(Received for publication, May 26, 1995; and in revised form, July 18, 1995)
From the
The integrin , a receptor for
members of the laminin family of basement membrane components,
contributes to the function of epithelial cells and their oncogenically
transformed derivatives. In our efforts to study
-mediated functions in more detail
and to assess the contribution of the
cytoplasmic
domain in such functions, we identified a rectal carcinoma cell line
that lacks expression of the
integrin subunit. This
cell line, termed RKO, expresses
but
not
, and it interacts with laminin-1
less avidly than similar cell lines that express
. We expressed a full-length
cDNA, as well as a mutant cDNA that lacks the
cytoplasmic domain, in RKO cells and isolated stable
subclones of these transfectants. In this study, we report that
subclones that expressed the full-length
cDNA in
association with endogenous
6 exhibited partial G
arrest and apoptosis, properties that were not evident in RKO
cells transfected with either the cytoplasmic domain mutant or the
expression vector alone. In an effort to define a mechanism for these
observed changes in growth, we observed that expression of the
integrin induced expression of the
p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases.
These data suggest that the
integrin cytoplasmic
domain is linked to a signaling pathway involved in cell cycle
regulation in the
transfected RKO cells.
The integrin is a receptor
for members of the laminin family of basement membrane components.
Initial studies established that
is
a receptor for laminin-1(1, 2, 3) , and
subsequent work has shown that it also functions as a receptor for
other laminin isoforms(4, 5) . In its capacity as a
laminin receptor,
is involved in the
formation and maintenance of hemidesmosomes (6, 7, 8) and in the dynamic adhesion and
migration of carcinoma cells(2, 3) . Most likely,
other functions of epithelial and carcinoma cells are dependent upon
because it plays such a pivotal role
in mediating their interactions with laminin matrices. It is widely
assumed that the unusually large and structurally unique cytoplasmic
domain of the
integrin subunit associates with
cytoskeletal and signaling molecules and that such associations provide
the basis for the distinct functions associated with
(5, 8, 9) .
In our efforts to study -mediated
functions in more detail and to assess the contribution of the
cytoplasmic domain in such functions, we identified a
rectal carcinoma cell line that lacks expression of the
integrin subunit. This cell line, termed RKO, expresses
but not
(2, 3) . In this
study, we report that RKO transfectants, which expressed the
full-length
cDNA in association with endogenous
exhibited G
arrest and a basal rate of
apoptosis, properties that were not evident in RKO cells transfected
with either a
cytoplasmic domain mutant or the
expression vector alone. In an effort to define a mechanism for these
observed changes in growth, we observed that expression of the
integrin induced expression of the
p21 (WAF1; Cip1) protein, an inhibitor of G
cyclin-dependent kinases(10, 11, 12) .
These data suggest that the
integrin cytoplasmic
domain is linked to a signaling pathway involved in cell cycle
regulation in the
-transfected RKO cells.
For propidium iodide staining, cells at approximately 50%
confluency were plated overnight on either tissue culture plastic,
EHS-laminin (4.2 µg/cm), or laminin-5 (0.2
µg/cm
; provided by R. Burgeson) and then harvested by
trypsinization. The cells were stained with propidium iodide (Sigma; 2
mg/ml in 4 mM sodium citrate containing 3% (w/v) Triton X-100
and RNase A (0.1 mg/ml)). The stained cells were analyzed by FACS.
Cells for immunohistochemistry
were plated for 18 h in complete growth medium on coverslips precoated
with either poly-L-lysine (10 µg/cm) or
EHS-laminin (5 µg/cm
). Cells were fixed for 8 min in 4%
paraformaldehyde, permeabilized for 2 min in 0.2% Triton X-100, and
stained with the p21 (10 µg/ml) and a fluorescein-conjugated donkey
anti-mouse IgG (Jackson Laboratories; 1:30). The cells were examined
using a confocal microscope (Bio-Rad MRC 600, Bio-Rad Microsciences,
Cambridge, MA) attached to a Zeiss Axiovert 35 equipped with a
63 Plan-Neofluar objective.
Representative RKO subclones from the full-length cDNA and the
-
CYT transfections that
expressed varying levels of
surface expression were
chosen for functional studies (Fig. 1A). No
expression was evident in subclones obtained from transfection of
the expression vector alone (Neo). The association of the transfected
subunits with endogenous
was
confirmed by immunoprecipitation of surface-biotinylated cells with the
A9 mAb (data not shown). Also, the full-length
transfectants exhibited increased adhesion, spreading, and
migration on laminin-1 providing evidence that the expressed
integrin is functional (18) .
