[CANCER RESEARCH 42, 1 142-1 146, March 1982]
0008-5472/82/0042-0000$02.OO
Endo-and EctocervicalHumanUterine EpithelialCells Distinguishedby
FibronectinProductionand Keratinizationin Culture'
K. Alitalo,
H. Halila, E. Vesterinen,
and A. Vaheri
Department of virology, University of Helsinki, Haartmaninkatu
3 (K. A., A. v.j, and Department of Obstetrics and Gynecology,
Helsinki University Central Hospital,
Haartmaninkatu
2 (H. H.,E. V.),00290Helsinki29, Finland
terectomyfor benignuterinetumorsat the Departmentof Obstetrics
ABSTRACT
and Gynecology,
Human ecto- and endocervical uterine epithelia grow in
culture as stratifying and nonstratifying cell layers, patterns
reflecting the differentiation of the corresponding cells in viva.
We found that both cultures synthesized and secreted fibro
nectin but very little other known glycoproteins
of basal lami
nae, laminin, and type IV collagen. Fibronectin production was
observed only in the lining marginal cells of the expanding
periphery of ectocervical
islands, but differentiating
and kera
Helsinki University
Central Hospital.
Only patients
with normal Papanicolaousmearswere accepted, and absenceof
atypiawas confirmedhistologically.Initiationand maintenanceof the
cultureswasessentiallyas describedpreviously(35).
Tissue strips were excised from the cervix, and the stroma was
separatedfromtheepitheliallayer.Eachstripwaswashedin PBS2and
cut into fragments
measuring
about 3 x 3 mm. To initiate explant
cultures, 8 to 10 fragmentswere seeded into 35-mm plastic Petri
dishes and incubatedin 3 ml of growth mediumcontainingHam's
MediumFl 0 (FlowLaboratories)supplementedwith 10% newborncalf
tinizing cells had lost fibronectin. Most cells in endocervical
serum and 100 @gof streptomycin, 100 IU of penicillin, 1.5 @zg
of
epithelial cultures grew as a monolayer and produced fibro
glutamine,and 5 @ig
of hydrocortisoneper ml. The cultures were
nectin. After stratification of the ectocervical cells, the cytoker
atm polypeptide pattern switched to the large-molecular-weight
type. In endocervical cultures, the low-molecular-weight keratin
polypeptides persisted. Fibronectin production thus appears to
be linked to the state of differentiation of the epithelial cells,
and fibronectin may function in the attachment of the basal
cells to underlying basal lamina matrix.
maintained in a humidified atmosphere with 5% CO2 at 37°, and the
medium was changed twice a week.
Subcultures were plated from actively growing cervical cell mono
INTRODUCTION
Epithelial cells in viva rest on basal lamina structures known
to promote epithelial differentiation (18, 19, 23) and to function
in the remodeling of tissue type-specific structures (36). In
culture conditions, the formation of basal lamina structure by
layersafter detachmentand disaggregationof the cells with 0.125%
trypsin and 0.01 5% EDTA (35). HeLa cells originating
The cells were labeled with 20 @Ci
of L-[5-3H]prolineand/or [23H)glycine in the presence of 50 @g
of both sodium ascorbate and of
,8-aminopropionitrile
fumarate per ml of regular medium or medium
lacking fetal calf serum but containing 0.05% bovine serum albumin.
Analysis of RadiolabeledProteins. Fibronectinwas isolatedfrom
the culturemediumby adsorptionto gelatin-agarosebeads(12). The
presenceof collagenousproteinwas testedby incubatingaliquotsof
culturemediumwith purifiedbacterialcollagenase(30 units/mI for 60
epithelia has been shown to require contact with mesenchymal
mm at 37°; Advance Biofactures,
tissues or with collagen (6, 15, 20, 22), but the biosynthetic
origin of defined basal lamina macromolecules, such as type
lv collagen, laminin, fibronectin, and proteoglycans, is known
only for few normal epithelia (3, 24, 27).
