Open Access

CpG island methylation of TMS1/ASC and CASP8 genes in cervical cancer

European Journal of Medical Research200914:71

DOI: 10.1186/2047-783X-14-2-71

Received: 6 February 2008

Accepted: 5 November 2008

Published: 18 February 2009

Abstract

Background

Gene silencing associated with aberrant methylation of promoter region CpG islands is an acquired epigenetic alteration that serves as an alternative to genetic defects in the inactivation of tumor suppressor and other genes in human cancers.

Aims

This study describes the methylation status of TMS1/ASC and CASP8 genes in cervical cancer. We also examined the prevalence of TMS1/ASC and CASP8 genes methylation in cervical cancer tissue and none - neo plastic samples in an effort to correlate with smoking habit and clinicopathological features.

Method

Target DNA was modified by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequently amplified by Methylation Specific (MS) PCR with primers specific for methylated versus unmethylated DNA. The PCR product was detected by gel electrophoresis and combined with the clinical records of patients.

Results

The methylation pattern of the TMS1/ASC and CASP8 genes in specimens of cervical cancer and adjacent normal tissues were detected [5/80 (6.2%), 3/80 (3.75%)-2/80 (2.5%), 1/80 (1.2%) respectively]. No statistical differences were seen in the extent of differentiation, invasion, pathological type and smoking habit between the methylated and unmethylated tissues (P > 0.05).

Conclusion

The present study conclude that the frequency of TMS1/ASC and CASP8 genes methylation in cervical cancer are rare (< 6%), and have no any critical role in development of cervical cancer.

Keywords

Methylation TMS1/ASC CASP8 cervical cancer

Introduction

Cervical cancer is the second most common cancer and an important cause of death in women worldwide [1]. Therefore, it is likely that host genetic and epigenetic events play an important role in cervical carcinogenesis. The term "epigenetic" is used to describe mitotically and meiotically heritable states of gene expression that are not due to changes in DNA sequence [2]. DNA methylation is an epigenetic mechanism used for long-term silencing of gene expression. The methylation pattern is established during development and is normally maintained throughout the life of an individual. It has been shown that such epigenetic mechanisms can be important in initiating tumorogenesis and supporting the malignant state of cancer cells [3].

Apoptosis is mediated by a family of cystine proteases called caspases. CASP8, located at chromosome 2q33-34, encodes caspase 8, an initiator caspase that plays an important role in the Fas-ligand pathway [4]. Alterations of these genes have been described in several neoplasias, such as mutations in colon cancer and promoter hypermethylation in medulloblastomas and neuroblastomas [5]. TMS1 gene located on 16p11.2-12 chromosome has function such as activates pro caspas-1-8, modulates NF-KappaB activation pathway [6]. TMS1/ASC is a bipartite protein comprising two protein-protein interaction domains, a pyrin domain (PYD) and a caspase recruitment domain (CARD). Proteins containing these domains play crucial roles in regulating apoptosis and immune response pathways, and mutations in a number of PYD-and CARDcontaining proteins have been linked to auto-inflammatory diseases and cancer [7]. This gene is also known as ASC (Apoptosis Speck like protein containing a CARD) [8]. So, the down regulation of TMS1/ASC in breast cancer cell lines correlates with dense methylation on the CpG islands [9]. Methylation of the promoter region of TMS1/ASC has also been identified in small cell lung cancer and non-small cell lung cancer [10], human glioblastoma [11], ovarian tumors [12], colorectal cancer [13], neuroblastoma [14], and melanoma [15]. It was appeared no correlation between methylation of the TMS1/ASC gene and acute lymphoblastic leukemia [16]. Tischoff et al. [17] reported that promoter methylation of the proapoptotic genes CASP8 and TMS1 are involved in the malignant epithelial liver tumor. The role of epigenetic (gene inactivation) in tumorigenesis in gynecologic malignancies have been poorly understood. So, we investigated the promoter methylation status in CASP8 and TMS1 genes and relationships between clinopathologic parameters and methylation status with risk of cervical cancer.