Figure 1:
A, surface
expression of the human integrin subunit in RKO
transfectants. Populations of transfected RKO cells expressing either a
full-length
integrin (A7, B8, D4, F10) or a
cytoplasmic domain mutant (3C1, 5A3) on the cell
surface were isolated by sequential FACS using UM-A9, a mAb specific
for the
integrin subunit, subcloned by single cell
sorting, and then analyzed by FACS. Mock transfectants (Neo 22 and Neo
26) were subcloned by limiting dilution analysis. For the mock
transfectants, the scans obtained with a nonspecific IgG and UM-A9 are
superimposed. For all of the other subclones, the UM-A9 scan (right-hand peak) is shown along with a mock transfectant scan (left-hand peak). B, analysis of DNA content in the
bulk
transfectants. RKO cells transfected with the
full-length
cDNA were enriched for
expression by sequential FACS using UM-A9, stained with propidium
iodide, and analyzed by FACS to assess DNA content. The A
,
G
/G
, S, and G
/M peaks are noted. C, analysis of DNA content in subclones of the
transfectants. Flow cytometry profiles obtained from propidium
iodide staining of three subclones described in A are shown.
For each profile, the percentage of cells in
G
/G
, S, and G
+ M is noted in
the upperright.
Initially, we observed that bulk sorts of the
full-length transfectants did not maintain high
levels of
surface expression for more than 3-4
days after sorting. To gain insight into the behavior of these
transfectants, we assessed their DNA content using propidium iodide. A
significant A
peak, characteristic of cells undergoing
apoptosis(19) , was evident in the bulk transfectants (Fig. 1B). Stable subclones that expressed full-length
cDNA maintained
surface expression,
although the level of expression was less than that seen in the initial
bulk sorts (Fig. 1A). These subclones grew noticeably
more slowly than the mock transfectants. Propidium iodide staining
revealed that the number of cells in G
was significantly
greater in these transfectants than in the mock transfectants (Fig. 1C). The D4 subclone, for example, exhibited
twice the percentage of total cells in G
compared with the
mock transfectants. However, subclones that expressed the
-
CYT subunit on the surface at levels comparable
with that of full-length
exhibited no increase in the
number of cells in G
. To extend these observations, the
doubling time for each of the subclones was determined (Table 1).
The doubling time for RKO cells, as well as for the mock transfectants,
is approximately 21 h. In contrast, the doubling times for subclones
that expressed full-length
ranged from 25 to 39 h,
and these times correlated with the level of
surface
expression (cf. Table 1and Fig. 1). However, the
-
CYT subclones exhibited doubling times similar
to the Neo subclones, i.e. 20-21 h. Neither DNA content
nor doubling times were affected significantly by growth of the
subclones on EHS laminin-1 or laminin-5 compared with tissue culture
plastic.
The results described above suggested that expression of
could induce partial G
arrest and possibly apoptosis in RKO cells. To examine this
possibility, we assayed ApopTag reactivity by FACS. ApopTag is a
fluorescein-conjugated antibody that recognizes digoxigenin-tagged
3`-OH DNA ends generated by DNA fragmentation, and its use for the
detection of apoptotic cells has been documented(20) . As shown
in Table 2, approximately 8% of the D4 transfectants and 3% of
the B8 transfectants were ApopTag positive. In comparison, fewer than
2% of the
-
CYT or mock transfectants were ApopTag
positive. Attachment to either EHS laminin-1 (data not shown) or
laminin-5 (Table 2) did not alter the pattern of ApopTag
staining. In addition, DNA fragmentation was evident in the B8 and D4
subclones but not in the
-
CYT transfectants (Fig. 2). These results indicate that a low, but significant,
rate of apoptosis occurs in the full-length
subclones.
Figure 2:
DNA fragmentation in the RKO
transfectants. DNA was extracted from the transfectants as described under ``Materials and
Methods'' and analyzed by agarose gel electrophoresis (1.2%). Lane1, 1-kilobase pair DNA ladder; lane2, 3C1, a
-
CYT subclone; lane3, B8, a full-length
subclone; lane4, D4, a full-length
subclone.
A possible mechanism for the observed changes in
growth that correlate with expression was suggested by the report that RKO cells express
relatively low levels of wild-type p53 (21) . Moreover, the
growth-suppressive function of p53 can result from its ability to
induce expression of p21 (WAF1; Cip1), an inhibitor of G
cyclin-dependent kinases(10, 11, 12) .
Based on these observations, we hypothesized that expression of the
integrin in RKO cells increases p21
expression. This hypothesis was assessed initially by immunostaining
using a p21-specific mAb. The results obtained revealed little p21
expression in either the Neo or
-
CYT subclones (Fig. 3, A and B). In these subclones, fewer
than 5% of the cells exhibited p21 staining. However, nuclear staining
of p21 was much more evident in both the B8 and D4 subclones (Fig. 3, C and D). The frequency of nuclear
staining was greater in the D4 subclone (30-40% cells stained)
than it was in the B8 subclone (15-20% cells stained), an
observation that correlates with the relative level of
expression in these two subclones.
