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(Received for publication, March 27, 1995; and in revised form, May 18, 1995) From the
WAF1 binds to cyclin-Cdk complexes and inhibits their activity,
causing cell cycle arrest. Previous studies have shown that expression
of WAF1 is induced through the p53-dependent pathway; WAF1 is induced
in cells with functional p53 but not in cells with either mutant p53 or
no p53. Human myeloblastic leukemia cells KG-1 had no constitutive
expression of p53, and irradiation did not induce p53. However,
irradiation increased WAF1 expression in KG-1 cells and other cell
lines containing mutant p53. The KG-1 cells constitutively produced low
levels of tumor necrosis factor (TNF); irradiation markedly increased
the production of TNF. Notably, induction of WAF1 mRNA by irradiation
was blocked by anti-TNF antibody. Furthermore, exogenously added TNF
increased levels of WAF1 mRNA in these cells. Irradiation increased the
rate of WAF1 transcription 3-fold, and the half-life (t) of
WAF1 mRNA in these cells increased from <1 h in unirradiated cells
to >4 h in irradiated cells. These findings indicate that increased
levels of WAF1 transcripts occur, at least in part, through a pathway
of TNF production and that the increase in WAF1 mRNA observed after
irradiation is regulated by both transcriptional and
posttranscriptional mechanisms. Our present study strongly suggests
that an alternative pathway of induction of WAF1 occurs independent of
activation by p53.
The molecular mechanism of cell proliferation is extremely
complex; deregulation results in neoplastic transformation. In
eukaryotes, proliferation of cells is finely regulated through the cell
cycle. Studies have shown that the cell cycle is regulated by a series
of enzymes known as cyclin-dependent kinases
(Cdks)(1, 2) . The activities of Cdks are controlled
by their association with regulatory subunits, cyclins; the expression
of cyclins and the activation of the different cyclin-Cdk complexes are
required for the cell to cycle(1, 2) . Thus, the cell
cycle is regulated by activating and inhibitory phosphorylation of the
Cdk subunits, and this program has internal check points at different
stages of the cell cycle(3) . When cells are exposed to
external insults such as DNA damaging agents, negative regulation of
the cell cycle occurs; arrest in either G Irradiation is one of the stresses that produce physical and
chemical damage to tissues; irradiation induces neoplastic
transformation as well as killing of cells. In the presence of oxygen,
irradiation increases the formation of radicals including superoxide
radicals (O)(10, 11) , and the importance of these
reactive oxygen species has been emphasized in irradiation-induced
tissue damage(12, 13) . The reaction of these radicals
with DNA results in DNA strand breaks, which may be a critical step in
radiation-induced transformation(14, 15) . In response
to these stresses, cells express or activate proteins that protect
themselves from external insults and also cause inhibition of
replicative DNA
synthesis(5, 6, 7, 8, 9, 16, 17, 18, 19, 20, 21, 22) .
Recent studies have shown that p53 (a tumor suppressor) plays an
important role as a cell cycle check point determinant following
irradiation; irradiation causes a transient inhibition of replicative
DNA synthesis, G In this study, we examined the effect of irradiation on
regulation of the WAF1 gene in a human myeloblast cell line
(KG-1) and other cell lines and also explored the possible mechanisms
of regulation of its expression. Our data show that irradiation induces WAF1 gene expression in cells containing either no p53 or
mutated p53 and that the induction occurs at both the transcriptional
and posttranscriptional levels. We also found that expression of the WAF1 gene by irradiation requires protein kinase C activation,
and increased levels of WAF1 transcripts are also regulated through a
pathway that requires production of tumor necrosis factor (TNF) (
Figure 1:
Expression of WAF1 in KG-1 cells after
irradiation. Cells were cultured for 8 h after irradiation at various
doses, as indicated. After cell lysis, 20 µg of whole cell protein
was electrophoresed in either a 12% (for WAF1) or 10% (for p53)
SDS-polyacrylamide gel, transferred to polyvinylidene difluoride
membranes, and analyzed for either WAF1 or p53 protein as described
under ``Materials and Methods.'' Arrows indicate the
WAF1 and p53 bands. SK-HEP-1, which is a hepatoma cell line, was used
as a positive control.
