Culture and transformation of human airway epithelial cells

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airway epithelial cell culture; methods of culture; transformed cell lines. THE TWO MAJOR FORCES ... (183) have b een successfully used to transform primary.
Culture and transformation of human airway epithelial

cells

DIETER C. GRUENERT, WALTER E. FINKBEINER, AND JONATHAN H. WIDDICOMBE Cardiovascular Research Institute, Gene Therapy Core Center, Departments of Laboratory Medicine, Pathology, and Physiology, University of California, San Francisco, California 94143 Gruenert, Dieter C., Walter E. Finkbeiner, and Jonathan H. Widdicombe. Culture and transformation of human airway epithelial cells. Am. J. Physiol. 268 (Lung CelZ. Mol. Physiol. 12): L347-L360, 1995.-The culture of human airway epithelial cells has played an important role in advancing our understanding of the metabolic and molecular mechanisms underlying normal function and disease pathology of airway epithelial cells. Recent advances in culturing primary epithelial cells and the development of transformed airway epithelial cell lines have been particularly important in enhancing our understanding of the pathology associated with cystic fibrosis and lung cancer. The establishment of conditions that enhance the proliferative capacity of airway epithelial cells in primary culture was the first technical hurdle overcome in the development of in vitro culture systems. Research is now being geared toward the development of cell culture conditions that facilitate the expression in culture of the differentiated characteristics found in the native epithelium. Aside from the advances that have been made in defining the growth media and extracellular matrixes that enhance the expression of differentiated features, the use of an air-liquid interface has been a significant advance in the culture of airway epithelial cells. The implementation of the in vitro cell culture systems that have now been established and the research into optimizing the conditions for the growth of airway epithelial cells have been and will continue to be essential in the development of therapies for airway disease. airway epithelial

cell culture;

methods

THE TWO MAJOR FORCES behind the development of human airway epithelial cell cultures have been cancer and cystic fibrosis (CF) research. Stimulated by the lack of tissue and the need to dissect the biochemical and genetic mechanisms underlying these and other diseases of the airways, researchers have now developed a number of defined cell culture systems. These cell culture systems start from isolated, dispersed cells or from explant outgrowth and offer a number of advantages over the earlier and more complex organ cultures (101, 181). However, development of cell culture systems that maintain differentiated morphol,ogical and biochemical features for long periods is in its infancy. Further research will be required to define conditions that result in expression of the specific differentiated features that distinguish one airway epithelial cell type from another. Primary cultures of human airway epithelial cells have been particularly important for understanding the mechanisms underlying diseases such as CF (3, 14-16, 19,54,64,80, 149,150,163, 165,166,169,177,179), for characterizing viral infection in the airway epithelium (7, 8, 136, 146, 147), for advancing our knowledge of 1040-0605/95

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of culture;

transformed

cell lines

airway inflammation (7, 73, 75, 105, 111, 114, 115, 121, 122, 154, 168), and for gaining insight into neoplastic progression in the airways (12,43,44,49,62,63,69,84, 93, 99, 126, 128, 131-133, 144, 171, 180). Differentiation of primary airway epithelial cultures, grown in conventional plastic tissue culture vessels, has been shown to be influenced by the extracellular matrix (39, 78, 101, 116, 167, 170, 176, 178), growth factors (1, 97-99, 101, 102, 113, 148, 157, 172, 174, 175), hormones (4, 29, 83-85, 101, 174), and the presence of an air-liquid interface (39, 164, 176, 178). In addition, airway epithelial cultures grown in a denuded tracheal xenograft can be reconstituted into a reasonable approximation of the native airway epithelium (46, 48, 49, 92). While primary cultures have played an important role in characterizing specific functions, they are limited by the number of viable cells that can be generated from the available tissue. This problem can be avoided by repeated subculturing of primary cultures, though this is generally accompanied by the loss or reduction of certain differentiated features. However, by manipulation of the culture conditions, certain differentiated

1995 the American

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features can be restored or maintained during subculture (51,62,64, 101,102,139,164,178,185). Development of permanent or immortal cultures of airway epithelial cell lines has augmented the types of analyses that can be carried out. Cell lines derived from lung carcinomas may retain functions resembling those of specific cell types of native epithelium (143). However, most studies have been carried out with primary cultures of airway epithelial cells that have been transformed in vitro (62, 69). A major advantage of such transformed cells over carcinoma lines is that the cell origin is known. While carcinogens (43) and oncogenes (183) have b een successfully used to transform primary airway epithelial cells, most airway epithelial cell lines generated by in vitro transformation were developed by using viruses or vectors containing the simian virus 40 (SV40) large T and small t antigens. The various SV40 transformation systems used include a plasmid containing origin of replication-defective SV40 virus (34,36,37, 62,63,67,94, 104), a wild-type adeno-SV40 hybrid virus (31,132, 140, 186) or one defective in the SV40 origin of replication (24), and a retrovirus vector containing the SV40 large T antigen (82,86). Transformed human airway epithelial cell lines have proved very useful in the study of the biochemical defects in CF (6, 23, 32, 42, 52, 67, 74, 76, 94-96, 104, 120, 135, 138, 149, 158-160), airway inflammation (7, 10, 11, 38, 112, 153, 154), and virus infection (8, 136, 147). While many transformed cell lines may lose certain differentiated functions, their cell proliferation properties make them excellent in vitro- models for

