(Received for publication, October 19, 1994; and in revised form, August 23, 1995)
From the
Integrin-mediated interactions between cells and the
extracellular matrix play a fundamental role in the development and
function of a variety of tissues by triggering intracellular signals
that regulate gene expression. In this study, mouse mammary epithelial
cells plated on tissue culture plastic were shown to dramatically
up-regulate the steady state levels of mRNA encoding the
,
,
,
,
,
,
, and
integrin subunits, in contrast
to cells cultured on a basement membrane matrix or cells in
vivo. This pattern of expression was also observed in a mouse
mammary epithelial strain, CID-9 and in other mouse cell lines such as
MMTE cells and K1735-M2 melanoma cells. The control of integrin
expression was mediated at different levels in different cell types. In
K1735-M2 cells, transcription of the
integrin gene
was influenced by the substratum, although the levels of integrin
protein remained similar. In mammary epithelial cells, the rates of
integrin gene transcription were similar, but mRNA
and protein levels were higher in cells cultured on plastic than those
on basement membrane. For both cell types, the rate of integrin protein
turnover was nearly identical in cells cultured on either substratum.
Our results demonstrate that extracellular matrix controls the
expression of
integrin subunits and that this
regulation is exerted at both transcriptional and post-transcriptional
levels.
During tissue formation, maintenance, and remodeling,
extracellular matrix (ECM) ()has an invaluable role not only
in promoting cell motility and anchorage, but also in inducing cell
activation and differentiation. It is becoming increasingly clear that
cell-matrix interactions, through specific adhesion receptors, trigger
biological responses similar to those transduced by growth factors,
hormones, or cytokines(1, 2) .
We previously showed
that interactions between mammary cells and the ECM could regulate the
expression of ECM molecules themselves(3) . In an in vivo environment, mammary epithelial cells interact with a basement
membrane, but on an inadequate substratum in tissue culture, such as on
a plastic surface, the cells attempt to recreate their basement
membrane by transcribing and translating genes coding for ECM proteins
such as fibronectin and laminin(3) . We have now asked whether
the type of cell-matrix interactions also induce a similar regulation
in the expression of cell surface integrin receptors
for ECM components. Any matrix-induced changes in the levels and
patterns of integrin subunits might alter the way that
microenvironmental signals are perceived, trigger the expression of new
sets of genes, and modify cell phenotype.
We therefore examined the
mRNAs coding for different subunits of the integrin
family in mammary epithelial cells and compared their expression levels
in cells cultured either on a plastic surface or on a laminin-enriched
reconstituted basement membrane matrix. We then assessed whether
changes in the amounts of mRNA were reflected at the protein level.
Our results demonstrate that the nature of the substratum controls
the expression of integrins at both the mRNA and protein levels. The
ECM-dependent modulation of integrin expression was
not ligand-specific since the levels of mRNA encoding a wide variety of
chains of the
integrins were altered. This
regulation was not restricted to mouse mammary epithelial cells but
also applied to other cell types, such as melanoma cells, and cells
from other species including epithelial cells from normal human breast.
We argue that this pattern of integrin gene expression reflects a
general control mechanism activated by cells in an inappropriate
environment, which is required for the establishment of sufficient
cell-matrix interactions to maintain survival and differentiation.
To induce differentiation in the mammary cultures, cells were first plated on tissue culture plastic or on EHS matrix (prepared as described previously) (12, 13) for 2 days in DMEM/F-12 medium containing 5 µg/ml insulin (Sigma) and 5% FCS, then washed three times with serum-free DMEM/F-12 medium and cultured in differentiation medium, DMEM/F-12 supplemented with 5 µg/ml insulin, 1 µg/ml hydrocortisone (Sigma), and 3 µg/ml prolactin (Sigma), as previously described(13, 14) .
To
establish cells stably expressing CAT under the control of integrin
promoters, CID-9 cells were transfected as described previously with
some minor changes(15) . M2 cells were plated in DMEM, 5% FCS
at 8 10
cells/85-mm dish 24 h prior to the
transfection. They were cotransfected by incubation for 16 h with
calcium phosphate precipitates containing 30 µg of the different
plasmids and 3 µg of pSV2neo. The cells were shocked with 25%
glycerol in 1
Hanks' balanced salts for 90 s. The
selection was started 36 h after transfection by addition of G418 (Life
Technologies) at 1 mg/ml. After
2 weeks, the G418-resistant clones
were pooled and expanded.
