(Received for publication, June 13, 1995; and in revised form, July 28, 1995)
From the
The ability of p53 species (wild-type and mutant) to modulate
the ``differentiated'' response of human hepatoma cell lines
Hep3B and HepG2 to interleukin-6 (IL-6) was investigated. Transient
transfection experiments were carried out in Hep3B and HepG2 cell
cultures in which IL-6 was used to activate a -fibrinogen
(
Fib) enhancer/reporter construct containing two copies of the
36-base pair IL-6-response element (IL-6RE) (p
FibCAT).
Cotransfection with constitutive expression vectors for wild-type (wt)
human or murine p53 inhibited the activation of the p
FibCAT
reporter by IL-6 in both Hep3B and HepG2 cells. Several mutant p53
species either did not inhibit the activation of p
FibCAT or
up-regulated the response. Hepatoma cell lines stably expressing the
Val-135 temperature-sensitive mutant of murine p53 (wt-like at 32.5
°C and mutant-like at 37 °C) were derived from Hep3B cells and
tested for the temperature-sensitive phenotype of their ability to
synthesize and secrete fibrinogen and
-antichymotrypsin in response to IL-6. In an
experimental protocol in which the parental Hep3B cells did not show a
significant difference in plasma protein secretion at the two
temperatures, hepatoma line 3 (p53Val-135
) had a
greater response to IL-6 at 37 °C than parental Hep3B cells, while
line 3 cells had a reduced response to IL-6 at 32.5 °C. Similarly,
hepatoma lines 1 and 2 (both p53Val-135
) had reduced
IL-6 responsiveness at 32.5 °C, whereas line 22 (transfected with
pSVneo alone) and the parental Hep3B cells did not. These data indicate
that mutations in p53 contained in tumor cells can modulate the
``differentiated'' response of these cells to cytokines.
Mutations in the transcription factor p53 are among the
commonest alterations observed in human cancer(1) . These tumor
cell-derived mutations in p53 can reflect both an abrogation of the
function(s) of wild-type (wt) ()p53 as well as
``gain-in-function''
mutations(2, 3, 4, 5, 6, 7) .
Mutational ``hot-spots'' have been demarcated within p53
derived from particular cancers, e.g. hepatocellular
carcinoma(1, 3) . The biological functions of p53 and
their alterations by mutations have been largely discussed in the
context of the regulation of cell proliferation, of apoptosis, and of
repair of DNA damage(1, 3, 5) . We have
investigated the ability of p53 species to modulate the
``differentiated'' function of tumor cells treated with
cytokines in a context distinct from that of the regulation of cell
proliferation or cell death. Can mutations in p53 in hepatoma cells
confer altered responsiveness of plasma protein gene expression to
cytokines such as interleukin-6 (IL-6)? The question posed takes on
particularly broad significance as cytokines enter the mainstream of
cancer therapy. To what extent do alterations in p53 present in tumor
cells alter cytokine-responsive gene expression in these cells?
Wild-type p53 can enhance transcription from promoters that contain DNA-binding sites for p53, whereas wt p53 can repress many promoters that do not contain p53-DNA-binding sites(1, 2, 3, 4, 5) . Transforming mutations in p53 alter the ability of this molecule to modulate transcription. Mutant p53 species lack the ability to enhance or repress transcription of test enhancer/reporter constructs. Additionally some tumor-derived mutants (e.g. the murine p53 mutants Val-135 and Phe-132) can exhibit a gain in function that is manifest as the ability to enhance transcription from reporter constructs that are otherwise repressed by wt p53 (6, also see (7) for a review of gain in function mutations in p53). Transcriptional modulation by p53, particularly repression, is thought to occur due to its interactions with cellular proteins such the TATA-binding protein (8) or CAAT-binding factor (9) (the ``squelching'' model). A variety of other cellular and viral proteins interact with p53 and these, in turn, affect the stability or the transcriptional activity of p53 (10, 11, 12, 13, 14, 15, 16, 17, 18) . There is little information about functional or structural interactions of p53 species with cell-surface receptors for cytokines or with molecules implicated in the signal transduction pathways triggered by cytokines or with transcription factors implicated in cytokine-mediated responses.
The negative regulation of some but not other cellular
promoters by wt p53 and the lack of repression by transforming mutants
was first pointed out in 1991(4, 30) . In transient
transfection experiments, we showed that reporter plasmids containing
regulatory elements from the IL-6, c-fos, -actin, or from
a major histocompatibility complex I promoter were strongly repressed
by wt p53 but less so by the transforming mutants tested(4) .