studies of neoplastic 62,69, 128). CELL =ES

OF

progression

AIRWAY

in human airways

EPITHELIUM

There are several excellent reviews on the types of epithelial cells found in airways (17, 68, 110). The basic types of surface airway epithelium are classified in Fig. 1. Ciliated cells comprise - 50% of the epithelial cell population in all airways [68,1 IO). In the larger airways, the second most common cell type is the basal cell, followed by the secretory mucous (goblet) cells. With increasing airway generations, the numbers of basal cells decline dramatically, and Clara cells become the predominant secretory cell type. Clara cells differ from mucous cells in that their cell secretory granules are smaller in size and number and in that they contain abundant granular endoplasmic reticulum (110). Other cell types (serous, brush, degranulated exocrine, preciliated, and neuroendocrine, for example) comprise < 5% of the total cell number in any airway. Basal cells contact the basal lamina, but not the airway lumen, and have been detected in all regions of human airways. This cell type has a relatively large nucleus with highly condensed chromatin. Considerable evidence suggests that basal cells can function as precur-sor cells for other more differentiated cell types (18, 77). In mixed dispersions of surface epitheliuq it is probably the basal cells that attach, divide, and grow in culture (53). Other studies have implicated a role for immature goblet cells as stem cells (5, 89). In the lower airways,

EPITHELIAL

/

CELLS

\

NO CONTACT WITH LUMEN

CONTACT WITH LUMEN

/

Fig. 1. A flow diagram correlating the function and location with respect to the airway lumen to specific cell types. The epithelial cells indicated are distributed throughout the airways. Specific ce22 types predominate in defined regions of the airways or are found only in specific tirways, e.g., Clara cells are found primarily in the small airways.

\ MACROMOLECULE SECRETING

SOLUTE TRANSPORTING

NON-CILIATED

dense-core

(25,

basal granulated

brush hydrotic

CILIATED

ciliated

goblet mucous S8fUUS

Clara neuroendocrine

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Clara cells appear to be the progenitor cells, since they are able to differentiate into ciliated cells, more Clara cells, or goblet cells (22, 47). The basal lamina and submucosa of the trachea and bronchi contain tracheobronchial glands. In adult humans there is approximately one tracheobronchial gland per square millimeter of tissue surface area (152), and they have a characteristic branching tubuloacinar structure (118). The gland acini are comprised primarily of serous cells, while the tubules are lined primarily with mucous cells (117). The tubules empty into a collecting duct, which communicates with the exterior via a ciliated duct (118). It has been estimated that the combined volume of the secreting cells in the gland is forty times that of the surface goblet cells (134). PRIMARY

CELL CULTURE:

METHODS

Cells from the surface epithelium of trachea or bronchus have been isolated by several different methods, including 1) explant culture (39, 64, lOl>, 2) enzymatic dissociation using a variety of enzymes (157, 167, 174, 175), and 3) bronchial brushing (90). The freshness of the tissue, the location of the cells in the airway, and the potential contamination by infectious agents such as bacteria and viruses are all deciding factors in choosing a methodology for isolation of epithelial cells. Tissue freshness can be an important factor when using a method that relies on enzymatic dissociation. Cell viability decreases dramatically if the tissue is older than 24 h (167). However, explants from tissue older than 48 h have been successfully used to generate epithelial cell cultures (64). Because of the direct contact of airway epithelial cells with the external environment, these cells are susceptible to contamination with bacteria and yeast. Thus the successof primary cultures is increased by pretreatment of tissue with antibiotics or disruption of the mucus blanket with dithiothreitol(167, 176). A primary factor affecting the viability of freshly isolated cells is the culture medium. The pioneering work of Lechner and colleagues (9%103,113) forms the basis for media formulations of more recent workers (64, 157, 175). In general, the growth media that have proven to be the best for freshly isolated cells are the LHC and the Dulbecco’s modified Eagle’s medium/ Ham’s F-12 based solutions. For long-term viability, the cells are exposed to serum in the medium for only a short period of time, if at all (64, 101, 174). This is because blood-derived serum contains tumor growth factor-l3 (TGF-P), which has been shown to inhibit airway epithelial cell proliferation and induce squamous differentiation (102, 113). When an appropriate number of cells has been generated, the media constituents can be manipulated so that the cells differentiate. Small amounts of serum have proven useful for stimulating morphological differentiation of primary cell cultures (91). Retinoic acid appears to be particularly important for cell differentiation (4, 21, 29, 83-85, 99, 101, 119, 124). Other compounds such as growth factors, phorbol esters, and steroid hormones can also change the differentiated phenotype of the cultures and can be used to elaborate specific

