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Regular Articles |
From the Department of Pathology,*
Leiden University
Medical Center, Leiden; and the Department of Obstetrics and
Gynecology,
Academic Hospital Utrecht,
Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-catenin is dramatically decreased in
high-grade SILs; 4) the level of ß-catenin is decreased during
progression of SILs, with plakoglobin suggestively becoming the
predominant catenin mediating connection of cadherins to the
cytoskeleton; 5) the assembly of desmosomes is affected during
progression of SILs and is accompanied by a dramatically decreased
expression for desmogleins and desmoplakins (I, II); and 6)
expression of differentiation markers (involucrin, CK13) in
high-grade SILs seems to be controlled by P-cadherin as opposed to
E-cadherin in the normal tissue counterpart. We conclude that during
development of cervical lesions substantial (both quantitative and
qualitative) changes occur in cell-cell junctions, making the
interactions of cells in lesions dissimilar from those of reserve
cells, basal cells, or cells of immature squamous
metaplasia, despite existing morphological similarity between
all of these cell types and cells of high-grade lesions.
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Introduction |
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In tumors, however, one deals with the final result of a multistage selection process of various cell geno- and phenotypes. The final tumor cell population usually consists of a number of subpopulations that originate from consecutive changes during tumor progression, based on immunohistochemical and genetic studies.9, 10 It is of considerable interest to pinpoint the early changes in intercellular adhesion, which are associated with the development of hyperplastic and neoplastic cell phenotypes.
The multistage nature of carcinogenesis in cervical epithelium makes it possible to follow the events for a number of intermediate stages leading from a disturbance in proliferation and differentiation of squamous metaplasia, via low- and high-grade squamous intraepithelial lesions (SIL), to carcinoma.11, 12 SILs are thought to arise from the "transformation zone," the squamocolumnar junction between ecto- and endocervical epithelium. Proliferation of the reserve cells and their differentiation via immature and mature squamous metaplasia into normal squamous epithelium serve the replacement of endocervical epithelium by ectocervical epithelium.12, 13 If the differentiation stimuli are blocked, these reserve cells may proliferate into an atypical immature squamous metaplastic epithelium or SIL.12 Suggestively originating from the reserve cells, SILs are believed to inherit some differentiation features of their progenitors. SILs express mostly cytokeratins 6, 8, 17, and 18; in this they resemble reserve cell phenotype/differentiation, although they also express some keratins (5, 14, 19) common to both the reserve cells and the basal cells of normal squamous epithelium.14
In this study, we have analyzed immunohistochemically whether the intercellular adhesions typical for the reserve and basal cells are reproduced in SILs cells during the development and progression of these lesions. We have also analyzed biochemically the composition of intercellular junctions and whether the different phenotypes are controlled by different cadherins in primary cultures of cervical keratinocytes from normal tissue and lesions. Both immunohistochemical and cell culture experiments have demonstrated that abnormal proliferation in cervical tissue and altered cell differentiation are accompanied by substantial changes in junctional proteins with respect to the type of cadherins mediating cell adhesion, the composition of catenins in cadherin junctions, and the composition of desmosomes. Despite certain similarities of SIL cells with respect to the organization of adhesions observed in reserve cells, the major changes occur in SILs with respect to the number and composition of adherens junctions and desmosomes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue samples (biopsies and surgical specimens of lesions of uterine cervix) were collected from the archive and tissue bank at the Department of Pathology (Leiden University Medical Center). Tissues were routinely fixed in 4% formalin and embedded in paraffin. Sections, subsequent to those used for immunostaining, were stained with hematoxylin/eosin. A total of 30 specimens were analyzed, containing normal squamous epithelium and metaplastic epithelia (immature squamous metaplasia and mature squamous metaplasia), and low- and high-grade SILs. Because most of the sections also contained normal squamous epithelium and sometimes contained metaplasia along with SILs, the numbers for each tissue/lesion type were normal cervical epithelium, n = 32; immature squamous metaplasia, n = 8; mature squamous metaplasia, n = 7; low-grade SILs, n = 8, and high-grade SILs, n = 9. In addition, several cases of reserve cell hyperplasia (RCH) were studied (n = 4).
