Stable depletion of RUNX1-ETO in Kasumi-1 cells induces expression and enhanced proteolytic activity of Cathepsin G and Neutrophil Elastase

Autoři: Caroline Schoenherr aff001;  Katharina Wohlan aff001;  Iris Dallmann aff001;  Andreas Pich aff002;  Jan Hegermann aff003;  Arnold Ganser aff001;  Denise Hilfiker-Kleiner aff004;  Olaf Heidenreich aff005;  Michaela Scherr aff001;  Matthias Eder aff001
Působiště autorů: Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany aff001;  Department of Toxicology, Research Core Unit Proteomics, Hannover Medical School, Hannover, Germany aff002;  Department of Functional and Applied Anatomy, Research Core Unit Electron Microscopy, Hannover Medical School, Hannover, Germany aff003;  Department of Cardiology and Angiology, Hannover Medical School, Hannover, Germany aff004;  Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle, United Kingdom aff005;  Princess Maxima Center for Pediatric Oncology, Utrecht, Netherlands aff006
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


The oncogenic fusion protein RUNX1-ETO is a product of the t(8;21) translocation and consists of the hematopoietic transcriptional master regulator RUNX1 and the repressor ETO. RUNX1-ETO is found in 10–15% of acute myeloid leukemia and interferes with the expression of genes that are essential for myeloid differentiation. The neutrophil serine protease Cathepsin G is one of the genes suppressed by RUNX1-ETO, but little is known about its impact on the regulation of other lysosomal proteases. By lentiviral transduction of the t(8;21) positive cell line Kasumi-1 with an RUNX1-ETO specific shRNA, we analyzed long-term effects of stable RUNX1-ETO silencing on cellular phenotypes and target gene expression. Stable anti RUNX1-ETO RNAi reduces both proliferation and apoptosis in Kasumi-1 cells. In addition, long-term knockdown of RUNX1-ETO leads to an upregulation of proteolytic activity in Kasumi-1 cells, which may be released in vitro upon cell lysis leading to massive degradation of cellular proteins. We therefore propose that protein expression data of RUNX1-ETO-silenced Kasumi-1 cells must be analyzed with caution, as cell lysis conditions can heavily influence the results of studies on protein expression. Next, a mass spectrometry-based approach was used to identify protease cleavage patterns in RUNX1-ETO-depleted Kasumi-1 cells and Neutrophil Elastase has been identified as a RUNX1-ETO candidate target. Finally, proteolytic activity of Neutrophil Elastase and Cathepsin G was functionally confirmed by si/shRNA-mediated knockdown in Kasumi-1 cells.

Klíčová slova:

Acute myeloid leukemia – Apoptosis – Cell differentiation – Gene expression – Lysosomes – Proteases – Protein expression – Proteolysis


1. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood. 1998 Oct 1;92(7):2322–33. 9746770

2. Byrd JC, Mrózek K, Dodge RK, Carroll AJ, Edwards CG, Arthur DC, et al. Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002 Dec 15;100(13):4325–36. 12393746

3. Erickson P, Gao J, Chang KS, Look T, Whisenant E, Raimondi S, et al. Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. Blood. 1992 Oct 1;80(7):1825–31. 1391946

4. Nisson PE, Watkins PC, Sacchi N. Transcriptionally active chimeric gene derived from the fusion of the AML1 gene and a novel gene on chromosome 8 in t(8;21) leukemic cells. Cancer Genet Cytogenet. 1992 Oct 15;63(2):81–8. doi: 10.1016/0165-4608(92)90384-k 1423235

5. Miyoshi H, Kozu T, Shimizu K, Enomoto K, Maseki N, Kaneko Y, et al. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J. 1993 Jul;12(7):2715–21. 8334990

6. Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA. Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO. Mol Cell Biol. 1998 Dec;18(12):7185–91. doi: 10.1128/mcb.18.12.7185 9819405

7. Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Davie JR, et al. ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol Cell Biol. 1998 Dec;18(12):7176–84. doi: 10.1128/mcb.18.12.7176 9819404

8. Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10860–5. 9724795

9. Shibata H, Spencer TE, Oñate SA, Jenster G, Tsai SY, Tsai MJ, et al. Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res. 1997;52:141–64; discussion 164–165. 9238851