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biochemical and genetic endpoints. Growth factors such as epidermal growth factor (EGF), TGF-P, bombesin, and fibroblast growth factor (FGF) primarily modulate the proliferative capacity of the cells (83, 100, 113, 119, 148, 172, 174, 176,181). Research has also been directed toward defining components of fibroblast secretions that influence epithelial cell differentiation and migration (78). The results provide important information regarding differentiation of epithelial cells in vivo. It may then be possible to use this information to determine which conditions will cause neoplastic cells to terminally differentiate. While TGF-P has already been determined to be an important factor in epithelial cell differentiation, there are obviously other components that play a role in this multifactorial process. Extracellular matrixes are another essential element in the differentiation of cultured airway epithelial cells. The type of surface on which the cells are grown influences cytoplasmic receptors as well as cellular architecture, two factors regulating gene expression and leading ultimately to the presentation of specific phenotypic characteristics. Primary airway epithelial cells in culture have been grown on fibroblast feeder layers (39, 91, 98), on collagen films or gels (36, 98, 139, 145, 155, 157, 167, 174, 176, 178, 181, 184), on bovine cornea1 endothelial cells ( 170)) and on collagen-fibronectinbovine serum albumin (FN/V/BSA) (64, 99, 101, 144). Growth on collagen gels provides the extracellular matrix components that both activate cell surface receptors and provide the elastic substratum that allows the assumption of a cuboidal rather than flattened epithelium (139). In addition, it appears that the epithelial cells will respond to secretory products from fibroblasts embedded in collagen gels (78, 79). One limitation of gels, however, is that they make the subculture of cells difficult. Though much attention has been devoted to culture media and growth supports, perhaps the most important factor in inducing differentiation of both primary cultures and transformed airway cell lines is the use of an air-liquid interface (39, 164, 176, 178). In this approach, cells are grown on a porous-bottomed insert placed in a tissue culture well. Medium is added to the cells’ basolateral aspect only (the outside of the insert). Under these circumstances, the mucosal face of the cells on the inside of the insert is covered with a thin layer of fluid - 15 km in depth (88). Levels of aerobic respiration in cells grown in this way are higher than when immersed in medium, and this could be the cause of their enhanced differentiation (88). In addition to the use of an air interface and the development of appropriate growth media and extracellular matrixes, a key factor in establishment of pure long-term airway epithelial cell cultures has been the removal of contaminating fibroblasts (64, 98, 101). Fibroblast contamination from explant culture has been mitigated by growth in serum-free medium (lOl), by growth in D-valine containing culture medium (58), and by selective trypsinization (64, 123).

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The surface epithelium of the nose resembles that of the lower airways (125). Isolation and culture of epithelial cells from nasal turbinates or from nasal polyps (especially from patients with CF) is similar to that described for the tracheobronchial epithelium. However, because of the greater ease of access, the ability to obtain fresh tissue, and the potential for repeated isolation from the same source, the nasal epithelium is a particularly attractive source of airway epithelial cells. In general, cultures of epithelial cells from the nasal turbinate or nasal polyps are established by the same methods as for tracheobronchial cells, i.e., explant outgrowth (64, 1851, enzymatic dissociation (64, 157, 175, Ml), or by brushing of the nasal mucosa (19, 20). The nasal brushing technique is appealing because it is less invasive than the other approaches but is limited by the number of cells that can be acquired in a given procedure. However, once the cells have been established in culture, they readily proliferate under the same culture

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conditions used for the tracheobronchial epithelial cells. Airway submucosal gland epithelial cells have recently been isolated and placed into primary culture (30, 145, 155, 177, 178). To obtain such cells, the surface epithelial layer is first stripped off. Gland-rich tissue is then removed from the adventitia by microdissection and minced. The fragments are then enzymatically digested in a solution containing collagenase, elastase, hyaluronidase, and DNAse (145, 155). After digestion and disaggregation, gland acini are placed in a culture dish in the presence of serum-containing medium to allow attachment. After attachment, outgrowths from explants are promoted in serum-free medium (145, 155, 177, 178). Cells can then be removed by trypsinization, dispersed, and replated. The submucosal gland epithelial cells can then be subcultured and grown in serumfree medium under conditions comparable to those used for the luminal or nasal mucosa cells.