Antibodies
Antibodies against E-cadherin (HECD-1), P-cadherin (NCC-CAD-299),
Ki-67 (MIB1), and cytokeratin 13 (1C7) were from Thamer Diagnostica BV
(Uithoorn, the Netherlands). Monoclonal antibodies (mAbs) against
E-cadherin (clone 36), P-cadherin (clone 56), -catenin (clone 5),
ß-catenin (clone 14), 
-catenin/plakoglobin (clone 15), and pp120
(clone 98) were all from Transduction Laboratories (Lexington, KY). mAb
against desmoplakins I+II (clone 115F) was kindly provided by Prof. D.
Garrod (University of Manchester, Manchester, England).
Anti-desmoglein antibody (clone DG3.10, recognizing desmoglein
"band 3" polypeptide of the desmosomal complex) was from Progen
(Heidelberg, Germany). Anti-involucrin mAb (clone SY5) was from Sigma
Immunochemicals (St. Louis, MO). mAb aCAT-7A4 to -catenin was from
Zymed Laboratories (San Francisco, CA). mAb HECD-1 was used for both
immunohistochemistry and blocking studies, mAb NCC-CAD-299 was used in
blocking studies only, mAb anti--catenin (no. 5) was used for
immunohistochemistry only. mAbs against E-cadherin (no. 36) and
-catenin (aCAT-7A4) were used for immunoblotting experiments.
Immunohistochemical Staining of Tissue Sections
From paraffin-embedded tissues 4-µm sections were cut, mounted on glass pretreated with 2% 3-aminopropyltriethoxysilane (Sigma), and air-dried overnight at 37°C. Sections were deparafinized in xylene, hydrated in a graded alcohol series, fixed in methanol (5 minutes room temperature, RT), incubated in 0.3% H2O2 in methanol (20 minutes), and subsequently washed with distilled water. To retrieve the antigen's reactivity, sections were pretreated before staining as described earlier.15 Briefly, sections were incubated for 10 minutes in boiling citrate buffer (0.01 mol/L citrate/0.01 mol/L Na-citrate, pH 6.0) or citrate-detergent buffer (0.01 mol/L citrate/0.01 mol/L Na-citrate, pH 6.0, 0.05% Dish Clean; Bosman Chemie, Heijningen, the Netherlands), cooled down in buffer for 2 hours, and subsequently incubated overnight at RT with the first antibody. Anti-involucrin mAb was used on nontreated sections. Binding of the mAb was detected using an anti-mouse ABC system (Dako, Glostrup, Denmark). Histology for all sections was analyzed by two pathologists independently (H.v.K., G.J.F.). Staining within the lesion, where possible, was related to the staining of normal tissue (ecto- and endocervical epithelium) within the same section.
Cells and Culture
For biochemical analysis of the cell-cell junctions in cervical keratinocytes we used primary cultures of cells from tissue samples of normal and abnormal cervical tissues, isolated as previously described (Van Dorst et al, manuscript submitted for publication, and Ref. 16 ). Cells were cultured until 80% confluence in keratinocyte-SFM culture medium supplemented with human recombinant epidermal growth factor and bovine pituitary extract (Gibco BRL, Paisley, Scotland). Before the experiments, medium was replaced for 24 hours with Dulbecco's minimum essential medium (DMEM)/HAM's F12 1:1 (BioWhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum. Cadherin-mediated adhesions were disrupted by culturing the keratinocytes in the presence of function-blocking antibodies for E-cadherin (HECD-1, IgG1) and P-cadherin (NCC-CAD-299, IgG1) at a concentration of 20 µg/ml for 24 hours in DMEM/HAM's F12 medium. Three cell cultures/lines representing normal cervical keratinocytes and two different stages of SIL development were used. Normal cervical keratinocytes (NCKs) were expanded immediately after isolation to yield a large quantity of cells (= passage 1), which were frozen in liquid nitrogen. For experiments the NCK cells were thawed and cultured for one extra passage (= passage 2). Two established cell cultures, one representing a low-grade SIL (612) and one a high-grade SIL (612.6E) were derived from histologically characterized biopsy specimens, as was previously described (Van Dorst et al, manuscript submitted for publication). All three cell types were able to reproduce the morphology of the respective tissue of origin in the presence of calcium in culture medium, as described (Van Dorst et al, manuscript submitted for publication).
