(Received for publication, July 25, 1995; and in revised form, October 18, 1995)
From the
Interleukin-1 (IL-1) causes G/G
phase
growth arrest in human melanoma cells, A375-C6. Because
hypophosphorylation of the retinoblastoma susceptibility gene product,
RB, is one of the key events responsible for G
/G
phase growth arrest, we investigated whether IL-1 altered the
phosphorylation status of RB protein in these cells. Exposure to IL-1
caused a time-dependent increase in hypophosphorylated RB that
correlated with an accumulation of cells arrested in the
G
/G
phase. The ability of IL-1 to cause
hypophosphorylation of RB and growth arrest was abrogated by the SV40
large T antigen, which binds preferentially to hypophosphorylated RB,
but not by the K1 mutant of the T antigen, which is defective in
binding to RB. Furthermore, the cells were protected from
IL-1-inducible growth inhibition by ectopic expression of
dominant-negative mutants of the Rb gene, or the transcription
factor E2F-1, which is a downstream target of RB. These results suggest
that hypophosphorylated RB mediates the growth arrest induced by IL-1.
Interleukin-1 (IL-1) ()and tumor necrosis
factor-
(TNF-
) are two important cytokines secreted primarily
by activated macrophages and
monocytes(1, 2, 3, 4, 5) .
These cytokines elicit diverse biological effects ranging from those
that are beneficial to the host, such as recruitment of immunological
and anti-tumor responses, to those that may have deleterious
consequences, such as cell proliferation and tissue injury, and
inflammatory responses that may lead to toxic shock and
sepsis(1, 2, 3, 4, 5) . In
cell culture, these cytokines show pleiotrophic effects on the growth
of mammalian cells. The growth regulatory effects of IL-1 and TNF-
are cell type-dependent: the cytokines stimulate the growth of
nontransformed cells such as fibroblasts (6, 7) and
kidney epithelial cells (8) and transformed cells, such as
glioblastoma (8, 9, 10) and renal
carcinoma(11) , but inhibit the growth of certain
nontransformed fibroblast cells(12) , and tumor cells, such as
melanoma(8, 13) , breast carcinoma(14) , lung
adenocarcinoma(15) , ovarian carcinoma (16) , and
myeloid leukemia(17, 18) . Although these cytokines
show overlapping biological activities, their action is mediated by
distinct cell surface receptors(19, 20, 21) .
Signal transduction and phenotypic alterations by IL-1 are mediated via
IL-1-receptor type I(22, 23, 24) , whereas
two different TNF-
receptors mediate distinct phenotypic responses
to TNF-
(25) . The action of each of these cytokines is
associated with diverse second messengers in the cytosol, which in turn
activate DNA binding proteins, particularly AP-1 and NF-
B, that
lead to induction of a number of immediate-early and delayed response
genes (cited in Refs. 24, 26, and 27). The mechanism of action of these
cytokines is, however, not known.
To understand the molecular basis
for the pleiotrophic effects of IL-1 on the growth of human cells, we
are studying the gene programs associated with the actions of this
cytokine(13) . Human melanoma cells, A375-C6, show growth
arrest in response to IL-1 or TNF- and provide an excellent
experimental model for exploring the molecular pathways that are
initiated by the binding of these cytokines to the cognate cell surface
receptors and that lead to a dose- and time-dependent growth arrest
response(8, 13, 24) . The action of IL-1 and
TNF-
in the melanoma cells is dependent on induction of
immediate-early genes(13) , which are expected to initiate a
cascade of events leading to growth arrest. Fluorescence-assisted cell
sorting (FACS) analysis has revealed that IL-1 causes growth arrest in
the G
/G
phase of the cell cycle (24) .
Studies on cell cycle regulation have identified the product, RB, of
the retinoblastoma susceptibility gene, Rb, as a key
checkpoint control protein responsible for imposing a block in the cell
cycle at the transition from the G to the S
phase(28, 29) . Mutations that functionally inactivate
the Rb gene have been identified in a variety of human
tumors(30) . Moreover, ectopic expression of the wild-type Rb gene can suppress the growth of tumor cells in culture or
the formation of tumors in animal models (31, 32) .
The characterization of RB as a tumor suppressor has been further
consolidated by the observations that three different transforming
proteins encoded by DNA tumor viruses, adenovirus E1A, papovavirus
large T antigen, and papillomavirus E7 protein, can bind to the RB
protein at a domain called the A/B pocket and that the transforming
functions of these proteins are linked to their ability to bind to RB
protein(33, 34, 35, 36) . The RB
protein is differentially phosphorylated during cell cycle
progression(37, 38, 39, 40, 41) .