Figure 2:
Dose-dependent effect of irradiation on
levels of WAF1 mRNA in KG-1 cells. Cells were cultured for 4 h after
irradiation. Total RNA (15 µg/lane) was prepared and
analyzed by formaldehyde-agarose gel electrophoresis and transferred to
a nylon membrane as described under ``Materials and
Methods.'' Hybridization was with
Figure 3:
Time-dependent effect of irradiation on
levels of WAF1 mRNA in KG-1 cells. Cells were cultured for various
durations (0-8 h) after irradiation at 40 Gy. Northern blot
analysis of mRNA was performed by blotting total RNA (15
µg/lane).
Figure 4:
A, expression of WAF1 and p53 mRNAs in
various cell lines. Total or cytoplasmic RNA was extracted from each
cell line and analyzed by formaldehyde-agarose gel electrophoresis (15
µg/lane) as described under ``Materials and
Methods.'' Hybridization was with
Figure 5:
Increased levels of TNF in KG-1 cells
exposed to irradiation. Cells were cultured for 8 h after various doses
of irradiation as indicated. Cells were harvested, and conditioned
media and cell lysates were assayed for TNF by enzyme-linked
immunosorbent assay as described under ``Materials and
Methods.'' Results represent mean and standard error of triplicate
assay. PanelA shows the concentration of TNF in
conditioned medium, and panelB demonstrates the
amount of TNF in cellular lysate of KG1. p < 0.01, control
and 10 Gy; p < 0.001, control and 20
Gy.
Figure 6:
Effect
of production of TNF on expression of WAF1 in KG-1 cells exposed to
irradiation. Cells were exposed to different concentrations of TNF (100
or 1000 units/ml) for 2 h. In parallel, cells were pretreated for 1 h
with anti-TNF antibody at a concentration that neutralizes 1000
units/ml of TNF. These cells were then irradiated at 40 Gy in the
presence of the antibody and cultured for 2 h. Untreated and treated
cells were harvested, and levels of WAF1 mRNA were
determined.
The KG-1 cells also expressed IL-1 mRNA upon
irradiation as shown in Fig.2, 3, and 6A. However,
exposure of the cells to IL-1 did not increase the levels of WAF1 mRNA
in these cells (data not shown).
Figure 7:
Effect of prolonged exposure to a phorbol
ester on expression of WAF1 mRNA induced by irradiation. KG1 cells were
pretreated with TPA (100 nmol/liter, 24 h), washed, and treated with
either TPA (50 nmol/liter) or irradiation (20 Gy). Two hours later,
total RNA was extracted, and Northern blotting was performed. As
controls, cells were cultured either with TPA alone (50 nmol/liter, 2
h) or irradiated (20 Gy, 2 h) alone.
Figure 8:
Transcriptional run-on analysis of WAF1 in
irradiated KG-1 cells. Cells were either untreated or irradiated at 20
Gy, and 2 h later nuclei were isolated as described under
``Materials and Methods.'' Newly elongated
Figure 9:
Stability of steady state WAF1 mRNA in KG1
cells exposed to irradiation. Untreated cells or cells irradiated at 40
Gy were cultured with actinomycin D (5 µg/ml) for 0.5-2.0 h.
Cytoplasmic RNA (30 µg/lane in untreated cells and 15
µg/lane in irradiated cells) was extracted and analyzed by
RNA blotting as described under ``Materials and Methods.''
Intensity of hybridization was determined by densitometry of several
different exposures of the autoradiograms. Untreated cells were assumed
to have 100% activity.
Many of the damaging effects of ionizing irradiation are
mediated by reactive free
radicals(10, 13, 53) . Irradiation increases
the production rate of these free, radicals which cause DNA breakage (15, 16) and transformation of cells(14) .
Cells inhibit their replicative DNA synthesis when exposed to DNA
damage such as irradiation. Mutations of the p53 gene are commonly
found in various human cancers(54) , and loss of normal p53
activity leads to uncontrolled cell growth(55) , suggesting
that p53 is a tumor suppressor(56) . Although the mechanism of
this suppression is not clear, p53 can bind to DNA in a
sequence-specific manner and stimulate the transcription of genes
downstream of the binding site(23, 57, 58) .