Immunoprecipitation and Immunoblotting
Cross-linking of the molecules of the cadherin-catenin complex was performed after the method of Hinck et al,17 using dithio-(bis)-succinimidylpropionate (DSP), a cleavable bifunctional linker (Pierce Chemical Co., Rockford, IL). Detergent extraction of cells (1% Triton X100 or 25 mmol/L CHAPS ([3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate]),18 nonextracted cell fraction in 1% sodium dodecyl sulfate, immunoprecipitation, and immunoblotting were as previously described.19, 20 In all experiments, an equal amount of protein was analyzed per cell type.
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E-Cadherin
In normal endocervical (glandular) epithelium E-cadherin was
present at the lateral domains of cells, with no detectable
intracellular presence, in contrast to reserve cells, which were
negative (Figure 1
, top). Expression of
E-cadherin in reserve cells was noted only in relation to the
development of a reserve cell metaplasia (Figure 1
, bottom). Actively
proliferating cells of immature squamous metaplasia were marked by a
dramatic increase in intracellular staining for E-cadherin (as compared
to glandular epithelium), although the presence of a large amount of
E-cadherin at cell-cell boundaries was still clearly evident (Figure 2)
. A slight increase in intercellular
E-cadherin was observed in areas where immature squamous metaplasia
cells undergo maturation, as shown by involucrin expression. No
substantial differences were found with respect to E-cadherin
expression and localization in mature squamous metaplasia (not shown),
as compared to normal squamous epithelium (Figure 3)
. A decreased presence of E-cadherin at
cell-cell boundaries was noted for cells of the basal layer of normal
squamous epithelium, with a subsequent increase in parabasal and,
especially, suprabasal layers (Figure 3)
. It should be noted that in
lower layers of normal squamous epithelium, in addition to lateral
staining, a substantial intracellular staining for E-cadherin was
observed. This intracellular staining was practically absent from
columnar epithelium (Figure 1
, top) and most likely reflects active
rearrangement of adherens junctions in lower layers of normal squamous
epithelium.
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.
Two SILs (2/8) showed a dramatic decrease in E-cadherin in the
basal/abnormally differentiated layers, with the molecule reappearing
in the upper, more differentiated layers, practically at the same
levels as in other low-grade SILs (Figure 4)
. These latter lesions were difficult
to place strictly in either the low- or high-grade SIL category.
According to morphology and degree of proliferation, as assessed by
Ki-67 expression and the frequency of mitosis, they were grouped as
low-grade SIL, but changes observed in the cadherins and catenins
affected approximately half of the thickness of the squamous tissue
(not shown).
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. P-Cadherin
P-cadherin was practically absent from endocervical (simple)
epithelium, in contrast to reserve cells, which were positive for
P-cadherin (Figure 1)
. We also noted that in areas of an enriched
presence of reserve cells, the upper layers of glandular epithelium
expressed low levels of P-cadherin, which disappeared in glandular
epithelium that was just slightly distant from this site (not shown).
Cells of reserve cell hyperplasia retained expression of P-cadherin
(Figure 1)
along with de novo expressed E-cadherin.
Development of immature squamous metaplasia was accompanied by a
dramatic increase in P-cadherin expression, present both at the
cell-cell boundaries and in the cytoplasm (Figure 2)
. During maturation
of immature squamous metaplasia, the expression of P-cadherin remained,
being present in morphologically normal (similar to normal cervical
epithelium) basal, parabasal, and suprabasal cells of mature squamous
metaplasia (not shown). Only further maturation of mature squamous
metaplasia to normal cervical epithelium resulted in the disappearance
of P-cadherin from all cell layers except for the basal layer (not
shown). In normal cervical epithelium, P-cadherin was present only in
cells of the basal layer, with some traces of expression in parabasal
cells (Figure 3)
, in accordance with patterns observed for normal skin
keratinocytes induced to differentiate,24
and in other
squamous epithelia.25
In low-grade SILs, an up-regulation of P-cadherin was observed in intermediate and upper layers (not shown). Staining was predominantly located at the cell-cell boundaries.