Materials and methods

Study subjects

The case -control study involved collection of tissue samples of 160 North Indian subjects. 80 cases were newly diagnosed, previously untreated and histologically confirmed as cervix cancer patients. The samples were collected from the Post graduate Institute of Medical Education and Research (PGIMER) Chandigarh and Government Medical College (GMC), Chandigarh. The control tissue samples (n = 80) were collected from the same institute with no history of cancer or pre cancer. Informed consent was obtained from all the cases and controls.

DNA Extraction

Genomic DNA was isolated from tissue samples by the procedure of Roe et al. [18]. For the DNA methylation studies, 1 μg of genomic DNA was processed and modified with sodium bisulfite using the Intergen CpGenome DNA modification kit (Intergen, Nor-cross, GA). Briefly, genomic DNA was modified by sodium bisulfite, desulfonated with sodium hydroxide, and then purified and resuspended in TE (10 mM Tris, 0.1 mM EDTA, pH 7.5). Negative control (no sample) and positive control (in vitro methylated and bisulfite treated human placenta DNA) were included in all reaction. The methylation-specific (MS) PCR conditions and the sequences of premiers are used for TMS1/ASC and Caspes-8 previously described by Liu et al. [19] and Lázcoz et al. [20] respectively.

The positions of the 191 bp PCR product representing the methylated and unmethylated 196 bp alleles for TMS1/ASC were separated on 3% agarose gels (Figure 1). In case of Caspes-8, the PCR products for the methylated and unmethylated alleles which separated on 3% were 321 and 320 bp respectively (Figure 2).
Figure 1

Methylation analysis of TMS1 / ASC in cervical cancer. Lane U: amplified product with primers recognizing unmethylated sequence; Lane M: amplified product with primers recognizing methylated sequence. NC, normal control; PC, positive control for methylation. L: ladder (100 bp).

Figure 2

Methylation analysis CASP8 in cervical cancer. Lane U: amplified product with primers recognizing unmethylated sequence; Lane M: amplified product with primers recognizing methylated sequence. NC, normal control; PC, positive control for methylation. L: ladder (100 bp).

Statistical analysis

The results combined with the clinical records of Patients were analyzed with the the Epi-Info software (Epi-Info, version 3.2, Centers for Disease Control and prevention, Atlanta, GA, USA) and the software package SPSS, version 10.0 (SPSS, Chicago, IL, USA). Significance was set at P ≤0.05.

Results

The methylation status of the TMS1/ASC and Caspase-8 in primary cervical cancer and control non-neoplastic cervix tissue specimens derived from north India population was analyzed.

There were no methylation frequency association for TMS1/ASC gene with various clincopathological parameters including: age, smoking, histological type, and stage (P > 0.05) (Table 1). Similarly, same result was observed for Caspase8 gene with various clinicopathological parameters (Table 2).
Table 1

The relationship between TMS1 promoter methylation and clinicopathology parameter of cervical cancer

Characteristic

overall

present

Absent

P Value

< 50

44

2(4.5%)

42(95.4%)

0.65

> 50

36

3(8.3%)

33(91.6%)

 

Smoker

5

1(20%)

4(80%)

0.28

Nonsmoker

75

4(5.3%)

71(94.6%)

 

Histological type SCC

68

4(5.8%)

64(94.1%)

0.56

AC

12

1(8.3%)

11(91.6%)

 

Stage

    

Ib

46

1(2.2%)

45(97.8%)

0.10

IIa

12

2(16.6%)IIb

10(83.3%)

 

IIb

19

2(10.5%)

17(89.4%)

 

IIIb

3

-

3(100%)

 
Table 2

The relationship between Caspase-8 promoter methylation and clinicopathology parameter of cervical cancer

Characteristic

overall

present

Absent

P Value

< 50

44

0

44(100%)

-

> 50

36

2(5.6%)

34(94.4%)

 

Smoker

5

1(20%)

4(80%)

0.12

Nonsmoker

75

1(1.3%)

74(98.6%)

 

Histological type SCC

68

2(2.9%)

66(97.05%)

 

AC

12

-

  

Stage

    

Ib

46

1(2.17%)

45(97.8%)

0.37

IIa

12

1(8.3%)

11(91.6%)

 

IIb

19

-

-

 

IIIb

3

-

-

 

The status of promoter methylation for TMS1/ASC and Caspase-8 genes in 80 primary cervical cancer tissue and 80 control non-neoplastic cervix tissue specimens were examined. The promoter methylation frequency for TMS1/ASC gene in cervical cancer and control were 6.2% (5/80), 3.75% (3/80) respectively.