Growth conditions have been developed that retain differ
entiated characteristics of epithelial cells from human uterine
cervix. Ectocervial epithelial cells exhibit a stratified pattern in
culture with terminal differentiation and exfoliation, whereas
endocervical cells grow essentially as a single layer with
smooth overlapping of adjacent cells (35). We now report that
(29).
cervical epithelial cells grown in culture synthesize and secrete
fibronectin and produce minor amounts of the basal lamina
glycoprotein laminin and basement membrane collagen. In
immunofluorescence staining, intracellular fibronectin is do
tected only in the lining marginal cells of the growing ectocerv
ical cell islands, while all cells in endocervical cultures produce
fibronectin.
MATERIALS
AND METHODS
from a cervical
adenocarcinoma and locally established human skin fibroblasts were
subcultured twice a week.
For treatment
Lynbrook,
with pepsin (0.1 mg/mI
N. V.) or with buffer only
for 6 hr at 1 5°), the pH of
the samplewasset to 3 with aceticacid and neutralizedfor inhibition
of pepsin activity with sodium hydroxide and pepstatin A (1 g@g/ml;
Sigma Chemical Co., St. Louis, Mo.), after which the sample was
dialyzedagainstneutralpH bufferand H20at 4°and lyophilized.
Labeled proteins were precipitated from the culture medium with
ammonium sulfate (176 mg/mI) in the presence of protease inhibitors
(3) or with 10% TCA at O@,followedby washeswith cold 5% TCA,
95% ethanol,andabsolutediethylether.
Cell layerswere homogenizedin 0.1 N acetic acid containing1 @g
pepstatinA per ml, dialyzed,clarified, lyophilized,and digestedwith
pepsin in 0.5 N acetic acid as were the medium proteins (10). Samples
of cell layers and mediawere solubilizeddirectly in electrophoresis
sample buffer, and the polypeptides
were analyzed by polyacrylamide
gel electrophoresisin the presenceof sodiumdodecyl sulfate(25),
followedby proteinstainingof the gels (13) andfluorography(5). For
determinationof apparentmolecularweights,nonradioactivemarkers
(Pharmacia,
Uppsala, Sweden), radioactive
markers (Radiochemical
Centre,Amersham,United Kingdom),or collagenousmarkerscova
lentlyprestainedaccordingto Griffith(17) wereused.
ImmunologicalProcedures.Theaffinity-purifiedantibodiesspecific
to procollagen
types I and Ill and collagen types I, II, and IV (kind gift
Cell Cultures and RadioactiveLabeling. Cervicaltissue samples of Dr. R.Timpl)wereusedas describedpreviously(1, 3, 4). Antiserum
were obtainedfrom patientsof childbearingage who underwenthys against human plasma fibronectin was produced as described (37) and
1 Supported
by
the
Orion
Corporation
Research
Foundation,
the
Medical
Research Council of the Academy of Finland, the Association of the Finnish Ufe
Assurance Companies. and the Finnish Cultural Foundation.
Received May 27, 1981 ; accepted November 5, 1981.
1142
2 The
abbreviations
used:
are
PBS,
0.01
M sodium
phosphate,
0. 1 4 M sodium
chloride, pH 7.4; TCA, trichloroacetic acid.
CANCERRESEARCHVOL. 42
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Basement Membrane Proteins and Epithelial Differentiation
@
@
was preabsorbedwith fibronectinpurifiedfrom fetal calf serum.Anti
Iamininrabbitserumwas producedas described(2). Doubleantibody
zv)
11@
z
LA@
U)
c'Z@
0
Z
immunoprecipitation from culture,medium was as described (1).
0
For immunofluorescencemicroscopy,the cell cultures grown on
glass coverslips were rinsed 3 times with PBS and reacted with
antibodies either in the cold (4°)or after fixation with 3% paraformal
dehyde(20 mm)at roomtemperaturefor 20 to 30 mmfor stainingof
extracellularantigens.To exposeintracellularstructuresas well, the
cultureswere treatedwith acetone(10 mmat —20°)
or 0.05% of the
NonidetP.40 detergent(26). After 3 washesin PBS,the cell layers
were stained with commerciallyobtained (Wellcome,Beckenham,
UnitedKingdom)fluoresceinisothiocyanate-conjugated
goat anti-rab
@
@roaI(IV)J#
proa2(!V) .
@$•
bit y-globulin at the recommended dilutions.