In high-grade SILs, P-cadherin was slightly decreased in comparison to
cells of immature squamous metaplasia and even low-grade SILs. However,
in contrast to the pattern observed for E-cadherin, P-cadherin was
present at cell-cell boundaries, with its expression increasing from
the lower to the upper layers of high-grade SIL tissue (Figure 3)
.
These observations suggest that P-cadherin actively participates in
cell-cell adhesion in SIL lesions, especially in those of a high grade.
-Catenin
Expression of -catenin, a molecule that mediates interactions
of classic cadherins with the actin cytoskeleton,26, 27
was
high in immature squamous metaplasia (Figure 2)
, during proliferation
into mature squamous metaplasia (not shown), as well as in normal
cervical epithelium (Figure 3)
.
In low-grade SILs, no dramatic decrease in -catenin expression was
observed. In general, at the levels of expression and subcellular
localization of -catenin (at cell-cell boundaries, but also slightly
in the cytoplasm), as observed in basal layer cells, most low-grade
SILs resembled basal cells of normal cervical epithelium (Figure 3)
. In
two low-grade SILs (with grossly disturbed E-cadherin expression), a
very strong decrease in -catenin staining was observed in the lower
basal layers (Figure 4)
. Remarkably, no reexpression of -catenin in
the upper layers of these lesions, as was seen for E-cadherin, was
observed.
For high-grade SILs, a strong reduction of -catenin expression was
observed in the majority (8/9) of the lesions (Figure 3)
. Only in the
very upper layers could the presence of some -catenin molecules be
detected at cell-cell boundaries.
ß-Catenin and Plakoglobin (-Catenin)
Both ß-catenin and plakoglobin can participate in the formation
of the cadherin connection to -catenin,26, 28
whereas
plakoglobin is additionally involved in desmosome formation by the
mediation of the connection of desmoglein to desmoplakins.29, 30
As compared to normal endocervical epithelium, no
quantitative change and/or relocation of ß-catenin was observed
during the development of immature squamous metaplasia (Figure 2)
. For
plakoglobin, however, an increased expression was observed in immature
squamous metaplasia cells, including enhanced cytoplasmic staining
(Figure 2)
. Mature squamous metaplasia showed a high level of
plakoglobin, both at the cell membranes and in the cytoplasm of less
differentiated cells (not shown). No decrease in or relocation of
either ß-catenin or plakoglobin was obvious during maturation of
mature squamous metaplasia into normal cervical epithelium (Figure 3)
.
In low-grade SILs both ß-catenin and plakoglobin were present, and
the level of expression, with an enhanced cytoplasmic staining in basal
layers cells, was similar to expression observed in basal layer cells
of normal cervical epithelium (Figure 3)
. In one SIL, a practically
complete reduction of ß-catenin expression was observed, accompanied
by a strong reduction in E-cadherin and -catenin, but no changes in
plakoglobin expression levels were observed (Figure 4)
. The other case
with a reduction of E-cadherin and -catenin described earlier,
expressed levels of ß-catenin similar to those observed in other
low-grade SILs.
In high-grade SILs a relative reduction in the level of ß-catenin
expression was observed (9/9), whereas the presence of ß-catenin at
cell-cell boundaries was also decreased (Figure 3)
, as has also been
observed for various other types of epithelial tumors.31
Some reduction of plakoglobin expression was seen in the majority of
high-grade SILs (6/9), but not as much as was observed for ß-catenin
(Figure 3)
.
pp120
pp120, a catenin-like molecule, is a tyrosine kinase substrate implicated in receptor-ligand-induced signaling by growth factors and in transformation by src.32 pp120 colocalizes with E-, P-, or N-cadherin32 and binds to the juxtamembrane domain of the E-cadherin cytoplasmic tail,33 not competing for binding with ß-catenin or plakoglobin.