Fig. 2. Low-power electron micrograph of cultured primary epithelial cells grown on Millicell-HA inserts (Millipore Products Division, Bedford, MA) coated with vitrogen (Collagen, Palo Alto, CA). Cells were grown in Dulbecco’s modified Eagle’s medium and Ham’s F-12 (1:l) containing 2% serum supplement Ultorser G with an air-liquid interface. (Reproduced with permission, Ref. 176). C, ciliated cells, G, goblet cells, and B, basal cells. Scale bars = 10 km.

INVITED PRIMARY

CULTURES:

CELL

BIOLOGY

Primary cultures of human tracheal and bronchial epithelium have proven very useful in demonstrating and elucidating defects in Cl secretion and Na absorption in CF (87, 151, 161, 162, 166). However, the cultures used in these studies were usually dedifferentiated and unsuitable for many types of functional analyses. When grown in primary culture on a FN/V/BSA or collagen extracellular matrix film, these cells tend to be flattened (63) rather than columnar. More recently, culture conditions have been developed that now allow primary cultures to display an ultrastructure essentially identical to that of native epithelium (Fig. 2). Functional properties such as ion transport and mucin immunochemistry also closely resemble those of the original tissue (176). Confluent primary cultures of surface epithelium can be essentially obtained at plating densities as low as 3 x lo5 cells/cm2 (176), can maintain differentiated function for up to 1 mo (176), and can be subcultured several times (when not grown on collagen gels) while retaining epithelial polarity (64). Thus even where access to viable human tracheas is limited, primary diploid cultures may still represent a reasonable approach to a variety of research questions. Primary cultures of gland acinar epithelium also retain many of their differentiated characteristics, as determined immunocytochemically or by electron microscopy (30,41,60,145,155,175,176,178). Cells that have not been subcultured have been found with mucous or serous cell granules [in some instances cells were shown to contain both serous and mucous cell granules (145, 155)]. These cells in culture have proven to be important in elucidating the mechanisms underlying secretion of mucus fluid (30,80,139,145,155,173,177-179) and in clarifying the role that gland cells play in CF (9,45, 177). Aside from their obvious secretory properties, these cells display unique ion transport properties distinct from those of the mucosal epithelium (177, 178). Although the numbers of gland cells that can be isolated per trachea are much fewer than the number of surface cells, we have found that the cells can be passaged to up to four times without changes in iontransport function (51). Thus there is potential for considerable expansion of the numbers of gland cells in primary culture. TRANSFORMED CONSIDERATIONS

CELL

LINES:

GENERAL

A disadvantage of primary epithelial cell cultures is their limited lifespan. While airway epithelial cells in culture can provide model in vitro systems for investigating certain biochemical and genetic mechanisms underlying airway epithelial cell function, limitations are often encountered because the cells senesce or terminally differentiate. One mechanism for overcoming this obvious drawback has been to rely on transformed airway epithelial cells. These cell lines can either be derived from carcinomas of the airways or they can be generated through exposure to various agents (viral, chemical, or physical). Once transformed cells have been

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established in culture, they will have an enhanced-tounlimited lifespan. This enhanced proliferative ability may in part be due to the karyotypic instability and aneuploid nature of the cells. In the early stages of transformation the cells retain many of the same properties found in primary diploid epithelial cells (35, 62, 63, 67, 86, 183). After the cells pass through “crisis” (59), they tend to become less differentiated and lose cell-type specific features. However, there are a few cell lines that have recently been identified as having properties characteristic of the native epithelium, especially in their ability to form polar monolayers (34,36, 104,143). In addition to their use as tools to investigate specific biochemical and genetic pathways associated with disease pathology and cell function, transformed cells can serve as vehicle for isolating large quantities of cellular macromolecules and are useful in the development of therapies for specific disease (Table 1). Because of their unlimited growth potential, transformed cells can be readily transfected with eukaryotic expression vectors and assayed for transgene expression over multiple generations (109). The potential for growing large numbers of cells makes it feasible to isolate endogenous secretory macromolecules or transgene-defined proteins en masse. This type of expression system can be very informative if the proteins coded for by the transgene are normally expressed in the airway epithelium. The protein can then be modified appropriately after transcription and thereby be functionally representative of the protein in its native state. Maintenance of specific differentiated features also makes transformed cells useful for development of chemotherapeutic and gene therapy protocols. Transformed cells have already played a significant role in genetic complementation studies that substantiate the efficacy of vector systems for expression of the cystic fibrosis transmembrane conductance regulator (CFTR) expression (42, 52, 138). The ensuing sections will describe some features of airway epithelial cell lines that have been generated from tumor tissue and by in vitro transformation.