Numerous investigators have confirmed and extended the ability of wt
p53 to repress various cellular promoters in transient transfection
experiments; these include the c-fos,
-actin, heat shock
protein-70, c-jun, c-myc, p53 itself, the
retinoblastoma gene promoter, proliferating cell nuclear antigen, and
the multi-drug resistant gene
promoters(8, 9, 30, 37, 38, 39, 40) .
Additionally a variety of viral promoters derived from the Rous sarcoma
virus long terminal repeat (RSV-LTR), herpes simplex virus (thymidine
kinase gene), simian virus 40 (early promoter), and human T cell
leukemia virus (p53-responsive element) have also been reported to be
repressed by wt but not mutant p53
species(41, 42, 43) .
In an earlier study,
we observed the up-regulation of the IL-6 promoter by the murine p53
mutants Val-135 and Phe-132(2) . An intact CAAT enhancer
binding protein (C/EBP)-binding site (alias nuclear factor-IL6 (NF-IL6)
or C/EBP site) in the IL-6 promoter or in the herpesvirus
thymidine kinase promoter was a requirement for up-regulation by these
p53 mutants suggesting that the transcription factor C/EBP
might
be a target for p53 modulation(2) . In functional experiments,
wt p53 blocked transcriptional activation of the IL-6 promoter by
C/EBP
. In contrast, the p53 mutant species Val-135 and Phe-132
enhanced C/EBP
-mediated gene activation. Stimulated by prior
investigations implicating the C/EBP family transcription factors in
the cascade of transcription factors activated by cytokines (e.g. by IL-1 or by the IL-6-type cytokines) in differentiated cells (e.g. the
hepatocyte)(44, 45, 46, 47) , we
posed a more general question: can p53 species modulate the response of cells to cytokines in a context distinct from the
regulation of cell proliferation? Could p53 species modulate
IL-6-induced activation of plasma protein gene expression in an
hepatoma cell? Could mutations in p53 alter the response of a hepatoma
cell to cytokines such as IL-6 that could contribute to an altered
ability of these cells to synthesize and secrete acute-phase plasma
proteins?
We have addressed these questions in two different ways.
First, in a series of transient transfection experiments in Hep3B and
HepG2 hepatoma cells we investigated the effect of p53 species on the
function of the 36-bp IL-6-response element (IL-6RE) derived from the
rat fibrinogen (
Fib) promoter. Second, we derived Hep3B
hepatoma lines stably transfected with a constitutive expression vector
for a temperature-sensitive mutant of p53 (``Val-135'';
wt-like at 32.5 °C and mutant-like at 37 °C), and investigated
the ability of IL-6 to enhance the synthesis and secretion of Fib and
of
-antichymotrypsin (ACT) at the two temperatures in
these p53Val-135
hepatoma cell lines.
Figure 1:
Schematic
diagram showing the structure of the enhancer/reporter constructs
pFibCAT and p50-2.
Figure 6:
Modulation of IL-6-induced fibrinogen
secretion as a function of temperature in hepatoma line 3
(p53Val-135). Parental Hep3B cells and line 3 cells
plated in 24-well multiwell plates were induced with IL-6 (1 or 10
ng/ml) and shifted to 32.5 °C or continuously kept at 37 °C
using the protocol described in the text. The IL-6-induced synthesis
and secretion of
, B
, and
fibrinogen chains into the
culture medium was monitored as described under ``Materials and
Methods'' and the autoradiograms quantitated in arbitrary units by
densitometry. Panel A, an illustrative autoradiogram showing
IL-6-induced synthesis and secretion of the B
fibrinogen chain. Panel B, numerical data depicting B
fibrinogen secretion
in response to IL-6 at 1 ng/ml; data from IL-6-treated cultures at the
two temperatures of each cell line are presented. Panel C,
numerical data depicting B
fibrinogen secretion in response to
IL-6 at 10 ng/ml; data from IL-6-treated cultures at the two
temperatures of each cell line are presented. The error bars indicate standard error of mean; n denotes the number of
individual cultures evaluated. The p values indicated in panels B and C correspond to comparisons between
response at 37and 32.5 °C for each of cell line and were derived
using the Student's t test (two-tailed). Additionally, *
represents p < 0.05 for the comparison between line 3 and
Hep3B both at 37 °C, and** represents p < 0.05 for the
comparison between line 3 and Hep3B both at 32.5
°C.