The expression of p21 in the subclones was also assessed by
immunoblotting detergent extracts prepared from the
subclones. Relatively little p21 expression was evident in either
the Neo or
-
CYT subclones based on these
immunoblots (Fig. 3E). The level of p21 expression in
these subclones is consistent with other reports of cells that express
low levels of wild-type p53(12) . In contrast, a substantial
increase in p21 expression was seen in both the B8 and D4
subclones. Attachment to EHS laminin-1 did not alter this pattern
of p21 expression (Fig. 3E).
Figure 3:
Expression of p21 in the RKO
transfectants. A-D, detection of p21 by
immunohistochemistry. A mock transfectant subclone (A), a
-
CYT subclone (B), and two full-length
subclones B8 and D4 (C and D) were
plated on poly-L-lysine and stained with a p21-specific mAb as
described under ``Materials and Methods.'' Bar in A equals 50 µM. E, immunoblot detection
of p21. RKO transfectants were plated on either tissue culture plastic (lanes 1, 3, 5, 7, and 9) or EHS laminin-1 (lanes
2, 4, 6, 8, and 10) for 18 h. After detergent
extraction, the samples were normalized for protein content, resolved
by SDS-polyacrylamide gel electrophoresis (12%), transferred to
nitrocellulose, and blotted with the p21-specific mAb. Bound protein
was detected by enhanced chemiluminescence. Lanes1 and 2, Neo-24, a mock transfectant subclone; lanes3 and 4, Neo-26, a mock transfectant subclone; lanes5 and 6, 3C1, a
-
Cyt subclone; lanes7 and 8, B8, a full-length
subclone; lanes9 and 10, D4, a full-length
subclone.
We have shown that expression of the
integrin in RKO cells, a
-deficient carcinoma cell line, results in partial
G
arrest as well as the apoptotic death of some
transfectants. A possible mechanism for these observed changes in
growth is provided by our finding that
also induces expression of the G
cyclin-dependent
kinase inhibitor p21. The specificity of the observed induction of p21
expression is provided by the finding that the
-
CYT integrin failed to affect
p21 expression or growth even though it was expressed on the cell
surface at levels comparable with the full-length integrin. In
addition, we had previously observed that expression of either the
E-cadherin (22) or galectin-3 cDNAs (
)in RKO cells
had no effect on their growth.
Our finding that
can trigger apoptosis in a
population of transfectants is intriguing because our efforts to
isolate transfectants with
surface expression greater
than that observed in the D4 subclone, for example, were not
successful. These transfectants did not survive for more than a few
days after their selection by FACS. A reasonable interpretation for
these data is that lower levels of
surface expression induce expression of p21 sufficient to induce
partial G
arrest and some apoptosis and that higher levels
of
expression result in more p21
expression and more widespread apoptotic death.
The
-dependent increase in growth arrest and p21
expression does not appear to depend on attachment to either a
laminin-1 or laminin-5 matrix. For this reason, we suggest that the
cytoplasmic domain is linked constitutively to the
p21 pathway of growth arrest and apoptosis in RKO cells. This
possibility of constitutive activation of adhesion-dependent signaling
pathways in transformed cells is supported, for example, by the recent
report that the binding of focal adhesion kinase to SH2-containing
proteins in v-src-transformed 3T3 cells is
independent of integrin engagement and cell attachment(23) .
The results of this study raise the issue of the role of
in the regulation of normal cell
growth and apoptosis. The intestinal epithelium may provide a superb
model for examining such a relationship. In this epithelium,
undifferentiated cells at the base of the crypts proliferate rapidly
and give rise to differentiated enterocytes that migrate to the tips
where they are sloughed off into the intestinal lumen. Apoptosis occurs
at the tips and may provide the mechanism for cell loss in this
structure(24) . Interestingly, p21 is expressed in mature
enterocytes but not in undifferentiated crypt cells(25) , and
there is evidence that
is not
expressed in the undifferentiated crypt cells. (
)For these
reasons, the possibility of a functional relationship between
expression and p21 induction merits
investigation.
Several reports have implicated cell adhesion as well as specific adhesion receptors in growth control and apoptosis(26, 27, 28, 29, 30) . The significance of our findings is that they establish a link between the cytoplasmic domain of a specific integrin subunit and the expression of a molecule known to be critical for growth suppression, apoptosis, and tumorigenesis. These findings should facilitate the elucidation of the signaling pathway(s) that results in the induction of p21 expression by cell surface receptors.