Table 1. Characteristics epithelial cells Immortalized

of immortalized Epithelial

Cell

Characteristics

Can be studied for an unlimited period Overcomes tissue shortage Establishes continuity in the system to be studied Allows for ongoing biochemical characterization Creates an in vitro system for assessment of therapies Act as factories for production of large quantities components Protein isolation Isolation of nucleic acids (DNA, RNA) Secretory macromolecules Necessary for stable gene transfer Required for genetic complementation Development of animal models Development of gene therapy methodologies

of cellular

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CARCINOMA-DERIVED

CELL

LINES

We recently screened 12 carcinoma-derived cell lines for ultrastructure, levels of airway secretory proteins and their mRNA, and ion transport function (50, 143). Grown under standard culture conditions, these cell lines showed somewhat dedifferentiated ultrastructure. Furthermore, the mRNA and protein content was not characteristic of any particular cell type of the native epithelium. Thus the usefulness of most of these lines for studies of airway epithelial function must be limited. However, one cell line, CALU-3, had features consistent with differentiated function of secretory gland cells (143). They also contained high levels of CFTR protein and formed polarized cell sheets with tight junctions and a transepithelial resistance of - 100 a/crnm2. Consistent with the presence of CFTR, they showed CAMPdependent transepithelial secretion of Cl. In addition, these cells have also proven useful in studying the kinetic properties of CFTR’s function as an apical membrane Cl channel (71). Though of predominantly serous phenotype, these cells have some characteristics of mucous gland cells. Thus they contain typical mucous granules and mRNAs for MUG-2, one of the secreted mucin protein backbones. These properties indicate that they may also prove useful in studies on the regulation of mucus secretion. IN VITRO

TRANSFORMED

CELL

LINES

Primary cultures of airway epithelium have been transformed with a variety of agents. The most commonly used are constructs containing the large T antigen of the SV40 virus. While most SV40-transformed airway epithelial cell lines have been able to maintain certain differentiated features, such as ion transport (34, 57, 63, 76, 82, 86, 94, 129, 130, 138, 141), secretion (6, 34, 37, 104), metabolic enzymes (6, 10, 13), and adhesion molecules (11, 108, 153, 154), they have generally lost the capacity to form tight junctions and polarized monolayers after the early stages of transformation. There is, however, a normal human airway epithelial cell line with wild-type Cl ion transport phenotype (16HBEl40-) (36,37, 72) and a CF nasal polyp cell line (CCFNPE14o-) (104) that form tight junctions, generate polar monolayers, and express other differentiated features characteristic of the native epithelium. The 16HBE140cell line is particularly interesting in that it not only forms polarized monolayers with intact tight junctions, but it also has levels of CFTR mRNA comparable to those seen in the colon carcinoma cell line, T84, which has been used as a standard for analysis of CFTR function (36). In addition, it will display cilia under defined growth conditions with an air-liquid interface and may provide a functional correlation for CFTR in ciliated cells (36). Studies measuring the modulation of CFTR expression have indicated that retinoic acid will increase the steady-state level of CFTR mRNA and that phorbol esters modulate CFTR mRNA differently in airway epithelial cells than in the T84 colon carcinoma cells. Previous studies in T84 cells have shown a significant reduction in CFTR expression after

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exposure to phorbol esters (142, 156). The suggestion was that this reduction in CFTR mRNA was directly correlated with a decrease in CAMP-dependent Cl transport. However, when similar measurements were made in the 16HBE140-, the results indicate that the phorbol ester effects CAMP-dependent Cl transport by another mechanism. A 24-h exposure to phorbol ester indicated that the level of CFTR mRNA remains relatively unaffected in the 16HBEl40cells, but is greatly reduced in T84 cells even when both cell types show a reduction in CAMP-dependent Cl transport (Gruenert, unpublished observations). This indicates that the phorbol ester 1) regulates steady-state CFTR mRNA levels in a cell-type specific fashion and 2) may be involved in inhibiting CAMP-dependent Cl transport by a mechanism independent of CFTR. Because activation of CAMP-dependent Cl secretion is a multistep process, it is possible that another element of this pathway, such as basolateral potassium channels (142), is affected by the phorbol ester. This result also points out the need to evaluate chemotherapeutic treatments for CF in airway epithelial cells to accurately assess efficacy for the treatment of CF airway disease. Thus this cell line, among other things, may be able to play an important role in defining chemotherapeutic regimens for regulating CFTR. By manipulation of the culture conditions, it has also been possible to cause some of these cell lines to express mucin (37) and secretory cell-specific antigens that indicate goblet cell features (104). Another cell line with tight junctions and properties of airway epithelial cells has also been developed (37). This cell line expresses mRNA for the HAM-l (MUC-2) gene, a secreted mucin associated with airway epithelial cells (81). In addition, these cells have been used to show enhanced mucin expression after exposure to retinoic acid, as determined by mucin-specific antibody immunohistochemistry (G. Place, personal communication). The CCFNPE14ocells have been shown to express secretory cell-specific antibodies that localize to mucous, serous, and goblet cells; mucous and goblet cells; and serous and goblet cells, but not antibodies that localize to either serous or mucous cells alone (104). Because both of these cell lines have tight junctions and display cell polarity, they can be employed to investigate vectoral secretion of cellular macromolecules and their regulation from the apical or basolateral compartments. To add to this repertoire of transformed airway cells, there is now a report that human papilloma virus can also be used to generate transformed human airway epithelial cells with differentiated features characteristic of the native epithelium (182). In addition, several airway epithelial cell lines, both CF and normal, have been generated using a temperature-sensitive mutant of the SV40 large T antigen (65,66). While these cells may provide important information regarding the expression of various airway epithelial cell proteins at conditions that are nonpermissive for cell growth, there does seem to be a reduction of CFTR protein expression at this nonpermissive temperature (40°C) (Gruenert, unpublished observations). The expression of CFTR in these cells appears to be optimal at 37”C, although CFTR

