(Received for publication, November 23, 1994 )
From the
Transforming growth factor- (TGF-
) inhibits cell
cycle progression of many types of human cells by arresting them in the
G
phase of the cell cycle. The arrest is mediated through
interactions of various cyclin-dependent protein kinases (CDKs) and
their inhibitors. We demonstrate that treatment with TGF-
induces
increased levels of WAF1/Cip1/p21, a potent inhibitor of
various cyclin-CDK kinase activities, in two colon cancer cell lines
(LS1034 and LS513), which are sensitive to TGF-
-induced growth
arrest. The induction in at least one of these cells lines (LS1034, p53-) is p53-independent. No WAF1 induction
was observed after TGF-
treatment in a third cell line (HT-29),
which is completely insensitive to the cytoinhibitory effect of
TGF-
. In both LS513 and LS1034, WAF1 induction correlated
with reduced cyclin E-associated kinase activity in vitro and
suppression of the retinoblastoma susceptibility gene (Rb)
protein phosphorylation in vivo. In addition, WAF1 was physically associated with cyclin E in the two sensitive cell
lines. These results suggest that WAF1/Cip1/p21 is a mediator
of cellular sensitivity to TGF-
.
In many mammalian cell types, TGF- (
)treatment
promotes growth arrest in G
(1, 2, 3) that is thought to be
mediated through the many elements of the complex cell cycle
machinery(4) . Among these elements, G
cyclin-CDK
complexes, which include cyclin E-CDK2, cyclin D-CDK4, and cyclin
D-CDK6, play important roles. It is now widely accepted that these
complexes are needed during G
to phosphorylate and thus
inactivate the retinoblastoma susceptibility gene (Rb), which
inhibits cell cycle progression in its underphosphorylated
state(4, 5) . It is thought that TGF-
causes
G
arrest by inhibiting G
cyclin-CDK kinase
activities, thereby suppressing Rb phosphorylation. Consistent
with this model, it has been shown that TGF-
can inhibit cyclin
E-CDK2 kinase activity (6) and cause the accumulation of Rb in the underphosphorylated form(7, 8) . In
addition, it has been reported that TGF-
treatment reduces the
level of CDK4 and that overexpression of the CDK4 gene precludes the
TGF-
cytoinhibitory effect in a sensitive cell line(9) .
The exact mechanism by which TGF-
inhibits CDK-associated kinase
activities has remained elusive, however, until the recent discoveries
of several mammalian CDK kinase inhibitors, which include WAF1 (also called p21/Cip1/Sdi1)(10, 11, 12, 13) , p16
(14, 15) , p27
(16, 17) , and p15
(18) . Two of these, p27
and p15
, have been implicated as
effectors of TGF-
-induced cell cycle arrest (16, 18) .
We sought to examine whether WAF1 is involved in mediating TGF--induced growth arrest in human
cells, as it is a potent inhibitor of a wide range of cyclin-CDK
complexes. To this end we employed three colon cancer cell lines
previously characterized as having varying degrees of sensitivity to
the cytoinhibitory effect of TGF-
1(19) : HT-29, which is
completely insensitive; LS513, which is moderately sensitive; and
LS1034, which is very sensitive to TGF-
1.
Dose-response curves for treatment of the three colon cancer
cell lines with TGF-1 are shown in Fig. 1. As can be seen
in Table 1, SELISA measurement of TGF-
1 production revealed
an inverse relationship between TGF-
1 sensitivity and its
production in these cells. However, the correlation may not be of
functional significance as none of the TGF-
1 produced was in its
biologically active form(19) . Other characteristics of these
cell lines, including their p53 status and cell cycle analysis
after treatment with TGF-
1 and ionizing radiation, are also shown
in Table 1.
Figure 1:
TGF- sensitivity of the three
colon cancer cell lines. The methods employed to measure the
cytoinhibitory effects of TGF-
in such cell lines have been
described in detail elsewhere(19) .
In order to determine sensitivity of these cell
lines to the induction of a G arrest, cell cycle analysis
after exposure to either TGF-
1 or ionizing radiation exposure was
carried out by analysis of fluorescence-activated cell sorting (Table 1). HT-29 demonstrated no G
arrest after
either TGF-
1 or radiation exposure, whereas LS513 showed a
significant G
arrest after
-irradiation but only a
moderate G
arrest after TGF-
1 treatment. LS1034 showed
no G
arrest after irradiation but a significant G
arrest in TGF-
1-treated cells. The results obtained with
ionizing radiation is consistent with previous reports that p53 status determines whether a G
arrest will
occur(22) . The TGF-
-induced G
arrest observed
in LS1034 and its sensitivity to TGF-
in the absence of p53 indicate that the TGF-
cytoinhibitory effect in this cell
line is not mediated by p53.
Cells exposed to either
radiation or TGF-1 were assayed for WAF1 mRNA expression
by Northern blot analysis (Fig. 2A). Almost no WAF1 mRNA was observed after either radiation or TGF-
1 exposure in
HT-29 cells, while in LS513 there was a clear induction of WAF1 mRNA in irradiated cells, consistent with the role of p53 as the positive transcriptional regulator of the WAF1 gene after irradiation. A small induction of WAF1 mRNA
was observed in LS513 after exposure to TGF-
1. In LS1034, on the
other hand, a significant induction occurred after TGF-
1
treatment, whereas no WAF1 expression was observed after
irradiation (Fig. 2A). As p53 is mutated in
LS1034, we conclude that the induction of WAF1 in this cell
line is p53-independent. Western analysis of the three cell
lines confirmed the Northern analysis (Fig. 2B). A time
course study of the induction of WAF1 expression in LS1034 by
TGF-
1 treatment was also performed (Fig. 3). As can be seen
in Fig. 3, induction is clearly visible beginning at 3 h after
exposure to TGF-
1, reaching its peak level by 6 h. Induction
remained steady up to at least 48 h, dropping to near background level
at 96 h.
