Dome Formation by a Human Colonie Adenocarcinoma Cell Line (HCA-7)1
Susan C. Kirkland
CflC Cell Proliferation Unit, Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 OHS, England
ABSTRACT
A new cell line, HCA-7, has been established from a primaryhuman colonie adenocarcinoma. The HCA-7 cells have an epithelial morphology by phase-contrast microscopy, they secretecarcinoembryonic antigen, and they form adenocarcinomaswhen injected s.c. into nude mice.
HCA-7 cells retain some of the morphological and functional
polarity exhibited by normal colonie epithelium. Cells form apolarized epithelial sheet when grown on tissue culture plastic instandard culture medium (Dulbecco's modification of Eagle's
medium with 10% fetal calf serum). The cells were shown to bestructurally polarized with sparse microvilli present at the apicalsurface but absent from the basolateral surface. Cells wereconnected by apical tight junctions and frequent desmosomes.Confluent monolayers of HCA-7 cells formed "domes" or "hemi-cysts" due to vectorial fluid transport resulting in fluid accumu
lation in localized areas of the monolayer.HCA-7 cells will provide a useful system for studying the role
of hormones and other factors in the control of transepithelialtransport and maintenance of cell polarity.
INTRODUCTION
The epithelial cell membrane can be divided into 2 distinctregions based on both structural and functional criteria. Themaintenance of this cell polarity is necessary for the performanceof specialized epithelial functions such as absorption or secretion. Ultrastructural studies have shown the apical surface tohave a characteristic structure such as the brush border seen inintestinal epithelium. The apical surface is separated from thebasolateral surface by a belt of tight junctions, and biochemicalstudies have shown each domain to have a characteristic set ofproteins and lipids (1-4).
Although many cell lines have been derived from human colo-rectal adenocarcinomas (5-7), very few retain the polarity of theirtissue of origin (8). This report describes the establishment andcharacterization of a human colonie adenocarcinoma cell linewhich retains some of the structural and functional polarity ofcolonie epithelium.
MATERIALS AND METHODS
Establishment of Cell Line HCA-7. HCA-7 cells were derived from a
primary adenocarcinoma of the colon (hepatic flexure) removed from a58-year-old woman in April 1981. The tumor was a moderately-well-
differentiated mucinous adenocarcinoma which had just penetrated thebowel wall with limited spread to the pericolic fat (Duke's B case). Shortly
after removal from the patient, a small piece of tumor, approximately 1sq cm, was placed in culture medium (see below for details) and trans-
1This work was supported by a Cancer Research Campaign grant awarded to
Professor N. A. Wright.Received 12/10/84; revised 3/20/85; accepted 3/27/85.
ported on ice to our laboratories. The tissue was rinsed repeatedly witha total of 20 ml culture medium by holding with a pair of forceps andflushing medium over the tissue with a Pasteur pipet. Cells were thengently teased from the tissue using 2 pairs of forceps. The resultant cellclumps were collected by centrifugation at 150 x g for 5 min at roomtemperature. Cells were plated into 25-sq cm culture flasks in Dulbecco'smodification of Eagle's medium (Gibco Europe, Ltd., Paisley, Scotland)
with glucose (4500 mg/liter). The medium was supplemented with 10%fetal calf serum [Gibco], kanamycin [100 /jg/ml; Bristol Laboratories,Langley, Slough, England), amphotericin B [2.5 M9/ml; E. R. Squibb,Morton, Cheshire, England], minocycline (1 pg/m\; a kind gift from LederleLaboratories, Gosport, Hampshire, England), gentamicin (100 ^g/ml;Roussel Laboratories, Ltd., London, England], and penicillin [50 t¡g/m\;Crystapen-Benzylpenicillin (sodium), Glaxo Laboratories, Ltd., Green-
ford, England], The antibiotic levels were reduced after several passagesso that, after passage 20, cells were maintained in the presence ofkanamycin (100 ng/m\) and amphotericin B (1.25 ng/ml).