Figure 1:
Influence of substrata on cell
morphology and spreading in culture. Cells were plated at 0.5
10
cells/cm
on EHS matrix or tissue culture
plastic. Primary mouse mammary epithelial cells and CID-9 cells were
cultured for 2 days in medium containing 5% FCS, then for 2 further
days in differentiation medium. MMTE and M2 cells were cultured for 2
days in serum-containing medium followed by 1 day in serum-free medium.
These images are phase contrast micrographs (bar = 1
mm).
In our initial studies on integrin expression, we focused on mammary
epithelial cells. These cells differentiate and synthesize milk
proteins in the presence of lactogenic hormones, but differentiation
requires integrin-mediated interaction with the
basement membrane(13) . By using cDNA probes to most of the
subunits that form heterodimers with the
chain,
we found that primary cultures of mammary epithelial cells express
,
,
,
,
, and
(Fig. 2A).
and
were undetectable (not shown). The expression of
was very low and was probably due to contamination of our
cultures by myoepithelial cells, which in humans are known to express
the
subunit strongly(19) . We did not examine
mRNA, but no
protein was detectable
in mammary epithelial cultures. The spectrum of integrin mRNA
expression compares well with our subsequent protein analysis and
defines for the first time the
integrin subunit expression
profile for alveolar epithelial cells isolated directly from pregnant
mouse mammary gland (see below).
Figure 2:
Expression of integrin subunit mRNAs in
mouse mammary epithelial cells in culture and in vivo. Total
cellular RNA (10 µg/lane) was isolated from cultured primary
mammary epithelial cells (A), mouse mammary gland at different
stages of development (B), and cultured CID-9 cells (C), electrophoresed, transferred to Hybond membrane, and
hybridized with ,
,
,
,
,
,
and
integrin,
and
-casein probes. Even loading of each gel was verified by
ethidium bromide staining (see B for representative example).
In each case, standard RNA size markers were included in the gel. A, primary mammary epithelial cells cultured on EHS matrix (E) or plastic (P) for 2 days in serum-containing
medium and 2 more days in differentiation medium. Up-regulation of
integrin mRNA in primary cells cultured on plastic was continuous and
sustained at day +1 and +2 in differentiation medium; there
were also no changes of integrin mRNA expression in cells plated on EHS
matrix between day +0 and +2 in differentiation medium. Note
that
-casein expression was higher in cells cultured on the EHS
matrix. B, integrin expression was assessed in virgin mouse
mammary gland (V), 14.5-day midpregnant mouse mammary gland (Pr), and purified mammary epithelial cells isolated from a
midpregnant mouse mammary gland (T
). Integrin mRNA
levels in vivo were compared with levels of the mRNA in
culture, in cells plated on EHS matrix (E) and plastic (P). C, CID-9 cells cultured as for primary mammary
epithelial cells. Total RNA was harvested after a 2-day culture in
differentiation medium. Note that the integrin chain mRNA intensities
shown in the autoradiographs are not representative of their absolute
cell content since the probes for the different integrin subunits used
were of different lengths, and were of variable activities, and the
autoradiographs were exposed for different
times.
When we compared the mRNA levels of
the subunits and the
chain in cells cultured on
the plastic and basement membrane substratum, we found that the cells
responded to plastic by up-regulating expression of these integrin
subunits (Fig. 2A). This was particularly dramatic for
the
,
, and
subunits, although the integrin mRNA levels were so low in cells
cultured on EHS matrix that they could not satisfactorily be
quantitated with a densitometer. The response was likely to represent
an up-regulation, since the integrin mRNA levels both in mammary gland
tissue isolated at different stages of development and in purified
alveolar epithelial (T
) cells isolated directly
from the mammary gland were low and comparable to the levels in cells
cultured on EHS matrix (Fig. 2B). The integrin subunit
expression profile and response to ECM were similar in a mammary
epithelial cell strain, CID-9, that retains its ability to
differentiate after stimulation with basement membrane and lactogenic
hormones (Fig. 2C). In contrast to the integrin
expression pattern, both primary mammary cells and CID-9 cells
expressed higher levels of the milk protein gene,
-casein, on the
basement membrane matrix, as expected (11, 14) (Fig. 2, A and C).