When needed for the labeling of DNA (31), the benzimidazole deny
!
ative(Hoechst33258)wasaddedon the coverslipsat a concentration
of 0.5 gig/mIfor 5 mmbeforethe final washesin PBS,distilledwater,
andwet mounting.Themountingmediumbetweenthe invertedcover
slips and objective glass was 50% glycerol in 0.1 M Veronal-buffered
saline(O.15 PA,pH 8.6). Fluorescence was observed with a Leitz Dialux
20 fluorescencemicroscopeequippedwith epiillumination.
@J
1
HI
EN
@“EC
Fig. 1. Immunoprecipitation, polyacrylamide gel analysis, and autofluorogra
RESULTS
phy of fibronectin (a-FN) from endocervical (EN) and ectocervical (EC) epithelial
cell cultures. Lanes NRS, control precipitation with normal rabbit serum; Lanes
Radioactive polypeptides were closely similar in media of
both ectocervical and endocervical cultures freed of tissue
explantswhenlabeledfor either8 or 24 hr in thepresenceor
absence of fetal calf serum or ascorbate. All major TCA-insol
uble radiolabeled polypeptides seen in 5% polyacrylamide gels
M, ammoniumsulfate-precipitated
proteinsof the culturemediaof endo-and
ectocervical cells; Lane HT, ammonium sulfate-precipitated proteins of the HT
1080 human tumor cells. The polypeptides of the latter identify the positions of
migration of fibronectin and procollagen type IV proal(lV) and proa2(IV) chains
(3).
wereprecipitatedby 30% ammoniumsulfate,differedfrom epithelial cells. The faint disulfide-linked polypeptide bands
those of human fibroblasts, and had apparent molecular
migrating in reducing conditions with a mobility similar to that
weights(in theorderof decreasingradioactivityincorporated of procollagen type IV chains were degraded by collagenase
from 3H-glycine) of 220,000, 130,000, 150,000 and 100,000,
in reducing conditions (Chart 1). In addition, 2 weakly labeled
bands were seen at MW. 185,000 and 200,000 (molecular
digestion (Chart 1, Lanes A and B). In contrast, in control
samples from human skin fibroblast cultures a major part of
[3H]glycine- and [3H]proline-derived radioactivity was con
weightdeterminations
basedonglobularproteinsmarkers).In
verted dialyzable in similar digestion conditions,
nonreducing conditions, all these polypeptides migrated in
disulfide-linked higher-molecular-weight forms (not shown).
The polypeptide at M.W. 220,000 was shown to be fibronec
tin by immunoprecipitation (Fig. 1) and by its affinity to gelatin
agarose. No radioactive polypeptides comigrating with those
of laminin were seen in the culture media of the cervical
capacity of collagenase treatment to digest collagens of fibro
blasts. Comparison of the polypeptides in the above culture
media indicated that the secreted polypeptides could probably
not be derived from cells other than the cervical epithelial cells.
Only a small fraction (less than 1%) of radioactivity in medium
proteins resisted pepsin digestion, and analysis of radioactive
polypeptides in this material after long exposure times in au
toradiography indicated that there was no or only negligible
amounts of radioactivity in either the culture media or in the
cell extracts comigrating with standard chains of collagen types
A
FN
130000
front
showing the
I, Ill, V, or pepsin-resistant fragments of type IV collagen. The
fact that collagen types I and Ill were not detected confirmed
that the cultures were free of fibroblasts. Since type IV collagen
may give rise to a heterogenous population of pepsin-resistant
fragments (1 1, 33) that are difficult to standardize, the char
acterization was complemented with analysis of collagenase
digested material from cell layers. Again, no indication for the
presence of substantial amounts of type IV collagen was ob
tamed. Analysis of radioactive polypeptides of HeLa cervical
carcinoma cells showed synthesis and secretion of small
amounts of type IV procollagen (Fig. 2) and laminin but no
fibronectin (data not shown).