The methylation status of the Caspase-8 gene was detected in cervical cancer tissue 2.5% (2/80) and normal tissue 1/80 (1.2%). There was no deference significant methylation frequency for the TMS1/ASC and Caspase-8 genes as compared to controls non-neo-plastic cervix.

Discussion

The effect of DNA hypermethylation in gene promoter regions is similar to genetic loss-offunction mutations [21]. Many cellular pathways are inactivated by this epigenetic event, including DNA repair, cell cycle, apoptosis, cell adherence, and detoxification [22]. The specific patterns of CpG island hypermethylation between tumor types may provide a useful signature for tumor diagnosis and prognosis [23]. TMS1/ASC gene was originally identified as a target of methylation-induced silencing using cell lines that over express DNA methyltransferase 1 (DNMT1). In another critical pathway mediating cell death via death receptors, CASP8 acts as a key apoptotic enzyme by serving as an "initiator CASP"; moreover, CASP8 was recently shown to be silenced by aberrant methylation [24]. However, because the 5' region of CASP8 does not contain a typical CpG island, the relevance of methylation to its silencing remains unclear [25]. TMS1/ASC is a novel proapoptotic gene previously identified as a target of DNA methylation in breast cancer [26]. Terasawa et al. [27] showed that aberrant methylation of the 5' region of TMS1/ASC is well correlated with loss of expression in ovarian cancer. Notably, decreasing TMS1/ASC expression reduces sensitivity to chemotherapeutic drugs [28]. The methylationmediated silencing of TMS1/ASC would be expected to contribute to a survival advantage for tumor cells, by enabling them to escape apoptosis, which supports a role for aberrant methylation in human ovarian tumorigenesis [29]. Approximately 40% (10/17) of primary breast tumors also exhibited aberrant methylation of the TMS1/ASC gene. Different studies have confirmed the frequency of aberrant methylation of TMS1/ASC in primary breast cancers, ranging from 10% to 40%. Methylation-associated silencing of the TMS1/ASC gene was observed in 11% of gastric carcinomas, 40%-41% of small cell and non small cell lung carcinomas, 50% of malignant melanomas, and 44% of primary glioblastomas [30, 31]. Jens et al. [32] reported frequency of methylation for Caspase-8 (1.2%) and TMS1 (5.1%) in ovarian cancer, and concluded that, methylation of these genes in regulation of apoptosis was no significant (P = 0.74). In line of our study Feng et al. [33] demonstrated the rate of hypermethylation for TMS1 to be 3.1% in controls and 6.7% in patient with cervical cancer. Liu et al. [34] found that aberrant methylation of the TMS1 gene was detected in tumor tissues %36.1 with Choangio carcinoma, and in normal tissue 8.3%. So, there were no statistical differences in age, gender, pathologic type between the methylated and unmethylated tissues. Aberrant methylation of TMS1 gene was detected in 15 of 80 ovarian cancer tissue (19%) but in none of the normal ovary specimens [35]. Michalowski et al. [36] observed, hypermethylation of Caspase-8 in 38% in neuroblastoma. Matinez et al. [37] strongly suggested that hypermethylation of the pro-apoptotic Caspase-8 in glioblastoma (P = 0.0035). The present study's results were consonance with those of Yang et al. [38] who carried out a similar study in Chinas women. However, our study has several potential limitations. First, we were not able to incorporate into the model information concerning the relative level of hypermethylation of each specific gene. It is possible that other gene combinations, which may have increased sensitivity and specificity, will be identified through the use of real-time PCRbased assays such as MethyLight [39], which provided information on the relative level of hypermethylation of each specific gene examined. Second, we limited our search for useful hypermethylated genes to an assessment of 2 genes that had been previously reported to be associated with cancers at other sites. Identification of additional novel CpG islands that are specifically associated with cervical cancer will be needed to construct a panel with higher sensitivity that maintains high specificity, and studies examining detection of such a panel of genes using recently developed quantitative assays should be undertaken. More study using a much larger samples size are needed to further define the potential role of methylated DNA marker in cervical cancer management.

