Targeting cAMP in chronic lymphocytic leukemia: A pathway-dependent approach for the treatment of leukemia and lymphoma

Expert Opinion on Therapeutic Targets

Volumn 17, Issue 8, 2013, Pages 937-949

  Murray, Fiona ^a,b   Insel, Paul A ^a,b  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA SAN DIEGO   (United States)

Author keywords

Adenylyl cyclase; Apoptosis; Chronic lymphocytic leukemia; Cyclic AMP; Epac; Lymphoma; Phosphodiesterase; Protein kinase A; S49 cells

Indexed keywords

ADENYLATE CYCLASE; ADENYLYL CYCLASE ACTIVATOR; ANTINEOPLASTIC AGENT; CYCLIC AMP; CYCLIC AMP DEPENDENT PROTEIN KINASE; EXCHANGE PROTEIN DIRECTLY ACTIVATED BY CAMP; EXCHANGE PROTEIN DIRECTLY ACTIVATED BY CAMP INHIBITOR; G PROTEIN COUPLED RECEPTOR; G PROTEIN COUPLED RECEPTOR AGONIST; G PROTEIN COUPLED RECEPTOR ANTAGONIST; PHOSPHODIESTERASE; PHOSPHODIESTERASE IV INHIBITOR; PHOSPHODIESTERASE VII INHIBITOR; PROTEIN; UNCLASSIFIED DRUG;

APOPTOSIS; CANCER CELL CULTURE; CELL DEATH; CHRONIC LYMPHATIC LEUKEMIA; DRUG EFFECT; GENE EXPRESSION; HUMAN; LYMPHOMA CELL LINE; MITOCHONDRION; NONHUMAN; PATHOPHYSIOLOGY; PROTEIN EXPRESSION; REVIEW; SIGNAL TRANSDUCTION;

ANIMALS; CELL LINE, TUMOR; CYCLIC AMP; HUMANS; LEUKEMIA, LYMPHOCYTIC, CHRONIC, B-CELL; LYMPHOMA;

EID: 84880371723     PISSN: 14728222     EISSN: 17447631     Source Type: Journal    
DOI: 10.1517/14728222.2013.798304     Document Type: Review

References (100)