10. Chen JD, Li H. Coactivation and corepression in transcriptional regulation by steroid/nuclear hormone receptors. Crit Rev Eukaryot Gene Expr. 1998;8(2):169–90. doi: 10.1615/critreveukargeneexpr.v8.i2.40 9714896

11. Hwang ES, Hong JH, Bae SC, Ito Y, Lee SK. Regulation of c-fos gene transcription and myeloid cell differentiation by acute myeloid leukemia 1 and acute myeloid leukemia-MTG8, a chimeric leukemogenic derivative of acute myeloid leukemia 1. FEBS Lett. 1999 Mar 5;446(1):86–90. doi: 10.1016/s0014-5793(99)00190-8 10100620

12. Linggi B, Müller-Tidow C, van de Locht L, Hu M, Nip J, Serve H, et al. The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia. Nat Med. 2002 Jul;8(7):743–50. doi: 10.1038/nm726 12091906

13. Zhuang W-Y, Cen J-N, Zhao Y, Chen Z-X. Epigenetic silencing of Bcl-2, CEBPA and p14(ARF) by the AML1-ETO oncoprotein contributing to growth arrest and differentiation block in the U937 cell line. Oncol Rep. 2013 Jul;30(1):185–92. doi: 10.3892/or.2013.2459 23673926

14. Wang L, Gural A, Sun X-J, Zhao X, Perna F, Huang G, et al. The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation. Science. 2011 Aug 5;333(6043):765–9. doi: 10.1126/science.1201662 21764752

15. Martinez-Soria N, McKenzie L, Draper J, Ptasinska A, Issa H, Potluri S, et al. The Oncogenic Transcription Factor RUNX1/ETO Corrupts Cell Cycle Regulation to Drive Leukemic Transformation. Cancer Cell. 2018 08;34(4):626–642.e8. doi: 10.1016/j.ccell.2018.08.015 30300583

16. Vangala RK, Heiss-Neumann MS, Rangatia JS, Singh SM, Schoch C, Tenen DG, et al. The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia. Blood. 2003 Jan 1;101(1):270–7. doi: 10.1182/blood-2002-04-1288 12393465

17. Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre G, et al. AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med. 2001 Apr;7(4):444–51. doi: 10.1038/86515 11283671

18. Choi Y, Elagib KE, Delehanty LL, Goldfarb AN. Erythroid inhibition by the leukemic fusion AML1-ETO is associated with impaired acetylation of the major erythroid transcription factor GATA-1. Cancer Res. 2006 Mar 15;66(6):2990–6. doi: 10.1158/0008-5472.CAN-05-2944 16540647

19. Zhang J, Kalkum M, Yamamura S, Chait BT, Roeder RG. E protein silencing by the leukemogenic AML1-ETO fusion protein. Science. 2004 Aug 27;305(5688):1286–9. doi: 10.1126/science.1097937 15333839

20. Amann JM, Nip J, Strom DK, Lutterbach B, Harada H, Lenny N, et al. ETO, a target of t(8;21) in acute leukemia, makes distinct contacts with multiple histone deacetylases and binds mSin3A through its oligomerization domain. Mol Cell Biol. 2001 Oct;21(19):6470–83. doi: 10.1128/MCB.21.19.6470-6483.2001 11533236

21. Klisovic MI, Maghraby EA, Parthun MR, Guimond M, Sklenar AR, Whitman SP, et al. Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells. Leukemia. 2003 Feb;17(2):350–8. doi: 10.1038/sj.leu.2402776 12592335

22. Shia W-J, Okumura AJ, Yan M, Sarkeshik A, Lo M-C, Matsuura S, et al. PRMT1 interacts with AML1-ETO to promote its transcriptional activation and progenitor cell proliferative potential. Blood. 2012 May 24;119(21):4953–62. doi: 10.1182/blood-2011-04-347476 22498736

23. Ptasinska A, Assi SA, Mannari D, James SR, Williamson D, Dunne J, et al. Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding. Leukemia. 2012 Aug;26(8):1829–41. doi: 10.1038/leu.2012.49 22343733