Chart 1. Collagenous and noncollagenous polypeptides in culture media of
ectocervical
cells.Thelabeledmediawereincubated
withbuffer(A)withcolla
In order to compare the state of differentiation of the epithelial
genase (B) for 60 mm at 37°.Medium proteins were precipitated with ammonium
cell
populations from endo- and ectocervices, their cytokeratin
sulfateandsubjected
to electrophoresis,
autofluorography,
andscanning
den
polypeptides synthesized during labeling experiments were
sitometry. Three minor radioactive peaks are abolished with bacterial collagen
ase. Two of thesehavethe mobilityof type IV procollagen[proal(IV), compared from the acetic acid-insoluble residues. The data,
prOa2(lV)], while the third one (X) has a mobility intermediate between type I
shown in Fig. 3, indicated that high-molecular-weight polypep
collagen al(l) and a2(I) chains. FN shows the mobility of cellular fibronectin, and
130,000 is the apparent molecular weight of another major polypeptide.
tides typical of cytokeratin of keratinizing epithelial cells (14)
‘@AAJ
MARCH
1982
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1143
K. Alitala et a!.
collagenase -
-
+
cells. Antibodies to procollagen types I and Ill showed no
staining of the epithelial or carcinoma cells, and only a weak
intracellular staining was seen with antibodies to laminin (Fig.
4, E and F) and type IV collagen (not shown). Staining for DNA
r
demonstrated mitoses in both the endocervical and the strati
fied ectocervical cell layers used for immunofluorescence stud
ies.
:@
DISCUSSION
1@
@
culture
media
cell
layers
Fig.2. Sodiumdodecylsulfate-polyacrylamide
gel analysisandautoradlog
raphy of [3H]glycine and proline-labeled proteins in culture media and cell layers
of HeLa cells. Four polypeptides in culture media are digested by bacterial
Two kinds of epithelial cell populations can be grown from
human cervical tissues. Ectocervical cells can be cultured from
explants of stratified squamous epithelium derived from the
uterine ectocervix, whereas endocervical cell cultures are ob
tamed from the single layer of the columnar secretory epithe
hum lining the cervical canal (35). The 2 epithelial cell popula
tions obtained are morphologically distinct and exhibit growth
properties characteristic of their origin. When considering the
results from the HeLa cervical carcinoma cells, it should be
noted that this cell line has been grown in vitro for extensive
periods
of time and may have undergone
selection
and
changed during that time.
collagenase.
Twoofthecollagenouspolypeptidescomigratewiththoseofmarker
The present study was undertaken to approach the biosyn
type IV procollagen [proal(1 V), proa2(1 v)]. Note that, in cell layers, no major
collagenouspolypeptidesaredetected.
thetic origin of the basal lamina proteins in the basement
membrane underlying the cervical epithelial cells and to further
characterize the differentiation of the 2 distinct normal cell
populations. The results showed synthesis and secretion of
200
fibronectin by all endocervical cells in culture, whereas only
some cells in the stratified layers of ectocervical cells were
found to produce fibronectin. Small amounts of laminin and
100
only minor amounts of basement membrane collagen were
produced apparently by all cells in both cultures.
c@
691111.
The failure to detect substantial production of glycoproteins
confined to basal laminae, laminin, and type IV collagen by
human cervical epithelial cells is not totally unexpected, since
46
@O
previous morphological and autoradiographic studies with hu
man keratinocytes have indicated that, for deposition of a basal
lamina structure in tissue culture, contact of the epithelial cells
with mesenchymal structures is required. Moreover, many mes
30
enchyme-derived cells lose their differentiated characteristics
upon isolation from their in viva connective tissue matrix (8,
KC ED EN EC
KC ED EN EC
21 ). Thus, the results presented may not fully reflect the
synthesis of the epithelial connective tissue matrix in viva.
.-@
@
@
@
@
@
I..
3
CBB
Mostcells,evenepithelialones,makecollagenin culture
(16, 28). The selective amino acid labeling with glycine and
docervical(EN)andectocervical(EC)celllayers.Two-week-old
growingprimary
praline and enzymatic tests for collagenous polypeptides
cultures were labeled with glycine and proline for 24 hr. Gels were first stained
among radiolabeled molecules indicated that only minor quan
with Coomassie blue (CBB) and then subjected to autofluorography (3H).Lanes
KCandEDshowcorrespondinganalysisof primaryculturesof humanepidermal tities of collagen were produced by the cervical epithelial cells.
keratinocytes and epidermal tissue, respectively. Note that, in the samples from
Laminin is located to the lamina rara of skin basal lamina and
endocervical cells, no polypeptldes are seen in the MW. 48,000 to 68,000
region, where mature keratln polypeptides typical of stratum comeum appear
is thought to mediate the adhesion of epithelial cells to collagen
Fig. 3. Polyacrylamide gel analysis of acetic acid-insoluble residues of en
(14). Arrows, origin of the electrophoretic migration.
type IV (32). Although
many epithelial cells synthesize
and
secrete laminin, it is also produced by, e.g. , the fibroblastic
were labeled in cultures of ectocervical cells but were not
found in endocervical cell layers, which contained only low
molecular-weight cytokeratin polypeptides.