Figure 7:
Modulation of IL-6-induced ACT secretion
as a function of temperature in hepatoma line 3
(p53Val-135). Parental Hep3B cells and line 3 cells
plated in 24-well multiwell plates were induced with IL-6 (1 or 10
ng/ml) and shifted to 32.5 °C or continuously kept at 37 °C
using the protocol described in the text. The IL-6-induced synthesis
and secretion of ACT into the culture medium was monitored as described
under ``Materials and Methods'' and the autoradiograms
quantitated in arbitrary units by densitometry. Panel A, an
illustrative autoradiogram showing IL-6-induced synthesis and secretion
of ACT; same experiment as illustrated in Fig. 6, panel
A. Panel B, numerical data depicting ACT secretion in
response to IL-6 at 1 ng/ml; data from IL-6-treated cultures at the two
temperatures of each cell line are presented. Panel C,
numerical data depicting ACT secretion in response to IL-6 at 10 ng/ml;
data from IL-6-treated cultures at the two temperatures of each cell
line are presented. The error bars indicate standard error of
mean; n denotes the number of individual cultures evaluated.
The p values indicated in panels B and C correspond to comparisons between response at 37 and 32.5 °C
for each of cell line and were derived using the Student's t test (two-tailed). Additionally, * represents p < 0.05
for the comparison between line 3 and Hep3B both at 37 °C, and**
represents p < 0.05 for the comparison between line 3 and
Hep3B both at 32.5 °C.
Figure 8: Temperature-sensitive phenotype of the response to IL-6 (ACT secretion) in line 1 and 2 hepatoma cells, but not in parental Hep3B and line 22 cells. Hepatoma lines 1, 2, and 22, as well as the parental Hep3B cells were evaluated for their response to IL-6 (10 ng/ml) at 32.5 °C compared to that at 37 °C in duplicate cultures using the procedure indicated in the legend to Fig. 7. For each cell line, the ACT secretion at 32.5 °C is expressed as a percent of that observed at 37 °C. The error bars depict the standard error of the mean; p < 0.05 using matched analysis of variance for all means; and p < 0.05 for all two-way comparisons between lines 1 and 2 on the one hand and line 22 or Hep3B on the other hand, but not when Hep3B was compared to line 22 using the exact paired means randomization test.
The fibrinogen and the
-antichymotrypsin genes are so-called Type II
acute-phase plasma protein genes in that their expression at maximal
levels in hepatocytes requires only IL-6 (reviewed in Refs. 6, 46).
More generally, the group of IL-6-type cytokines that include IL-6,
leukemia inhibitory factor, oncostatin M, interleukin-11, ciliary
neurotrophic factor, and cardiotrophin-1 can stimulate hepatic
fibrinogen gene expression provided that the hepatoma cell lines tested
harbor the appropriate
chain or cytokine-binding component of the
cell-surface receptor complexes (reveiwed in Refs. 6, 46, 56, 57). In
each instance the signal-transducing chain is the gp130
chain of
the receptor (reviewed in Refs. 57, 58). IL-6 enhances fibrinogen gene
expression in both Hep3B and HepG2 cells. The 36-bp IL-6RE in the rat
-fibrinogen gene consists of the consensus CTGGGA sequence present
in all Type II plasma protein genes that constitute binding sites for
various stat-family transcription
factors(59, 60, 61) . The
Fib IL-6RE is
also functionally responsive to C/EBP family transcription
factors(44, 45, 46) . Thus the reporter
construct p
FibCAT (Fig. 1) allows two questions to be
answered: (i) the ability of p53 species to modulate a response driven
by IL-6, and (ii) the ability of p53 species to modulate a response
driven by C/EBP
,
, or
per se following
cotransfection of appropriate constitutive expression vectors.
Figure 2:
Modulation by p53 species of IL-6-induced
activation of pFibCAT in hepatoma cells. Cultures of Hep3B (panel A) or HepG2 (panels B and C) cells in
100-mm plastic dishes cotransfected with the enhancer/reporter
p
FibCAT (10 µg) and various p53 expression vectors (5 µg)
were treated with IL-6 (30 ng/ml) for 24 h and the level of CAT
expression monitored (
gal normalized). The level of CAT expression
is depicted as fold change (± standard deviation) with reference
to the CAT activity in cells not treated with IL-6 or any p53 vector
taken as 1. n represents the number of replications of each
test combination; statistical evaluation was carried out using the
Student's two-tailed t test. Panel C is an
autoradiogram that illustrates CAT assays corresponding to one
experiment. p53 constructs: SN3, human wt; CX3, human
mutant; Nc9, murine wt; c5, Val
, and Phe
, murine
mutants.
