[CANCER RESEARCH 40, 4403-4409.
0008-5472/80/0040-OOOOS02.00
December 1980]
Isolation and Spontaneous Transformation of Cloned Lines of Hamster
Trachea! Epithelial Cells1
Brooke T. Mossman,2 Elizabeth B. Ezerman, Kenneth B. Adler, and John E. Craighead
Department of Pathology. University of Vermont College of Medicine, Burlington,
Telaco, Bar Harbor, Maine) and dissected in Hanks' balanced
ABSTRACT
In respiratory carcinogenesis studies using rodents, the tra
chéalepithelium is the target tissue for the induction of tumors
after exposure of animals to chemical carcinogens. In the
studies described below, trachea! epithelial cells were isolated
to evaluate their biological and biochemical features. Epithelial
cells derived from the trachéalmucosa of Syrian golden ham
sters were established in culture. Three morphological types of
polygonal cells were observed as mixed populations in four
clonally derived lines. One type of cell is mucin secreting since
membrane-bound vesicles that stain positively using the alcian
blue:periodic acid-Schiff reaction are present in the cytoplasm,
and increased amounts of mucin constituents are demonstrable
in the culture medium. Cells of a second type possess both
intracytoplasmic and surface cilia, but they lack mucin vesicles.
The third type exhibits no differentiating features. Four densitydependent inhibited cloned cell lines were established. After
repeated passage, these cells: (a) grew in soft agar; (b) re
leased proteases that were activators of plasminogen; (c) dem
onstrated measurable basal and inducible aryl hydrocarbon
hydroxylase activity; and (d) produced anaplastic carcinomas
in syngeneic hamsters. Factors affecting the transformation
and differentiation of respiratory epithelial cells have not been
elucidated. The availability of these cell lines will permit studies
that focus on these questions.
INTRODUCTION
Although embryonal and mesenchymal cell systems are
available to study the processes of neoplastic transformation
in vitro (1, 5, 9), there are relatively few studies of viral and
chemical carcinogenesis using epithelial cells (25). Efforts to
culture epithelial cells from parenchymal organs have met with
limited success since repeated passage results in an apparent
loss of differentiated characteristics. Cells exhibiting properties
of the differentiated respiratory epithelium would serve as
appropriate models for studies of carcinogenesis since most
lung tumors are of bronchogenic origin. To allow investigation
of the cellular mechanisms involved in respiratory carcinogen
esis, we isolated, propagated, and cloned lines of epithelial
cells derived from the hamster trachéal mucosa. This report
describes our methods and the properties of the cell lines thus
far established.
MATERIALS
Vermont 05405
AND METHODS
Preparations of Cultures. Tracheas were excised from neo
natal and weanling female Syrian hamsters (87.20 BIO strain,
salt solution containing gentamicin (100 /ig/ml) and nystatin
(25 units/ml). Two techniques were utilized to separate the
epithelium from the subjacent mesenchymal tissue, (a) Organ
cultures were prepared in plastic dishes from trachéal seg
ments (19) and maintained in Waymouth's MAB 87/3 medium
(Grand Island Biological Co., Grand Island, N. Y.) containing
10 to 20% fetal calf serum. This encouraged the outgrowth and
proliferation of epithelial cells on the plastic surface at the
margins of the tissue, (b) The trachea was divided into hemisections, and the epithelium was a stripped from the basement
membrane by dissection with a scalpel. Epithelial strips were
planted in either 35-mm Falcon dishes or Linbro multiwell
dishes. Contamination by fibroblasts occurred occasionally.
These cultures were either treated with hydrocortisone hemisuccinate (0.2 mg/ml) to deter fibroblastic growth (3) or dis
carded.
Feeder layers of hamster fibroblasts irradiated at 4000 to
5000 rads or substrates of either 0.01% gelatin or rat tail
collagen were utilized for primary cultures. Ham's Nutrient
Medium F12 or F12K with addition of 10% fetal calf serum,
gentamicin (50 fig/ml), and nystatin (25 units/ml) was used
routinely. All cultures were maintained at 36 to 37°in a humid
ified environment containing 5% CO2:95% air, and the medium
was changed at 3- or 4-day intervals.
Crystalline trypsin (0.25%) and collagenase (0.7 mg/ml) in
Ca2+-Mg2+-free Hanks' solution were used to dissociate cells.
