Fractalkine is a CX3C-type chemokine that induces chemotaxis of monocytes and cytotoxic T cells. Using the differential display method for examining gene expression in p53-defective cells transfected by adenovirus containing wild-type p53, we observed that fractalkine was induced by ectopic expression of p53. An electrophoretic mobility shift assay showed that a potential p53-binding site present in the promoter of the fractalkine gene could bind to p53 protein. Moreover, a heterogeneous reporter assay indicated that this promoter sequence possessed p53-dependent transcriptional activity. The strong induction of fractalkine when p53 protein was expressed by a wild-type transgene in p53-defective cells brought to light a novel role for p53; that is, potential elimination of damaged cells by the host immune response system through transcriptional regulation of fractalkine. Our results imply a pivotal role of p53 in immunosurveillance to prevent cells from undergoing malignant transformation.

The p53 gene is mutated in more than half of the cancers examined by numerous investigators (1). The protein it encodes functions as a transcription factor when it binds to specific DNA sequences, exerting a tumor-suppressive function through transcriptional regulation of target genes that possess its binding site(s) (2). Several such target genes have been isolated already; usually they appear to function as mediators of either cell cycle arrest or apoptosis (2). One of them,p21Waf1, is considered one of the most important p53 targets because its protein product is essential for cell cycle arrest (3, 4); another, BAX, might mediate p53-dependent apoptosis (5). These two functions of p53 are believed to constitute the core mechanisms of p53-dependent tumor suppression(2). Recently, however, we reported evidence that p53 might be involved directly in the repair of damaged DNA(6). Moreover, given the fact that by now so many p53-target genes have been reported by our group and others(3–12) and because at least 100 potential binding sites for p53 are present in the human genome (13), we predict that p53 might achieve tumor suppression through not a few but multiple physiological functions. Before the precise mechanism(s) of p53 can be clarified, identification of additional target genes would seem to be unavoidable.

Chemokines, secreted proteins of low molecular weight, provide important signals for migration of leukocytes. Chemokines are classified into three major subfamilies, CXC, CC, and C, on the basis of the number and spacing of the first two cysteines in a conserved structural motif. CXC molecules target neutrophils and, to some degree,lymphocytes; CC molecules target monocytes, lymphocytes, basophils, and eosinophils with variable selectivity; and the C chemokine seems to act only on lymphocytes (14). A fourth type of chemokine,designated fractalkine (CX3C), differs from all three of those families in that it is a unique transmembrane molecule, a mucin/chemokine hybrid, which is expressed on the surfaces of endothelial cells activated by cytokines (14, 15). In contrast to other chemokines, fractalkine has multiple functions; it transduces signals through a CX3CR1 receptor and plays a role in adhesion of monocytes,NK3cells and T cells (14–16). In addition, the soluble form of fractalkine is chemotactic for monocytes, NK cells, and T lymphocytes (14). Cleavage of CX3C from the membrane might serve to modulate trafficking at local sites of infection or inflammation.

Here we report evidence to suggest that fractalkine is a direct transcriptional target of p53. The p53-directed regulation of this chemokine provides a novel model of p53 participation in immunosurveillance, i.e., interaction with the host immune system to prevent damaged cells from undergoing malignant transformation.

Cell Lines.

Human cancer cell lines U373 MG (glioblastoma), MCF7 (breast cancer),and H1299 (lung carcinoma) were purchased from American Type Culture Collection. All cells were cultured under conditions recommended by their respective depositors.

Differential Display.