In order to determine whether this pattern of integrin regulation
was specific for differentiating mammary epithelial cells or not, we
examined the dependence on ECM of integrin expression in one other
mouse epithelial cell type, MMTE, and one other cell line of
nonepithelial lineage, the melanoma cell line K1735-M2 (or M2) (Fig. 3). In MMTE cells, the influence of substrata was
dramatic, notably for the ,
, and
subunits, and was serum-independent (Fig. 3A). The majority of integrin mRNAs tested were
also up-regulated in M2 cells cultured on plastic (Fig. 3B). These results indicate that this
substratum-dependent control of integrin expression was not confined to
mammary epithelial cells either of a differentiating phenotype (primary
cultures and CID-9) or of a tumor phenotype (MMTE), but was also seen
in cells of a completely different lineage (M2).
Figure 3: Regulation of integrin mRNA levels in mouse cell lines. Mouse cell lines MMTE (A) and M2 (B) were cultured for 2 days in serum-containing medium. RNA was harvested after cells were cultured for one more day in serum free- or in FCS-containing medium. Northern blots of equal amounts of total mRNA (10 µg/lane) were exposed as described in Fig. 2.
Figure 4:
Substratum effect on transcriptional
regulation of 1 integrin. A, schematic representation of
the constructs containing the human
integrin promoter
regions. pEMBL-D carries the distal promoter contained in a 630-bp PstI-ApaI fragment spanning nucleotides 842-1471 of a
4.5-kb genomic clone containing the 5`-flanking region of the
gene, and pEMBL-P carries the proximal promoter
contained in a 360-bp ApaI fragment spanning nucleotides
1471-1828. Nomenclature for nucleotide numbering is as in
Cervella et al.(7) . Black squares, GC-rich
regions; black triangle, potential octamer binding site; black circle, potential AP-1 binding site. B and D, CAT activity in M2 cells (B) and CID-9 cells (D) transfected with the constructs pEMBL-D, pEMBL-P, and
pSV2-CAT and cultured for 2 days on EHS (white columns) or
plastic (gray columns). Cells were harvested, and 10 µg of
total cell protein was assayed for CAT activities as described under
``Materials and Methods.'' In each case 1 µg of the same
protein extract was separated by SDS-PAGE and analyzed by silver
staining to confirm accurate estimation of protein concentrations. In
this study, only the distal promoter sequence and proximal promoter
sequence were used since the complete sequence (nucleotides 842-1828)
was previously shown to have the same activity as that of the distal
region(7) . The increased activity of the SV40 promoter in
CID-9 cells cultured on plastic is similar to that noted
previously(42) . C and E, transcription
activity assessed by nuclear run-on. 5 µg of
integrin and GAPDH cDNAs were immobilized on nylon membrane using a
multiwell blot apparatus. The cDNAs were probed with equal counts of
trichloroacetic acid-precipitated,
P-labeled nuclear
transcripts from nuclei of M2 cells (C) and primary mammary
cells (E) cultured under the same conditions as for the CAT
assays. Relative transcription activities in cells cultured on plastic (gray columns) are shown as a percentage of transcription
activities in cells cultured on EHS taken equal to 100% (white
columns).
To
confirm that transcription was regulated by cell-matrix interactions,
nuclear run-on assays were performed. Our results show that
transcription from the endogenous integrin promoter
in M2 cells was increased by culture on a plastic substratum (Fig. 4C), suggesting that a matrix-dependent control
element lies within the promoter sequence for this integrin. However,
in contrast to M2 cells,
integrin transcription in
mammary epithelial cells was not up-regulated on plastic, as assessed
either by CAT assay (Fig. 4D) or by nuclear run-on (Fig. 4E). This suggests that the ECM-dependent
transcription machinery in mammary epithelial cells is different from
that in M2 cells.
Thus, at least in some cell types, part of the
ECM-dependent control of integrin expression occurs at the
transcriptional level. The difference between rates of transcription
and levels of integrin mRNA suggests that additional
controls occur at the level of mRNA stability, although we have not
been able to assess this owing to the negligible quantity of integrin
mRNA present in any of the epithelial or melanoma cell cultures on EHS
matrix.
Figure 5:
Characterization of integrin subunits in mammary epithelial cells. Mouse mammary
epithelial cells were cultured for 2 days on plastic, and steady
state-labeled with Tran
S-label for 24 h. Rabbit antisera
against synthetic peptides corresponding to C-terminal sequences in
,
,
,
integrin subunits were used in immunoprecipitations with (+)
or without(-) the immunizing peptides. Immunoprecipitates were
separated under nonreducing conditions. Positions of the 200- and
97-kDa size markers and the
,
,
and
integrin subunits are
shown.