WAF1 has been recently identified as an inhibitor of the kinase
activity of the cyclin-Cdk complex. The upstream region of the gene
contains several p53 binding sites(59, 60) . The data
strongly suggest that p53 can bind to the WAF1 promoter region and
enhance transcription of the gene. Furthermore, DNA damage to cells
activates p53 to induce expression of WAF1, which plays an important
role in G In the
present study, we showed that irradiation could induce increased levels
of WAF1 transcripts in KG-1, MOLT-4, and SK-HEP-1 cells. Further
studies showed that the increased levels of WAF1 mRNA in the KG-1 cells
is at least in part explained by significantly increasing the rate of
WAF1 transcription. Studies have shown that KG-1 cells have no
transcripts or protein of p53; five bases are inserted between codons
224 and 225 of the p53 coding sequence with no wild-type p53 cDNA
sequence detected(42, 43, 44) . Our Western
blot using monoclonal anti-p53 antibody PA-1, which reacts with both
wild-type and mutated p53(62, 63) , detected no p53 in
KG-1 cells. These p53 results are consistent with previous
studies(42, 43, 44, 45) . The MOLT4
and SK-HEP-1 cells also contain a mutated p53. These mutations inhibit
DNA binding by p53 and therefore abrogate its ability to transactivate
WAF1(64, 65) . Taken together, our studies clearly
indicate that irradiation can increase the accumulation of WAF1
transcripts through a p53-independent pathway, and our results with
KG-1 cells show that at least in part this regulation occurs by an
increased levels of WAF1 transcription. A recent study has reported
that WAF1 mRNA was induced in fibroblasts derived from ``p53
knock-out'' mice(66) . This WAF1 expression was induced by
exposure of cells to platelet-derived growth factor, fibroblast growth
factor, and epidermal growth factor but not irradiation. The present
study is the first to show that irradiation can cause transcription of
WAF1 independently of p53. Irradiation has been shown to increase the
expression of a number of genes. For example, recent studies showed
that either X or UV irradiation increased expression of reporter gene
through the long terminal repeat of Moloney murine sarcoma
provirus(67) . Also the long terminal repeat of the human
immunodeficiency virus has been also shown to be activated by UV
irradiation in the absence of the Tat
transactivator(68, 69) . Irradiation in addition can
increase the expression of the transcriptional factors FOS, JUN, and
the early growth response family of genes(70, 71) .
The nuclear factor The steady
state level of mRNAs in the cell is dependent on both the rates of
transcription and degradation. The t of WAF1 RNA was less than
1 h in untreated KG-1 cells; irradiation markedly stabilized WAF1 mRNA
in these cells (t > 4 h). How various extracellular signals
can result in mRNA stabilization of key transcripts is unknown. Prior
studies have shown that another cell cycle related protein, c-Myc, is
modulated in part by changes in the stability of its
mRNA(74, 75) . Various extracellular stimuli such as
protein synthesis inhibitors and stimulator of protein kinase C are
able to stabilize the c-Myc RNA(76, 77) . Indeed,
proteins important in the cell cycle must be able to undergo rapid
changes as the cell comes in contact with various stimulators and
inhibitors of cellular proliferation. Changes in the stability of
specific mRNA afford an extremely rapid mechanism to change the levels
of a critical cell cycle-related protein. Another interesting
finding of this study is that accumulation of WAF1 mRNA after exposure
to irradiation occurs through, at least in part, production of TNF. The
KG-1 cells constitutively produced TNF mRNA and protein at low levels;
this constitutive expression of TNF was markedly increased after
irradiation. Previously, we have found that irradiation altered the
expression of cytokines in cells(17) . Other investigators have
also demonstrated that irradiation induced TNF mRNA in myeloid cells