Replication-deficient recombinant viruses Ad-p53 and Ad-LacZ, encoding p53 and LacZ, respectively, under control of the human cytomegalovirus promoter, were generated as described previously (17). Human glioma cell line U373 MG, in which no wild-type p53 is expressed,was infected with Ad-p53 or Ad-LacZ at an multiplicity of infection of 80. Total RNA was isolated with TRIzol Reagent (Life Technologies,Inc., Rockville, MD), following the manufacturer’s instructions, on a time course of 0, 6, 12, 24, and 48 h after infection. Poly(A) RNA was purified from each total RNA with Oligotex-dT30 (JSR, Tokyo,Japan). Each isolated poly(A)+ RNA (0.2 μg) was mixed with 25 pmol of 3′-anchored oligo(dT) primer (GT15 MG, GT15 MA, GT15 MT, or GT15 MC,where M represents a mixture of G, A, and C) in 8 μl of diethylpyrocarbonate-treated water, and heated at 65°C for 5 min. To this solution were added 4 μl of 5× first-strand buffer (Life Technologies, Inc.), 2 μl of 0.1 m DTT, 1 μl each of 250 μm deoxynucleotide triphosphates, 1 μl of RNase inhibitor (40 units; TOYOBO), and 1 μl of Superscript II reverse transcriptase (200 units; Life Technologies, Inc.), to a final volume of 20 μl. After the mixture was incubated at 37°C for 1 h, it was diluted 2.5-fold by addition of 30 μl of diethylpyrocarbonate-treated water and stored at −20°C until use.

The reversely transcribed cDNA mixtures (2 μl each) were amplified by the PCR, in a mixture containing 2 μl of 10× EX Taq buffer (TaKaRa),1.5 μl of each 2.5-mm deoxynucleotide triphosphate, 0.25μl of EX Taq (5 units; TaKaRa), 4.25 μl of water, 10 pmol each of 3′-anchored oligo(dT) primer, and [33P]ATP (10 mCi/ml;Amersham) labeled 5′-primer (12-mer deoxyoligonucleotide primer with arbitrary sequences). Amplification was performed under the following conditions: 2 min at 94°C, then 40 cycles of 20 s at 94°C,30 s at 45°C, and 1 min at 72°C, followed by 5 min at 72°C. Amplified cDNAs were separated on 6% sequencing gels. Subcloning of fragments and DNA sequencing were performed as described previously(11).

Semiquantitative RT-PCR Analysis and Northern Blotting.

RT-PCR experiments were carried out using cDNA generated from 0.2 μg of total RNA. The PCR was performed for 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min. Two μg of poly(A)+ RNA were separated on a 1% agarose gel containing 1×4-morpholinepropanesulfonic acid and 2% formaldehyde and transferred onto a nylon membrane. The membrane was hybridized with random-primed 32P-labeled fractalkine cDNA, washed with 0.1×SSC, 0.1% SDS at 65°C, and exposed for autoradiography at −80°C.

Electrophoretic Mobility Shift Assay.

Synthesized oligonucleotides 5′-GGGCATGTTCCCAGCTTGTGGGGGCATGTTCCCAGCTTGTGG-3′ were annealed and labeled with [γ32P]dATP. Nuclear extracts from lung cancer cell line H1299 infected with Ad-p53 were incubated with the radiolabeled double-stranded oligomer for 30 min at room temperature, in a reaction volume containing 2.0 μg of sonicated salmon sperm DNA, EMSA buffer [0.5 × TBE, 20 mm HEPES (pH 7.5), 0.1 m NaCl, 1.5 mm MgCl2, 10 mm DTT, 20%glycerol, 0.1% NP40, 1 mm phenylmethylsulfonyl fluoride,10 mg/ml pepstatin, and 10 mg/ml leupeptin]. In some cases, monoclonal anti-p53 antibodies, pAb421 (Oncogene Science), and/or pAb1801 (Santa Cruz Biotechnology) were present in the mixture. After incubation, each sample was electrophoresed in a native 4% polyacrylamide gel using 0.5× TBE. The gels were dried and exposed for autoradiography at−80°C for 3 h.

Luciferase Assay.