It is unphosphorylated (RB) during the early and mid G
phase, then phosphorylated by cyclin/cyclin-dependent kinase
complexes during late G
and further in the S phase. Late in
mitosis, it is enzymatically dephosphorylated, and cells entering the
next G
phase contain primarily RB. These observations
coupled with the facts that the large T antigen preferentially binds to
RB and induces G
-arrested cells to enter the S
phase(42) , suggest that RB causes G
phase growth
arrest and RB phosphorylation is linked to rescue from such an arrest.
The A/B pocket domain of RB can bind and inhibit the function of
several cell cycle regulators such as the E2F family of transcription
factors(28, 33, 35, 36, 43, 44, 45, 46) .
The ability of RB protein to arrest the cell cycle depends on sequences
necessary for its interaction with
E2F(28, 33, 35, 36, 43, 44, 45, 46) .
Ectopic overexpression of E2F-1, a member of the E2F-family, can rescue
cells from RB-imposed G phase growth arrest, presumably by
transactivating E2F-1 target genes that are necessary for entry and
progression through the S phase (47, 48, 49) . Moreover, a recent study has
identified a carboxyl-terminal region within the A/B pocket domain,
designated the C pocket, which binds to the tyrosine kinase,
c-Abl(50, 51) . RB can simultaneously bind to c-Abl
and E2F; however, in the absence of RB protein, c-Abl does not bind to
E2F(51) . In this manner, RB acts a molecular
``matchmaker'' bringing together proteins that would not
otherwise interact. Cotransfection studies suggest that coexpression of
full-length RB along with an RB deletion mutant designated SE, which
contains only the C pocket region(51) , disrupts the ability of
RB to suppress growth. The SE mutant binds to, and inhibits the
function of, E2F, c-Abl, and perhaps other as yet unknown proteins.
Despite inhibition of these protein activities, the SE mutant protects
the cells from RB-imposed growth arrest(51) . In other words,
RB acts as a growth suppressor only when the A/B and the C pockets are
in the cis configuration, suitable for molecular matchmaking,
and RB function is inhibited by coexpression of the C pocket domain.
Thus, the SE mutant acts as a dominant-negative inhibitor of RB
function.
Because IL-1 and TNF- often induce overlapping signal
transduction and phenotypic effects, and because IL-1 causes cell cycle
arrest in the G
/G
phase, we sought to examine
whether their action is mediated by RB. Data presented here suggest
that growth arrest by IL-1 is dependent on RB function, but that by
TNF-
is mediated by an RB-independent pathway.
Figure 1:
IL-1 or TNF- cause
hypophosphorylation of RB, which correlates with accumulation of cells
in G
/G
phase of the cell cycle. a-c, A375-C6 cells were left untreated (UT) or
treated with IL-1 (100 units/ml) or TNF-
(100 units/ml) for 24,
48, or 72 h. The cells were harvested, and either whole cell protein
extracts were prepared and 20 µg of the indicated extract protein
was subjected to Western blot analysis using the anti-RB antibody, C15 (a) or the cells were subjected to FACS analysis, to determine
the percentage of cells in the G
/G
(b)
or the S phase (c) at each exposure time. d,
phosphorylation status of RB protein. Extracts from cells exposed to
IL-1 for 48 h were treated in vitro with vehicle (v)
or potato acid phosphatase (PAP) for 15 min and then subjected
to Western blot analysis using the anti-RB antibody. These experiments
used protein extracts from cells exposed to IL-1 for 72 h as a control
for the fast-migrating RB species seen with IL-1 treatment. The
slow-migrating differentially phosphorylated forms of RB (pRB)
and the fast-migrating unphosphorylated form of RB (RB) are
indicated (a and d).