Declarations

Acknowledgements

The authors are grateful to University Grant Commission (UGC) -New Delhi, India, and University of Sistan and Baluchistan - Zahedan, Iran, for supporting this project financially.

Authors’ Affiliations

(1)
Department of Biotechnology, Panjab University
(2)
Department of Biology, Sistan and Baluchistan University
(3)
Department of Genetic, Hormozgan University of Medical Sciences
(4)
Department of Obstetrics and Gynaecology, Government Medical College and Hospital

References

  1. Jemal A, Murray T, Samkuels A, Ghafoor A, Ward E, Thun MJ: Cancer statistics. CA Cancer J Clin 2003, 53: 5–26. 10.3322/canjclin.53.1.5PubMedView ArticleGoogle Scholar
  2. Adcock IM, Ford P, Barnes PJ, Ito K: Epigenetics and airways disease. Respir Res 2006, 6: 21.View ArticleGoogle Scholar
  3. Chen W, et al.: Epigenetic and genetic loss of Hic1 function accentuates the role of p53 in tumorigenesis. Cancer Cell 2004, 6: 387–398. 10.1016/j.ccr.2004.08.030PubMedView ArticleGoogle Scholar
  4. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999, 274: 20049–20052. 10.1074/jbc.274.29.20049PubMedView ArticleGoogle Scholar
  5. Gonzalez-Gomez P, Bello MJ, Inda MM, Alonso ME, Arjona D, Aminoso C, Lopez-Marin I, de Campos JM, Sarasa JL, Castresana JS, Rey JA: Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma. Oncol Rep 2004, 12: 663–666.PubMedGoogle Scholar
  6. Philchenkov A, Zavelevich M, Tadeusz KroczakJ: Caspases and cancer: mechanisms of inactivation and new treatment modalities. Exp Oncol 2004, 26: 82–97.PubMedGoogle Scholar
  7. McConnell BB, Vertino PM: Activation of a caspase-9-mediated apoptotic pathway by subcellular redistribution of the novel caspase recruitment domain protein TMS1 . Cancer Res 2000, 60: 6243–6247.PubMedGoogle Scholar
  8. McConnell BB, Vertino PM: TMS1 / ASC : the cancer connection. Apoptosis 2004, 9: 5–18.PubMedView ArticleGoogle Scholar
  9. Grenier JM, Wang L, Manji GA, Huang WJ, Al-Garawi A, Kelly R, Carlson A, Merriam S, Lora JM, Briskin M, DiStefano PS, Bertin J: Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-kappaB and caspase-1. FEBS Lett 2002, 530: 73–78. 10.1016/S0014-5793(02)03416-6PubMedView ArticleGoogle Scholar
  10. Virmani A, Rathi A, Sugio K, Sathyanarayana UG, Toyooka S, Kischel FC, Tonk V, Padar A, Takahashi T, Roth JA, Euhus DM, Minna JD, Gazdar AF: Aberrant methylation of TMS1 in small cell, non small cell lung cancer and breast cancer. Int J Cancer 2003, 106: 198–204. 10.1002/ijc.11206PubMedView ArticleGoogle Scholar
  11. Stone AR, Bobo W, Brat DJ, Devi NS, Van Meir EG, Vertino PM: Aberrant methylation and down-regulation of TMS1 / ASC in human glioblastoma. Am J Pathol 2004, 165: 1151–1161. 10.1016/S0002-9440(10)63376-7PubMed CentralPubMedView ArticleGoogle Scholar
  12. Dhillon VS, Aslam M, Husain SA: The contribution of genetic and epigenetic changes in granulosa cell tumors of ovarian origin. Clin Cancer Res 2004, 10: 5537–5545. 10.1158/1078-0432.CCR-04-0228PubMedView ArticleGoogle Scholar
  13. Yokoyama T, Sagara J, Guan X, Masumoto J, Takeoka M, Komiyama Y, Miyata K, Higuchi K, Taniguchi S: Methylation of ASC / TMS1 , a proapoptotic gene responsible for activating procaspase-1, in human colorectal cancer. Cancer Lett 2003, 202: 101–108. 10.1016/j.canlet.2003.08.027PubMedView ArticleGoogle Scholar