24. Rhoades KL, Hetherington CJ, Harakawa N, Yergeau DA, Zhou L, Liu LQ, et al. Analysis of the role of AML1-ETO in leukemogenesis, using an inducible transgenic mouse model. Blood. 2000 Sep 15;96(6):2108–15. 10979955

25. Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hetherington CJ, et al. AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations. Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10398–403. doi: 10.1073/pnas.171321298 11526243

26. Heidenreich O, Krauter J, Riehle H, Hadwiger P, John M, Heil G, et al. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells. Blood. 2003 Apr 15;101(8):3157–63. doi: 10.1182/blood-2002-05-1589 12480707

27. Martinez N, Drescher B, Riehle H, Cullmann C, Vornlocher H-P, Ganser A, et al. The oncogenic fusion protein RUNX1-CBFA2T1 supports proliferation and inhibits senescence in t(8;21)-positive leukaemic cells. BMC Cancer. 2004 Aug 6;4:44. doi: 10.1186/1471-2407-4-44 15298716

28. Dunne J, Cullmann C, Ritter M, Soria NM, Drescher B, Debernardi S, et al. siRNA-mediated AML1/MTG8 depletion affects differentiation and proliferation-associated gene expression in t(8;21)-positive cell lines and primary AML blasts. Oncogene. 2006 Oct 5;25(45):6067–78. doi: 10.1038/sj.onc.1209638 16652140

29. Scherr M, Battmer K, Ganser A, Eder M. Modulation of gene expression by lentiviral-mediated delivery of small interfering RNA. Cell Cycle Georget Tex. 2003 Jun;2(3):251–7.

30. Dawodu D, Patecki M, Hegermann J, Dumler I, Haller H, Kiyan Y. oxLDL inhibits differentiation and functional activity of osteoclasts via scavenger receptor-A mediated autophagy and cathepsin K secretion. Sci Rep. 2018 Aug 2;8(1):11604. doi: 10.1038/s41598-018-29963-w 30072716

31. Surdziel E, Cabanski M, Dallmann I, Lyszkiewicz M, Krueger A, Ganser A, et al. Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways. Blood. 2011 Apr 21;117(16):4338–48. doi: 10.1182/blood-2010-06-289058 21368288

32. Jochim N, Gerhard R, Just I, Pich A. Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI. Proteome Sci. 2011 Aug 17;9:48. doi: 10.1186/1477-5956-9-48 21849038

33. Wang J, Takeuchi T, Tanaka S, Kubo SK, Kayo T, Lu D, et al. A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J Clin Invest. 1999 Jan;103(1):27–37. doi: 10.1172/JCI4431 9884331

34. Riggs AC, Bernal-Mizrachi E, Ohsugi M, Wasson J, Fatrai S, Welling C, et al. Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis. Diabetologia. 2005 Nov;48(11):2313–21. doi: 10.1007/s00125-005-1947-4 16215705

35. Akiyama M, Hatanaka M, Ohta Y, Ueda K, Yanai A, Uehara Y, et al. Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice. Diabetologia. 2009 Apr;52(4):653–63. doi: 10.1007/s00125-009-1270-6 19190890

36. Masciarelli S, Capuano E, Ottone T, Divona M, De Panfilis S, Banella C, et al. Retinoic acid and arsenic trioxide sensitize acute promyelocytic leukemia cells to ER stress. Leukemia. 2018;32(2):285–94. doi: 10.1038/leu.2017.231 28776567

37. Jin W, Wu K, Li Y-Z, Yang W-T, Zou B, Zhang F, et al. AML1-ETO targets and suppresses cathepsin G, a serine protease, which is able to degrade AML1-ETO in t(8;21) acute myeloid leukemia. Oncogene. 2013 Apr 11;32(15):1978–87. doi: 10.1038/onc.2012.204 22641217

38. Schuster B, Hendry L, Byers H, Lynham SF, Ward MA, John S. Purification and identification of the STAT5 protease in myeloid cells. Biochem J. 2007 May 15;404(1):81–7. doi: 10.1042/BJ20061877 17300217

39. Azam M, Erdjument-Bromage H, Kreider BL, Xia M, Quelle F, Basu R, et al. Interleukin-3 signals through multiple isoforms of Stat5. EMBO J. 1995 Apr 3;14(7):1402–11. 7537213