Single cells were cloned from cultures after 10 to 20 passages
and plated on plastic dishes. Cell lines were tested sporadically
for mycoplasma by the method of Hayflick (12). Contaminants
were not demonstrated using these procedures.
Morphological Studies. Cultured cells were examined using
a Zeiss inverted-stage phase-contrast microscope. For trans
mission electron microscopy, cells in plastic dishes were fixed
in situ in 2.5% glutaraldehyde (0.1 N sodium cacodylate buffer),
postfixed with 1% osmium tetroxide, and embedded in Epon.
Clusters of plastic-embedded cells then were identified by
phase-contrast microscopy and cut from the cast of the plate,
and the blocks were glued to plastic stubs. Thin sections were
stained with uranyl acetate and lead citrate and examined with
a Philips 300 electron microscope.
Selected cultures on glass coverslips were stained for acid
mucopolysaccharides
using the AB-PAS3 (pH 2.5) reaction
(23). The Gomori method for demonstration of acid phosphatase was utilized to delineate lysosomes (11). To exclude the
possibility of fibroblastic contamination, clonal lines were
stained for collagen using the technique described by Berman
ef al. (4). Cultures of mouse skin fibroblasts (L-cells) (21 ) were
used as a positive control for these studies.
' Supported by Contract NO1 CP 33360 from the Lung Cancer Division of the
National Cancer Institute.
2 To whom requests for reprints should be addressed.
Received August 15. 1979; accepted August 19. 1980
DECEMBER
3 The abbreviations
aryl hydrocarbon
used are: AB-PAS. alcian blue:periodic
acid-Schiff;
AHH,
hydroxylase.
1980
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4403
6. T. Mossman et al.
Assays for Glycoproteins. Medium was collected from cul
tures at 3- to 4-day intervals and tested for selected constitu
ents of mucin after cellular debris was removed by passage
through a Millipore filter (0.22-fim pore diameter). Sialic acid
was analyzed by the thiobarbituric acid method of Warren (24);
determination of /V-acetylhexosamine was done using the
method described by Reissig et al. (20). Normal hamster fibroblasts and L-cells were utilized as negative controls in these
studies.
Assessment for Tumorigenicity. At each tenth consecutive
passage (routinely a 1:4 split) after cloning, cells (approxi
mately 1 and 2 x 106) from clonal lines were introduced s.c.
into young adult syngeneic hamsters. Animals were palpated
at 3-week intervals for the appearance of tumors. Thereafter,
masses of approximately 1-cm diameter were excised and
fixed in Bouin's solution. Five-micron histological sections of
tumors were stained routinely with hematoxylin and eosin and
selectively for mucin (1 3, 15), reticulin (26), and collagen (18).
For electron microscopy, tissues were fixed in 4% glutaraldehyde and processed as described above.
Several markers of neoplastic transformation in vitro have
been described for mesenchymal cells. These include growth
in soft agar (16) and production of plasminogen activators (2).
To determine whether or not our cell lines possessed properties
in common with transformed fibroblasts, assays were per
formed using cloned cells.
In brief, a stock of Difco Bacto agar (1.25 g/100 ml distilled
water; Difco, West Molesey, United Kingdom) was mixed 1:1
with complete Ham's F12 medium (2-fold concentration) and
dispensed into 35-mm dishes. Cell suspensions in complete
medium then were added 1:1 to the agarmedium and layered
on the gel (approximately 10" cells/dish). Dishes were incu
bated at 37°and moistened every 4 days with 3 to 4 drops of
complete medium. After 3 weeks, colonies (>0.1 -mm diameter)
were evaluated with an inverted phase microscope and microm
eter. A transformed rat liver epithelial cell line (22) and normal
hamster lung fibroblasts isolated in this laboratory were used
as positive and negative controls, respectively.
The casein overlay assay for the detection of plasminogen
activator was a modification of the procedures described by
Goldberg (10). Single-cell suspensions were plated in replicate
60-mm dishes at 103 cells/dish. Cells were fed routinely for 2
weeks and then overlaid with agar (1.2% in 1% casein) in
Ham's F12 containing 10% dog serum. After 4 hr at 37°,
medium was eluted, and dishes were observed for plaques.
