Mutagenesis, Vol. 15, No. 2, 127-132,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
p53 mutations experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those found in human skin cancers in PUVA-treated patients
1 Mutagenesis Laboratory, National Cancer Institute (IST), Largo Rosanna Benzi, 10, 16132-Genova, 2 Department of Oncology, Biology and Genetics, University of Genova, Genova, and 3 Dermatology Unit, National Cancer Institute (IST) Genova, Italy
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Abstract |
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8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis. PUVA therapy has been associated with an increased risk of developing skin squamous cell carcinoma (SCC). In order to determine the PUVA-induced p53 mutation spectrum, a yeast expression vector harbouring a human wild-type p53 cDNA was incubated with 8-MOP, and UVA irradiated in vitro. PUVA-damaged and undamaged DNA was transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. An 8-MOP concentration-dependent decrease in survival and increase in mutant frequency were observed. At a fixed 8-MOP concentration, survival decreased and mutant frequency increased as UVA irradiation increased. Eleven mutant clones contained 11 mutations: 10 were single base pair substitutions, the remaining one being a complex mutation. All eight T:A-targeted mutations were at 5'-TpA sites, hallmark mutations of PUVA mutagenesis. Through a rigorous statistical test, the PUVA-induced p53 mutation spectrum appears to differ significantly (P < 0.0002) from that observed in SCC in PUVA-treated patients. The present work demonstrates that a specific PUVA-induced mutational fingerprint could be obtained and recognized on human p53 cDNA. This result may suggest that PUVA therapy can be a risk factor for the development of SCC in psoriasis patients through a mechanism not involving the induction of p53 mutations.
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Introduction |
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8-Methoxypsoralen (8-MOP) plus UVA irradiation (PUVA therapy) has been used for the treatment of psoriasis (Parrish et al., 1974
). 8-MOP is ingested, and 2 h later the patient is exposed to long wavelength UV irradiation (UVA, 
= 320–400 nm). Neither the drug nor the UVA irradiation is effective alone. Given the penetration characteristics of UVA (the absorption of photons is confined to the skin), PUVA therapy is a form of targeted chemotherapy. Several studies have shown that PUVA is a mutagen and carcinogen (Bredberg and Nachmansson, 1987; Sage et al., 1993; Yang et al., 1994; Gunther et al., 1995; Nataraj et al., 1996) and may have immunosuppressive effects (Kraemer et al., 1981; Moscicki et al., 1982; Bredberg et al., 1983; Kripke et al., 1984; Gasparro et al., 1997). Clinical follow-up studies have shown that psoriasis patients who received extensive PUVA therapy had an increased risk of developing skin squamous cell carcinoma (SCC) over that of the general population (Stern et al., 1984, 1988, 1994; Bruynzeel et al., 1991; Lindelöf et al., 1991; Lever and Farr, 1994; Maier et al., 1996). On the other hand, the overall risk of non-cutaneous malignant tumours was nearly identical to that expected in the general population (Stern et al., 1997a). Stern et al. (1997b) have shown that the risk of malignant melanoma also increases in PUVA patients, especially among those who received at least 250 PUVA treatments. These results raise the question of whether PUVA should be abandoned (Wolff, 1997).
8-MOP intercalates between DNA base pairs and, following UVA irradiation, undergoes photocycloaddition with pyrimidines in a sequence-dependent manner to generate monoadducts and interstrand crosslinks at 5'-TpA sites (Cimino et al., 1985; Sage and Moustacchi, 1987). Monoadducts induced by a bifunctional psoralen like 8-MOP are derived by photoaddition between the 5,6 double bond of a pyrimidine and a double bond in the furan-(4',5') or pyrone-(3,4) rings of the psoralen. At 5'-TpA sites a monoadduct can absorb a second UVA photon with the formation of an interstrand crosslink. Mutagenesis studies in rodent or human cell lines reveal that 8-MOP leaves a highly specific mutation signature (T:A-targeted base pair substitutions at 5'-TpA sites) (Sage et al., 1993; Yang et al., 1994; Gunther et al., 1995; Laquerbe et al., 1995).