Figure 6:
Effect of substratum on integrin subunit expression in mouse mammary epithelial cells,
CID-9 cells, and human mammary epithelial cells. A, mouse
mammary epithelial cells and CID-9 cells were cultured for 2 days on
EHS matrix (E) or plastic (P) in differentiation
medium and radiolabeled for 24 h. Equal amounts of trichloroacetic
acid-precipitable counts were immunoprecipitated with an
anti-
integrin antibody and analyzed by gel
electrophoresis under nonreducing conditions and fluorography. To
confirm that equal amounts of newly synthesized cell proteins were
being used, equal volumes of each immunoprecipitation reaction mix were
separated by SDS-PAGE and analyzed by fluorography (not shown). B, mouse mammary epithelial cells grown for 6 days on EHS
matrix (E) or plastic (P) were radiolabeled for 24 h,
and cell extracts were immunoprecipitated using rat anti-mouse
mAb GoH3 or rat anti-mouse
mAb
346-11A as in A. C, primary human mammary epithelial
cells were cultured, radiolabeled, and then
integrins
were immunoprecipitated using anti-
integrin antiserum
as in A. Immunoprecipitations with chain-specific antibodies
indicated that the subunit partners for
integrin in
these cells included
,
,
, but not
chains (not shown). In
each case (A, B, and C) the levels of
integrin subunit were significantly higher in cells cultured on plastic
than on EHS matrix.
To determine whether
ECM regulated the expression of any integrin subunits at the protein
level, cells cultured on the different substrata were steady state
labeled and equal amounts of trichloroacetic acid-precipitable newly
synthesized proteins were used for immunoprecipitations. We could not
examine the relative abundance of surface-labeled integrin due to the
harsh procedure required to dissociate cells from EHS matrix. An
anti- integrin peptide antibody that precipitates the
chain together with its associated
subunits
revealed that, in first passage mammary cultures and in CID-9 cells,
the
chains as well as the
chain were
synthesized at higher levels on plastic than on EHS matrix (Fig. 6A). Immunoprecipitations with
-
and
-specific monoclonal antibodies showed that
expression of
and
protein was also
up-regulated on plastic (Fig. 6B). Scanning
densitometry and PhosphorImager analysis demonstrated that the
up-regulation varied from 3-fold (
chain) to 5-fold
(
and
chains). A 3-fold
up-regulation of integrin synthesis was also observed, for both the
and
chains, in primary cultures of luminal epithelial cells
isolated from normal human breast (Fig. 6C). This
result suggests that matrix-dependent control of
integrin expression is conserved across species and may therefore
be of widespread importance.
Although we found that different cell
types cultured on plastic dishes all showed increased levels of
integrin mRNA, this was not always reflected at the protein level. For
example, in M2 cells, which expressed ,
, and
integrin subunits (Fig. 7A), as well as
(not shown),
integrin protein synthesis was either similar on the different
substrata (
and most of the
subunits), or for
the
subunit, was 3 times lower on plastic than on
basement membrane matrix (Fig. 7B).
Figure 7:
Effect of substratum on integrin subunit expression in M2 cells. A, M2 cells
cultured on plastic for 2 days in FCS-containing medium and for 2
further days in serum-free medium, then radiolabeled for 24 h. Cell
extracts were immunoprecipitated using rabbit-anti
,
,
, or
integrin
antisera in presence (+) or absence(-) of immunizing
peptides. Immune precipitates were separated by SDS-PAGE under
nonreducing conditions and analyzed by fluorography. Positions of the
integrin subunits are noted in the margin. The
subunit was present only in
immunoprecipitates. B, M2 cells were cultured on EHS matrix (E) or
plastic (P), and extracts were immunoprecipitated with rabbit
anti-
chain antiserum and analyzed as in A.
The levels of newly synthesized
and
subunits were
quantitated after exposure to storage PhosphorImager
plates.