AHH Activity. To determine whether or not our cell lines
possessed the ability to convert polycyclic hydrocarbons to
metabolically active derivatives, cloned lines were assayed by
the fluorometric procedure described by Chuang and Bresnick
(7) using 3,4-benzo(a)pyrene
as the substrate. In replicate
assays, individual cell lines were plated onto twelve 100-mm
dishes at 106 cells/dish. Three days after plating, 10 ^l of 3methylcholanthrene
in dimethyl sulfoxide were added to 6
dishes (final concentration, 0.5 mg 3-methylcholanthrene
per
ml medium). Six control dishes received dimethyl sulfoxide
alone. After 24 hr, dishes were pooled, cells were scraped
from the surfaces using a rubber policeman, and cell pellets
were sonicated in 0.05 M Tris buffer (pH 7.5). The average
AHH activity (pg of 3-hydroxy-3,4-benzo(a)pyrene
per min per
mg protein) was determined from 6 replicate incubations at 2
or 3 levels of enzyme protein.
4404
Plating Efficiencies. Cloned cell lines were plated in five 60mm dishes at 103 cells/dish. After 8 hr, adherent cells were
stained with Giemsa solution and counted using an inverted
phase microscope. The average plating efficiency was deter
mined as the percentage of cells per total cells plated.
Chromosomal Analyses. Chromosomal spreads were pre
pared for cloned cell lines at various passages (3). Evaluated
for each preparation were 130 to 200 metaphase plates.
RESULTS
Primary Cultures and Subcultures
Cells having epithelial characteristics were observed in pri
mary cultures prepared from both mucosal strips and organ
cultures. These populations were relatively static for they were
maintained for periods of as long as 10 to 12 weeks without
passage. Differentiated cells with beating cilia and clusters of
polygonal cells with granular surface accumulations appeared
at scattered sites. Despite repeated attempts, it was possible
to subculture only a few lines of cells (less than 1 in 100) from
the primary outgrowth (Fig. 1). Four lines (A to D) were cloned
after 15 to 20 passages of the primary culture and character
ized further for the purposes of this study.
Characterization
of Cell lines
Assay for Glycoproteins. The concentrations of sialic acid
and N-acetylhexosamine in the medium of cultures of trachéal
epithelial cells were severalfold greater than the amounts dem
onstrated in fluids in cultures of both L-cells and hamster
N-Acetylhexosamine
04
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-s
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02
9
Sialic acid
.§
o '16
§
u
•12
•08
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i
2
3
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4
567
Control
Chart 1. Media from uncloned trachéalcell cultures (Bars ) fo 4) contained
relatively larger amounts of glycoprotein components (sialic acid. /V-acetylhexo
samine) as compared to normal hamster fibroblasts (Bars 5 to 7). Each bar
represents 3 to 5 duplicate determinations and shows the average amounts (mg)
of material secreted per monolayer culture per day (± S.E.). The values of the
media controls (F12:10% serum) were subtracted from the totals prior to graph
ing; thus, the oars represent only the contributions of the cells. ', medium was
collected at 3-day intervals.
CANCER
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40
TrachéalEpithelial Cells in Culture
Characterization
Table 1
of cloned cell lines derived from the hamster trachéalepithelium
activity0Basal11
AHH
acti
vator(plaques/dish)20404558473530NTAv.
effi
(tumors/no.
sagesaftercloning1020102010201020Chromosomes344
in
ciency(%)40485080708570NT
of ani
softb
agar0257101513NTPlasmino-gen
duced75
mals)0/60/60/61/40/65/60/66/6Growthin
CelllineABCDPas
(33-63)44
±0.721
9.390
±
(27-46)44
4.5110
±
0.935
±
(32-52)44
11.980
±
2.436
±
(30-79)44
.836±1
2.463
±
(40-53)48
4.618
±
11.188
±
(20-96)48
2.516
±
4.071
±
(30-69)NT0Tumori-genicity
3.4NTPlating
±
2.8NT3-Methylchol-anthrene
±
" The modal number and chromosomal distribution are presented.
b Colonies (>0.1 mm diameter) per 10" cells plated.
0 Pg of 3-hydroxy-3.4-benzo(a)pyrene
per min per mg protein ±S.E.
d NT, not determined; assays were not performed on cell line because of microbial contamination
fibroblasta (Chart 1). Many cells in each of the 4 lines exhibited
dense peripheral granules that stained with AB-PAS (Fig. 2).
We concluded that these structures were mucin granules and
not lysosomes, as acid phosphatase activity was restricted to
smaller perinuclear vesicles in degenerating cells (Fig. 3).