Oligonucleotides 5′-CGCGTGGGCATGTTCCCAGCTTGTGGGGGCATGTTCCCAGCTTGTGGC-3′ (sense) and 5′-TCGAGCCACAAGCTGGGAACATGCCCCCACAAGCTGGGAACATGCCCA-3′ (antisense)were annealed and ligated into MluI- and XhoI-digested pGL3-promoter vector (Promega Corp., Madison, WI). The plasmid was designated pGL3-FKNBS2. Reporter plasmids pGL3-FKNPro1 and pGL3-FKNPro2 were constructed by excising the gene and subcloning them into pGL3-Basic (Promega). Three oligonucleotides were designed as follows: 5′-AAAACGCGTGGCCTTTTGTGTGTTGCCCACTTA-3′ (F1),5′-AAAACGCGTCAACATCCTGAGGAATCCAGCGGC-3 (F2), and 5′-AAACTCGAGAGGCGGCTAGAGCCAGGCGGC-3′ (R1). H1299 cells were plated in 60-mm tissue culture dishes (1 × 105 cells/dish) 24 h before cotransfection of 1 μg of pGL3-FKNBS2 or pGL3-Control vector and p53-wt or p53-mt vectors in combination with 1 μg of pRL-TK vector, according to the manufacturer’s optimized methodology (FuGENETM6 Transfection Reagent; Roche). To make pGL3-FKNPro1-mt1 or pGL3-FKNPro1-mt2, a point mutation “T” was inserted into the site of either the fourth nucleotide “C” or the seventh nucleotide“G” of p53BS using the QuickChange site-directed mutagenesis kit (Stratagene). For the promoter assay, pGL3-FKNPro1, pGL3-FKNPro2,pGL3-FKNPro1-mt1, or pGL3-FKNPro1-mt2 vector was cotransfected the same way. Thirty-six h after transfection, cells were lysed in 250 μl of a passive lysis buffer (Promega). The lysates were forwarded directly to the Dual Luciferase assay system (Promega), which depends on sequential measurements of firefly and Renilla luciferase activities in specific substrates (beetle luciferin and coelenterazine,respectively). Quantification of both luciferase activities and calculations of relative ratios were carried out manually with a luminometer.

Cell Treatments by Gamma Irradiation.

We used two cell lines, MCF7 and H1299. Cells were seeded 24 h prior to treatment and were 50–70% confluent at the time of treatment. Cells were irradiated (0, 10, 20, 30, 40, 50, and 60 Gy) at∼1 Gy/min. RNAs were isolated from cells incubated for different periods of time (0, 6, 12, 24, and 48 h). To examine expression of the fractalkine gene in the damaged cells, we performed RT-PCR according to the regime described above. The PCR products were transferred to nylon membranes, and the blots were hybridized with an internal oligonucleotide probe. The blots were washed with 6× SSC at 50°C and exposed for autoradiography at −80°C for 3 h.

To isolate target genes that are inducible by wild-type p53, we applied a differential display method using mRNAs isolated from p53-deficient U373 MG cells transfected with adenovirus containing either wild-type p53 or Lac Z genes. A DNA fragment corresponding to one of the bands that showed stronger intensity in p53-transfected cells than in the Lac Z controls was excised, cloned, and sequenced. A BLAST search for homologies between the cloned DNA fragment and archived sequences indicated that this fragment was identical, according to the sequence of the fractalkine cDNA, and we performed semiquantitative RT-PCR analysis. The results clearly indicated that expression of the fractalkine gene was dramatically increased by transfection of Ad-p53, but not Ad-LacZ,in a time-dependent manner (data not shown). The results were confirmed by Northern blotting (Fig. 1 A). The early and strong induction by wild-type p53 suggested that fractalkine was likely to be a direct target of transcriptional activation by p53. To address this hypothesis, we searched for p53-binding site(s) within the 20-kb genomic sequence of the fractalkine gene, obtained from GenBank. We found four possible p53-binding sequences in the promoter region, each of which revealed a >75% match to the consensus p53-binding sequence proposed by El-Deiry et al.(18).

To verify whether p53 could in fact bind to any of these four candidate sequences, we applied an EMSA using the nuclear extract purified from H1299 cells infected by an adenoviral vector containing p53. Oligonucleotides were synthesized according to the four candidate sequences, and each was analyzed separately by EMSA. One of the four candidates showed specific binding to p53 protein. This sequence,designated p53BS, is located 279 bp upstream of the first exon (Fig. 1, B and C). Of the 20 bp of p53BS (with a 1-bp spacer), 17 matched the consensus p53-binding sequence (Fig. 2,A). As shown in Fig. 2B, p53BS was bound (Lanes 1and 2), and a super-shifted band in the presence of mouse monoclonal anti-p53 antibody Pab421 clarified that the bound structure actually included p53 protein (Fig. 2,B, Lane 3). This evidence was clarified by specific competition with self-DNA but not TL(Fig. 2,B, Lanes 4 and 5) and by the super-super-shifted band in the presence of mouse monoclonal anti-p53 antibody Pab1801 (Fig. 2 B, Lane 6).