  14. Alaminos M, Davalos V, Cheung NK, Gerald WL, Esteller M: Clustering of gene hypermethylation associated with clinical risk groups in neuroblastoma. J Natl Cancer Inst 2004, 96: 1208–1219. 10.1093/jnci/djh224PubMedView ArticleGoogle Scholar
  15. Guan X, Sagara J, Yokoyama T, Koganehira Y, Oguchi M, Saida T, Taniguchi S: ASC/TMS1, a caspase-1 activating adaptor, is downregulated by aberrant methylation in human melanoma. Int J Cancer 2003, 107: 202–208. 10.1002/ijc.11376PubMedView ArticleGoogle Scholar
  16. Roman-Gomez J, Jimenez-Velasco A, Castillejo JA, Agirre X, Barrios M, Navarro G, Molina FJ, Calasanz MJ, Prosper F, Heiniger A, Torres A: Promoter hypermethylation of cancer-related genes: a strong independent prognostic factor in acute lymphoblastic leukemia. Blood 2004, 104: 2492–2498. 10.1182/blood-2004-03-0954PubMedView ArticleGoogle Scholar
  17. Tischoff , Witzgmann H, Hauss J, Wittekind C, Tannapfel A: CpG island methylation of proapoptotic genes in hepatocellular carcinoma and cholangiocarcinoma. Z Gastroenterol 2006., 44: DOI: 10.1055/s-2006–931705Google Scholar
  18. Roe BA, Crabtree JS, Khan AS: Methods for DNA isolation. Part III. In Protocols for recombinant DNA isolation, cloning, and sequencing [Internet edition]. Norman, OK: University of Oklahoma; 1995. Available from: ; also available in paper copy as: DNA isolation and sequencing; New York: Wiley; 1996 http://www.genome.ou.edu/protocol_book/protocol_index.html Google Scholar
  19. Liu X, Zhu S, Zhang H, Xu H, Su H, Li S, Zhou X: The methylation status of the TMS1 / ASC gene in cholangiocarcinoma and its clinical significance. Hepatobiliary Pancreat Dis Int 2006, 5: 449–453.PubMedGoogle Scholar
  20. Lázcoz P, Muñoz J, Nistal M, Pestaña A, Encío I, Castresana JS: Frequent promoter hypermethylation of RASSF1A and CASP8 in neuroblastoma. BMC Cancer 2006, 6: 254. 10.1186/1471-2407-6-254PubMed CentralPubMedView ArticleGoogle Scholar
  21. Jones PA: Epigenetics in carcinogenesis and cancer prevention. Ann N Y Acad Sci 2003, 983: 213–219. 10.1111/j.1749-6632.2003.tb05976.xPubMedView ArticleGoogle Scholar
  22. Esteller M: CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene 2002, 21: 5427–5440. 10.1038/sj.onc.1205600PubMedView ArticleGoogle Scholar
  23. Laird PW: The power and the promise of DNA methylation markers. Nat Rev Cancer 2003, 3: 253–266. 10.1038/nrc1045PubMedView ArticleGoogle Scholar
  24. Teitz T, Wei T, Valentine MB, et al.: Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nat Med 2000, 6: 529–535. 10.1038/75007PubMedView ArticleGoogle Scholar
  25. Banelli B, Casciano I, Croce M, et al.: Expression and methylation of CASP8 in neuroblastoma: identification of a promoter region. Nat Med 2002, 8: 1333–1335. 10.1038/nm1202-1333PubMedView ArticleGoogle Scholar
  26. Conway KE, McConnell BB, Bowring CE, et al.: TMS1 , a novel proapoptotic caspase recruitment domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res 2000, 60: 6236–6242.PubMedGoogle Scholar
  27. Terasawa K, Sagae S, Toyota M, Tsukada K, Ogi K, Satoh A, et al.: Epigenetic Inactivation of TMS1 / ASC in Ovarian Cancer. Clin Cancer Res 2000, 10: 2000–2006.View ArticleGoogle Scholar