Morphology. The majority of the cell types in each of the 4
clonal lines appeared polygonal and undifferentiated by phase
microscopy (Fig. 4). However, examination of cultures by elec
tron microscopy showed that occasional cells had either mucinlike granules (Fig. 5) or cilia (Fig. 6) in the cytoplasm. These
differentiated cell types were found after repeated passage of
the lines. Collagen fibrils were not detected in cloned lines by
ultrastructure or by histochemical staining (4).
Assays for Tumorigenicity and AHH Activity. These assays
in trachea! cell lines are described in Table 1. All lines grew in
soft agar after 20 passages (although numbers of colonies >
0.1 mm varied), showed detectable plasminogen activators,
and exhibited both basal and inducible AHH activity. The modal
number of lines A and B was 44 (2 n = 44), indicative of a
Syrian hamster diploid cell line, whereas the highly tumorigenic
lines C and D were aneuploid (2 n = 48) at 20 passages after
cloning.
At 10 passages after cloning, injection of cell lines (approx
imately 2 x 106 cells) into syngeneic hamsters failed to cause
neoplasms. Thereafter, anaplastic carcinomas (Fig. 7) ap
peared in animals inoculated with 3 of 4 lines that had been
passaged 20 times after cloning (Table 1). These tumors could
be cultured and transplanted in animals subsequently. Histo
chemical staining failed to reveal collagen and reticulin fibers
in the lesions. Occasional tumor cells contained either cilia or
mucin granules when examined ultrastructurally.
DISCUSSION
We isolated epithelial cells from the trachéal mucosa to
explore the processes of differentiation and carcinogenesis. It
was of further interest to determine whether or not the differ
entiated cells of the trachéalepithelium persisted in monolayer
after passaging of the cultures. Three cell types were seen in
all 4 cloned lines. The first cell, comprising the majority of cells
in all cultures, was apparently undifferentiated. The second
cell, found most infrequently, possessed cilia of the character
istic mammalian "9-2" centriolar structure. Our histochemical,
ultrastructural,
DECEMBER
and biochemical observations strongly suggest
in frozen stocks.
that the third cell type is mucin secreting, as media from
cultures contain components of mucin qualitatively similar to
those secreted by the human trachéal mucosa in vitro (14).
These results have been confirmed by Last ef a/.4 using our
cloned cell lines. Assay of medium from cell lines shows both
neutral and acidic mucin glycoproteins.
As populations of all 3 cell types appeared in all 4 cloned
cell lines, one might hypothesize that, in each case, the undif
ferentiated cloned cell was a basal or stem cell capable of
giving rise, under appropriate conditions, to both ciliated and
mucin-secreting cells. Although precise quantitation of ciliated
cells was not feasible using ultrastructural techniques, numbers
of ciliated and mucin-secreting cells appeared to vary in each
cell line with passage. An exciting possibility is the use of the
cells to study factors influencing the secretion of tracheobronchial mucin. We are now attempting to determine, using ABPAS staining to delineate mucin granules, if administration of
exogenous factors, such as vitamin A, affects the relative
proportions of mucin-secreting cells appearing in cultures. The
vitamin A analog, retinyl methyl ether, increases secretion of
labeled glycoproteins in cell line B when added at 10~7 M.5
The development of neoplasms after implantation of 3 of the
4 clonal lines (B to D) into syngeneic hamsters indicates that
the respiratory epithelial cell possesses the capacity to spon
taneously transform in vitro after continuous passaging. Curi
ously enough, the tumorigenic potential of the transformed
lines varied. Two (C and D) of the 4 lines were strongly
tumorigenic, causing tumors in the majority of animals inocu
lated in 10 to 14 weeks. Line B seemed weakly tumorigenic as
only one in 4 animals developed a tumor after s.c. injection of
2 x 106 cells at 20 passages after cloning. The latency period
of this neoplasm was approximately 52 weeks.
Assays of in vitro transformation were performed to deter
mine whether or not the cloned cells exhibited many of the
features associated with neoplastic fibroblastic lines. Our stud
ies indicate that increased fibrinolytic activity and plating effi
ciency of lines were not related directly to their relative tumorigenicity. In contrast, the number of colonies > 0.1 mm ap
pearing in soft agar correlated with tumorigenic potential. Like
wise, studies by Marshall et al. (17) have shown that epithelial
* J. A. Last. T Kaizu. and B. T. Mossman.