1. Gaidano G, Foa R, Dalla-Favera R. Molecular pathogenesis of chronic lymphocytic leukemia. J Clin Invest 2012;122:3432-8
2. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008;111:5446-56
3. Ma S, Rosen ST. Signal transduction inhibitors in chronic lymphocytic leukemia. Curr Opin Oncol 2011;23:601-8
4. Balakrishnan K, Gandhi V. Protein kinases: emerging therapeutic targets in chronic lymphocytic leukemia. Expert Opin Invest Drug 2012;21:409-23
5. Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626-9 (Pubitemid 39006738)
6. Falschlehner C, Schaefer U, Walczak H. Following TRAIL's path in the immune system. Immunology 2009;127:145-54
7. Packham G, Stevenson FK. Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia. Immunology 2005;114:441-9 (Pubitemid 40478137)
8. Kitada S, Andersen J, Akar S, et al. Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with in vitro and in vivo chemoresponses. Blood 1998;91:3379-89 (Pubitemid 28193890)
9. McConkey DJ, Chandra J, Wright S, et al. Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of Bcl-2 and BAX. J Immunol 1996;156:2624-30 (Pubitemid 26091593)
10. Petlickovski A, Laurenti L, Li X, et al. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood 2005;105:4820-7 (Pubitemid 40807309)
11. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Nat Acad Sci USA 2005;102:13944-9 (Pubitemid 41377683)
12. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Nat Acad Sci USA 2002;99:15524-9 (Pubitemid 35403967)
13. Daniel V, Litwack G, Tomkins GM. Induction of cytolysis of cultured lymphoma cells by adenosine 3':5'-cyclic monophosphate and the isolation of resistant variants. Proc Natl Acad Sci USA 1973;70:76-9
14. Insel PA, Bourne HR, Coffino P, et al. Cyclic AMP-dependent protein kinase: pivotal role in regulation of enzyme induction and growth. Science 1975;190:896-8
15. Mentz F, Merle-Beral H, Ouaaz F, et al. Theophylline, a new inducer of apoptosis in B-CLL: role of cyclic nucleotides. Br J Haematol 1995;90:957-9
16. Kim DH, Lerner A. Type 4 cyclic adenosine monophosphate phosphodiesterase as a therapeutic target in chronic lymphocytic leukemia. Blood 1998;92:2484-94 (Pubitemid 28452992)
17. Ogawa R, Streiff MB, Bugayenko A, et al. Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21 (WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells. Blood 2002;99:3390-7 (Pubitemid 34525321)
18. Smith PG, Wang F, Wilkinson KN, et al. The phosphodiesterase PDE4B limits cAMP-associated PI3K/AKTdependent apoptosis in diffuse large B-cell lymphoma. Blood 2005;105:308-16 (Pubitemid 40053098)
19. Insel PA, Zhang L, Murray F, et al. Cyclic AMP is both a pro-apoptotic and anti-apoptotic second messenger. Acta Physiol (Oxf) 2012;204:277-87
20. Tasken K, Andersson KB, Erikstein BK, et al. Regulation of growth in a neoplastic B cell line by transfected subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase. Endocrinology 1994;135:2109-19
21. Zhang L, Insel PA. The pro-apoptotic protein Bim is a convergence point for cAMP/protein kinase A-and glucocorticoid-promoted apoptosis of lymphoid cells. J Biol Chem 2004;279:20858-65 (Pubitemid 38656482)
22. Ji Z, Mei FC, Miller AL, et al. Protein kinase A (PKA) isoform RIIbeta mediates the synergistic killing effect of cAMP and glucocorticoid in acute lymphoblastic leukemia cells. J Biol Chem 2008;283:21920-5
23. Dong H, Zitt C, Auriga C, et al. Inhibition of PDE3, PDE4 and PDE7 potentiates glucocorticoid-induced apoptosis and overcomes glucocorticoid resistance in CEM T leukemic cells. Biochem Pharmacol 2010;79:321-9
24. Tiwari S, Dong H, Kim EJ, et al. Type 4 cAMP phosphodiesterase (PDE4) inhibitors augment glucocorticoid-mediated apoptosis in B cell chronic lymphocytic leukemia (BCLL) in the absence of exogenous adenylyl cyclase stimulation. Biochem Pharmacol 2005;69:473-83
25. Meyers JA, Taverna J, Chaves J, et al. Phosphodiesterase 4 inhibitors augment levels of glucocorticoid receptor in B cell chronic lymphocytic leukemia but not in normal circulating hematopoietic cells. Clin Cancer Res 2007;13:4920-7 (Pubitemid 47294800)
26. van Daalen Wetters T, Coffino P. Cultured S49 mouse T lymphoma cells. Methods Enzymol 1987;151:9-19
27. Bourne HR, Coffino P, Tomkins GM. Selection of a variant lymphoma cell deficient in adenylate cyclase. Science 1975;187:750-2