including monocytes from human peripheral
blood(19, 20) . We have found that treatment of the
KG1 cells with anti-TNF antibody inhibited the induction of WAF1 mRNA
by irradiation. Moreover, exogenously added TNF increased levels of
WAF1 transcripts in these cells. On the other hand, IL-1 has also been
implicated as an important factor in the inflammatory response and has
similar biological activities as TNF(78) . The induction of
IL-1 mRNA was also observed by irradiation. However, IL-1 was unable to
induce WAF1 mRNA, and treatment with anti-IL-1 antibody did not reduce
the irradiation-induced elevation in WAF1 mRNA (data not shown). Our
studies suggest that irradiation may induce WAF1 expression through an
autocrine loop of TNF production. Protein kinase C is involved in
signal transduction by coupling receptor-mediated inositol phospholipid
turnover with a variety of cellular functions(79) . Phorbol
esters that activate protein kinase C have been reported to stimulate
accumulation of WAF1 mRNA in fibroblasts from p53 knock-out
mice(66) . In this study, TPA induced the accumulation of WAF1
mRNA in KG1 cells. Furthermore, we took advantage of the fact that
prolonged exposure of cells to TPA leads to inactivation of protein
kinase C(51, 52) . Prolonged exposure to TPA (100
nmol/liter, 24 h) blocked accumulation of WAF1 mRNA after reexposure of
cells to TPA. Under the same conditions, accumulation of WAF1
transcripts upon irradiation was blocked after reexposure of the
TPA-treated cells to irradiation. Our findings suggest that induction
of WAF1 mRNA by irradiation is likely to be mediated through protein
kinase C. In summary, the present investigation demonstrated that
the levels of WAF1 mRNA can be increased by irradiation in human
myeloblasts (KG-1) and other cell types that have mutated p53; in
addition, irradiation also increased the levels of WAF1 protein. This
increased expression of WAF1 appears to occur at least in part
secondary to stimulation of production of TNF by exposure to
irradiation and the TNF inducing expression of WAF1. The
p53-independent induction of WAF1 occurred both by an increase in WAF1
transcription and stabilization of these transcripts. The data suggest
that alternative pathways of induction of WAF1 exist that are
independent of activation by p53.
Volume 270,
Number 32,
Issue of August 11, pp. 19181-19187, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or G
stages is induced to prevent the cells from prematurely entering
into the next stage before DNA is repaired (4, 5, 6, 7, 8, 9) . arrest in cells having wild-type p53,
while the inhibition did not occur in cells without a functional p53 (23, 24, 25, 26) . More recently, a
potent inhibitor of Cdks, which inhibits the phosphorylation of
retinoblastoma susceptibility gene product by cyclin A-Cdk2, cyclin
E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes, has been
identified(27, 28, 29, 30) . This
protein, named WAF1, Sdi1, Cip1, or p21 (a protein of M
21,000), contains a p53-binding site in its promoter, and studies
have reported that the expression of WAF1 was directly regulated by
p53(29, 31) ; cells with loss of p53 activity due to
mutational alteration were unable to induce
WAF1(27, 29, 31, 32) . However,
little is known about the p53-independent pathway of WAF1 induction by
irradiation.)in KG-1 cells.
Cells and Culture
All cell lines used in the
present study were obtained from American Type Tissue Culture
Collection. Cells were cultured in 
-medium (Cosmo Bio Co., Ltd.,
Tokyo, Japan) supplemented with 7% fetal calf serum (Mitsubishi Kasei
Co., Tokyo, Japan) in a humidified atmosphere containing 5%
CO. Nonadherent cell lines were cultured at an initial
concentration of 2 10
cells/ml, and flasks
containing 1 10
cells/ml were used for experiments.
For experiments using adherent cell lines, subconfluent cultures were
employed.Irradiation
Cells were irradiated with -ray
by a
Cs source emitting at a fixed dose rate of 12
Gy/minute as determined by dosimetry.