The p53 protein regulates expression of a number of genes playing an important role in the control of cell cycle progression and apoptosis (Ko and Prives, 1996). This main but not unique function requires selective recognition and binding to specific DNA sequences by the DNA-binding domain of the protein and a functional oligomerization domain that mediates the formation of active tetramers. Alterations in the p53 gene are the most common genetic defects known to occur in human tumours (Greenblatt et al., 1994). About 80% of such alterations are missense mutations, the most informative alterations in mutation spectrometry studies. Similarities have been observed between the features of p53 mutations observed in specific tumours and the mutagenic specificity (determined in different genes) of aetiological agents known to be a risk factor for such tumors. This evidence suggests a role for aflatoxin B1, UV light and tobacco smoke in the development of hepatocellular carcinomas (Bressac et al., 1991; Hsu et al., 1991), non-melanoma-skin cancers (NMSC) (Brash et al., 1991; Ziegler et al., 1994) and lung carcinomas (Greenblatt et al., 1994; Brennan et al., 1995), respectively. A limitation inherent in these studies is the failure to experimentally reproduce the observed p53 mutation spectra. Only recently has a yeast-based approach to select p53 mutants for the transactivation function become available (Inga et al., 1997a). Using this system we confirmed that carcinogens induce different experimental p53 mutation fingerprints and we also found that the UV-induced p53 mutation spectrum selected in yeast is indistinguishable from that observed in NMSC (Inga et al., 1998).
Recently p53 mutations in tumours developed in PUVA-treated mice (Nataraj et al., 1996) and in psoriasis patients previously treated with PUVA have been determined (Nataraj et al., 1997; Wang et al., 1997). In mice, with no exposure to any other carcinogen, PUVA acted as a complete carcinogen. Furthermore, ~40% of all p53 mutations in PUVA-induced murine skin tumours occurred at 5'-TpA sites. In contrast, in skin tumours which developed in PUVA-treated patients the incidence of T:A-targeted p53 mutations at 5'-TpA sites was greatly reduced (5-fold with respect to the murine study). In other experimental systems the PUVA-induced mutation fingerprint was far more recognizable [e.g. 26/27 mutations occurred at crosslinkable TpA sites at the hprt locus in CHO cells treated with 8-MOP + UVA (Sage et al., 1993)]. Why is the PUVA fingerprint less recognizable in humans? One hypothesis is based on the fact that mutation spectra may be dependent on DNA sequence context. Although well conserved among different species at the protein level, p53 coding sequences differ at the DNA level. Since induced mutation spectra can be dependent on DNA sequence context (Holmquist and Gao, 1997; Pfeifer and Holmquist, 1997), murine and human p53 spectra can differ significantly.
The lack of a mutation spectrum experimentally induced by PUVA for the human p53 locus prevents us from answering the question of whether the mutations found in vivo in tumours can be attributable to such therapy.
Here we report the determination of a PUVA-induced mutation spectrum for human p53 cDNA using a yeast functional assay. We also tested whether the molecular features of the p53 mutations observed in yeast support the notion of a direct role of PUVA therapy in inducing tumour-associated p53 mutations.
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Materials and methods |
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Compounds
8-MOP was purchased from Sigma (St Louis, MO).