Studies in other cell types have attributed differences in
the migration of RB protein in gel electrophoresis to the extent of
underphosphorylation of the protein(52) . Since the RB protein
is completely dephosphorylated upon treatment with potato acid
phosphatase, such a treatment has been used to identify
unphosphorylated RB(52) . To help determine whether the slow-
and fast-migrating forms of RB represent phosphorylated and
differentially underphosphorylated forms, respectively, of RB, we
treated the protein extract from A375-C6 cells that had been exposed to
IL-1 for 48 h, with vehicle or potato acid phosphatase, and then
subjected these extracts to Western blot analysis. Data shown in Fig. 1d indicated that the vehicle-treated extract
contained both the slow- and fast-migrating forms of RB (Fig. 1d), similar to those seen after 48-h exposure to
IL-1 (compare with Fig. 1a). On the other hand,
treatment of the extract with potato acid phosphatase resulted in
disappearance of the slow-migrating species and simultaneous appearance
of a fast-migrating species (Fig. 1d). This
fast-migrating species of RB protein that formed after potato acid
phosphatase treatment of the extract comigrated with the fast-migrating
species of RB protein that was seen in the extract from cells treated
with IL-1 for 72 h (Fig. 1d). These data indicate that
the slow-migrating forms of RB represent differentially phosphorylated
RB (pRB), whereas the fast-migrating form represents unphosphorylated
RB (RB). Similar observations were made when extracts from
TNF--treated cells were subjected to potato acid phosphatase (data
not shown). These results indicate that IL-1 and TNF-
cause a
time-dependent hypophosphorylation of RB.
Figure 2:
Overexpression of SV40 large T antigen
rescues cells from IL-1- or TNF--inducible growth inhibition. a, transfectant cells express T antigen or K1 mutant. A375-C6
cells were transfected with pSG5-SV40 large T antigen plasmid encoding
the SV40 large T antigen, or pSG5-K1 plasmid encoding the K1 mutant of
T antigen, and stable transfectant cell lines were selected. Protein
extracts from cell lines C6/T.L1 or C6/K1.L1 transfected with
constructs encoding the T antigen or the K1 mutant, respectively or
parent A375-C6 cells (C6) for a control were subjected to
Western blot analysis using an anti-T antigen antibody. The bands
corresponding to the large T antigen and K1 mutant are indicated. b, transfectant cells expressing T antigen do not show
increased hypophosphorylation of RB in response to IL-1 or TNF-
.
The transfectant cells were left untreated (UT) or treated
with IL-1 or TNF-
for 48 or 72 h as indicated, and whole cell
protein extracts were examined by Western blot analysis for RB
expression. As a control for the unphosphorylated form of RB, we used a
protein extract (50 µg of protein) from A375-C6 cells (C6)
which were exposed to IL-1 for 72 h (right panel). The
differentially phosphorylated (pRB) and unphosphorylated (RB) forms of RB are shown. C6/T.L1 cells (right
panel), which were left untreated or treated with IL-1 or
TNF-
, expressed primarily pRB, whereas K1.L1 cells (left
panel) showed hypophosphorylation of RB protein upon exposure to
IL-1 or TNF-
. c, overexpression of T antigen abrogates
the growth arrest response to IL-1 or TNF-
. The transfectant cell
lines were left unexposed or exposed to IL-1 or TNF-
for 48 or 72
h, and the effect on growth was examined by
[
H]thymidine incorporation studies. Percent
growth inhibition is expressed as a function of cytokine exposure
time.
To address whether ectopic
overexpression of the T antigen or K1 mutant affected the RB
phosphorylation status in response to IL-1 or TNF-, the
transfectant cell lines, confirmed to express the large T antigen or
the K1 mutant, were left unexposed or exposed to IL-1 or TNF-
for
48 or 72 h, and whole cell protein extracts were examined by Western
blot analysis for RB expression. Because the extracts from the SV40 T
antigen transfected cells were resolved on a separate gel, we used
protein extract from nontransfected A375-C6 cells exposed to IL-1 for
72 h, as a control for the fast-migrating unphosphorylated form of RB (Fig. 2b, right panel). As seen in Fig. 2b (right panel), the control extract from A375-C6 cells
exposed to IL-1 for 72 h primarily showed the fast-migrating
unphosphorylated form of RB. On the other hand, C6/T.L1 transfectant
cells, primarily expressed the phosphorylated form of RB protein
whether or not they were exposed to IL-1 or TNF-
for 72 h (Fig. 2b, right panel). By contrast, C6/K1.L1
transfectant cells showed hypophosphorylation of RB upon exposure to
IL-1 or TNF-
for 48 or 72 h (Fig. 2b, left panel).
Each of three different transfectant lines expressing the T antigen or
K1 mutant were examined in this manner by Western blot analysis for RB
phosphorylation status, and the results (not shown) were similar to
those presented in Fig. 2b. These studies indicated
that the SV40 large T antigen inhibited the ability of IL-1 or
TNF-
to evoke hypophosphorylation of RB.