Reagents
The p53 monoclonal antibody PAb1801
(Ab-2) and the WAF1 monoclonal antibody EA10 (Ab-1) were purchased from
Oncogene Science (Cambridge, MA). The neutralizing antibody against
human TNF (polyclonal rabbit anti-human TNF) was purchased from
Genzyme (Cambridge, MA). One microliter of this antibody neutralizes
1000 units of TNF. The antibody against human IL-1
(number 297)
was a polyclonal rabbit antibody and was kindly provided by Dr. Tsutomu
Nishida (Otsuka Pharmaceutical Co., Tokushima, Japan). Human
recombinant TNF and IL-1 were also from Genzyme, and the specific
activities were 1.08 10
units/mg protein and 5
10
units/mg protein, respectively. Actinomycin D
and cycloheximide were purchased from Sigma.Western Blot Analysis
Cells were lysed in buffer
containing 50 mmol/liter Tris-HCl (pH 8.0), 150 mmol/liter NaCl, 0.02%
NaN
, 0.1% SDS, 100 mg/ml phenylmethylsulfonyl fluoride, 1
mg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate. After
centrifugation, the protein concentrations in each sample were
determined by the method of Bradford(33) . Samples, each
containing 20 µg of cell lysates in SDS-polyacrylamide gel
electrophoresis loading buffer were electrophoresed in 12%
polyacrylamide gels and transferred to a polyvinylidene difluoride
membrane (Immobilon, Millipore, Bedford, MA). Then immunoblotting was
performed using anti-human p53 or anti-human WAF1 antibody (10
µg/ml and 1 µg/ml final concentrations, respectively). After
washing the blots, alkaline phosphatase-conjugated goat anti-human
mouse immunoglobulin G (IgG) diluted 1:2000 was added to the blots.
Imunoreactivity on the blots was detected by nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Life Technologies,
Inc.).Assay for TNF
The concentration of TNF in either
the culture supernatants or cell lysates was measured by enzyme-linked
immunosorbent assay. Conditioned media from cultures of either control
or irradiated KG-1 cells were prepared by centrifuging the supernatants
at 1000 g for 10 min. Cell lysates were made by
sonicating the cells in ice-cold phosphate buffered saline after
irradiation and debris was removed by centrifugation. The content of
protein in cellular lysates was measured by the method of Bradford (33) . Standard curves of TNF protein were plotted by serial
dilution of purified recombinant human TNF as a standard. This assay
was specific and did not detect other cytokines including GM-CSF,
G-CSF, M-CSF, IL-1, IL-3, transforming growth factor
, IL-6, and
IFN- (Genzyme).
DNA Probes
The plasmid containing human WAF1 cDNA
(2.1 kb, NotI) was kindly provided by Dr. B Vogelstein (Johns
Hopkins University, Baltimore, MD)(29) , and the p53 cDNA was
purified from the pR4-2 plasmid (0.5 kb, NcoI). The TNF
cDNA fragment (0.8 kb, EcoRI) was from
pSPl42-2(34) . IL-1 cDNA was from
pA-26(35) . B-actin DNA probe (0.7 kb, EcoRI-BamHI) was from pHFb A-3` ut
plasmid(36) . These probes were P-labeled by a
random priming method (37) . The specific activity was
approximately 2
10
cpm/µg of DNA.Isolation and Blotting of RNA
Total RNA from cells
was obtained by the guanidinium/hot phenol
method(38, 39) . Nonadherent cells were lysed in a
guanidinium isothiocyanate mixture (4 M guanidinium
isothiocyanate, 50 mM Tris-HCl (pH 7.6), 20 mM EDTA,
2% (v/v) sodium lauryl sarcosinate, and 140 mM 2
-mercaptoethanol. The lysed cells were treated with proteinase K,
and then their total RNA was extracted by phenol/chloroform. For
cytoplasmic RNA from adherent cells, cells were lysed with hypotonic
buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM KCl,
3 mM MgCl, and 0.3% Nonidet P-40. Cytoplasmic RNA
was extracted by the phenol/chloroform method as described
previously(40) . After denaturation at 65 °C, RNA was
electrophoresed in agarose-formaldehyde (1%) and transferred to a nylon
membrane filter (Amersham Corp.)(41) . Filters were hybridized
with P-labeled probe for 16-24 h at 42 °C in 50%
formamide, 2
SSC (1
SSC: 150 mmol/liter NaCl, 15
mmol/liter sodium citrate), 5
Denhardt's solution, 0.1%
SDS, 10% dextran sulfate, and 100 µg/ml salmon sperm DNA. Filters
were washed to a stringency of 0.1
SSC for 10 min at 65 °C
and exposed to x-ray film (RX, Fuji Photo Film Co. Ltd., Kanagawa,
Japan). Autoradiograms were developed at different exposures. For a
quantitative analysis, the relative densities of the bands of
hybridization of WAF1 in different lanes were scanned by the LKB
UltroScan XL laser densitometer.