In normal endocervical epithelium, pp120 was present at the lateral
domains of cells, with no pronounced intracellular presence (not
shown). In immature squamous metaplasia, both a high level of
membranous as well as cytoplasmic staining was observed (Figure 2)
,
suggesting a certain increase during cell proliferation in metaplasia.
In mature squamous metaplasia a strong intercellular staining was
observed (not shown) that was similar to normal cervical epithelium
(Figure 3)
.
In low-grade SILs, a slight increase in expression was observed for
pp120 that was also seen in high-grade SILs (Figures 3)
. In all cases
of SIL, a normal membranous staining with no increased cytoplasmic
staining was observed, as was observed for other catenins, as far as
can be judged by immunohistochemistry.
Desmosomes (Desmocollins and Desmogleins)
Desmosomes provide the cells with binding domains for intermediate filaments of the cytokeratin network and are thus required for tissue organization.34 Desmosomes are formed by two different types of transmembrane proteins belonging to the cadherin family of proteins, desmogleins and desmocollins, associating with various cytoplasmic proteins, like desmoplakins and plakoglobin, and some other molecules.35 Three of the desmosomal proteins, desmocollin, desmoglein, and desmoplakin, show different protein forms, either derived from alternative splicing or coded by different genes, which are either tissue specifically or ubiquitously expressed.36
In immature squamous metaplasia, an increased intracellular staining
for desmoplakin and an absolute absence of desmoglein were observed
(Figure 5)
. During maturation of mature
squamous metaplasia (not shown) into normal cervical epithelium,
formation of desmosomes resembled the pattern observed in normal
cervical epithelium. Both desmoglein as well as desmoplakins (I and II)
showed hardly any expression in the basal layer of normal cervical
epithelium, whereas both suprabasal cells and differentiating cell
layers showed a membranous staining, except for the superficial layers,
which were largely negative (Figure 5)
.
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. A punctate staining for desmoplakins as observed
in some high-grade lesions probably suggests the presence of
"half-desmosomes" on the cell surface. All changes were
consistently present in all SILs analyzed. Biochemical Analysis of the Composition of Cell-Cell Junctions in Established Cell Cultures Representing Various Stages of Cervical Lesion Development
Changes in cadherin and catenin expression patterns during SIL development and progression suggest that the roles of E- and P-cadherin, as well as their involvement in adherens junction formation, are changing. The results also suggest that the composition of cadherin adhesion complexes may differ greatly between normal and neoplastic keratinocytes. To analyze biochemically the components of cell-cell junctions present in cervical keratinocytes, we used three different cell types: NCKs and two cell cultures, one representing a low-grade (612) and one a high-grade (612.6E) SIL. The latter two were derived from biopsy specimens from patients as previously described (Van Dorst et al, manuscript submitted for publication).
To analyze whether culturing the cells did induce squamous/terminal
differentiation to the degree observed in situ, total cell
lysates of cells cultured in the presence of normal (1.8 mmol/L)
Ca2+ concentration were probed for involucrin and
cytokeratin 13 (Figure 6A)
. Both markers
were expressed at the highest level in NCKs, at a decreased level in
612, and at an even lower level in 612.6E cells, confirming that, at
least with respect to expression of these differentiation markers,
cells were similar to the lesions in situ. In another study
we have also analyzed the expression pattern of other cytokeratin
chains in these cells and concluded from the expression of both
squamous-specific and differentiation-specific cytokeratins that the
various cells resembled the respective tissues in situ (Van
Dorst et al, manuscript submitted for publication).
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-catenin, ß-catenin, pp120), did not show any remarkable
differences between the cell lines when total cell lysates were
analyzed (Figure 6B)
, with the exception of plakoglobin, that was
clearly increased in 612.6E cells.