Immunofluorescence studies of the cell layers confirmed the
results of metabolic labeling. Fibronectin was stained intracel
lularly in the ectocervical cells. However, fibronectin was found
only in cells close to the borders of epithelial cell islands (Fig.
3T3 cells (34). By virtue of its large size, laminin could span
the entire basal lamina, and it has not been established whether
mesenchymal tissue contributes to the Iaminin in basal lamina.
The biosynthetic origin of the laminin found in the lamina rara
can thus not be inferred from the present results.
It seems possible that only cells corresponding to the state
4, A and B) and at places pericellularly as faint matrix fibers
under the islands. Fibronectin staining was also seen in all
of differentiation of the basal cells facing the basement mem
brane show fibronectin production in vitro. In this respect, it is
interesting to note that Couchman et a!. (9) have localized
endocervical cells (Fig. 4, C and 0) but not in HeLa carcinoma
fibronectin to the peripheral lamina lucida layer of basal lamina
1144
CANCER RESEARCH VOL. 42
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Basement Membrane Proteins and Epithelial Differentiation
of rodentskin.Quaroniet a!. (30)havepostulateda rolefor
fibronectin in the adhesion of intestinal epithelial cells to basal
Iaminae. From the studies of Carlsson et a!. (7), it appears that
laminin and fibronectin may have distinct roles in mediating the
adhesion of resting and migrating/proliferating epithelial cells
to extracellular substrata.
Cancerof the cervixoriginatesin the stratifiedepithelium,
usually very close to its junction with the columnar epithelium
of thecervicalcanal,a sitewherealsohighmitoticactivityand
metaplasia occur. This fact makes it easy to study the prema
lignant changes that precede the appearance of cervical car
Worst, P. K. PA.Keratinization and structural organization in epidermal cell
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17. GrIffith, J. P. Immediatevisualization of proteins in dodecyl sulfate-polyacryl
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18. Grobstein, C. Mechanisms of organogenetic tissue interaction. Nati. Cancer
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19. Hay, E. D. Role of basement membranes in development and differentiation.
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pp. 1 19—1
36. New York: Academic Press, Inc., 1978.
20. Hay,E. D.,andDodson,J. W. Secretionof collagenby cornealepithelium.
cinoma.Ourcurrentstudiesare directedinto establishinga
graded series of epithellal cell cultures from dysplastic and
carcinoma
tissues
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the
cervix
in
order
to
study
their
21.
Biochem.,105:63-74, 1980.
produc
tion of basal lamina proteins and hydrolytic enzymes, plasmin
22. Hintner, H., Frisch, 0., Foidart, J.-M., Stingl, G., schuier, G., and Katz, S.
I. Expression of basement membrane zone antigens at the dermoepibolic
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ogenactivator,
andcollagenases.
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@
@:
K. Alitala et a!.
4A
,@
a,
*
40
4
.@
@p'
‘I
@1
4E
Fig. 4. Immunofluorescence for fibronectin (A and C) and phase-contrast microscopy (B and D) of ectocervical (A and B) and endocervical (C and 0) cell layers.
For staining, the cells were fixed with paraformaldehyde and treated with non-ionic detergent as described in Materials and Methods.“
Laminin was similarly stained
(EandF).Noteslightpericellularstainingof laminin.
1146
CANCERRESEARCHVOL. 42
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Endo- and Ectocervical Human Uterine Epithelial Cells
Distinguished by Fibronectin Production and Keratinization in
Culture
K. Alitalo, H. Halila, E. Vesterinen, et al.
Cancer Res 1982;42:1142-1146.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1982 American Association for Cancer Research.