For subculture, cells were treated with trypsin (Worthington Biochem-
icals; 3 times crystallized and dialyzed), 0.05% (w/v) in Versene (Glasgowformula). Fibroblasts were removed mechanically by scraping culturesviewed with an Olympus OM-2 phase-contrast microscope and by dif
ferential trypsinization with 0.025% trypsin (w/v) in Versene.Cells at passages 25, 31, and 92 were shown to be free of Myco-
plasma using the Mycotect Kit (Bethesda Research Lab., U.K., Ltd.,Cambridge, England). In addition, cells at passage 20 were independentlytested for Mycoplasma using the Hoechst staining method by Dr. M. J.O'Hare (Ludwig Institute for Cancer Research, Surrey, England).
Growth Characteristics. Cells were plated into 35-mm Petri dishes(Nunc, Gioco-Europe, Ltd.). Each day, 3 dishes were washed 3 timeswith 0.01 M phosphate-buffered 0.9% NaCI solution and frozen at -20°C
for a maximum of 7 days before DNA assay. The DMA assay was amodification of the fluorimetrie assay of Karsten and Wollenberger (9).
Cells were thawed, and 5 ml Pronase (36 ¿ig/mlin 0.01 M phosphate-
buffered 0.9% NaCI solution) were added to each dish. Dishes wereincubated at 37°C for 15 min to remove cells from the plastic. The
remainder of the assay, before final incubation, was performed on ice.Cells were homogenized using a Polytron (Northern Media Supply, Ltd.,Brough, North Humberside, England). The final incubation mixture contained cell homogenate, Pronase (27.5 Mg/ml), and RNase (12.5 jig/ml)and was incubated at 37°Cfor 1 h. Ethidium bromide was added to a
final concentration of 5 ^g/ml, and the fluorescence of samples wasmeasured using a Perkin-Elmer fluorimeter (540 nm excitation; 580 nm
emission). Calf thymus DNA was used as a standard. (All reagents werepurchased from Sigma Chemical Co., Ltd., Poole, Dorset, England.)
Carcinoembryonic Antigen. Carcinoembryonic antigen radioimmu-noassay was kindly performed by Dr. Morag Ellison (Ludwig Institute forCancer Research, Surrey, England) (10).
Karyotypic Analysis. Cytogenetic studies were performed an exponentially growing cultures using standard Giemsa trypsin G-banding
techniques. Metaphase spreads were counted to determine chromosomenumber.
Electron Microscopy. For electron microscopy, cells were grown onglass coverslips and fixed in 3% glutaraldehyde in 0.1 M phosphate bufferat 4°Cfor 1 h. Cells were postfixed in osmium tetroxide for 1 h at 4°C,
dehydrated, and embedded in TAAB resin (TABB Laboratories, Reading,Berks, England). Thin sections were stained with uranyl acetate and leadcitrate and viewed with an AEI 801 microscope at 60 kV.
Induction of Tumors in Nude Mice. Cells were trypsinized, washed
In Hanks' balanced salt solution, and injected s.c. into both flanks of
nude mice (approximately 2 x 106 cells/site). Tumors were removed and
fixed in neutral buffered formalin for histological examination. Sectionswere stained with hematoxylin and eosin, periodic acid-Schiff, and Alcian
blue (pH 2.5). Some cells from the xenograft were reestablished in vitroto compare morphology with that of the established cell line.
Quantitation of Domes. Cells were grown in 35-mm dishes for dome
quantitation. After the experimental period, each dish was washedcarefully 3 times with 2 ml 0.1 M phosphate buffer at 37°C and fixed
with 3% glutaraldehyde in 0.1 M phosphate buffer for 30 min at roomtemperature. Dishes were washed with phosphate buffer and twice indistilled water, before staining for 1 h with 10% Giemsa (v/v) in 0.01 MSorensen's phosphate buffer. Dishes were washed and stored in distilled
water at 4°Cbefore counting. Total domes per dish were counted with
an Olympus OM-2 microscope.
RESULTS
Establishment of the HCA-7 Cell Line. After dissociated
tumor tissue was plated into culture flasks, some clumps of cellshad adhered to the plastic within 24 h. Both tumor cells andfibroblasts grew out of these clumps. The fibroblasts proliferatedrapidly in these cultures and had to be repeatedly removed untilthey became senescent. Cultures were initially only subculturedwhen tumor cell colonies became very confluent. Cells hadreached passage 10 after 1 year in vitro. After this time, the cellswere subcultured weekly and had a doubling time of approximately 36 h during the exponential growth phase (Chart 1).Culture medium (5 ml) exposed to a confluent 25-sq cm mono-
layer (passage 8) for 24 h contained carcinoembryonic antigen(91 ng/ml).