Transcriptional Run-on Assay
KG-1 cells were
exposed to irradiation, the nuclei were isolated by suspending in a
hypotonic buffer (10 mmol/liter Tris-HCl (pH 7.4), 10 mmol/liter KCl, 3
mmol/liter MgCl), and lysis was accomplished in 0.5%
Nonidet P-40. Nuclei were harvested by centrifugation (500 g, 5 min), washed in a hypotonic buffer, resuspended in
nuclear storage buffer (40% glycerol, 50 mM Tris-HCl (pH 8.3),
5 mM MgCl
, 0.1 mM EDTA). Nuclei were
incubated for 30 min at 30 °C in a reaction buffer containing 150
mM KCl, 3 mM MgCl, 0.25 mM ATP,
0.25 mM GTP, 0.25 mM CTP, and 200 µCi of
[-P]UTP (3000 Ci/mmol). The reaction was
terminated by adding DNase I (10 min at 30 °C), and 30 µg of
carrier tRNA was added. The reaction mixture was digested by 40
µg/ml proteinase K in a solution containing 10 mM EDTA and
1% SDS, followed by phenol/chloroform extraction. The aqueous phase was
precipitated at -70 °C with 70% ethanol in the presence of
0.3 M sodium acetate, and the precipitate was collected by
centrifugation and dissolved in TE (10 mmol/liter Tris-HCl (pH 8.0), 1
mmol/liter EDTA). After denaturation in 0.2 N NaOH (ice-cold)
and neutralization in 0.2 mol/liter HEPES, nuclear RNA was run through
a Sephadex G50 spun column to remove unincorporated
[
P]UTP. Plasmid DNA containing the cDNA coding
inserts was denatured by heat and alkalization (0.3 N NaOH).
Denatured plasmids (5 µg for WAF1 and p53 and 2 µg for
-actin) were bonded to nylon membranes (Hybond-N) using BIO-DOT SF
(Bio-Rad) and immobilized by UV cross-linker. Newly elongated nuclear
RNA was hybridized to the filters containing plasmids. Hybridizations
were performed with 10
cpm of P-labeled RNA/ml
in 3
SSC, 5 mmol/liter EDTA, 0.1% SDS, 10
Denhardt's solution, 50% formamide, 10 mM NaH
PO
(pH 7.0), 200 µg/ml of yeast
tRNA, and 100 µg/ml of salmon sperm DNA for 3 days at 42 °C.
After hybridization, filters were rinsed in 2 SSC at room
temperature and then in 2
SSC and 0.1
SSC at 42 °C.
The relative density of bands of hybridization of WAF1 and
-actin
in untreated and irradiated lanes was scanned by the LKB UltroScan XL
laser densitometer.
Induction of WAF1 Protein by Irradiation in KG-1
Cells
We determined whether irradiation affected the expression
of WAF1 in p53 mutated KG-1 cells by Western blotting using a WAF1
monoclonal antibody (Fig.1). The KG-1 cells were cultured for 8
h after exposure to irradiation at different doses (0, 5, 10, 20, and
40 Gy). Cells were harvested, and the level of WAF1 in total cellular
protein was examined. Unirradiated KG-1 cells constitutively expressed
a very low level of WAF1 protein. A significant increased level of WAF1
was observed at 5 Gy of irradiation and continued to increase in a
dose-dependent fashion. At 40 Gy of irradiation, the level of WAF1 was
approximately 6 times greater than that from unirradiated cells. In
contrast, p53 expression was not detected in untreated KG-1 cells, and
irradiation did not induce p53 in these cells.
Dose-dependent Effect of Irradiation on Levels of WAF1
mRNA in KG-1 cells
The KG-1 cells were cultured for 2 h after
exposure to various doses of irradiation as indicated (Fig.2).