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protein can still be detected at the permissive temperature (34°C). Cells transformed in vitro are no longer under the same growth controls found in nontransformed diploid cells (Table 2). Although transformed epithelial cells have an extended life span, they may terminate their proliferation in the early stages of the neoplastic process, a phenomenon known as “crisis.” Crisis is characterized by a slowing of cell growth, vacuolization of cells, cell senescence, and general deterioration of the cultures (59). During crisis a subpopulation of cells survive to form proliferating colonies of cells. It is these cells that have progressed through crisis. While the underlying cause of crisis is not clearly understood, it is thought to be related to the normal course of cell senescence (59, 132). Airway epithelial cells can enter into crisis as early as 6 and as late as 25 subcultures posttransformation; however, crisis is generally observed at 15 to 20 subcultures after transformation (Gruenert, unpublished observations). Escape from crisis and subsequent neoplastic progression is directly correlated with the potential to proliferate and appears to be dependent upon a number of genetic factors, some of which have been defined by the transfer of various oncogenes or suppressor genes (126, 128, 132, 133). It appears as if the association of the SV40 large T antigen with tumor suppressor genes such as p53 (107) and the retinoblastoma (rb) gene product (40) influences the proliferative potential of transformed cell, and this association may be a factor in escaping from crisis. Among the oncogenes involved in neoplastic progression, postcrisis studies have indicated that v-Kirsten-ras (v-Ki-ras) (133) or c-~$1 and c-m;yc (126, 12S), in conjunction with the SV40 T antigens, can play a role in the tumorigenic potential of the transformed cells. If the transformed cells progress through crisis (59, 62), they generally acquire an unlimited growth capacity and are, in effect, immortal. Postcrisis cells are often more advanced neoplastically, as defined by their growth properties properties are usually (Table 2) (62). Th ese growth incompatible with the expression of differentiated features, such as cell polarity, tight junction formation, vectoral ion transport, cilia, and secretory granules. However, the airway epithelial cell lines with many of Table 2. Progressive transformed cells

growth

characteristics Comment

Action

Enhanced

growth

Loss of contact

of

inhibition

Immortalization Anchorage independent growth Tumorgenicity in nude mice Metastatic growth

Cell proliferation beyond senescence and apoptosis, escape from terminal differentiation Multilayered growth of cells, no inhibition of growth due to cell-cell contact Unlimited growth potential usually established after crisis Clonal growth in soft agar Formation cells into Dispersion to other animal

of tumors after injection of nude mouse of cells from a solid tumor organ sites within a host

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the differentiated features mentioned above (36, 37, 104) should prove useful for correlating the expression of specific differentiated functions with different stages of neoplastic progression, and for investigating both the cell and cancer biology of the airway epithelium in general. TRANSFORMED CELL LINES: DISEASE PHENOTYPE