  28. Masumoto J, Taniguchi S, Ayukawa K, et al.: ASC , a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic 60 cells. J Biol Chem 1999, 274: 33835–33838. 10.1074/jbc.274.48.33835PubMedView ArticleGoogle Scholar
  29. Stimson KM, Vertino PM: Methylation-mediated silencing of TMS1 / ASC is accompanied by histone hypoacetylation and CpG islandlocalized changes in chromatin architecture. J Biol Chem 2002, 277: 4951–4958. 10.1074/jbc.M109809200PubMedView ArticleGoogle Scholar
  30. Martinez R, Setien F, Voelter C, Casado S, Quesada MP, Schackert G, Esteller M: CpG island promoter hypermethylation of the pro-apoptotic gene caspase-8 is a common hallmark of relapsed glioblastoma multiforme. Carcinogenesis 2007, 28: 1264–1268. 10.1093/carcin/bgm014PubMedView ArticleGoogle Scholar
  31. Ohtsuka T, Liu XF, Koga Y, Kitajima Y, Nakafusa Y, Ha CW, et al.: Methylation-induced silencing of ASC and the effect of expressed ASC on p53-mediated chemosensitivity in colorectal cancer. Oncogene 2006, 25: 1807–1811. 10.1038/sj.onc.1209204PubMedView ArticleGoogle Scholar
  32. Teodoridis JM, Hall J, Marsh S, Kannall HD, Smyth C, et al.: CpG Island methylation of DNA damage response genes in advanced ovarian cancer. Cancer Res 2005, 65: 8961–8967. 10.1158/0008-5472.CAN-05-1187PubMedView ArticleGoogle Scholar
  33. Feng Q, Balasubramanian A, Hawes SE, Toure P, Sow PS, Dem A, Dembele B, et al.: Detection of hypermethylated genes in women with and without cervical neoplasia. J Natl Cancer Inst 2005, 97: 273–282. 10.1093/jnci/dji041PubMedView ArticleGoogle Scholar
  34. Liu X, Zhu S, Zhang H, Xu H, Su H, Li S, Zhou X: The methylation status of the TMS1 / ASC gene in cholangiocarcinoma and its clinical significance. Hepatobiliary Pancreat Dis Int 2006, 5: 449–453.PubMedGoogle Scholar
  35. Terasawa K, Sagae S, Toyota M, Tsukada K, Ogi K, Satoh A, Mita H, et al.: Epigenetic Inactivation of TMS1 / ASC in ovarian cancer. Clin Cancer Res 2000, 10: 2000–2006.View ArticleGoogle Scholar
  36. Michalowski MB, de Fraipont F, Plantaz D, Michelland S, Combaret V, Favrot MC: Methylation of tumor-suppressor genes in neuroblastoma: the RASSF1A gene is almost always methylated in primary tumors. Pediatr Blood Cancer 2008,50(1):29–32. 10.1002/pbc.21279PubMedView ArticleGoogle Scholar
  37. Martinez R, Setien F, Voelter C, Casado S, Quesada MP, Schackert G, Esteller M: CpG island promoter hypermethylation of the pro-apoptotic gene caspase-8 is a common hallmark of relapsed glioblastoma multiforme. Carcinogenesis 2007, 28: 1264–1268. 10.1093/carcin/bgm014PubMedView ArticleGoogle Scholar
  38. Yang HJ, Liu VWS, Wang Y, Chan KYK, Tsang PCK, Khoo US, Cheung ANY, Ngan HYS: Detection of hypermethylated genes in tumour and plasma of cervical cancer patients. Gynecol Oncol 2004, 93: 435–440. 10.1016/j.ygyno.2004.01.039PubMedView ArticleGoogle Scholar
  39. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al.: MethyLight: a highthroughput assay to measure DNA methylation. Nucleic Acids Res 2000, 28: E32. 10.1093/nar/28.8.e32PubMed CentralPubMedView ArticleGoogle Scholar

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© I. Holzapfel Publishers 2009

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