Detergent extractability (25 mmol/L CHAPS) analysis showed that with an
increasing grade of SIL, more and more -catenin, ß-catenin (Figure 6C)
, E-cadherin, and pp120 (not shown) was detectable in the soluble
fraction. In contrast, both P-cadherin (Figure 6D)
and plakoglobin
(Figure 6C)
showed a decreased solubility with increasing grade of SIL.
This suggested that an increasing fraction of P-cadherin was involved
in mediating cell-cell adhesions in high-grade SIL cells, which was
possibly connected to the actin cytoskeleton via plakoglobin, which
also showed a decreased solubility (Figure 6C)
. In contrast to some
observations published earlier,37, 38
extraction with
Triton X100 extracted all cadherins from cervical keratinocytes. The
pellets obtained did not contain traces of E-cadherin, as was tested in
immunoblotting (not shown).
To investigate the association of E-cadherin molecules with catenins in
these SIL-derived cells, the adherens junction complexes were
cross-linked by the use of a bifunctional reagent (as earlier described
by Hinck et al).17
Immunoprecipitated E-cadherin complexes
were analyzed for the presence of catenins in immunoblotting (Figure 6E)
. In E-cadherin immunoprecipitates a tremendous decrease was
detected for -catenin, ß-catenin, and plakoglobin associated with
E-cadherin, increasing with grade of SIL. Small differences between
results obtained with the cultured cells and our immunohistochemical
data may be related to the short time of the differentiation.
Regulatory Roles of Cadherins in the Expression of the Markers for Terminal and Squamous Differentiation
To further analyze the roles of the cadherin junctions in cultured SIL cells, function-blocking antibodies for E-cadherin and P-cadherin were used. Cells were incubated for 24 hours in the absence or presence of function-blocking antibodies, after which cell lysates were analyzed for expression of the terminal differentiation marker involucrin.
Morphologically, the strongest effects were seen when the cells from
both low- and high-grade SILs were cultured in the presence of
anti-E-cadherin mAb or the mix of anti-E- and P-cadherin antibodies
(Figure 7)
. For the mix, the effect on
cell morphology was more pronounced than that for either antibody
alone. Cells no longer formed tight cell-cell adhesions, as observed
during culturing without antibodies. Instead, loose clumps of cells
were seen, floating on top of each other, with hardly any cell-cell
contact. The effect of antibodies on low-grade SIL cells (612) was more
pronounced than on high-grade SIL cells (612.E6).
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Cadherins, the transmembrane components of adherens junctions, mediate via homotypic interactions and binding to the cytoplasmic catenin molecules the interaction with the actin cytoskeleton, and in this way they play an important role as morphoregulatory molecules.2, 4, 39, 40 In squamous epithelia, E-cadherin is one of the major cell adhesion molecules defining the architecture and differentiation of keratinocytes.41, 42 Evidence is accumulating that E-cadherin may perform as a tumor-suppressor and invasion-suppressor molecule7, 8 and is functionally disturbed during carcinogenesis of various epithelial tissues, including cervical lesions and carcinomas.43, 44 In addition to changes in cell-adhesion molecules like cadherins,43-45 changes in catenins31, 46 have been observed in squamous epithelium, but not in great detail for cervical epithelium.
In this study we compared the expression pattern and organization of various molecules comprising the adherens junctions and desmosomes, as expressed in normal cervical epithelium, during transdifferentiation and in SIL development and progression. In this way we wanted to establish whether SILs are derived from reserve cells, and therefore whether a reserve cell phenotype (with respect to intercellular adhesion) is reproduced in SILs, similar to that reported for cytokeratins.14
To some extent the adherens junctions of SILs do reproduce
intercellular junctions of the reserve cell. A clear decrease in
E-cadherin and an increase in P-cadherin observed during SIL
progression may be viewed as a turn to the reserve cell phenotype, with
a prevalence of P-cadherin. The basal layers of cells of low-grade SILs
reproduce, in general, the type of adherens junctions and desmosomes
that are found in basal cells of normal squamous epithelium or in
immature squamous metaplasia. However, the expression and functionality
of the individual molecules comprising the adherens junctions and
desmosomes in SILs differed quite substantially from the junctions of
normal progenitor cells. The major differences were a nearly complete
disappearance of -catenin in SIL cells, along with a decrease in
ß-catenin and an increase (!) in plakoglobin. Among desmosomal
proteins, not only was desmoglein low in SILs, similar to the cells of
immature squamous metaplasia, but so was desmoplakins. Most of the
lesions showed a uniform pattern of changes. The immunohistochemical
results were all confirmed at the biochemical level, using cell
cultures of normal cervical keratinocytes and SIL cells.