40. Azam M, Lee C, Strehlow I, Schindler C. Functionally distinct isoforms of STAT5 are generated by protein processing. Immunity. 1997 Jun;6(6):691–701. doi: 10.1016/s1074-7613(00)80445-8 9208842

41. Meyer J, Jucker M, Ostertag W, Stocking C. Carboxyl-truncated STAT5beta is generated by a nucleus-associated serine protease in early hematopoietic progenitors. Blood. 1998 Mar 15;91(6):1901–8. 9490672

42. Chakraborty A, Tweardy DJ. Granulocyte colony-stimulating factor activates a 72-kDa isoform of STAT3 in human neutrophils. J Leukoc Biol. 1998 Nov;64(5):675–80. doi: 10.1002/jlb.64.5.675 9823774

43. Xia Z, Salzler RR, Kunz DP, Baer MR, Kazim L, Baumann H, et al. A novel serine-dependent proteolytic activity is responsible for truncated signal transducer and activator of transcription proteins in acute myeloid leukemia blasts. Cancer Res. 2001 Feb 15;61(4):1747–53. 11245492

44. Kato T, Sakamoto E, Kutsuna H, Kimura-Eto A, Hato F, Kitagawa S. Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases. J Biol Chem. 2004 Jul 23;279(30):31076–80. doi: 10.1074/jbc.M400637200 15145953

45. Sherman MA, Secor VH, Brown MA. IL-4 preferentially activates a novel STAT6 isoform in mast cells. J Immunol Baltim Md 1950. 1999 Mar 1;162(5):2703–8.

46. Sherman MA, Powell DR, Brown MA. IL-4 induces the proteolytic processing of mast cell STAT6. J Immunol Baltim Md 1950. 2002 Oct 1;169(7):3811–8.

47. Suzuki K, Nakajima H, Kagami S-I, Suto A, Ikeda K, Hirose K, et al. Proteolytic processing of Stat6 signaling in mast cells as a negative regulatory mechanism. J Exp Med. 2002 Jul 1;196(1):27–38. doi: 10.1084/jem.20011682 12093868

48. Bovolenta C, Testolin L, Benussi L, Lievens PM, Liboi E. Positive selection of apoptosis-resistant cells correlates with activation of dominant-negative STAT5. J Biol Chem. 1998 Aug 14;273(33):20779–84. doi: 10.1074/jbc.273.33.20779 9694822

49. Lokuta MA, McDowell MA, Paulnock DM. Identification of an additional isoform of STAT5 expressed in immature macrophages. J Immunol Baltim Md 1950. 1998 Aug 15;161(4):1594–7.

50. Lee C, Piazza F, Brutsaert S, Valens J, Strehlow I, Jarosinski M, et al. Characterization of the Stat5 protease. J Biol Chem. 1999 Sep 17;274(38):26767–75. doi: 10.1074/jbc.274.38.26767 10480881

51. Piazza F, Valens J, Lagasse E, Schindler C. Myeloid differentiation of FdCP1 cells is dependent on Stat5 processing. Blood. 2000 Aug 15;96(4):1358–65. 10942378

52. Xia Z, Sait SN, Baer MR, Barcos M, Donohue KA, Lawrence D, et al. Truncated STAT proteins are prevalent at relapse of acute myeloid leukemia. Leuk Res. 2001 Jun;25(6):473–82. doi: 10.1016/s0145-2126(00)00158-2 11337019

53. Gupta N, Hixson KK, Culley DE, Smith RD, Pevzner PA. Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry. Proteomics. 2010 Aug;10(15):2833–44. doi: 10.1002/pmic.200900821 20597098

54. Colaert N, Helsens K, Martens L, Vandekerckhove J, Gevaert K. Improved visualization of protein consensus sequences by iceLogo. Nat Methods. 2009 Nov;6(11):786–7. doi: 10.1038/nmeth1109-786 19876014

55. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 2018 04;46(D1):D624–32. doi: 10.1093/nar/gkx1134 29145643

56. O’Donoghue AJ, Jin Y, Knudsen GM, Perera NC, Jenne DE, Murphy JE, et al. Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase. PloS One. 2013;8(9):e75141. doi: 10.1371/journal.pone.0075141 24073241