28. Coffino P, Bourne HR, Tomkins GM. Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants. J Cell Physiol 1975;85:603-10
29. Sibley CH, Yamamoto KR. Mouse lymphoma cells: mechanisms of resistance to glucocorticoids. Monogr Endocrinol 1979;12:357-76
30. Ross EM, Howlett AC, Ferguson KM, et al. Reconstitution of hormone-sensitive adenylate cyclase activity with resolved components of the enzyme. J Biol Chem 1978;253:6401-12 (Pubitemid 9025319)
31. Gilman AG. Silver spoons and other personal reflections. Annu Rev Pharmacol Toxicol 2012;52:1-19
32. Albert DA. The effect of cyclic-AMP on the regulation of c-myc expression in T lymphoma cells. J Clin Invest 1995;95:1490-6
33. Honeysett JM, Insel PA. Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase activities of S49 lymphoma cells by agents increasing cyclic AMP. J Cyclic Nucleotide Res 1981;7:321-32 (Pubitemid 12149761)
34. Lemaire I, Coffino P. Cyclic AMP-induced cytolysis in S49 cells: selection of an unresponsive "deathless" mutant. Cell 1977;11:149-55 (Pubitemid 8126062)
35. Hughes RJ, Howard MJ, Allen JM, et al. Decreased beta 2-adrenergic receptor mRNA expression in receptor-deficient S49 lymphoma cells. Mol Pharmacol 1991;40:974-9
36. Johnson GL, Bourne HR, Gleason MK, et al. Isolation and characterization of S49 lymphoma cells deficient in beta-adrenergic receptors: relation of receptor number to activation of adenylate cyclase. Mol Pharmacol 1979;15:16-27 (Pubitemid 9150355)
37. Bourne HR, Brothers VM, Kaslow HR, et al. Genetic analysis of cyclic nucleotide phosphodiesterases in S49 mouse lymphoma cells. Adv Cyclic Nucleotide Protein Phosphorylation Res 1984;16:185-94
38. Haga T, Ross EM, Anderson HJ, et al. Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells. Proc Natl Acad Sci USA 1977;74:2016-20 (Pubitemid 8123600)
39. Zambon AC, Zhang L, Minovitsky S, et al. Gene expression patterns define key transcriptional events in cell-cycle regulation by cAMP and protein kinase A. Proc Natl Acad Sci USA 2005;102:8561-6 (Pubitemid 40862775)
40. Zhang L, Insel PA. Bcl-2 protects lymphoma cells from apoptosis but not growth arrest promoted by cAMP and dexamethasone. Am J Physiol Cell Physiol 2001;281:C1642-7
41. Zhang L, Zambon AC, Vranizan K, et al. Gene expression signatures of cAMP/protein kinase A (PKA)-promoted, mitochondrial-dependent apoptosis. Comparative analysis of wild-type and cAMP-deathless S49 lymphoma cells. J Biol Chem 2008;283:4304-13
42. Guo Y, Wilderman A, Zhang L, et al. Quantitative proteomics analysis of the cAMP/protein kinase A signaling pathway. Biochemistry 2012;51:9323-32
43. Zambon AC, Wilderman A, Ho A, et al. Increased expression of the pro-apoptotic protein BIM, a mechanism for cAMP/protein kinase A (PKA)-induced apoptosis of immature T cells. J Biol Chem 2011;286:33260-7
44. Zhang L, Yun H, Murray F, et al. Cytotoxic T lymphocyte antigen-2 alpha induces apoptosis of murine T-lymphoma cells and cardiac fibroblasts and is regulated by cAMP/PKA. Cell Signal 2011;23:1611-16
45. Mentz F, Mossalayi MD, Ouaaz F, et al. Theophylline synergizes with chlorambucil in inducing apoptosis of B-chronic lymphocytic leukemia cells. Blood 1996;88:2172-82 (Pubitemid 26307920)
46. Monahan TM, Marchand NW, Fritz RR, et al. Cyclic adenosine 3':5'-monophosphate levels and activities of related enzymes in normal and leukemic lymphocytes. Cancer Res 1975;35(9):2540-7
47. Samuel E, Chung C, Scher N, et al. Protein kinases in chronic lymphocytic leukemia. Blood 1980;55:618-24 (Pubitemid 10138845)
48. Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5:993-6 (Pubitemid 44835126)
49. Insel PA, Snead A, Murray F, et al. GPCR expression in tissues and cells: are the optimal receptors being used as drug targets? Br J Pharmacol 2012;165:1613-16
50. Frey UH, Nuckel H, Sellmann L, et al. The GNAS1 T393C polymorphism is associated with disease progression and survival in chronic lymphocytic leukemia. Clin Cancer Res 2006;12:5686-92 (Pubitemid 44629596)
51. Kaderi MA, Murray F, Jansson M, et al. The GNAS1 T393C polymorphism and lack of clinical prognostic value in chronic lymphocytic leukemia. Leuk Res 2008;32:984-7 (Pubitemid 351389548)
52. Nuckel H, Frey U, Aralh N, et al. The CC genotype of the C825T polymorphism of the G protein beta3 gene (GNB3) is associated with a high relapse rate in patients with chronic lymphocytic leukaemia. Leuk Lymphoma 2003;44:1739-43 (Pubitemid 37221049)