During SIL progression the role of E-cadherin in cell-cell adhesion
diminishes, and the molecule is practically absent in high-grade SILs.
The impression is that the cells switch to another type of functional
adherens junction, ie, P-cadherin, which seems to employ mainly
plakoglobin instead of ß-catenin (see Figures 3 and 6
) as the
connector to the actin cytoskeleton complex. Determination of whether
the observed down-regulation of either E-cadherin or the catenins, via
functional mutations or at the transcriptional level, results in
down-regulation/inactivation of the other, as has been shown in
vitro,38, 47
needs further investigation. However,
observations in this study suggest that inasmuch as the existing
coordination between E- and P-cadherin is disturbed only in high-grade
SILs, down-regulation of E-cadherin must be at the transcriptional
level.
A switch of E-cadherin to P-cadherin adhesions during SIL progression might explain the lack of terminal and squamous differentiation in SILs. Both cadherins are partially redundant in epidermal stratification in skin.48 Basal cells, which express both cadherins, could use either cadherin to induce stratification; however, superficial cells are dependent on E-cadherin for further stratification.48 In high-grade SIL, the expression of P-cadherin was observed throughout the lesion, suggesting that squamous and terminal differentiation cannot take place because of the presence of P-cadherin. In squamous carcinoma cell lines an expression pattern similar to that in the cervical lesions studied was observed. Expression levels of E- and P-cadherin were about the same in normal squamous keratinocytes, whereas reduced E-cadherin and high P-cadherin expression was observed in squamous carcinoma cell lines.49 This might indicate that upon malignant transformation of cells, expression of P-cadherin inhibits or induces specific signals, resulting in a lack of squamous and terminal differentiation, as indicated by the lack of CK13 and involucrin expression.
The most remarkable observation in SIL cells is the nearly complete
absence of -catenin and its decreased association with the
E-cadherin/ß-catenin complex. Normally, -catenin allows the
cadherin molecules to anchor to F-actin filaments. It is not absolutely
clear whether the reduced amount of cellular -catenin is still
sufficient to provide the anchor for P-cadherin adhesions as observed
in this study, or whether another molecule (ie, vinculin, relatively
homologues to -catenin)50, 51
takes its place. The
latter possibility was shown for some tumor cells by Hazan et
al,37
and more recently it was shown that vinculin can form
a complex with -catenin and thereby participate in the formation of
adherens junctions.52
However, we did not find any evidence
for this in our in vitro cultures, because no vinculin was
coimmunoprecipitated with E-cadherin (data not shown). Whether the
absence of -catenin was caused by inactivating mutations in SIL
cells remains to be investigated. However, the decrease in this catenin
observed for all high-grade SILs suggests that regulatory mechanisms
are responsible for this. Our observations are substantiated by the
fact that -catenin is also decreased in some other types of
carcinomas.46
During the progression of cervical lesions, we observe an increase in
plakoglobin expression. It is plausible that overexpression of
plakoglobin in SIL cells may lead to substantial changes in the
composition of adherens junctions, switching from
E-cadherin/ß-catenin/-catenin to P-cadherin/plakoglobin complexes.
Forced overexpression of plakoglobin was shown to induce a decrease in
cadherin-complexed ß-catenin, probably regulated via the
transcriptional pathway with degradation of noncomplexed
extrajunctional ß-catenin.53
Furthermore, both
ß-catenin and plakoglobin are capable of binding LEF-1, a
transcription factor, and participate in the transcriptional activation
of other genes.
The role of pp120, shown to be highly expressed in both normal and SIL cells, remains obscure. It has been shown that pp120 can form complexes with the cadherin-catenin cell adhesion complexes and is necessary for both adhesion formation and lateral clustering,33 but its complete function in cell-cell adhesion and signaling is not yet known.