Figure 8:
integrin turnover in
mammary epithelial cells cultured on EHS matrix or plastic. Mouse
mammary epithelial cells were cultured and labeled for 24 h as in Fig. 6A, after which they were washed and incubated
with excess (6 mM) unlabeled methionine. Cells were harvested
at chase times 0, 12, 24, and 48 h, and equal volumes corresponding to
equivalent trichloroacetic acid-precipitable counts at chase time 0
were immunoprecipitated with a rabbit anti-
chain
antiserum. Immunoprecipitated integrins were analyzed by gel
electrophoresis and fluorography, and were quantified using storage
PhosphorImager plates. A, time course of
integrin levels and their associated
subunits in cells
cultured on EHS matrix and plastic. B, the relative levels of
radioactivity in
subunits and the
subunit at
different times of the chase were measured, and their levels were
plotted as a percentage of the total radioactivity incorporated into
each subunit at time 0. In each case the levels of activity in
subunits and the
subunit were 3 times higher in cells
cultured on plastic than on EHS matrix, but their decay rates were
almost identical.
Together, our results indicate that the amount of integrin subunits expressed by cells is controlled at the transcriptional and post-transcriptional levels, but the mechanisms employed appear to be cell type-specific.
Our study establishes that ECM controls the expression of
integrins in mammary epithelial cells. When these
cells were cultured on a basement membrane matrix, integrin mRNA levels
were comparable to those in the mammary gland. By contrast, culturing
cells on a plastic substratum triggered a dramatic and sustained
expression of the mRNA coding for various
integrin
subunits. It is likely that multiple mechanisms operate since only a
proportion of the increase in mRNA was reflected at the level of
protein synthesis. This control of integrin expression was also
observed in epithelial cells from normal human breast and in cells from
other lineages such as melanoma cells, which suggests that it stems
from a general matrix-dependent regulatory mechanism. However, not all
of the transcribed mRNAs behaved like those of integrin subunits since
the amount of
-casein mRNA was reduced, not increased, in mammary
cells cultured on plastic, and we have recently shown that the mRNA
levels of some transcription factors required for mammary
differentiation (2) are not regulated by matrix. (
)
This work extends previous studies where we have shown
that the expression of ECM molecules themselves are controlled by the
type of interactions between mammary cells and their
substratum(3) . In the absence of a suitable ECM, these cells
up-regulate expression of laminin both at the mRNA and protein levels,
but once the cells interact with a basement membrane, laminin synthesis
is suppressed(3) . Since mammary epithelial cells normally
interact with a basement membrane in vivo, it is possible
that, when they are deprived of an interaction with this type of
matrix, as occurs on tissue culture plastic, they increase the levels
of both ECM protein and integrin receptor in order to maximize their
chances of establishing further contacts with the ECM. Precisely why
this should be so important to mammary cells is not clear at this time,
but it may reflect a generic wounding response that often occurs when
cells are placed in tissue culture. This type of control has been
discussed in relation to the wound response of keratinocytes, where
integrin receptors are greatly increased following
removal from skin to tissue culture(21) ; here it has been
proposed that a shift in expression of
integrin in stable contacts between hemidesmosomes and the
underlying dermis, to an expression of
heterodimers
in migratory keratinocytes, is critical for wound
repair(22, 23) . Alternatively, and perhaps more
appropriately for mammary epithelium, it may represent an effort to
maintain extracellular survival signaling, since mammary epithelial
cells, either in vivo or in culture, undergo apoptosis in the
absence of appropriate ECM, and survival is maintained only by cell
interactions with basement membrane. (
)
Substratum-dependent control on integrin gene expression has been noted previously in other cell systems. In addition to that already mentioned for keratinocytes, integrin mRNA levels were increased rapidly in osteosarcoma and cervical carcinoma cell lines that were deprived of adhesion to any substratum (24) . This regulation is likely to be different to that reported here since in our study increased integrin expression occurred when cells formed intact monolayers on tissue culture plastic, most likely through a fibronectin and/or vitronectin interaction, rather than in the complete absence of any ECM. However, the well documented change in adhesion receptors that occurs during terminal differentiation of keratinocytes (as opposed to the wound response) may be relevant(25) . In this instance, reduced transcription of integrin subunits and transport of functionally active heterodimers to the cell surface correlates with the expression of differentiation markers. Although this may be mechanistically related to the reduction of integrin expression in mammary epithelial cells induced to differentiate by culture on EHS matrix, in the latter case integrins are present at the cell surface, and indeed functional integrins are required for basement membrane to induce casein production(13) .