53. Burger JA. Nurture versus nature: the microenvironment in chronic lymphocytic leukemia. Hematol Am Soc Hematol Educ Program 2011;2011:96-103
54. Drost AC, Seitz G, Boehmler A, et al. The G protein-coupled receptor CysLT1 mediates chemokine-like effects and prolongs survival in chronic lymphocytic leukemia. Leuk Lymphoma 2012;53:665-73
55. Burger JA, Burger M, Kipps TJ. Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells. Blood 1999;94:3658-67
56. Cuesta-Mateos C, Lopez-Giral S, Alfonso-Perez M, et al. Analysis of migratory and prosurvival pathways induced by the homeostatic chemokines CCL19 and CCL21 in B-cell chronic lymphocytic leukemia. Exp Hematol 2010;38:756-64; 64 e1-4
57. Ferretti E, Bertolotto M, Deaglio S, et al. A novel role of the CX3CR1/CX3CL1 system in the cross-talk between chronic lymphocytic leukemia cells and tumor microenvironment. Leukemia 2011;25:1268-77
58. Jones D, Benjamin RJ, Shahsafaei A, et al. The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia. Blood 2000;95:627-32 (Pubitemid 30041679)
59. Mahadevan D, Choi J, Cooke L, et al. Gene expression and serum cytokine profiling of low stage CLL identify WNT/PCP, Flt-3L/Flt-3 and CXCL9/CXCR3 as regulators of cell proliferation, survival and migration. Hum Genomics Proteomics 2009;1:453634
60. Wong S, Fulcher D. Chemokine receptor expression in B-cell lymphoproliferative disorders. Leuk Lymphoma 2004;45:2491-6 (Pubitemid 40081196)
61. Burger JA, Tsukada N, Burger M, et al. Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1. Blood 2000;96:2655-63
62. Burger M, Hartmann T, Krome M, et al. Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells. Blood 2005;106:1824-30 (Pubitemid 41208600)
63. Burkle A, Niedermeier M, Schmitt-Graff A, et al. Overexpression of the CXCR5 chemokine receptor, and its ligand, CXCL13 in B-cell chronic lymphocytic leukemia. Blood 2007;110:3316-25 (Pubitemid 350106325)
64. Mamani-Matsuda M, Moynet D, Molimard M, et al. Long-acting beta2-adrenergic formoterol and salmeterol induce the apoptosis of B-chronic lymphocytic leukaemia cells. Br J Haematol 2004;124:141-50 (Pubitemid 38122185)
65. Kamp T, Liebl B, Haen E, et al. Defects of beta 2-adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression. Eur J Clin Invest 1997;27:121-7 (Pubitemid 27101383)
66. Mentz F, Merle-Beral H, Dalloul AH. Theophylline-induced B-CLL apoptosis is partly dependent on cyclic AMP production but independent of CD38 expression and endogenous IL-10 production. Leukemia 1999;13:78-84 (Pubitemid 29043720)
67. Ostrom RS, Post SR, Insel PA. Stoichiometry and compartmentation in G protein-coupled receptor signaling: implications for therapeutic interventions involving G(s). J Pharmacol Exp Ther 2000;294:407-12 (Pubitemid 30490809)
68. Meyers JA, Su DW, Lerner A. Chronic lymphocytic leukemia and B and T cells differ in their response to cyclic nucleotide phosphodiesterase inhibitors. J Immunol 2009;182:5400-11
69. Carpentieri U, Minguell JJ, Gardner FH. Adenylate cyclase and guanylate cyclase activity in normal and leukemic human lymphocytes. Blood 1981;57:975-8 (Pubitemid 11096493)
70. Mendelsohn J, Nordberg J. Adenylate cyclase in thymus-derived and bone marrow-derived lymphocytes from normal donors and patients with chronic lymphocytic leukemia. J Clin Invest 1979;63:1124-32 (Pubitemid 9193206)
71. Gao MH, Hammond HK. Unanticipated signaling events associated with cardiac adenylyl cyclase gene transfer. J Mol Cell Cardiol 2011;50:751-8
72. Mabed M, Aref S, Fouda M, et al. Chlorambucil plus theophylline vs chlorambucil alone as a front line therapy for B-cell chronic lymphatic leukemia. Leuk Lymphoma 2004;45:2029-35 (Pubitemid 39232851)
73. Willis CR, Goodrich A, Park K, et al. A phase I/II study examining pentostatin, chlorambucil, and theophylline in patients with relapsed chronic lymphocytic leukemia and non-Hodgkin's lymphoma. Ann Hematol 2006;85:301-7 (Pubitemid 43732652)
74. Wiernik PH, Paietta E, Goloubeva O, et al. Phase II study of theophylline in chronic lymphocytic leukemia: a study of the Eastern Cooperative Oncology Group (E4998). Leukemia 2004;18:1605-10 (Pubitemid 39386337)
75. Moon EY, Lerner A. PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A. Blood 2003;101:4122-30 (Pubitemid 36857894)