Chromosome numbers in HCA-7 cells ranged from 41 to 82
with a modal chromosome number of 43; 75% of cells had achromosome number in the range 43 to 46.
130-
120—
110-
100—
_ 90-£J°°-
I60-~ 50-
î^30-
20-
10-
I
1 8
Time (Days after plating)Chart 1. Growth curve of HCA-7 cells in Dulbecco's modification of Eagle's
medium with 10% fetal calf serum. Cells were plated into 35-mm dishes on Day 0.On each of the subsequent 8 days, the DMAcontent of 3 dishes was determined.Monolayers became confluent on Day 6 of this experiment. Points, means; oars,SE.
Morphology of HCA-7 Cells. HCA-7 cells grew as monolayers
with a typical epithelial morphology. The cells had large nucleiand distinct nucleoli (Fig. i A). Confluent monolayers formed"domes" or "hemicysts" (Fig. 1, B and C).
Transmission electron microscopic studies showed that thecells formed a polarized epithelial sheet (Fig. 2A). Cells werelinked by apical tight junctions (Fig. 2, ßto D); numerous des-mosomes and interdigitations were also observed (Fig. 2, B andC). Microvilli were present only at the apical surface and wereabsent from the basolateral membrane (Fig. 2A). These microvilliwere sparse but possessed a dense core of microfilaments whichextended into the apical cytoplasm (Fig. 2D).
Late-passage cells had a similar morphology except that manyrounded cells were present in monolayer cultures. Also, "cords"
of cells were observed both attached to the monolayer andfloating in the medium.
Morphology of Xenografts. Tumors were removed from thenude mice 3 to 4 months after injection of HCA-7 cells. Xeno
grafts were often lobular, and histological examination showedthem to be adenocarcinomas resembling the structure of theoriginal tumor (Fig. 3). Extracellular Alcian blue staining materialwas detected both in the original tumor and the xenograft. Noevidence for metastasis was obtained.
Cells mechanically removed from these xenografts could bereestablished in monolayer culture. The morphology of thesecells was indistinguishable from the parent cell line, and the cells
Fig.3. Section of a xenograft obtained by growing HCA-7 cells s.c&E, mice, x 130.
Chart 2. Kinetics of dome formation in monolayercultures of HCA-7 cells. Cellswere plated into 35-mm dishes in Dulbecco's modification of Eagle's medium with
10% fetal calf serum. Medium was changed daily. Each day, 3 dishes were fixedand stained with Giemsa for assessment of dome number. Because the domeswere not evenly distributed over the monolayer, the whole dish was counted fordome numbers. On Days 1, 3, 5, 9, and 13 of the experiment, DMA assay wasperformed on 3 dishes. Points, means (n = 3); oars, SE. • •,total number ofdomes/35-mm dish; • »,DNA (Mg/35-mmdish).
Table 1Totalnumber of domes in confluent monolayersof HCA-7cells after incubation for
24 h with the factors shownThe control medium for these experiments was Dulbecco's modification of
Eagle's medium with 10% fetal calf serum. The results are from 2 experiments.
No. of domes/35-mm dish
ControlOuabain(10-«M)Control
No serum450
±17s
77±7451
±43127 ±32
•Mean ±SE (n = 3).
proliferated at a similar rate after an initial lag phase.Dome Formation by HCA-7 Cells. Postconfluent monolayers
of cells produced "domes" or "hemicysts." To examine the
kinetics of dome formation, a time course analysis was performed on cells at passage 21 (Chart 2). No domes were observed before cells reached confluency. After the cells becameconfluent, the number of domes steadily increased to reach amaximum of 1300 domes/35-mm dish at 12 days. Cell number,
as determined by DNA assay, was unchanged after Day 3;therefore, changes in dome formation could not be attributed todifferences in cell density. However, this does not mean that thecultures were not proliferating, because many cells were shedfrom the monolayer during the course of this experiment.