Glycoprotein
secretion
by an
established cell line from hamster trachéalepithelium. Exp. Lung Res, in press,
1980.
5 K. B. Adler, B. T. Mossman, and J. E. Craighead, unpublished data.
1980
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4405
S. T. Mossman et al.
lines from human bladder and colon tumors possess some, but
not all, of the properties associated with in vitro transformation
of fibroblasts. In their experiments, 4 of 6 cell lines grew in soft
agar, and all produced plasminogen activators. Colony-forming
efficiency in soft agar correlated with neither increased fibrinolytic activity nor the ability of human tumor cells to induce
neoplasms in nude athymic mice. The inability to extrapolate
the results of in vitro assays of transformation to consequent
tumorigenicity also has been demonstrated for epithelial lines
derived from murine mammary carcinomas (6, 8).
A paucity of epithelial cell lines exists to explore the cellular
and molecular mechanisms involved in spontaneous transfor
mation and carcinogenesis (25). The ability of our untransformed and early-passage cell lines to metabolize polycyclic
hydrocarbons, as demonstrated by both basal and inducible
levels of AHH, suggests their use as a suitable system for the
bioassay of potential chemical carcinogens.
ACKNOWLEDGMENTS
We thank C. Cathcart, B. Clements, L. DiCesare, P. Halloran, L. Howard, P.
Kimberly. J. Kessler, B. MacLeod, and M. White for their excellent technical
assistance. Dr. H. Chuang performed the AHH assays. Dr. N. Heintz assisted
with the m vitro assays of transformation. Dr. J. C. Barren provided useful
suggestions and discussion.
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11. Gomori, G. Histochemical methods for acid phosphatase. J. Histochem.
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12. Hayflick, L. Tissue cultures and mycoplasma. Tex. Rep. Biol. Med., 23.
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13. Humanson, G. L. Animal Tissue Techniques, pp. 301-308. San Francisco:
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14. Kaizu, T., Lyons. S. A., Cross, C. E., Jennings. M. D., and Last, J. A.
Composition of glycoproteins secreted by trachéal explants from various
animal species. Comp. Biochem. Physiol., 62. 195-200. 1979.
15. Luna, L. G. (ed.). Manual of Histologie Staining Methods of the Armed
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Fig. 1. A, polygonal, granulated trachéalepithelial cells in monolayer. Cells were isolated from a mucosal strip and maintained in culture for 6 months after 4
passages. Phase contrast, x 450. B, monolayer culture with retractile droplets (arrow) on surfaces. The cells originated from the same explant as did the cells
illustrated in Fig. 1A and were maintained in culture for 5 passages over an elapsed period of 7 months. Phase contrast, x 800.
Fig. 2. Occasional cells from cloned lines stained with AB-PAS (pH 2.5). Granules staining positively for acid mucopolysaccharides
(arrow) were located at the
cell periphery, x 1,200.
Fig. 3. Cells from the culture depicted in Fig. 2 were stained for acid phosphatase to demonstrate lysosomes. Acid phosphatase-positive vesicles (arrow) were
distributed in a perinuclear array in degenerating giant cells, x 1,200.
Fig. 4. Comparative phase-contrast micrographs of 4 cloned lines A. B. C. and D. respectively. All lines were photographed at passage 16. Note the variations in
phenotype between lines, x 600.
Fig. 5. Electron micrograph of a cell with numerous membrane-bound granules. Uranyl acetate and lead citrate, x 12.000. Inset, a mucin-secreting cell in an
organ culture of hamster trachea maintained 1 week in vitro. Note the similarity in the mucin granules (arrows). Uranyl acetate and lead citrate, x 7.500.
Fig. 6. Cell from an uncloned culture after 15 passages showing an apparent intracellular cilium in association with a centriole (arrow). Uranyl acetate and lead
citrate, x 27.000.
Fig. 7. An anaplastic carcinoma developing in a weanling 87.20-strain golden hamster given an injection s.c. with 1 x 106 cells from cloned line C at passage 20.
H & E. x 1,300.
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40
TrachéalEpithelial Cells in Culture
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Isolation and Spontaneous Transformation of Cloned Lines of
Hamster Tracheal Epithelial Cells
Brooke T. Mossman, Elizabeth B. Ezerman, Kenneth B. Adler, et al.
Cancer Res 1980;40:4403-4409.
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