Because the large T
antigen preferentially binds and sequesters
RB(35, 52) , and overexpression of T antigen resulted
in lack of RB accumulation in response to IL-1 (Fig. 2b), we reasoned that if RB was involved in
IL-1-inducible growth inhibition, overexpression of the T antigen would
protect the cells from IL-1 action. To test this hypothesis, the
transfectant cell lines that expressed the large T antigen or K1 mutant
were left unexposed or exposed to IL-1 or TNF- for 48 or 72 h, and
the effect on growth was examined by [
H]thymidine
incorporation studies. The transfectant cell line, C6/K1.L1, which
expressed the K1 mutant, showed a time-dependent growth arrest response
to the cytokines: in response to IL-1, the cells showed about 50 and
80% growth inhibition in 48 and 72 h, respectively; and in response to
TNF-
, these cells showed about 45% and 80% growth inhibition in 48
and 72 h, respectively (Fig. 2c). The extent of growth
inhibition resulting from exposure of C6/K1.L1 cells to IL-1 or
TNF-
for 48 or 72 h was similar to that seen in parent A375-C6
cells exposed to IL-1 or TNF-
for the corresponding time periods
(data not shown and (8) ). On the other hand, the transfectant
cell line, C6/T.L1, which expressed the SV40 large T antigen, was
relatively resistant to IL-1, i.e. about 20 and 35% growth
inhibition was seen in 48 and 72 h, respectively, and to TNF-
, i.e. about 20 and 30% growth inhibition was seen in 48 and 72
h, respectively (Fig. 2c). These data suggest that the
SV40 large T antigen can rescue the cells from the growth arresting
actions of IL-1 and TNF-
.
Figure 3:
SE protects A375-C6 cells from
IL-1-inducible growth arrest. a, transfectant cells express Rb-deletion plasmids. A375-C6 cells were transfected with
plasmids encoding SE, SE
, or ME, and G418 sulfate-resistant
transfectant cell lines were selected. Whole cell protein extracts were
prepared from parent A375-C6 cells (C6) or the transfectant
cell lines SE.L1 (C6/SE), SE
.L1 (C6/SE
),
and ME.L1 (C6/ME) and subjected to Western blot analysis using
the anti-RB antibody, C15. Arrows indicate position of SE or
SE
(upper arrow;
18 kDa) and ME (lower
arrow;
12 kDa) proteins. b and c, SE or
SE
abrogates IL-1-inducible growth arrest. The transfectant cell
lines, SE.L1, SE
.L1 or ME.L1, were left unexposed or exposed to
TNF-
(b) or IL-1 (c) for 48 or 72 h, and the
effect on growth was examined by [
H]thymidine
incorporation studies. Percent growth inhibition in the transfectant
cell lines is expressed as a function of cytokine exposure time. d, IL-1 causes hypophosphorylation of RB in SE
transfectant cells. SE
.L1 cells were left untreated (UT)
or treated with IL-1 for 48 or 72 h, and whole cell protein extracts
were subjected to Western blot analysis for RB protein. The
phosphorylated (pRB) and unphosphorylated RB (RB)
forms are shown.
To determine whether ectopic
expression of SE, SE, or ME could rescue the cells from growth
arrest by IL-1 or TNF-
, the transfectant cell lines, SE.L1,
SE
.L1, or ME.L1, were left unexposed or exposed to IL-1 or
TNF-
for various intervals of time, and the effect on growth was
examined by [
H]thymidine incorporation studies.
ME.L1 cells showed growth inhibition in response to IL-1 and TNF-
with kinetics similar to those seen in parent A375-C6 cells (Fig. 3, b and c). Similarly, the growth of
SE.L1 and SE
.L1 transfectant cells was inhibited by TNF-
(Fig. 3b). By contrast, SE.L1 and SE
.L1
transfectant cells were relatively resistant to the growth arresting
action of IL-1: a maximum of about 25-35% growth arrest was seen
in 48 or 72 h (Fig. 3c). Three different transfectant
cell lines for SE, SE
, and ME were examined for expression of the Rb derivatives and susceptibility to IL-1 or TNF-
, and
the observations (data not shown) were similar to those described for Fig. 3, a-c. These results suggest that the
dominant-negative mutants of RB, SE, and SE
, protect cells from
IL-1 but not from TNF-
action. Thus, RB plays a role in the
growth-arresting action of IL-1, but may be a dispensable component of
the TNF-
-inducible growth arrest pathway. The accumulation of
hypophosphorylated RB upon exposure to TNF-
, therefore, seems to
be a consequence of G
/G
phase growth arrest
mediated by another checkpoint control protein. Further studies will
examine whether the RB-related proteins, p107 or p130, mediate the
action of TNF-
in these cells.
