then selecting clones that develop functional tight junctions (and a transepithelial resistance) was useful in identifying a cell line with highly differentiated phenotypic properties. Cell lines that do not form transepithelial resistances may be valuable for studies that do not depend on cell polarization. Initial evidence suggests that some of the differentiated properties of CFfr43, i.e., formation of functional fight junctions and a transepithelial resistance, are lost at late passages. Although these properties may be a function of the culture medium constituents and the nature of the culture substmte, it is possible that the transforming gene caused further mutations that resulted in subclones that have increased growth rate but different phenotypic properties. The continuing expression of the CFTR messenger RNA in these late passage cells indicates that they will be an important tool for future CF research. 4~ j. y . Chou, Proc. Natl. Acad. Sci. U.S.A. 75, 1409 (1978). 42 O. Hurko, L. McKee, and J. G. E. M. Zuurveld, Ann. Neurol. 20, 573 (1986).
[37] C e l l C u l t u r e o f B o v i n e C o r n e a l E n d o t h e l i a l C e l l s a n d Its A p p l i c a t i o n t o T r a n s p o r t S t u d i e s By
M I C H A E L WIEDERHOLT a n d THOMAS J. JENTSCH
Introduction Ion transport studies using cultured epithelial cells have become increasingly popular in the past few years. Advantages of studies of cultured cells as opposed to investigations of the tissue in situ include the availabilMETHODS IN ENZYMOLOGY,VOL. 192
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