©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Irradiation Induces WAF1 Expression through a p53-independent Pathway in KG-1 Cells (*)

(Received for publication, March 27, 1995; and in revised form, May 18, 1995)

Makoto Akashi (§) Misao Hachiya Yoshiaki Osawa Konstantin Spirin (1) Gen Suzuki H. Phillip Koeffler (1)

From theDivision of Radiation Health, National Institute of Radiological Sciences, Chiba, 263 Japan and Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(1) or G(2) 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(1) 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(r) 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) (^1)in KG-1 cells.


MATERIALS AND METHODS

Cells and Culture

All cell lines used in the present study were obtained from American Type Tissue Culture Collection. Cells were cultured in alpha-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(2). Nonadherent cell lines were cultured at an initial concentration of 2 10^5 cells/ml, and flasks containing 1 10^6 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 TNFalpha) was purchased from Genzyme (Cambridge, MA). One microliter of this antibody neutralizes 1000 units of TNF. The antibody against human IL-1beta (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-1beta were also from Genzyme, and the specific activities were 1.08 10^8 units/mg protein and 5 10^8 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(3), 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 beta, 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-1beta 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 10^8 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 beta-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(2), 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(2)), 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(2), 0.1 mM EDTA). Nuclei were incubated for 30 min at 30 °C in a reaction buffer containing 150 mM KCl, 3 mM MgCl(2), 0.25 mM ATP, 0.25 mM GTP, 0.25 mM CTP, and 200 µCi of [alpha-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 beta-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 10^7 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(2)PO(4) (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 beta-actin in untreated and irradiated lanes was scanned by the LKB UltroScan XL laser densitometer.


RESULTS

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.


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.



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.


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.



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-1beta RNA in a manner almost parallel to that of WAF1.


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).



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).


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.



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).


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.



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).


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).

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.


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.



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.


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, beta-actin, or the control plasmid, pUC118. The relative density of bands of hybridization of WAF1 and beta-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.


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.




DISCUSSION

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(1) 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 kappaB (NF-kappaB) 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.


FOOTNOTES

*
This work was supported in part by Grant-in-Aid for Science Research 04671542 from the Ministry of Education, Science, and Culture. Further support was provided by National Institutes of Health Grants CA43277, CA26038, CA33936, DK41936, and DK42792 as well as generous gifts from Concern Foundation and the Parker Hughes Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Division of Radiation Health, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-city, CHIBA, 263 Japan. Fax: 81-43-284-1736.

^1
The abbreviations used are: TNF, tumor necrosis factor; Gy, gray; IL, interleukin; kb, kilobase(s); TPA, 12-O-tetradecanoylphorbol-13-acetate.


ACKNOWLEDGEMENTS

We thank Ikuko Furusawa for assistance.


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