Figure 3:
Modulation by p53 species of the
muscle-specific creatine kinase-derived enhancer/reporter p50-2
in IL-6-treated hepatoma cells. Duplicate cultures of Hep3B cells (panels A and B) in 100-mm plastic dishes
cotransfected with the enhancer/reporter p50-2 (10 µg) and
various p53 expression vectors (5 µg) were treated with IL-6 (30
ng/ml) for 24 h and the CAT expression monitored essentially as
described in legend to Fig. 2. p53 constructs: SN3,
human wt; CX3, human mutant; Nc9, murine wt; c5, Val, and Phe
, murine mutants.
Figure 4:
Modulation by p53 species of
C/EBP-induced activation of pFibCAT in hepatoma cells. HepG2
cultures in 100-mm dishes were cotransfected with p
FibCAT (10
µg), expression vectors for C/EBP
,
, or
(5 µg)
together with various p53 expression vectors (5 µg) in duplicate.
Cells were harvested 24 h after the beginning of transfection and the
level of CAT expression monitored essentially as described in legend to Fig. 2. Inset in panel C shows an
autoradiogram of a CAT assay from one experiment. p53 constructs: SN3, human wt; CX3, human mutant; Nc9,
murine wt; c5, Val
, and Phe
, murine mutants.
In order to confirm that
the inhibitory effect of wt p53 on activation of the FibCAT
enhancer by the C/EBP transcription factors was specific, the control
reporter p50-2 was used in HepG2 cells. The reporter p50-2
was activated to a small extent by cotransfection with C/EBP
,
, or
expression vectors in HepG2 cells. However, the
combination of wt p53 species (SN3 or Nc9) with C/EBP isoforms enhanced
this expression further (data not shown).
The binding of recombinant
baculovirus expression vector-derived murine wt p53 to the IL-6RE in
FibCAT was tested in electrophoretic mobility shift assays. The
data obtained indicate that when compared to authentic p53-binding DNA
elements, such as p50-2, the binding of p53 to the
Fib
IL-6RE oligonucleotide in gel-shift assays was, at best, relatively
weak (data not shown).
Figure 5:
p53 immunostaining in hepatoma cell
lines. Parental Hep3B cells (panel A), three
p53Val-135-expressing hepatoma lines (lines
1-3, respectively, panels B-D), and a hepatoma line
transfected with pSVneo alone (line 22, panel D) were
tested for p53 by immunostaining using a panreactive anti-p53
monoclonal antibody (Pab 240) (reactive with wt and mutant human and
murine p53 species). The length marker equals 20
µm.
In order to address this question, it was
first necessary to devise an experimental protocol for a ts shift
experiment that would lead to a minimal, if any, difference in the
response of the parental Hep3B cells to IL-6. Briefly, Hep3B cells
first plated at 37 °C at a density of 2.5 10
cells/well in 24-well plates for 24 h, washed with
phosphate-buffered saline at 37 °C, incubated at 32.5 °C for 4
h with medium containing insulin and dexamethasone that was at 37
°C when it was added to the cultures (thus ensuring a slow
transition of the cells from 37 to 32.5 °C), then adding IL-6 to
that medium for 18-20 h, followed by subsequent labeling with
[
S]methionine also at 32.5 °C, revealed
little difference in the response of parental Hep3B cells to IL-6 at
32.5 °C compared to cells kept at 37 °C throughout ( Fig. 6and Fig. 7). Under these experimental conditions,
it was possible to investigate the ts phenotype of the
p53Val-135
hepatoma cell line 3. Fig. 6and Fig. 7represent a summary of data from a series of experiments
in which the ts phenotype of hepatoma line 3 with respect to the
synthesis and secretion of fibrinogen and ACT were investigated. In
both instances, line 3 exhibits a greater sensitivity to IL-6 at 37
°C compared to the parental Hep3B cells, but a reduced sensitivity
to IL-6 at 32.5 °C compared to parental Hep3B cells or its own
response at 37 °C. These observations were extended to the
p53Val-135
hepatoma cell lines 1 and 2. Both line 1
and 2 exhibited a reduced response to IL-6 at 32.5 °C compared to
that at 37 °C, in contrast to the absence of a ts phenotype in the
parental Hep3B cells or of the pSVneo-containing hepatoma line 22 (Fig. 8). The data in Fig. 6Fig. 7Fig. 8suggest that mutant p53 species
contained in hepatoma cells can alter the ability of these cells to
secrete acute-phase plasma proteins in response to cytokines such as
IL-6.