Figure 2:
A, Northern analysis of WAF1/p21 mRNA level in cell lines HT-29, LS513, and
LS1034. For radiation exposure, the cells were given doses of 2 and 10
gray (Gy). For TGF- treatment, the cells were incubated
with 60 ng/ml TGF-
1. RNA were extracted 5 h after exposure to
either radiation or TGF-
. Twenty µg of the total RNA were
loaded into each lane for analysis. Equal loading of the RNAs
is indicated in the lowerpanel, in which the 18 S
ribosomal RNA band is shown prior to capillary transfer to a
nitrocellulose filter (Schleicher and Schuell). B, Western
analysis of WAF1 protein levels. Proteins were isolated at the
same time when RNA was isolated. About 50 µg of protein was loaded
into each lane.
Figure 3:
A Northern analysis of the time course of
induction of WAF1 mRNA by TGF-1. RNA was extracted at
various time points after incubation with TGF-
(as indicated at
the top of each lane). Twenty µg of the total RNA
were loaded into each lane for
analysis.
As WAF1 has been shown to be induced by radiation
and implicated as an inhibitor of cyclin kinase activities in
irradiated cells(23, 24) , the cyclin E-CDK2 complex
was assayed for its kinase activity after immunoprecipitation against
cyclin E by use of histone H1 as the substrate. As can be seen in Fig. 4, no reduction in the kinase activity of the complex was
observed after irradiation in the HT-29 cell line. In LS513, the
activity of the cyclin complex was reduced significantly by radiation
and to a lesser extent by TGF-1 treatment. In LS1034, the kinase
activity was almost completely abolished after TGF-
1 treatment,
while the change was much less significant in the irradiated cells.
Figure 4:
Histone H1 kinase activity of the cyclin E
complex after 16 h of either radiation or TGF-
treatment.
Immunoprecipitation of [S]methionine-labeled
cell lysates using a monoclonal antibody against cyclin E indicated
that a protein of molecular mass 21 kDa (as determined by
polyacrylamide gel electrophoresis) coprecipitated with cyclin E in
both LS1034 and LS513 but not in HT-29 cells (data not shown).
In
order to further examine cyclin-CDK kinase activity and to determine if
TGF-1 sensitivity of these colon cancer cell lines is mediated
through the Rb protein, Western analysis was performed using a
monoclonal antibody that recognizes both the underphosphorylated and
hyperphosphorylated forms of Rb. As can be seen in Fig. 5, the Rb protein was in both the hyper- and
hypophosphorylated forms in the untreated control cultures of all three
cell lines, with the majority in the hyperphosphorylated form. This is
consistent with the fact that most of the cells are actively
proliferating. In HT-29, which has a mutated form of p53 and
is insensitive to TGF-
1, Rb remained in the
hyperphosphorylated form in the majority of the cells after both
TGF-
1 and radiation exposure, similar to control untreated cells (Fig. 5). In LS513, most of the cells showed an
underphosphorylated form of the Rb gene product 16 h
postirradiation, which is consistent with the wild type p53 status of the cell line. After TGF-
1 treatment, a significant
albeit smaller increase in the amount of the underphosphorylated form
of Rb was observed, which correlates well with the moderate
sensitivity of the cell line. In LS1034, however, the majority of the
cellular Rb protein is converted into the underphosphorylated
form after TGF-
1 treatment, while no significant change in the
distribution of Rb was observed after radiation (Fig. 5). Thus, the mechanisms of growth arrest induced by both
ionizing radiation and TGF-
1 are consistent with a model in which
both WAF1 and Rb are essential players.
Figure 5:
Western analysis of the retinoblastoma
protein (Rb). In each cell line, there were three different
treatment groups: control, TGF--treated, or irradiated. The
protein was extracted 16 h after radiation and TGF-
exposure. A
monoclonal antibody to Rb (G1-245) from Pharmingen was
used as the primary antibody. The polyacrylamide gel was used at
8%.
Northern
analysis of p16 mRNA expression revealed no
correlation with either the p53 status or cellular sensitivity
to TGF-
1 (data not shown). In fact, the mRNA expression of the p16
gene was detected only in HT-29, which is
completely insensitive to TGF-
1 and has no normal p53 activity. Expression was not altered in either radiation or
TGF-
1-treated cells. Experiments are also under way to test
whether the newly identified p27
and p15
kinase inhibitors are involved in
mediating the TGF-
sensitivity of these colon cancer cell lines.
It is possible that all these factors participate in mediating the
cellular sensitivity to TGF-
1 by inhibiting different cyclin-CDK
complexes.
In summary, these results provide evidence that
TGF-1 can induce WAF1 in a manner that correlates with
its cytoinhibitory effect and that cell cycle arrest induced by both
radiation and TGF-
1 appears to be mediated through WAF1,
but the upstream signal transduction pathways are different as the
former is mediated through p53 and the latter not. Further
understanding of the various cell cycle regulators involved will
greatly facilitate our understanding of the complex pathways
constituting cellular sensitivity to TGF-
.