Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

BMC Cancer

Gabriele Siegert²  Graeme Eisenhofer²  Renate Lehnert²  Claudia Schönefeldt¹  Christian Thiede¹  Margot Vogel²  Albert Hagelgans²  Uwe Platzbecker¹  Mario Menschikowski² 

[1]Medizinische Klinik und Poliklinik I, Technische Universität Dresden, Dresden, Germany

[2]Institut für Klinische Chemie und Laboratoriumsmedizin, Technische Universität Dresden, Fetscherstrasse 74, D-01307, Dresden, Germany

关键词: Leukemia;    High resolution melting analysis;    DNA methylation;    Phospholipase A₂ receptor;   

Others  :  1080038
DOI  :  10.1186/1471-2407-12-576

 received in 2012-08-16, accepted in 2012-11-28,  发布年份 2012

【 摘 要 】

Background

The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia.

Methods

Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA.

Results

Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia.

Conclusions

The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.

【 授权许可】

   
2012 Menschikowski et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Waite M: The Phospholipases. Plenum Press, New York; 1987.
• [2]Dennis EA, Deems RA, Yu L: Extracellular phospholipase A2. Adv Exp Med Biol 1992, 318:35-39.
• [3]Kudo I, Murakami M: Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat 2002, 68–69:3-58.
• [4]Menschikowski M, Hagelgans A, Siegert G: Secretory phospholipase A2 of group IIA: Is it an offensive or a defensive player during atherosclerosis and other inflammatory diseases? Prostaglandins Other Lipid Mediat 2006, 79:1-33.
• [5]Triggiani M, Granata F, Balestrieri B, Petraroli A, Scalia G, Del Vecchio L, Marone G: Secretory phospholipases A2 activate selective functions in human eosinophils. J Immunol 2003, 170:3279-3288.
• [6]Saegusa J, Akakura N, Wu CY, Hoogland C, Ma Z, Lam KS, Liu FT, Takada YK, Takada Y: Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins alphavbeta3 and alpha4beta1 and induces proliferation of monocytic cells in an integrin-dependent manner. J Biol Chem 2008, 283:26107-26115.
• [7]Yagami T, Ueda K, Asakura K, Nakazato H, Hata S, Kuroda T, Sakaeda T, Sakaguchi G, Itoh N, Hashimoto Y, Hori Y: Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons. J Neurochem 2003, 85:749-758.
• [8]Yagami T, Ueda K, Asakura K, Hata S, Kuroda T, Sakaeda T, Kishino J, Sakaguchi G, Itoh N, Hori Y: Group IB secretory phospholipase A2 induces cell death in the cultured cortical neurons: a possible involvement of its binding sites. Brain Res 2002, 949:197-201.
• [9]Lambeau G, Barhanin J, Schweitz H, Qar J, Lazdunski M: Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom. J Biol Chem 1989, 264:11503-11510.
• [10]Hanasaki K, Arita H: Phospholipase A2 receptor: a regulator of biological functions of secretory phospholipase A2. Prostaglandins Other Lipid Mediat 2002, 68–69:71-82.
• [11]Hanasaki K, Arita H: Biological and pathological functions of phospholipase A2 receptor. Arch Biochem Biophys 1999, 372:215-223.
• [12]Taylor ME, Conary JT, Lennartz MR, Stahl PD, Drickamer K: Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains. J Biol Chem 1990, 265:12156-12162.
• [13]Valentin E, Lambeau G: Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins. Biochim Biophys Acta 2000, 1488:59-70.
• [14]Beck LH, Bonegio RGB, Lambeau G, Beck DM, Powell DW, Cummins TD, Klein JB, Salant DJ: M-Type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 2009, 361:11-21.
• [15]Platzbecker U, Wermke M, Radke J, Oelschlaegel U, Seltmann F, Kiani A, Klut IM, Knoth H, Röllig C, Schetelig J, Mohr B, Graehlert X, Ehninger G, Bornhäuser M, Thiede C: Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial. Leukemia 2012, 26:381-389.
• [16]Li LC, Dahiya R: MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002, 18:1427-1431.
• [17]Frommer ML, McDonald E, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. P Natl Acad Sci USA 1991, 89:1827-1831.
• [18]Clark SJ, Harrison J, Paul CL, Frommer M: High sensitivity mapping of methylated cytosines. Nucleic Acids Res 1994, 22:2990-2997.
• [19]Warnecke PM, Stirzaker C, Song J, Grunau C, Melki JR, Clark SJ: Identification and resolution of artifacts in bisulfite sequencing. Methods 2002, 27:101-107.
• [20]Kim HJ, Kim KS, Kim SH, Baek SH, Kim HY, Lee C, Kim JR: Induction of cellular senescence by secretory phospholipase A2 in human dermal fibroblasts through an ROS-mediated p53 pathway. J Gerontol A Biol Sci Med Sci 2009, 64:351-362.
• [21]Augert A, Payré C, de Launoit Y, Gil J, Lambeau G, Bernard D: The M-type receptor PLA2R1 regulates senescence through the p53 pathway. EMBO Rep 2009, 10:271-277.
• [22]Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, Schlegelberger B, Stein H, Dörken B, Jenuwein T, Schmitt CA: Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 2005, 436:660-665.
• [23]Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Pandolfi PP: Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 2005, 436:725-730.
• [24]Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011, 144:646-674.
• [25]Jiang Y, Dunbar A, Gondek LP, Mohan S, Rataul M, O'Keefe C, Sekeres M, Saunthararajah Y, Maciejewski JP: Aberrant DNA methylation is a dominant mechanism in MDS progression to AML. Blood 2009, 113:1315-1325.
• [26]Follo MY, Finelli C, Mongiorgi S, Clissa C, Bosi C, Testoni N, Chiarini F, Ramazzotti G, Baccarani M, Martelli AM, Manzoli L, Martinelli G, Cocco L: Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS. Proc Natl Acad Sci USA 2009, 106:16811-16816.
• [27]Costa-Junior HM, Hamaty FC: da Silva Farias R, Einicker-Lamas M, da Silva MH, Persechini PM. Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2. Cell Tissue Res 2006, 324:255-266.
• [28]Lee C, Park DW, Lee J, Lee TI, Kim YJ, Lee YS, Baek SH: Secretory phospholipase A2 induces apoptosis through TNF-α and cytochrome c-mediated caspase cascade in murine macrophage RAW 264.7 cells. Eur J Pharmacol 2006, 536:47-53.
• [29]Mora R, Maldonado A, Valverde B, Gutiérrez JM: Calcium plays a key role in the effects induced by a snake venom Lys49 phospholipase A2 homologue on a lymphoblastoid cell line. Toxicon 2006, 47:75-86.
• [30]Petrovic U, Sribar J, Matis M, Anderluh G, Peter-Katalinić J, Krizaj I, Gubensek F: Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces cerevisiae. Biochem J 2005, 391:383-388.
• [31]Putz T, Ramoner R, Gander H, Rahm A, Bartsch G, Bernardo K, Ramsay S, Thurnher M: Bee venom secretory phospholipase A2 and phosphatidylinositol-homologues cooperatively disrupt membrane integrity, abrogate signal transduction and inhibit proliferation of renal cancer cells. Cancer Immunol Immunother 2007, 56:627-640.
• [32]Hanasaki K, Yokota Y, Ishizaki J, Itoh T, Arita H: Resistance to endotoxic shock in phospholipase A2 receptor-deficient mice. J Biol Chem 1997, 272:32792-32797.
• [33]Lambeau G, Lazdunski M: Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol Sci 1999, 20:162-170.