Strain, vectors and media
The pLS76 yeast expression vector harbouring human wild-type p53 cDNA under the control of an ADH1 promoter has the LEU2 gene as a selectable marker in yeast cells. pRDI-22, the expression vector used for gap repair, is identical to pLS76 except for the presence of a linker cloned between the p53 BsmI and StuI sites. pRDI-22 cut with HindIII and StuI is used for gap repair assay without gel purification, and gives a very low number of colonies containing self-ligated vector. The haploid strain yIG397 (MATa ade2-1 leu2-3,112 trp1-1 his3-11,15 can1-100, ura3-1 URA3 3xRGC::pCYC1::ADE2) containing the ADE2 reporter gene under p53 control (Flaman et al., 1995) was used as recipient for p53 expression vectors. Standard yeast manipulations were performed as described (Guthrie and Fink, 1991). Complete medium supplemented with 200 mg/l adenine was used for routine cultures, minimal medium lacking leucine and containing 5 mg/l adenine was used to test p53 status, and minimal medium lacking leucine and adenine was used to confirm the ade– phenotype. Synthetic minimal medium lacking leucine and containing 200 mg/l adenine was used to grow ade– clones.
DNA modification, transfection and plasmid recovery
pLS76 (300 ng/µl) was treated for 30 min with different concentrations of 8-MOP in the dark. Drops of 8-MOP-treated plasmid (20 µl) were then placed in a Petri dish and UVA irradiated using a Multiband UV lamp, model UVGL-15 UV (maximum emission at = 365 nm) at 5 or 10 kJ/m2. Radiation flux was measured using a UV meter, model UVX-Digital radiometer (UVP Inc., San Gabriel, CA). DNA was purified by three ethanol precipitations, washed with 70% ethanol and resuspended in sterile water. Damaged or undamaged vectors were then transfected into yIG397 cells by electroporation and transformants were plated on selective synthetic medium containing 1 M sorbitol. After 3 days incubation at 30°C, colonies appeared and were analysed. Selection for the plasmid marker (LEU2) allowed an indirect determination of the lethal effect of the damaging treatment as number of transformants scored in transfections with damaged plasmids with respect to number obtained with undamaged vector. As transformation plates contained a minimal amount of adenine, adenine auxotrophs were able to achieve only a few cell divisions and thus produced smaller red colonies. Spontaneous and induced mutant frequency were defined as the number of small red colonies with respect to the total number of transformants. The fold mutant induction was defined as the ratio between the mutant frequency observed with damaged vector and the spontaneous frequency.
DNA amplification, gap repair and sequencing
From each yeast plasmid preparation, the p53 open reading frame between nucleotide positions 125 and 1122, including the entire sequence coding for the DNA-binding domain, was PCR amplified. Briefly, PCR was performed with primers P3 and P4 (Flaman et al., 1995) for 35 cycles of 94°C for 30 s, 65°C for 60 s and 72°C for 80 s with Taq polymerase (Promega, Madison, WI). Unpurified PCR products and HindIII + StuI linearized pRDI-22 were co-transfected by electroporation into yIG397. Gap repaired transformants were selected on suitable plates and the percentage of small red colonies was determined. p53 mutants, giving ~100% red colonies, were re-amplified with primers P3 and P4 and Taq DNA polymerase (Promega, Madison, WI). PCR products were purified with Microcon 100 (Amicon Inc., Beverly, MA) and directly sequenced on both strands with an ABI PRISMTM dye terminator cycle sequencing Ready Reaction Kit (Perkin Elmer, Milano, Italy) with primers P3, P4, P5 and P6 (Inga et al., 1997a) on an Applied Biosystems 377 Automated Sequencer (Perkin Elmer, Milan, Italy). (For a general scheme of the methodology of the yeast system see Figure 1 in Inga et al., 1997a).