To determine whether ectopic
expression of SE, which protected the cells from IL-1- but not
TNF-
-inducible growth inhibition, affected the phosphorylation
status of RB in response to the cytokines, SE
.L1 cells were left
unexposed or exposed to IL-1 for 48 or 72 h, and whole cell protein
extracts were examined by Western blot analysis for RB expression. As
seen in Fig. 3d, IL-1 caused a time-dependent
hypophosphorylation of RB in the SE
.L1 cells, in a manner similar
to that seen in the parent cells (compare with Fig. 1). Since
IL-1-inducible growth arrest was abrogated in SE
.L1 cells, these
results suggest that SE
provides protection from IL-1 action
without interfering with the ability of the cytokine to cause
hypophosphorylation of RB.
Figure 4:
Ectopic expression of E2F-1 rescues
A375-C6 cells from IL-1 action. a, transfectant cells express
full-length E2F-1 or E2F-1(1-368) deletion protein. A375-C6 cells
were left nontransfected or were transfected with the full-length E2F.1
expression construct or the transactivation-deficient deletion
construct E2F-1(1-368), and stable transfectant cell lines
E2F-1.L1 and E2F-1(1-368).L1 were selected. Protein extracts were
prepared from parent A375-C6 cells (C6) or the transfectant
cell lines C6/E2F-1.L1 and C6/E2F-1(1-368).L1 and subjected to
Western blot analysis, using anti-E2F-1 antibody. Arrows indicate position of E2F-1 (upper thick arrow) and
E2F-1(1-368) mutant (lower thin arrow). The right and left panels represent different immunoblots. b and c, overexpression of E2F-1 abrogates IL-1- or
TNF--inducible growth inhibition. Transfectant cells, E2F-1.L1 and
E2F-1(1-368).L1, were exposed to IL-1 or TNF-
for 24, 48, or
72 h and then subjected to [
H]thymidine
incorporation assays. Percent growth inhibition is expressed as a
function of IL-1 or TNF-
exposure
time.
We then exposed the transfectant cells to
IL-1 or TNF- for 24, 48 or 72 h and performed
[
H]thymidine incorporation experiments, to
determine their susceptibility to the cytokines. The E2F-1 transfectant
cell line, E2F-1.L1, showed 5, 10, or 30% growth inhibition in response
to IL-1 in 24, 48, or 72 h, respectively (Fig. 4b), and
<5, 15, or 35% growth inhibition in response to TNF-
in 24, 48,
or 72 h, respectively (Fig. 4c). On the other hand, the
E2F-1(1-368) transfectant cell line E2F-1(1-368).L1 showed
about 20, 50, or 80% growth inhibition in response to the cytokines in
24, 48, or 72 h, respectively (Fig. 4, b and c). Three different transfectant cell lines for E2F-1 or
E2F-1(1-368) were examined in this manner and the results (not
shown) were similar to those described above in Fig. 4, a-c. These data suggest that ectopic expression of E2F-1
can rescue the melanoma cells from growth arrest by IL-1 and are
consistent with the view that RB plays a role in IL-1 action. However,
the rescue of cells expressing E2F-1 from the growth arresting action
of TNF-
is particularly interesting, because the data in Fig. 3suggest that the action of this cytokine is not mediated
via RB hypophosphorylation. It is possible, therefore, that the action
of TNF-
is mediated by an RB-related protein, perhaps p107, p130,
or another checkpoint control protein whose expression or function may
be modulated or circumvented by expression of E2F-1. As the
transactivation-deficient mutant of E2F-1 did not rescue the cells from
IL-1 or TNF-
action, the data imply that the transactivation
function of ectopically expressed E2F-1 is required for protection from
IL-1-evoked hypophosphorylation of RB or TNF-
-mediated growth
arrest via an RB-related protein.