NORMAL

AND

Transformed airway epithelial cells have a stable Cl ion transport phenotype, and therefore have facilitated CF research by providing a cell system where studies could be carried out over an extended period using the same cells. In particular, it has been possible to investigate ways in which the cellular Cl ion transport pathways can be manipulated to overcome the defect in CAMP-dependent activation of Cl secretion, which is characteristic of this disease. Thus, using transformed epithelial cell lines, three different pathways for the regulation of Cl current have been defined: 1) CAMP dependent (34, 36, 37, 63, 72, 82, 86, 94, 138, 141, 143, 158), 2) Ca2+ dependent (28, 36, 94, 129, 141, 158), and 3) volume regulated (55, 57, 129, 130, 158). These results indicate that activation of alternate Ca2+dependent transport pathways could compensate for the CAMP-dependent defect. It has also been shown that heterotrimeric G proteins can play a role in the regulation of CAMP-dependent Cl transport (141). Of particular importance is the finding that inhibition of Gai-2 corrects the defect in CAMP-dependent Cl secretion in CF cell lines, probably by allowing mutant CFTR to traffic to the cell membrane where it is able to function as a Cl channel (106). In addition to investigations of the ion-transport defect in CF, the K+ currents of transformed cells have been studied (56). In addition to the studies investigating the hormonal regulation of CFTR mentioned above, these findings open up the possibility of pharmacological treatment of CF and modulation of ion transport pharmacologically. The secretory cell-specific properties of transformed cells include expression of mucin mRNA (37), antigenitally detectable mucin (G. Place, personal communication), and the presence of antigens that localize to secretory cells in vivo (34, 104). It should therefore be possible to study further the mechanisms regulating airway epithelial cell secretion and secretory cell macromolecular biochemistry. In addition, the role that these macromolecules play in bacterial infections in CF can potentially be elucidated by comparing adhesion to CF cells and the same CF cells corrected with wild-type CFTR. The role of the airway epithelium in infection and inflammation has been investigated in the context of viral replication and neutrophil adhesion (146, 147, 154). Infection of transformed airway epithelial cells with influenza virus A (136) and parainfluenza virus (147) has been very important for defining parameters influencing neutrophil adhesion. Studies in transformed airway epithelial cells have shown that intercellular adhesion molecule-l (ICAM-1) is an important factor in neutrophil adhesion (153, 154).

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An important mediator of airway inflammation, interleukin-8 (IL-@, has been shown to be expressed in both primary (7,33) and transformed (38,112) airway epithelial cells and is increased by viral (8) and bacterial (112) infection. In the case of a Pseudomonas aeruginosa infection, a novel, low-molecular-weight bacterial product appears to enhance the production of IL-8 by the epithelial cells (112). The chemotactic activity of IL-8 can thereby recruit neutrophils to the site of infection, resulting in the inflammatory response associated with this type of infection. TRANSFORMED

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CANCER

BIOLOGY

In vitro transformation of human tracheobronchial epithelial cells presents an ideal means for investigating the cellular and genetic changes that occur during carcinogenesis in the airways. It has been possible to monitor spontaneous neoplastic progression by assessing changes in the growth properties of cells at different times after transformation (62, 70, 128, 183). In addition, it is possible to influence the transformed state of cells by 1) changing growth conditions, 2) introducing oncogenes, and 3) exposing them to chemical or physical carcinogens. Neoplastic progression can be defined in terms of changes in the growth characteristics of cells as defined by specific growth endpoints (Table 2). These different stages can be designated as 1) enhanced growth, 2) anchorage independent growth, 3) tumorgenicity in animal models, 4) invasiveness, and 5) metastasis (62). Once transformed, cells tend to lose their contact inhibition, become aneuploid, and often no longer respond to certain agents that stimulate differentiation (62, 69, 70, 102, 113, 128). This does not mean that specific functional characteristics cannot be retained. However, it is difficult to predict which agent will be an effective inducer of differentiation in any given cell line. Introduction of oncogenes into transformed cells has been shown to cause progression to a phenotype that indicates a less differentiated, less growth-regulated cell. The Ki-ras oncogene has been associated with lung cancer (27, 137), and immortalized cells exposed to the Kirsten murine sarcoma virus (Ki-MSV) progressed neoplastically in that they now form tumors in nude mice (133). This oncogene also makes the cells less sensitive to agents that induce terminal differentiation, such as TGF-P (132). Infection with a recombinant retrovirus containing the v-Harvey-ras (v-Ha-ras) oncogene also led to the neoplastic transformation of an immortalized bronchial cell line (2). Other studies have shown that the c-raf-1 and the c-myc oncogenes act cooperatively in inducing neoplastic transformation of airway epithelial cells (127). The resulting tumors appear to have phenotypic features generally associated with small-cell lung carcinoma (126). Finally, immortalized cells in the early stages of transformation can be used to study neoplastic progression after exposure to chemical and physical agents associated with lung carcinogenesis (128). Studies have been carried out analyzing the effect of exposure to asbestos (61) and ionizing radiation (128, 171, 180) on

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transformed airway epithelial cells in the context of proliferative capacity and biochemical changes associated with lung pathology. Asbestos was shown to be involved with fibrinolytic activity of the lung resulting from stimulation of urokinase-type plasminogen activator activity. This finding implies a mechanism whereby asbestos inhalation leads to chronic inflammation, which results in fibrosis of the lung. The chronic infiltration of inflammatory cells could also be involved in recruitment of growth factors important in activation of preneoplastic and neoplastic growth. Ionizing radiation directly decreases cell viability and can cause airway epithelial cells to increase their growth potential (180). In addition, ionizing radiation can cause specific chromosomal aberrations that appear to be linked to different stages in the neoplastic process and are associated with specific growth phenotypes (171). The loss of chromosome 13 appears to be associated with regulation of cell proliferation in that it causes the cells to overgrow, but not to become tumorigenic. Loss of chromosomes 3p, llp, and/or 22 after ionizing radiation also changes the growth characteristics of the immortalized cells and may be related to the presence of tumor suppressor genes on these chromosomes. Immortalized airway epithelial cells that have been neoplastically transformed can further be studied for the effect of agents that enhance differentiation. In general, transformed cells tend to be less responsive to agents that normally induce epithelial differentiation (128). Further studies are required to determine the conditions under which neoplastic cells can be induced to differentiate. Numerous agents have been shown to influence gene expression and differentiation. A systematic analysis of these agents in neoplastically transformed cells could identify agents that might be useful for stimulating terminal differentiation (62, 128). Retinoic acid has emerged as one such agent. It has been implicated in airway epithelial differentiation and may prove to play an important role in altering neoplastic progress in the airways (4,26,84,85). CONCLUSIONS