To examine further whether p53BS does possess p53-dependent transcriptional activity, we performed a heterogeneous-reporter assay using a pGL-FKN BS2 luciferase vector prepared by cloning two copies of p53BS upstream of the minimal SV40 promoter in the pGL3 promoter vector(Promega; Fig. 3,A). Luciferase activity was strongly enhanced by cotransfection with wild-type p53 expression vector but not with mutant p53 (Fig. 3,B). We also prepared pGL3-FKNPro1, which contained the native promoters of the fractalkine gene, and pGL3-FKNPro2, in which p53BS was deleted (Fig. 4,A). Luciferase activity was induced more strongly by wild-type p53, but not by mutant p53, when we used pGL3-FKNPro1 (Fig. 4,B). However, no induction was observed when pGL3-FKNPro2 was transfected (Fig. 4,C). The p53-dependent transcriptional activity of pGL3-FKNPro1 was clearly blocked by a point mutation, which changes either the fourth nucleotide C or seventh nucleotide G of the p53 binding sequence of fractalkine to T (Fig. 4,C). We further examined whether endogenous fractalkine could be induced by cell stress, such as DNA damage by gamma radiation. Fractalkine mRNA was induced by gamma radiation in a dose-dependent manner in MCF7 p53+/+ cells but not H1299 p53−/− cells. Fig. 5 demonstrates that fractalkine is induced by 50 Gy of gamma radiation in a p53-dependent manner. Taken together, these results clearly indicated that fractalkine is a direct target for p53, and that severe cell stress might be required for its induction.

Chemokines play important roles in immunological surveillance to protect host cells from invasion by foreign molecules or infectious pathogens (14, 19, 20). To target leukocytes and direct them toward the infected or inflammatory site, chemokines are secreted locally from many cell types including fibroblasts, endothelial cells,leukocytes, and others (14, 19, 20). Migration of leukocytes occurs in several steps, each of which is regulated by chemokines (14, 19, 20). Circulating leukocytes attach to endothelium with the mediation of integrin molecules; then, following a chemotactic gradient, they pass through the endothelial layer to extravascular spaces and move toward the tissues where the chemokines are being produced. The major role of chemokines is believed to be the host immune response against foreign pathogens or against inflammation in general.

The fact that fractalkine is directly regulated by p53 provides an attractive hypothesis concerning a role of p53 in the immune response. Specifically, p53 may bring about elimination of abnormal (dangerous) cells that have a high potential for malignant transformation, by causing NK cells to target them. Fractalkine secreted from an abnormal cell would spread through the tissues around it, enter blood vessels, and circulate throughout the body, forming a chemotactic gradient as if sending an “SOS” signal. Dangerous cells might be eliminated by self-killing through p53-dependent apoptosis and/or through p53-dependent targeting of NK cells. This model could explain one of the ways cancer cells escape from host immunosurveillance; if membrane-bound fractalkine is present on the surface of a cancer cell, CTLs and NK cells should attach firmly to that cell because they strongly express CX3CR receptor on their surfaces (16). However, if p53 is mutated, this unusual chemokine will not be produced; in consequence, such a cell would have a greater likelihood of escaping the host immune response.

Several studies have exploited the leukocyte-chemoattractant properties of chemokines to enhance a host antitumor response through augmentation of leukocyte infiltration into the tumor. For example IP-10, a CXC chemokine, can elicit a thymus-dependent antitumor response in vivo(21); TCA3, the β chemokine, can also inhibit tumor growth in vivo(22). Others have shown that induction of MCP-1 expression in cancer cells results in suppression of tumor growth and metastasis (23), and that lymphotactin combined with IL-2 can reduce the size of a tumor(24). These results, and our discovery of direct regulation of fractalkine by p53, shed light on a mechanism by which p53 guards cells from malignant transformation and suggest the possibility of developing a novel form of cancer therapy.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by “Research for the Future” Program Grant 96L00102 of the Japan Society for the Promotion of Science.

The abbreviations used are: NK, natural killer;RT-PCR, reverse transcription-PCR; EMSA, electrophoretic mobility shift assay.

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