To determine the effect of irradiation on WAF1 gene
expression, we performed Northern blot analysis of total RNA using P-labeled WAF1 cDNA probe (Fig.2). The KG-1 cells
constitutively contained low but detectable concentrations of mRNA
coding for WAF1. Irradiation with a dose of 10 Gy increased the WAF1 gene expression to a detectable level; the induction of
WAF1 RNA was dependent on the dose of irradiation. The induction of the WAF1 gene was maximal after exposure to 40 Gy of irradiation
(greater than 10-fold stimulation over base-line levels). Irradiation
also induced TNF and IL-1 mRNAs in these cells in a dose-dependent
fashion.
P-labeled WAF1 cDNA
(2.1-kb band of hybridization), TNF cDNA (1.7-kb band), and IL-1 cDNA
(1.6-kb band). The bottompanel shows the picture of
the ethidium bromide-stained formaldehyde gel before Northern blotting;
levels of 28 and 18 S ribosomal RNA were comparable in each lane.
Kinetics of Induction of WAF1 mRNA after Irradiation in
KG-1 Cells
The KG-1 cells were irradiated at 40 Gy and harvested
sequentially at different durations. Northern blot analysis showed that
irradiation markedly increased the level of WAF1 mRNA by 2 h after
irradiation, and then these levels slightly decreased by 8 h (Fig.3). On the other hand, an increase of TNF mRNA was
observed 1 h after irradiation. By 2 h, expression of TNF RNA reached
nearly plateau levels, and then the levels returned to base line by 8 h
after irradiation. Irradiation also increased the levels of IL-1
RNA in a manner almost parallel to that of WAF1.
Induction of the WAF1 Gene by Irradiation in Various Cell
Lines Including Those Having either Mutant p53 or No p53
In
order to investigate whether irradiation was capable of inducing WAF1
RNA in cells having either mutant p53 or no p53, a variety of cell
lines were investigated. Studies that are shown on Fig.4A analyzed for constitutive expression of p53 and WAF1 mRNA by the
various cell lines. The KG-1, THP-1, K562, U937, and SK-OV-3 cells did
not express p53 RNA, and these results were consistent with previous
studies(42, 43, 44, 45) . The
MOLT-4, CEM, and SK-HEP-1 cells have been reported to have mutated p53
gene; these cells expressed these mutant p53 transcripts (Fig.4A)(46, 47, 48, 49) .
The p53 gene in IMR32 cells is known to be normal(50) , and p53
mRNA was easily detectable. These cell lines, except SK-HEP-1 cells,
constitutively had either very low or undetectable levels of WAF1.
These cells were irradiated at different doses and cultured for 4 h.
Cells were harvested and examined by Northern blotting. Exposure to
irradiation clearly induced expression of WAF1 RNA in MOLT-4 cells that
have a mutation at codon 248 of p53 (49) and SK-HEP-1 cells
that have a partial deletion of the p53 gene (48) (Fig.4, B and C). However,
irradiation failed to induce WAF1 expression in K562, U937, CEM, HL60,
and SK-OV-3 cells; each of these lines contains a mutant p53 gene (data
not shown).
P-labeled WAF1 cDNA
(2.1-kb band of hybridization) and p53 cDNA (2.8-kb band). The bottompanel shows the picture of the ethidium
bromide-stained formaldehyde gel before Northern blotting; levels of 28
and 18 S ribosomal RNA are comparable in each lane. The upperparts of panelsB and C show
induction of WAF1 mRNA by different doses of irradiation in MOLT4 and
SK-HEP-1 cells, respectively. The bottomparts show
levels of p53 protein.
Induction of TNF Protein by Irradiation
As shown
in Fig.2and 3, irradiation increased levels of TNF mRNA in
KG-1 cells. To investigate whether irradiation affects translation of
these transcripts, KG-1 cells were cultured for 8 h after exposure to
irradiation at different doses (10-80 Gy) (Fig.5). As a
control, unirradiated cells were cultured for 8 h. Conditioned media
and cells were harvested; levels of TNF in conditioned media and cell
lysate were determined by enzyme-linked immunosorbent assay. The TNF
protein in supernatants of untreated cells was negligible (Fig.5A). Irradiation induced levels of TNF in a
dose-dependent fashion. A significant increase of TNF production was
observed at 10 Gy of irradiation (p < 0.01). At 80 Gy of
irradiation, the level of TNF was approximately 7 times greater than
levels from 10 Gy-irradiated cells (p < 0.001). In
parallel, the study of TNF in cell lysates showed that untreated KG1
cells constitutively contained low levels of TNF; irradiation markedly
increased the TNF levels in a dose-dependent manner. At a dose of 10
Gy, levels of intracellular TNF were 3 times greater than that in
untreated cells (p < 0.01), and at 80 Gy levels were 9-fold (Fig.5B).
