(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 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) .
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 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 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) ()in KG-1 cells.
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 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.
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 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.
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 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.
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 arrest of these
cells(1, 31, 32, 61) .
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 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.
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.