The assembly of desmosomes in all SILs was grossly affected, showing a
strong decrease in expression of both desmoplakins and desmoglein,
similar to observations in squamous cell carcinomas.36, 54
A decrease in both desmoplakins and desmoglein in oral squamous cell
carcinomas was correlated with poor differentiation, invasiveness, and
lymph node metastases.55
Clearly, by itself the disturbance
in desmosomal plaques does not lead to invasive properties of cells,
because in the case of SILs, it was observed for noninvasive
precancerous lesions. The assembly of desmosomes is believed to
be dependent on E-cadherin and proper adherens junction
formation56-58
and an intact actin and cytokeratin
network.57
Down-regulation of E-cadherin was shown to
result in a reduction of desmosome formation and normal
stratification.56, 59
Apparently, P-cadherin cannot take
over the function of E-cadherin in desmosome formation.48
Furthermore, desmosome formation is largely dependent on free
plakoglobin.56, 60, 61
During SIL progression, we observed
a relative decrease in the level of free plakoglobin (Figure 6D)
. In
the studied cervical lesions we thus observe changes in all of the
relevant molecules necessary for proper desmosome formation, ie,
improper formation of adherens junctions using P-cadherin instead of
E-cadherin, a large pool of cytoplasmic - and
ß-catenins, occupied plakoglobin, as well as an impaired
cytokeratin network (Van Dorst et al, manuscript submitted for
publication). All of these changes contribute to the lack of desmosome
formation during SIL progression.
Various mechanisms, probably all working together, are possibly
involved in SIL development. A genetic mechanism, regulating the
expression of either E-cadherin or P-cadherin during normal cervical
differentiation, might be disturbed during SIL progression, like
mutations leading to gene inactivation or promotor silencing. Because
P-cadherin can still form adherens junctions but is not able to induce
squamous/terminal differentiation, this might lead to uncontrolled
proliferation of cells.48
We previously reported that
overexpression of Ep-CAM in cells interconnected by classic cadherins
leads to abrogation of adherens-type junctions and a reduction in the
total cellular -catenin.19
In our previous
study22
we showed Ep-CAM overexpression during SIL
progression, and in this study we observe a down-regulation of
-catenin. Both phenomena possibly result in a down-regulation and/or
abrogation of E-cadherin-mediated adherens junctions. Another option
might be the involvement of oncoproteins. It was demonstrated that the
retinoblastoma protein was able to induce E-cadherin
expression.62
In nearly all cases of SIL, human papilloma
virus (HPV) is involved, and HPV protein E7 is able to inactivate the
retinoblastoma protein. This could thus lead to a decreased activation
of E-cadherin expression. Because the overall impression is that
genetic inactivation of E-cadherin, as observed for some
tumors,15
does not take place during SIL progression, the
suppression of E-cadherin rather takes place at the regulatory level,
probably via inactivation of the retinoblastoma protein.
In conclusion, cell adhesion complexes are dramatically changed,
especially in high-grade SIL, as compared to both normal squamous
epithelium and reserve cells, which are both proliferating and
undergoing transdifferentiation. Despite the fact that a rather
organized cell structure is formed during the onset of cervical
lesions, great disturbances in cell-cell interactions are present. For
pathological purposes, both the expression of P-cadherin and the
absence of -catenin and desmoplakin expression throughout the lesion
can be used in the classification of SIL.
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Footnotes |
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Supported by a grant from the Dutch Cancer Society (NKB-KWF) (RUL 95-1107).
Accepted for publication April 11, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex. Proc Natl Acad Sci USA 1995, 92:18813-18817
-catenin expression in human cancers. Am J Pathol 1994, 144:667-674[Abstract]
-catenin fusion molecules. J Cell Biol 1994, 127:235-245-catenin is a vinculin homologue. Proc Natl Acad Sci USA 1991, 88:9156-9160-catenin and vinculin. Mol Cell Biol 1998, 141:755-764
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