Incubation of cells for 24 h in the presence of ouabain (10~e
M) was shown to decrease dome formation (Table 1). Removalof serum also inhibited dome formation (Table 1).
The ability of confluent monolayers to form domes declinedbetween passage 25 and 30, until domes were no longer observed in these cultures. For this reason, all doming experimentswere performed on cells at passage 21.
DISCUSSION
The cell line described in this report was derived from a primaryhuman adenocarcinoma of the colon. The cells retain some ofthe morphological and functional features of colonie epithelium.
These include ultrastructural features such as apical tight junctions, desmosomes, and microvilli. The microvilli have densecores of microfilaments which extend into the apical cytoplasmof the cell. These ultrastructural features are frequently observedin sections of well- and moderately-differentiated colorectal ad-
enocarcinomas (11) but are infrequently retained by cell linesderived from these tumors.
HCA-7 cells form a polarized epithelial sheet when grown on
tissue culture plastic. The cells transport vectorially, which results in the formation of "domes" or "hemicysts," as fluid accu
mulates in localized areas between the monolayer and the cultureflask. The morphology of these domes resembles those observed in cultures of lung adenocarcinoma cells, mammary adenocarcinoma cells, kidney epithelial cells (12, 13), and colonieadenoma cells (14). As found in these other systems, domeformation by HCA-7 cells is inhibited by ouabain, an inhibitor ofNa+-K+-ATPase (13). In addition, dome formation by HCA-7 cells
was shown to be serum dependent, a finding previously obtainedwith Madin Darby canine kidney cells and RAMA 25 cells (ratmammary adenocarcinoma cells) (13). "Spontaneous" dome for
mation is greater in HCA-7 monolayers than observed with either
Madin Darby canine kidney cells (13) or RAMA 25 cells (13),which show a dome formation of less than 10 domes/sq cm.
Few other colorectal adenocarcinoma cell lines retain thetransport functions of HCA-7 cells. CaCo-2 cells form domes in
confluent cultures (8), and T84 cells have been shown to transport fluid vectorially (15). Preliminary evidence shows that transport by T84 cells can be controlled by a variety of secretagogues(15). Recently, cells with the ability to form domes have beenisolated after sodium butyrate treatment of HT29 cells, which donot normally form domes (16).
HCA-7 cells slowly lose the ability to form domes between
passages 25 and 30, which may be due to selection for a celltype which is unable to form domes. Some evidence for thishypothesis has been obtained from an experiment using cellsfrom passage 12. These cells were seeded at very low densityinto culture flasks, and the morphology of the resulting colonieswas examined. In these cultures, colonies were present which,although they contained only a few hundred cells, were alreadyforming domes. The majority of colonies were devoid of domes.This does suggest that some heterogeneity exists in early passages of this cell line, and experiments are in progress to isolatethe different cell types.
In conclusion, the HCA-7 cell line of human colonie adenocar
cinoma cells retains some of the features of normal colonieepithelium. These cells will serve as a useful model system forstudying hormonal and other factors controlling transepithelialtransport and cell polarity.
ACKNOWLEDGMENTS
I am grateful to I. Bailey for technical assistance, to Dr. T. Krausz for help withthe chromosome analysis, and to V. Emons for preparation of cells for electronmicroscopy.
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BFig. 1. A, phase-contrast micrograph of an HCA-7 monolayer. x 130. B and C, phase-contrast micrographs of confluent monolayers of HCA-7 cells showing dome
formation B. focus on the cell monolayer. x 310. C, focus on the top of the domes, x 310.
cFig.2. Transmission electron micrographs of HCA-7 cells grown on plastic culture flasks. A. low-magnification electron micrograph to demonstrate cell shape and
polarity, x 14,490. B, higher-magnificationmicrograph of the border between 2 HCA-7cells showing apical tight junctions, interdigitations,and a desmosome. x 34,200.C, tight junction between HCA-7cells,also showing microfilamentsenteringa desmosome. x 72,000.D, microvilliat the apicalsurfaceof an HCA-7cellwith microfilamentsextending into the apical cytoplasm, x 68,000.
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