The present study revealed that in melanoma cells, A375-C6,
IL-1, or TNF-, invoke hypophosphorylation of RB, and the extent of
hypophosphorylation correlates with inhibition of growth and
accumulation of cells in the G
/G
phase of the
cell cycle. Since un(der)phosphorylated RB has been implicated in the
G
/G
phase growth arrest caused by another
cytokine, transforming growth factor-
(52, 53) ,
we sought to determine whether RB was functionally involved in the
action of IL-1 or TNF-
. We began by asking whether the SV40 large
T antigen, which has been shown to preferentially overcome the growth
suppressor action of hypophosphorylated RB, could abrogate the growth
arresting action of the cytokines. Previous studies (52, 53) evaluating the role of hypophosphorylated RB
in TGF-
-inducible G
/G
growth arrest in
other cell types have shown that the SV40 large T antigen can rescue
the cells from the G
/G
block. The rescue action
of the large T antigen is attributed to its ability to displace the key
proteins, particularly those of the E2F family, that are sequestered in
the A/B pocket domain by hypophosphorylated RB, to sequester
hypophosphorylated RB, and to trigger the phosphorylation of
RB(35, 52) . Our studies in the melanoma cells
indicated that the large T antigen, but not the K1 mutant, which cannot
bind to RB, could rescue the cells from the action of IL-1 or
TNF-
. This observation suggested that hypophosphorylated RB, a
target of the large T antigen, was likely to be involved in the action
of these cytokines. However, because the large T antigen is known to
bind to other key G
/G
checkpoint control
proteins, such as the RB-related proteins p107 and p130, the
experiments with the large T antigen did not conclusively identify RB
as a functional mediator of either IL-1 or TNF-
action. To
definitively identify RB as a mediator of IL-1- or TNF-
-inducible
growth arrest, we used dominant-negative mutants of RB. The SE
mutant selectively inhibits the function of RB, but not of the other
key G
/G
checkpoint control proteins, by
interfering with the ability of RB to act as a molecular matchmaker and
assemble protein complexes(51) . Ectopic expression of SE
abrogated the ability of IL-1, but not of TNF-
, to cause growth
arrest in the melanoma cells, implying that RB is an important mediator
of IL-1-inducible growth arrest. However, because growth arrest by IL-1
was not completely blocked by SE
, we cannot exclude the
possibility that other checkpoint control proteins are also involved in
IL-1 action. By contrast, IL-1 causes growth stimulation in human
fibroblast cells, WI-38, or human glioblastoma cells, U373-MG, and does
not invoke hypophosphorylation of RB. (
)The cell surface
receptor, IL-1-receptor type I, is involved in both the growth
inhibitory and growth stimulatory actions of IL-1 in different cell
lines(22, 23, 24) . The induction of RB
hypophosphorylation in the melanoma cells, but not in the fibroblasts
or glioblastoma cells, suggests that cell type-specific differences
characterized by the signaling components that are located downstream
of the receptor and that lead to the differential regulation of RB
phosphorylation are responsible for the pleiotrophic growth effects of
IL-1. Further studies directed toward identifying the individual
components of the signaling pathway responsible for hypophosphorylation
of RB will help delineate the molecular basis of the pleiotrophic
growth effects of IL-1.
The mechanism(s) by which RB mediates growth arrest is an area of intense investigation. The dominant-negative mutants of RB disrupt the capacity of RB to form protein complexes, but they do not alter the hypophosphorylation status of RB ((51) ; this study), nor do they restore E2F transactivation function in transfectant cells(51) , suggesting that rescue from hypophosphorylated RB-mediated growth inhibition can occur in the absence of functional E2F. Nevertheless, ectopic overexpression of one of the RB-target proteins, E2F-1, provides protection from the growth inhibitory action of hypophosphorylated RB ((46) ; this study). It is obvious, therefore, that the dominant-negative mutants of RB and ectopically overexpressed E2F-1 use different mechanisms to overcome the effect of hypophosphorylated RB. This hypothesis is being tested so as to elucidate the mechanism(s) by which IL-1-inducible accumulation of hypophosphorylated RB causes growth inhibition.
Although the
biological actions of IL-1 and TNF- are initiated by the binding
of the cytokines to distinct cell surface receptors (22, 23, 24, 25) , these cytokines
generally show overlapping biological activities and the induction of
similar immediate-early gene programs (13) . However, by using
the dominant-negative mutants of RB, the present study has revealed
that the G
/G
growth arresting action of IL-1 is
dependent on the hypophosphorylation of the RB protein, but the action
of TNF-
is independent of RB hypophosphorylation. Because E2F-1
overexpression rescues the cells from the action of TNF-
, an
RB-related protein like p107 or p130 may possibly mediate the action of
this cytokine. The ability of the dominant-negative mutants of RB to
selectively overcome the action of IL-1, and not that of TNF-
,
indicates the usefulness of these mutants in dissecting the growth
inhibitory pathways regulated by RB and other related checkpoint
control proteins.