One possible effector of the
increased expression of both ECM protein and receptor in the mammary
system is TGF-. In addition to substratum-dependent control of ECM
and integrin, we previously demonstrated that TGF-
mRNA levels were low in mammary epithelial cells cultured on a
basement membrane matrix but were up-regulated in cells on
plastic(26) . Since TGF-
is known to increase
the expression of various ECM proteins as well as
integrins(27, 28, 29) , it might behave as an
autocrine regulator when cells are placed in an inappropriate culture
environment. This is an attractive mechanism, but is likely to
represent only part of the story since first, TGF-
has previously
only been shown to increase integrin mRNA expression by a small amount
but we detected a very dramatic ECM-dependent up-regulation of integrin
mRNA(27, 28) , and second, TGF-
is a normal
contaminant of EHS matrix preparations(30) , but there were
only very low levels of integrin mRNA in cells cultured on this
substratum.
Because the integrin expression pattern that we have reported is not restricted to normal mammary epithelium, but also applies to tumor lines and cells from other developmental lineages, an alternative explanation for its regulation is that the effector mechanism is a general one. Changes in cell shape, for example, represent such a mechanism, and morphological alterations are evident in our cultures. It has long been proposed that shape contributes to the phenotype of a cell by regulating gene expression(31, 32) , but it is still unclear whether it acts directly through cytoskeleton and alteration of nuclear structure, for example via tensegrity(33) , or indirectly through adhesion receptor-directed second messenger signals such as focal contact associated kinases (1) or cadherins. Since cells cultured on EHS matrix are more cohesive than those on plastic, cadherin-mediated signals may indeed be responsible for integrin mRNA and protein down-regulation, as has been shown in differentiating keratinocytes (34) .
The intracellular mechanism of
substratum-dependent control on integrin gene expression is complex,
and since it occurs at the transcriptional and post-transcriptional
levels, appears to be multifactorial. In addition, mammary epithelial
cells and M2 cells respond differently to ECM, with only the latter
showing altered transcription of the integrin gene,
indicating that the pathways triggered in response to signals from the
matrix are cell type-dependent. The low level of transcriptional
activation by basement membrane in mammary epithelial cells suggests
that the dramatic up-regulation of steady state integrin mRNA is very
likely accounted for by an increase of integrin transcript stability.
The discrepancy between the up-regulation of integrin mRNA and
protein levels in this system suggests that post-translational controls
are operative. This is especially evident for M2 melanoma cells, where
the levels of subunit were higher in cells cultured
on EHS matrix than on plastic, but the reverse was true for the amounts
of mRNA. Although this may be related to the
up-regulation observed in human
melanoma cells cultured in collagen gels(35) , the mechanism
for this type of control is not clear. However, two possibilities
should be considered because they act as useful pointers for future
studies. (i) Evidence for a possible translational control is visible
in the structure of at least the
and
chain mRNAs, where the 5`-untranslated region is extremely rich
in GC sequences(7, 36) . These highly structured
sequences are known to reduce translation of a variety of mRNAs coding
for regulatory or cytoskeletal proteins whose translation has to be
tightly controlled(37) . (ii) Comparison of integrin mRNA and
protein levels in this study indicates that translation of integrin
mRNA is relatively more efficient in cells cultured on the basement
membrane matrix than on plastic. This may be due to segregation of
integrin mRNA into separate cytoplasmic pools that are either
translatable or nontranslatable, as occurs for some cytoskeletal
genes(38) , or alternatively, since mRNA apparently needs to be
translated in order for RNA degradation to occur(39) , the high
level of integrin chain mRNA in cells cultured on plastic might result
from a poor translation and thus a low degradation.
In summary, this
study extends our understanding of ECM-dependent gene regulation. We
have now shown that expression of ECM proteins (3) and their
integrin receptors, and cytokines such as
TGF-(26) , can all be regulated by the type of
matrix with which cells interact. We propose that ECM-dependent
regulation of all these proteins is part of a general mechanism whereby
normal cells react to an inappropriate substratum and attempt to
reestablish suitable cell-matrix interactions necessary for maintaining
survival and for progressing through a differentiation program. It is
significant that the only cell type in our study that did not conform
to this pattern was the transformed, highly metastatic M2 cells. The
lack of integrin up-regulation at the protein level in these cells
cultured on plastic might be related to their anchorage-independent
growth (40) and very likely results from the diversification
that takes place during tumor progression when clones with traits most
adapted for immortality and metastasis are selected(41) .