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Statistical analysis
The Adams and Skopek algorithm uses a Monte Carlo method to simulate a P value for the standard hypergeometric test for a contingency table (Adams and Skopek, 1987
). Unlike the 
2 test, which can also be applied to contingency tables and requires that all cells contain five or more events, the hypergeometric test is appropriate when applied to sparse data sets, as are often found in mutational spectra analysis. The Cariello program (Cariello et al., 1994) uses a random number generator to produce a large number of simulated spectra based on the hypergeometric probability of the experimentally observed input spectra. The degree to which the simulated spectra differ from the input spectra is used to estimate the probability that the two input spectra were derived from the same population. A P value <0.05 leads one to reject the null hypothesis and conclude that the input spectra are different. |
Results |
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Induction of p53 mutations by 8-MOP + UVA irradiation
The pLS76 yeast expression vector harbouring human wild-type p53 cDNA was treated with two concentrations of 8-MOP in the dark and then UVA irradiated (
= 365 nm) at two fluences. This split treatment was expected to allow non-covalent interaction of 8-MOP with DNA, followed by the formation of 8-MOP monoadducts and crosslinks after UVA irradiation. Both damaged and undamaged DNA were transfected into yIG397 cells by electroporation. UVA irradiation (10 kJ/m2) had no effect on survival per se, while it slightly increased mutant frequency (Table I). An 8-MOP concentration-dependent decrease in survival and an increase in mutant frequency were observed (Table I). At a fixed 8-MOP concentration survival decreased and mutant frequency increased as UVA irradiation increased. Molecular analysis was limited to mutant clones isolated at 9.2 µM 8-MOP, when mutant frequency was at least 96-fold above background level, assuring the determination of a 8-MOP + UVA-specific fingerprint since 98.5% of mutations are expected to result from the damaging treatment.
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Molecular characterization of p53 mutations
The p53 expression plasmids were rescued from 31 ade– leu+ colonies and the p53 open reading frame was PCR amplified. Surprisingly, the majority (20/31, 64%) of the phenotypically confirmed p53 mutants were not amplifiable: these samples gave no PCR products in at least two independent PCR reactions. The presence of a p53 expression plasmid in these 20 mutant clones was also searched for by direct transformation in Escherichia coli. None of these mutants gave transformants in E.coli, suggesting that no plasmid carrying the ß-lactamase gene (AmpR) was present. Due to the fact that all these clones were selected on plates lacking leucine, we reasoned that possible recombination events could have directed chromosomal integration of the entire or part of the p53 expression plasmid. Universal primers for the yeast pRS series (progenitor of the pLS76 plasmid) were used in order to verify the presence of at least portions of the p53 expression vector. Again, none of these 20 clones were amplifiable. We concluded that in the majority of phenotypically mutant clones p53 cDNA was lost along with most of the plasmid, except for the LEU marker, which was probably chromosomally integrated. It is possible that the presence of PUVA-induced interstrand crosslinks strongly inhibited plasmid replication and concomitantly favoured recombination events, causing the loss of p53 cDNA.
PCR fragments obtained from the 11 amplifiable samples tested by gap repair invariably gave 100% red colonies after gap repair and showed p53 mutations by DNA sequencing. Table II reports the complete list of the 11 mutations identified, with information on the type of mutation and the amino acid change. All 10 single mutations were base pair substitutions, mainly (8/10, 80%) T:A-targeted (Table III). The remaining mutation is a complex mutation (tandem mutation and a –1C deletion). All T:A-targeted mutations were in 5'-TpA-3' sequences (Table III).
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Discussion |
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One of the tasks of molecular epidemiology is to help in identifying carcinogens that induce a specific cancer. Since most carcinogens leave specific mutation fingerprints, the mutation spectrometry approach can be used in identifying factors involved in tumorigenesis (Harris, 1993
).
PUVA therapy is an effective treatment for severe psoriasis, but was unfortunately shown to be mutagenic and carcinogenic in experimental systems (Bredberg and Nachmansson, 1987; Sage et al., 1993; Yang et al., 1994; Gunther et al., 1995; Nataraj et al., 1996). PUVA therapy constitutes a risk factor for the development of SCC of the skin in psoriasis patients treated with it (Stern et al., 1984, 1988, 1994, 1997a; Bruynzeel et al., 1991; Lindelöf et al., 1991; Lever and Farr, 1994; Maier et al., 1996). It was estimated that high dose PUVA (>300 treatments) caused an 80-fold increase in the overall risk of SCC, with lower risks of 20- and 10-fold for medium and low dose exposures, respectively (Stern et al., 1994). Surprisingly, p53 mutations in skin tumours which developed in PUVA-treated human patients (Nataraj et al., 1997; Wang et al., 1997) or in mice (Nataraj et al., 1996) were only partially consistent with the known mutational specificity for PUVA. In the present work we demonstrate that a clean PUVA-induced p53 mutation fingerprint was obtainable using a yeast p53 functional assay.