The development of human airway epithelial cell culture systems has made an important contribution to our knowledge of airway epithelial cell and cancer biology. These in vitro cell systems have made it possible to dissect the biochemical and genetic mechanisms underlying multiple cellular pathways. Furthermore, these cell culture systems have given us the ability to discern some of the causes of airway disease. The isolation and culture of primary diploid airway epithelial cells (as opposed to aneuploid transformed epithelial cells) has provided a convenient model system for looking at the airway epithelium and its function, devoid of the myriad influences found in native tissue. Current technology now provides a means of systematically evaluating culture medium components, extracellular matrixes, and overall growth conditions in terms of the differentiated features expressed by the cells within a given culture (39, 176, 178). As further research defines individual agents that modulate the expression

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of specific phenotypic endpoints, it will be possible to optimize the conditions for growth and subsequent differentiation of a given epithelial cell type. Characterization of specific airway epithelial cell types has to some degree been facilitated by the transformation of primary diploid epithelial cells. Isolating clones of transformed cells leads to cultures that are theoretically derived from a single cell. Because primary cultures of epithelial cells are heterogeneous in terms of the types of cells (Fig. l), it should be possible to generate clones of specific cell origin. Although transformed epithelial cells often dedifferentiate in culture, under well-defined isolation procedures and growth conditions it has been possible to generate transformed airway epithelial cell lines with phenotypic characteristics similar to those of primary cultures and native epithelium (36,37). Thus the currently developed airway epithelial cell both nontransformed and transculture systems, formed, have been responsible for significant progress in the understanding of various aspects of airway pathology and function. As more is discovered about the culture and extracellular matrix components required for the growth and differentiation of epithelial cells, it will be possible, to some extent, to reconstitute the native epithelium in vitro, thus increasing the usefulness of these cell systems as adjuncts to animal systems for studies on the physiology, pathology, and toxicology of human airway epithelium. We thank Linda Escobar and Dr. Dachuan Lei for their assistance with the cell culture and the phenotypic characterization of the epithelial cells and Dr. Graham Place for sharing his unpublished data with us. We also acknowledge Rebecca Salzer for her assistance with the typing and editing of this manuscript. The work described here was supported by National Institutes of Health Grants DK-46002, DK-39619, HL-41928, HL-42368, and DK-47766, grants from the National Cystic Fibrosis Foundation, and a grant from the Tobacco-Related Disease Research Program of the state of California. Address for reprint requests: D. C. Gruenert, SU203, Box 0911, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94143. REFERENCES 1. Albright, C. D., P. M. Grimley, R. T. Jones, J. A. Fontana, K. P. Keenan, and J. H. Resau. Cell-to-cell communication: a differential response to TGF-beta in normal and transformed (BEAS-2B) human bronchial epithelial cells. Carcinogenesis 12: 1993-1999,1991. 2. Amstad, P., R. R. Reddel, A. Pfeifer, L. Malan-Shibley, G. E. Mark, and C. C. Harris. Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding for viral Harvey ras. Mol. Carcinog. 1: 151-160, 1988. 3. Anderson, M. P., and M. J. Welsh. Calcium and CAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc. NatZ. Acad. Sci. USA 88: 6003-6007,199l. 4. Ann, D. K., M. M. J. Wu, T. Huang, D. M. Carlson, and R. Wu. Retinol-regulated gene expression in human tracheobronchial epithelial cells. J. Biol. Chem. 263: 3546-3549, 1988. 5. Ayers, M. M., and P. K. Jeffery. Proliferation and differentiation in mammalian airway epithelium. Eur. Respir. J. 1: 58-80, 1988. 6. Barasch, J., B. Kiss, A. Prince, L. Saiman, D. Gruenert, and Q. Al-Awqati. Defective acidification of intracellular organelles in cystic fibrosis [see comments]. Nature Lond. 352: 70-73, 1991. 7. Becker, S., H. S. Koren, and D. C. Henke. Interleukin-8 expression in normal nasal epithelium and its modulation by

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