The hypothesis that p53 species may have a role in modulating
the response of differentiated cells to cytokines (2) was
tested in a context devoid of considerations of the regulation of cell
proliferation. The effect of p53 species on the rapid activation by
IL-6 of the enhancer derived from the acute-phase plasma protein gene
-fibrinogen was tested in hepatoma cell lines Hep3B and HepG2 in
transient transfection assays. Wild-type p53 inhibited the activation
of the IL-6-responsive enhancer, whereas several tumor cell-derived
mutants of p53 had lost this inhibitory property. Since the 36-bp
IL-6RE derived from
Fib is also activated by C/EBP transcription
factors, this experimental system was used to investigate the
functional interaction between p53 species and C/EBP transcription
factors. Again, wt p53 species inhibited the activation of the
Fib
IL-6RE by C/EBP
,
, or
, whereas several tumor
cell-derived mutants had lost this inhibitory property. Some mutants, e.g. p53Val-135 and p53Phe-132, appeared to enhance C/EBP
species-driven activation of the
Fib IL-6RE.
The biological
consequences of modulation by p53 of the acute-phase plasma protein
response of hepatoma cells were examined by deriving Hep3B cell lines
constitutively expressing p53Val-135. This mutant of p53 is previously
known to have a ts phenotype(28) . Derivation of
p53Val-135 hepatoma lines allowed us to examine the
influence of p53 on synthesis and secretion of plasma proteins by
hepatoma cells as a function of temperature. At 37 °C, hepatoma
line 3 had an increased response to IL-6 than did the parental Hep3B
cells, whereas at 32.5 °C, line 3 had a reduced response than did
the parental cells. Two additional p53Val-135
hepatoma
lines (lines 1 and 2), but not the p53Val-135
line 22
displayed a similar ts phenotype with respect to their response to
IL-6. The data show that certain tumor-derived mutations in p53 can
alter the ability of hepatoma cells to respond to IL-6. From a broader
perspective, the data suggest that mutations in p53 can modulate gene
expression elicited in tumor cells in response to cytokines.
In
previous experiments we had observed that the p53 mutants Val-135 and
Phe-132 enhanced C/EBP-driven activation of the IL-6 promoter and
of the herpesvirus thymidine kinase promoter(2) . Mutagenesis
studies showed that an intact C/EBP-binding site in the two target
promoters was required for this enhancing effect of Val-135 and
Phe-132. In the present study, Fig. 4shows that Val-135 and
Phe-132 also enhanced C/EBP-driven p
FibCAT expression in a
C/EBP-species and cell type-specific manner. While we have observed
that recombinant baculovirus-derived p53 can coprecipitate recombinant
C/EBP-
,
or
species in protein interaction studies in vitro, (
)it is not yet clear that this
interaction represents a regulatory event that occurs in intact cells.
That p53 species in stably expressing lines can modulate the
response of hepatoma cells to IL-6 such as depicted in Fig. 7and Fig. 8leads to a broad range of questions with
respect to the interactions of p53 species with molecules and
transcription factors involved in the signal transduction pathways
triggered by cytokines. That plasma protein secretion in response to
IL-6 can be modulated by the presence of a mutant p53 species in a
hepatoma cell is clear from our data. The
p53Val-135-expressing hepatoma cell lines developed by
us provide a unique substrate for further studies of the interplay
between mutations in p53 and the biological response of tumor cells to
cytokines.
The molecular mechanisms by which p53 modulates the
response to IL-6 in the p53Val-135-expressing hepatoma
lines may well include interactions between p53 and target molecules at
the level of the cytokine receptor, at the level of the signal
transduction cascade, and/or at the level of the transcription factors
activated by IL-6. A systematic exploration of each of the these
possibilities now becomes feasible because of the availability of the
p53Val-135
-expressing Hep3B-derived lines and their ts
phenotype with respect to the response to IL-6. With cancer patients
being increasingly administered various cytokines for their direct or
indirect antitumor effects or for their immunosupportive effects, the
consequences of mutations in p53 contained in tumor cells (admittedly a
quite frequent event) upon cytokine-elicited alterations in gene
expression in these tumor cells need to be understood.