8-MOP + UVA-induced mutations are targeted to TpA sites
Although a small number of 8-MOP + UVA-induced p53 mutants were analysed, the mutation fingerprint that emerged is clearly consistent with the known properties of 8-MOP + UVA determined in different experimental systems (Cimino et al., 1985; Sage and Moustacchi, 1987; Sage et al., 1993; Yang et al., 1994; Gunther et al., 1995; Laquerbe et al., 1995): mutations were mainly (8/10, 80%) T:A-targeted, located at 5'-TpA sites. The genotoxicity of 8-MOP has been attributed to its ability to covalently bind to DNA upon UVA irradiation. 8-MOP intercalates between DNA base pairs and, following UVA irradiation, undergoes photocycloaddition with pyrimidines (mainly thymine) in a sequence-dependent manner (Cimino et al., 1985; Sage and Moustacchi, 1987) to generate monoadducts and/or interstrand crosslinks. The 5'-TpA site is the preferred target for the formation of monoadducts and the exclusive one for interstrand crosslinks (Sage and Moustacchi, 1987). Our results also confirmed the strand bias observed in previous studies (Sage et al., 1993; Yang et al., 1994; Gunther et al., 1995; Laquerbe et al., 1995). The strand bias is consistent with the observation that in mammalian cells psoralen interstrand crosslinks and/or monoadducts are preferentially removed from the transcribed strand of actively transcribed genes compared with the genome overall (Vos and Hanawalt, 1987; Islas et al., 1991). In our system, where transcription of the target gene (p53) is necessary for the mutation assay, we observed that 75% (6/8) of 5'-TpA-targeted mutations can be interpreted as derived from a lesion present on the non-transcribed strand.
The PUVA fingerprint in yeast is significantly different from those induced in the same experimental system by UV light (P < 0.05) (Inga et al., 1998) and by chloroethyl cyclohexyl nitrosourea (P < 0.02) (Inga et al., 1997a) when comparisons are performed by considering both mutation type and location of the alteration in the p53 gene through a rigorous statistical test that provides a precise measure of the relatedness of two spectra (Cariello et al., 1994).
8-MOP + UVA-induced p53 mutation spectra in yeast and in skin cancer developed in PUVA-treated human patients are different
The mutation spectrum induced by PUVA in the p53 cDNA in yeast (this work) was compared with that obtained in mice (Nataraj et al., 1996) and with that obtained in SCC induced in PUVA-treated patients (Nataraj et al., 1997; Wang et al., 1997). Comparisons were performed by class of mutations and, when applicable, through the Cariello program, which provides a precise measure of the relatedness of two spectra (Cariello et al., 1994).
When classes of mutations were considered (Table IV), only the yeast system shows 100% T:A-targeted mutations at 5-TpA sequences, the hallmark of 8-MOP + UVA mutagenesis, an incidence significantly higher than in mice (P < 0.05, Fisher's exact test) and in human tumours (P < 0.0001, Fisher's exact test). Consistently, the incidence of T:A-targeted mutations at TpA sites is non-significantly different between mouse and yeast.
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No p53 mutations induced by 8-MOP + UVA in yeast were found in skin tumours induced in PUVA-treated patients (Nataraj et al., 1997
; Wang et al., 1997). Overall, statistical comparison using the Cariello program considering both type and site of mutations revealed that the two p53 mutation spectra are clearly different (P < 0.0002) (Figure 1
). We could not perform a Cariello analysis between the yeast and the mice spectra since the two genes have different nucleotide sequences. The difference between the yeast and the human p53 spectra is significant and interesting, but some caution in the conclusions has to be exercised. Certainly the yeast p53 functional assay has some interesting features (e.g. it is based on a pivotal function of p53, whose gene is frequently mutated in tumours). However, it also has some drawbacks: (i) the damaging treatment is done in vitro and the influence, for example, of chromatin structure is not taken into account; (ii) the target is cDNA and not the entire gene; (iii) p53 is basically a transcription factor but it also exerts transcription-independent functions. Futhermore, when one compares p53 mutations obtained in yeast with those observed in tumours another level of complexity is added.