76. Zhang L, Murray F, Zahno A, et al. Cyclic nucleotide phosphodiesterase profiling reveals increased expression of phosphodiesterase 7B in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2008;105:19532-7
77. Moon E, Lee R, Near R, et al. Inhibition of PDE3B augments PDE4 inhibitor-induced apoptosis in a subset of patients with chronic lymphocytic leukemia. Clin Cancer Res 2002;8:589-95 (Pubitemid 34193981)
78. Houslay MD. Underpinning compartmentalised cAMP signalling through targeted cAMP breakdown. Trends Biochem Sci 2010;35:91-100
79. Page CP, Spina D. Selective PDE inhibitors as novel treatments for respiratory diseases. Curr Opin Pharmacol 2012;12:275-86
80. Schett G, Sloan VS, et al. Apremilast: a novel PDE4 inhibitor in the treatment of autoimmune and inflammatory diseases. Ther Adv Musculoskelet Dis 2010;2:271-8
81. Lee R, Wolda S, Moon E, et al. PDE7A is expressed in human B-lymphocytes and is up-regulated by elevation of intracellular cAMP. Cell Signal 2002;14:277-84 (Pubitemid 34102457)
82. Bloom TJ, Beavo JA. Identification and tissue-specific expression of PDE7 phosphodiesterase splice variants. Proc Natl Acad Sci USA 1996;93:14188-92 (Pubitemid 26424267)
83. Gantner F, Gotz C, et al. Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation. Br J Pharmacol 1998;123:1031-8 (Pubitemid 28120497)
84. Peiro AM, Tang CM, Murray F, et al. Genetic variation in phosphodiesterase (PDE) 7B in chronic lymphocytic leukemia: overview of genetic variants of cyclic nucleotide PDEs in human disease. J Hum Genet 2011;56:676-81
85. Zhang L, Murray F, Rassenti LZ, et al. Cyclic nucleotide phosphodiesterase 7B mRNA: an unfavorable characteristic in chronic lymphocytic leukemia. Int J Cancer 2011;129:1162-9
86. Tiwari S, Felekkis K, Moon EY, et al. Among circulating hematopoietic cells, B-CLL uniquely expresses functional EPAC1, but EPAC1-mediated Rap1 activation does not account for PDE4 inhibitor-induced apoptosis. Blood 2004;103:2661-7 (Pubitemid 38393022)
87. Mantovani G, Bondioni S, Lania AG, et al. High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells. Oncogene 2008;27:1834-43 (Pubitemid 351430825)
88. Mantovani G, Lania AG, Bondioni S, et al. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: relationship with cell proliferation. Exp Cell Res 2008;314:123-30 (Pubitemid 350180780)
89. Grandoch M, Roscioni SS, Schmidt M. The role of Epac proteins, novel cAMP mediators, in the regulation of immune, lung and neuronal function. Br J Pharmacol 2010;159:265-84
90. Klein U, Tu Y, Stolovitzky GA, et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 2001;194:1625-38 (Pubitemid 33126813)
91. Grandoch M, Lopez de Jesus M, Oude Weernink PA, et al. B cell receptor-induced growth arrest and apoptosis in WEHI-231 immature B lymphoma cells involve cyclic AMP and Epac proteins. Cell Signal 2009;21:609-21
92. Longo PG, Laurenti L, Gobessi S, et al. The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells. Blood 2008;111:846-55
93. Raymond DR, Wilson LS, Carter RL, et al. Numerous distinct PKA-, or EPAC-based, signalling complexes allow selective phosphodiesterase 3 and phosphodiesterase 4 coordination of cell adhesion. Cell Signal 2007;19:2507-18 (Pubitemid 47531931)
94. Almahariq M, Tsalkova T, Mei FC, et al. A novel EPAC-specific inhibitor suppresses pancreatic cancer cell migration and invasion. Mol Pharmacol 2013;83:122-8
95. Courilleau D, Bisserier M, Jullian JC, et al. Identification of a tetrahydroquinoline analog as a pharmacological inhibitor of the cAMP-binding protein Epac. J Biol Chem 2012;287:44192-202
96. Calin GA, Croce CM. Chronic lymphocytic leukemia: interplay between noncoding RNAs and protein-coding genes. Blood 2009;114:4761-70
97. Stangherlin A, Zaccolo M. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system. Am J Physiol Heart Circ Physiol 2012;302:H379-90
98. Yamamoto S, Sugahara S, Ikeda K, et al. Amelioration of collagen-induced arthritis in mice by a novel phosphodiesterase 7 and 4 dual inhibitor, YM-393059. Eur J Pharmacol 2007;559:219-26 (Pubitemid 46295404)
99. Copsel S, Garcia C, Diez F, et al. Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. J Biol Chem 2011;286:6979-88.
100. Regard JB, Sato IT, Coughlin SR. Anatomical profiling of G protein-coupled receptor expression. Cell 2008;135:561-71