Effect of TNF Production on Expression of WAF1 mRNA after
Irradiation
To investigate the involvement of endogenously
produced TNF in the expression of WAF1 mRNA by irradiation, KG-1 cells
were preincubated with antibody against human TNF for 1 h, which
neutralizes 1000 units/ml of TNF, and then cells were irradiated with
20 Gy in the presence of anti-TNF antibody (Fig.6). After 2 h,
cells were harvested, and levels of WAF1 mRNA were compared with those
in control cells, which were cultured in medium alone. Treatment with
anti-TNF antibody blocked the increase in irradiation-induced WAF1
transcripts by 80%. These experiments were repeated twice with similar
results. Treatment with exogenously added TNF (100-1000 units/ml)
alone for 4 h induced WAF1 mRNA expression in a dose-dependent fashion (Fig.6).
Effect of Prolonged Exposure of Cells to Protein Kinase C
Activator on WAF1 mRNA Expression by Irradiation
Cells exposed
for prolonged durations to
12-O-tetradecanoylphorbol-13-acetate (TPA) reduce their
protein kinase C activity, thus making them resistant to repeated
exposure to TPA(51, 52) . KG1 cells cultured with TPA
(50 nmol/liter) for 4 h had markedly increased accumulation of WAF1 RNA
compared with untreated cells, and prolonged exposure (24 h) to TPA
(100 nmol/liter) did not affect the increased level of WAF1 RNA (Fig.7). However, reexposure of these cells to TPA (50
nmol/liter) for 4 h failed to increase the level of WAF1 transcripts as
compared with cells cultured with TPA for 24 h alone. Irradiation did
not increase the accumulation of WAF1 mRNA in cells exposed to TPA for
24 h.
Transcriptional Regulation of WAF1 in Irradiated KG-1
Cells
Transcriptional run-on assays were performed to help to
determine the mechanisms responsible for the accumulation of WAF
transcripts by irradiation (Fig.8). WAF1 was constitutively
transcribed in untreated KG-1 cells. Exposure of the cells to
irradiation (20 Gy, 2 h) increased the transcriptional rate of WAF1
3-fold.
P-labeled transcripts were hybridized to the linearized
plasmid containing inserts of WAF1,
-actin, or the control
plasmid, pUC118. The relative density of bands of hybridization of WAF1
and -actin in untreated and irradiated lanes was scanned by the
LKB UltroScan XL laser densitometer.
Stability of Steady State WAF1 mRNA in Irradiated KG-1
Cells
To examine posttranscriptional regulation of WAF1 mRNA in
irradiated KG-1 cells, either unirradiated or 20 Gy-irradiated cells
were cultured for 2 h and actinomycin D (5 µg/ml) was added to
cultures. Cells were further cultured for an additional 1-4 h and
were sequentially harvested and examined for level of WAF1 mRNA (Fig.9). In order to make clear the constitutive levels of WAF1
mRNA in untreated cells, the blot with WAF1 probe was exposed to x-ray
film longer than usual (96 h). The half-life (t) of steady
state WAF1 mRNA in unirradiated KG-1 cells was <1 h, while t of WAF1 mRNA after irradiation was >4 h.
arrest of these
cells(1, 31, 32, 61) .
B (NF-B) is also known to be activated by
irradiation(72, 73) . Studies have shown that
expression of WAF1 was induced through functional
p53(29, 30) . The mechanism by which irradiation
increases the level of transcription of the WAF1 gene will
require dissection of its promoter and enhancer region using reporter
gene studies in irradiated cells containing mutant p53.
We thank Ikuko Furusawa for assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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