Why is the PUVA fingerprint not found in SCC induced in PUVA-treated patients? Gasparro et al. (1998) hypothesized that a bias against the detection of mutations at T:A sites could exist. Our results clearly show that a PUVA-induced mutation fingerprint can be obtained at the human p53 cDNA locus and that there is no bias against T:A-targeted mutations. An alternative hypothesis would be that specific PUVA-induced mutants cannot be selected for in vivo. Only one of the mutations found in this study has not been previously reported in the p53 database (Hainaut et al., 1998), suggesting that this is not the case. Furthermore, we evaluated the dominant potential of PUVA-induced p53 mutants using a simple yeast functional assay (Inga et al., 1997b) and found that many of them were dominant (Pro177His, Cys238Trp and Tyr163His), a result which would suggest a selective advantage for those mutants in vivo.
Another hypothesis would be that the p53 mutation spectrum found in SCC induced in PUVA-treated patients could not show a PUVA-specific fingerprint because those cancers were initiated by other carcinogens and PUVA therapy promoted their development. Gasparro et al. (1998) noted that 62% of psoriasis patients included in the p53 mutation studies (Nataraj et al., 1997; Wang et al., 1997) were previously treated with UVB therapy, while others may have intentionally sought solar exposure because it is known to have palliative effects. It has been reported that even sun-exposed normal skin contains populations of cells harbouring p53 mutations (Nakazawa et al., 1994; Jonason et al., 1996). Therefore, PUVA may have acted as a promoter for those cells without leaving a specific mutational fingerprint. The fact that many p53 mutations found in SCC induced in PUVA-treated patients are consistent with UVB mutagenesis support a promoting rather than initiator role for PUVA therapy.
All the above-mentioned hypotheses are based on the notion that p53 mutations are a determinant factor for the development of SCC. However, this may not always be the case. Wrone-Smith et al. (1995) showed that psoriatic skin and SCC contain increased amounts of bcl-x and bcl-2, respectively, which could counteract apoptotic signals, promoting cell survival even in presence of wild-type p53. Finally, PUVA treatment may cause an immunological alteration that permits the growth of skin cancers induced by other carcinogens (Gasparro et al., 1997). The fact that the p53 mutational fingerprint induced in yeast is completely different from that observed in PUVA-treated patients strongly suggests that PUVA therapy is a risk factor for the development of skin tumours through a mechanism other than one involving its p53 mutagenicity.
In conclusion, we have shown that the generation of a mutation fingerprint for the human p53 cDNA sequence by the yeast functional assay can represent a useful tool in the field of cancer molecular epidemiology (Vogelstein and Kinzler, 1992; Harris, 1993). In fact, a comparison of the experimentally obtained p53 mutation fingerprints with spectra obtained in human tumours can take into account all the informational richness contained in a p53 mutation spectrum, i.e. simultaneously consider both type and position of all mutations. The present approach can be used to pinpoint the aetiological agent of specific tumours (e.g. UV or NMSC; see Inga et al., 1998) or can contribute in elucidating the molecular mechanism by which an agent is carcinogenic.
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Acknowledgments |
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This work was partially supported by the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST). P.M. was supported by an Adriano Buzzati-Traverso fellowship.
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Notes |
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4 To whom correspondence should be addressed: Tel: +39 010 5600292; Fax: +39 010 5600992; Email: fronzagi{at}hp380.ist.unige.it
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Received on August 9, 1999; accepted on October 14, 1999.
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