57. Vizovišek M, Vidmar R, Van Quickelberghe E, Impens F, Andjelković U, Sobotič B, et al. Fast profiling of protease specificity reveals similar substrate specificities for cathepsins K, L and S. Proteomics. 2015 Jul;15(14):2479–90. doi: 10.1002/pmic.201400460 25626674

58. Adkison AM, Raptis SZ, Kelley DG, Pham CTN. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest. 2002 Feb;109(3):363–71. doi: 10.1172/JCI13462 11827996

59. Lausen J, Liu S, Fliegauf M, Lübbert M, Werner MH. ELA2 is regulated by hematopoietic transcription factors, but not repressed by AML1-ETO. Oncogene. 2006 Mar 2;25(9):1349–57. doi: 10.1038/sj.onc.1209181 16247445

60. Oelgeschläger M, Nuchprayoon I, Lüscher B, Friedman AD. C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter. Mol Cell Biol. 1996 Sep;16(9):4717–25. doi: 10.1128/mcb.16.9.4717 8756629

61. Nuchprayoon I, Shang J, Simkevich CP, Luo M, Rosmarin AG, Friedman AD. An enhancer located between the neutrophil elastase and proteinase 3 promoters is activated by Sp1 and an Ets factor. J Biol Chem. 1999 Jan 8;274(2):1085–91. doi: 10.1074/jbc.274.2.1085 9873055

62. Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, et al. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta. 2012 Jan;1824(1):68–88. doi: 10.1016/j.bbapap.2011.10.002 22024571

63. Nishimura Y, Kawabata T, Furuno K, Kato K. Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes. Arch Biochem Biophys. 1989 Jun;271(2):400–6. doi: 10.1016/0003-9861(89)90289-0 2658811

64. Rowan AD, Mason P, Mach L, Mort JS. Rat procathepsin B. Proteolytic processing to the mature form in vitro. J Biol Chem. 1992 Aug 5;267(22):15993–9. 1639824

65. Dalet-Fumeron V, Guinec N, Pagano M. In vitro activation of pro-cathepsin B by three serine proteinases: leucocyte elastase, cathepsin G, and the urokinase-type plasminogen activator. FEBS Lett. 1993 Oct 18;332(3):251–4. doi: 10.1016/0014-5793(93)80643-9 8405467

66. Laurent-Matha V, Derocq D, Prébois C, Katunuma N, Liaudet-Coopman E. Processing of human cathepsin D is independent of its catalytic function and auto-activation: involvement of cathepsins L and B. J Biochem (Tokyo). 2006 Mar;139(3):363–71.

67. Jayakumar A, Kang Y, Frederick MJ, Pak SC, Henderson Y, Holton PR, et al. Inhibition of the cysteine proteinases cathepsins K and L by the serpin headpin (SERPINB13): a kinetic analysis. Arch Biochem Biophys. 2003 Jan 15;409(2):367–74. doi: 10.1016/s0003-9861(02)00635-5 12504904

68. Boyapati A, Ren B, Zhang D-E. SERPINB13 is a novel RUNX1 target gene. Biochem Biophys Res Commun. 2011 Jul 22;411(1):115–20. doi: 10.1016/j.bbrc.2011.06.107 21723253

69. Meier HL, Heck LW, Schulman ES, MacGlashan DW. Purified human mast cells and basophils release human elastase and cathepsin G by an IgE-mediated mechanism. Int Arch Allergy Appl Immunol. 1985;77(1–2):179–83. doi: 10.1159/000233779 3924838

70. Bangalore N, Travis J. Comparison of properties of membrane bound versus soluble forms of human leukocytic elastase and cathepsin G. Biol Chem Hoppe Seyler. 1994 Oct;375(10):659–66. doi: 10.1515/bchm3.1994.375.10.659 7888078

71. Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol. 1995 Nov;131(3):775–89. doi: 10.1083/jcb.131.3.775 7593196

72. Döring G. The role of neutrophil elastase in chronic inflammation. Am J Respir Crit Care Med. 1994 Dec;150(6 Pt 2):S114–117. doi: 10.1164/ajrccm/150.6_Pt_2.S114 7952645

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden