摘要
目的:研究WAVE1在儿童急性淋巴细胞性白血病(ALL)患者中的表达及其与病程的关系;研究儿童ALL中还原型辅酶Ⅱ(NADPH)氧化酶亚基p22phox的表达及血浆SOD与GSH-Px的活性;探讨儿童ALL WAVE1的表达与氧化应激的相关性。
方法:采用荧光定量PCR(real-time PCR)法测定不同阶段ALL儿童外周血单个核细胞(PBMCs)WAVE1和NADPH氧化酶亚基p22phox的表达水平。黄嘌呤氧化酶法(羟胺法)和二硫代二硝基苯甲酸法(DTNB法)分别测定不同阶段ALL儿童外周血血浆总SOD活性和GSH-Px活性。
结果:
1.儿童ALL活动期组(初治+复发)外周血单个核细胞(PBMCs)中WAVE1和NADPH氧化酶亚基p22phox的表达与正常对照组和完全缓解组相比均显著增高,P＜0.05;ALL完全缓解组PBMCs中,WAVE1和NADPH氧化酶亚基p22phox的表达高于正常对照组,P＜0.05。
2.ALL活动期组患儿外周血血浆总SOD、GSH-Px活性较完全缓解组和正常儿童明显降低,P＜0.01;ALL完全缓解组患儿外周血血浆总SOD、GSH-Px活性较正常儿童明显降低,P＜0.01。
3.WAVE1的表达与NADPH氧化酶亚基p22phox呈正相关,与SOD、GSH-Px水平呈负相关,P＜0.05;NADPH氧化酶p22phox表达与SOD、GSH-Px水平呈负相关,P＜0.05。
结论:
1.WAVE1、NADPH氧化酶亚基p22phox在儿童ALL PBMCs中表达增高,而且其变化与ALL病程密切相关,表现为活动期增高,缓解期接近到正常水平。
2.儿童ALL中WAVE1表达水平与NADPH氧化酶亚基p22phox表达呈正相关,与SOD、GSH-Px活性呈负相关。
3.ALL患儿氧化应激水平升高可能参与了PBMCs WAVE1表达的调控。
Objective: Researching the expression of WAVE1 in childhood acute lymphocytic leukemia (ALL) and the relationship with courses of ALL. Studying the expression of reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase subunit p22phox and the activity of SOD and GSH-Px in childhood acute lymphocytic leukemia. Analyzing the relationship between WAVE1 and oxidative stress. Methods: The expression of WAVE1 and p22phox in peripheral blood monouclear cells (PBMCs) were detected by real-time PCR .The activity of SOD and GSH-Px were separately tested by hydroxylamine test and dithio-nitrobenzene test (DTNB).
Results:
1. The expression of WAVE1 and p22phox in active phase group were detected higher than that in complete remission (CR) group and normal control, P < 0.05. The expression of WAVE1 and p22phox in CR group were higher compared with the normal control, P < 0.05.
2. The activities of plasma SOD and GSH-Px in active phase group were significantly lower than that in CR group and normal control, P < 0.05.
3. There was positive correlation between the expression of WAVE1 and p22phox, P < 0.05. Inverse association was found between the expression of WAVE1 and SOD, P<0.05. The inverse association was also found between WAVE1 and GSH-Px, P<0.05. The expression of p22phox had inverse correlations with SOD and GSH-Px, P<0.05.
Conclusion:
1. The expression of WAVE1 and p22phox were detected in childhood ALL and had significant relationship with the courses of ALL—high expression in active group, low expression in CR group.
2. In PBMCs of childhood ALL, the expression of WAVE1 and p22phox had positive correlation. The expression of WAVE1 had inverse correlations with SOD and GSH-Px in childhood ALL.
3. The higher levels of oxidative stress may participate to the up-regulation of WAVE1.
引文
[1]袁聿军。细胞骨架的基本成分与功能。生物学教学,2005,31(4):5-8
[2]P.J.Ancliff,M.P.Blundell,G.O.Cory,et al.Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia[J].Blood,2006,108(7):2182-2189
[3]Y.Kim,J.Y.Sung,I.Ceglia,et al.Phosphorylation of WAVE1 regulates actin polymerization and dendritic spine morphology[J].Nature,2006,442(7104):814-817
[4]S.Eden,R.Rohatgi,A.V.Podtelejnikov,et al.Mechanism of regulation of WAVE 1- induced actin nucleation by Rac1 and Nck[J].Nature,2002,418(6899):790-793
[5]D.Yamazaki,T.Oikawa,T.Takenawa.Rac-WAVE-mediated actin reorganization is required for organization and maintenance of cell-cell adhesion[J].Cell Sci,2007,120(1):86-100
[6]P.A.Zipfel,S.C.Bunnell,D.S.Witherow,et al.Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling[J].Curr Biol,2006,16(1):35-36
[7]C.W.Gourlay,K.R.Ayscough.The actin cytoskeleton:a key regulator of apoptosis and aging? Nat Rev Mol Cell Biol,2005,6(7):583-589
[8]C.W.Gourlay,L.N.Carpp,P.Timpson,et al.A role for the actin cytoskeleton in cell death and aging in yeast[J].Cell Biol,2004,164(6):803-809
[9]康睿,曹励之,俞燕等。WAVE1基因在K562/A02白血病细胞多药耐药中的作用。中华血液学杂志,2007,28(6):379-382
[10]Y.R.Hong,L.Han,J.Huang,et al.Elimination of false negative results in the two hybrid system in the phagocyte NADPH oxidase[J].Kao Hsiung I Hsueh Ko Hsueh Tsa Chih,1996,12:301-305
[11]M.C.Dinauer,E.A.Pierce,G.A.Bruns.Human neutrophil cytochrome B light chain(p22phox),gene structure,chromosomal location,and mutations in cytochrome negative autosomal recessive chronic granulo matous disease[J].Clin Invest,1990,86:172921737
[12]Pedruzzi E,Guichard C,Ollivier V,et al.NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells [J]. Mol Cell Biol 2004, 24:10703-10717
[13]Martyn KD, Frederick LM, von Loehneysen K, et al. Functional analysis of Nox4 reveals unique characteristics compared with other NADPH oxidases [J]. Cell Signal 2006,18:69-82
[14] Robert J. McKallip, Wentao Jia, Jerome Schlomer, et al. Cannabidiol-Induced Apoptosis in Human Leukemia Cells: A Novel Role of Cannabidiol in the Regulation of p22phox and Nox4 Expression [J]. The American Society for Pharmacology and Experimental Therapeutics. 2006(3);70, 897-908
[15]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative pcr and the 2 method [J]. METHODS, 2001,25:402-408
[16] M.Innocenti, S.Gerboth, K.Rottner, et al. Abil regulates the activity of N-WASP and WAVE in distinct actin-based processes [J]. Nat Cell Biol, 2005,7(10):969-976
[17] H.Miki,H.Yamaguchi,S.Suetsugu, et al. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of embrane ruffling [J]. Nature, 2000,408(6813):732-735
[18]H.Miki, S. Suetsugu, T. Takenawa. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. Embo [J], 1998,17(23):6932-6941
[19] Yamazaki D, Kurisu S, T akenawa T. Regulation of cancer cell motility through actin reorganization [J],Cancer Sci,2005,96(7):379-386
[20] Kazunori Yoshimitsu, Yohnosuke Kobayashi, Tomofusa Usui, et al. Superoxide Dismutase Activity in Leukemic Blasts of Children with Acute Leukemia [J]. Acta Paediatrica, 2002;73 (1), 92-96
[21] OzcanME, GulecM, Ozerol E, et al. Antioxidant enzyme activities and oxidative stress in affective disorders [J]. Int Clin Psychopharmacol, 2004; 19: 89 - 95
[22] Vaquero EC, Edderkaoui M, Pandol SJ, et al. Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells [J]. Biol Chem 2004,279:34643-34654
[23] Edderkaoui M, Hong P, Vaquero EC, et al . Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase. Am [J]. Physiol, 2005, 289:1137-1147
[24] Mary C. Perianayagam, Orfeas Liangos, Alexey Y. Kolyada, et al. NADPH Oxidase p22phox and Catalase Gene Variants Are Associated with Biomarkers of Oxidative Stress and Adverse Outcomes in Acute Renal Failure [J] . Am Soc Nephrol 2007,18: 255-263
[25] San Jose G, Fortuno A, Beloqui O, et al. NADPH oxidase CYBA polymorphisms, oxidative stress and cardiovascular diseases [J]. Clin Sci (Lond),2008 Feb,114(3): 173-182
[26] K. G Santosl, L.H. Canani2, J.L. Gross, et al. Relationship of p22phox C242T polymorphism with nephropathy in type 2 diabetic patients [J] . NEPHROL 2005, 18:733-738
[1]Athar M.Oxidative stress and experimental carcinogenesis[J].Indian Exp Bio,2002;40:656-66
[2]Ishii T,Yasuda K,Akatsuka A,et al.A mutation in the SDHC gene of complex Ⅱincreases oxidative stress,resulting in apoptosis and tumorigenesis[J].Cancer Res,2005;65:203-209
[3]Ray G,Batra S,Shukla NK,et al.Lipid peroxidation,free radical production and antioxidantstatus in breast cancer [J]. Breast Cancer Res Treat, 2000; 59: 163-170
[4] SaygiliEI, Akcay T, Konukoglu D,etal. Glutathione and glutathi-one-related enzymes in colorectal cancer patients [J]. ToxicolEnviron Health A, 2003; 66: 411-415
[5] Burdon R H. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation [J]. Free Radic Biol Med, 1995,18(4): 775 - 794
[6] Vasilev D, Karadimov D, Vogt J, Santak B. Nitric oxide--a basic mediator of vasodilation and septic shock [J]. Khirurgiia (Sofiia). 1996;49(4):14-6.
[7] Leeder RG, Rafeiro E, Brien JF, Mandin CC, Massey TE. Evaluation of reactive oxygen species involvement in amiodarone pulmonary toxicity in vivo and in vitro [J]. Biochem Toxicol. 1996; 11 (3): 147-60.
[8] Ray RS, Mehrotra S, Prakash S, Joshi PC. Ultraviolet radiation-induced production of superoxide radicals by selected antibiotics [J]. Drug Chem Toxicol. 1996;19(1-2):121-30.
[9] Urbanek H, Gajewska E, Karwowska R, Wielanek M. Generation of superoxide anion and induction of superoxide dismutase and peroxidase in bean leaves infected with pathogenic fungi [J]. Acta Biochim Pol. 1996;43(4):679-85.
[10] Devi GS, Prasad MH, Saraswathi I, Raghu D, Rao DN, Reddy PP. Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias [J]. Clin Chim Acta. 2000 Mar;293(1-2):53-62
[11] Devi SG, Prasad MH , Devi ChVR , et al. Free radicals antioxidant ezymes and lipid perioxidation profile in untreated leukemias [J]. Med Sci Res , 1998 ; 26:423-425
[12] Fujisawa S, Atsumi T, Satoh K, et al. Radical generation, radical-scavenging activity, and cytotoxicity of eugenol-related compounds [J]. In Vitr Mol Toxicol. 2000 Winter; 13(4):269-80
[13] Yamanaka N. Biochemical and medical Aspect of Active Oxygen [J]. Biology of Reproduction. 1993 ;5(9): 1037-1046
[14] Oltra AM, Carbonell F, Tormos C, et al. Antioxidant status, erythrocyte membrane lipid peroxidation and osmotic fragility in malignant lymphoma patients [J]. Clin Chem Lab Med. 2000 Aug;38(8):737-42.
[15] Abou-Seif MA, Rabia A, Nasr M. Antioxidant enzyme activities and the production of MDA and 8-oxo-dG in chronic lymphocytic leukemia [J]. Free Radic Biol Med. 2001 Jun 1;30(11): 1286-92.
[16] Saito H, Kuroki T, Nose K. Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells [J]. FEBS Lett. 1989 Jun 5;249(2):253-6,
[17]Auwerx JH, Chait A, Wolfbauer G, et al. Loss of copper-zinc superoxide dismutase gene expression in differentiated cells of myelo-monocytic origin [J]. Blood. 1989 Oct;74(5): 1807-10.
[18] Kazunori Yoshimitsu, Yohnosuke Kobayashi, Tomofusa Usui, et al. Superoxide Dismutase Activity in Leukemic Blasts of Children with Acute Leukemia Acta Paediatrica [J]. 2002;73 (1), 92-96
[19] Masahiko Kato, Hisanori Minakami, Minoru Kuroiwa, et al. Superoxide radical generation and Mn- and Cu-Zn superoxide dismutases activities in human leukemic cells [J]. Hematological Oncology, 2002,21(1): 11-16
[20] Danesch U , Weber PC , Sellmayer A. Arachidonic acid increases c2fos and Egr21 mRNA in 3T3 fibroblasts by formation of prostaglandin E2 and activation of protein kinase C [J]. Biol Chem, 1994 ; 269 :27258 - 27263
[21] Hii CS , Ferrante A , Edwards YS , et al . Activation of mitogen2 activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C2dependent mechanism [J]. Biol Chem, 1995 ; 270 :4201 - 4204
[22] Tang DG, Honn KV. Apoptosis of W256 carcinosarcoma cells of the monocytoid origin induced by NDGA involves lipid peroxidation and depletion of GSH: role of 122lipoxygenase in regulating tumor cell survival [J]. Cell Physiol , 1997 ; 172:155-170
[23] Piazza GA, Rahm AL, Krutzsch M , et al. Antineoplastic drugs sulindac sulfide and sulfone inhibit cell growth by inducing apoptosis [J]. Cancer Res , 1995 ; 55:3110-3116
[24] Shiff S. J , Koutsos M, Qiao L , et al . Nonsteroidal antiinflammatory drugs inhibit the proliferation of colon adenocarcinoma cells : effects on cell cycle and apoptosis. Exp Cell Res , 1996 ; 222 :179 -188
[25] O'Donnell VB , Spycher S , Azzi A. Involvement of oxidants and oxidant generating enzyme(s) in tumour necrosis factor alpha mediated apoptosis : role for lipoxygenase pathway but not mitochondrial respiratory chain [J]. Biochem, 1995; 310:133-141
[26] Rizzo MT , Regazzi E , Garau D , et al . Induction of apoptosis by arachidonic acid in chronic myeloid leukemia cells. Cancer Res, 1999 ; 59 :5047 - 5053
[27] Takeya R, Ueno N, Kami K, et al. Novel Human homologues of p47phox and p67phox participate in activation superoxide-producing NADPH oxidase [J] . Biol Chem, 2003, 278( 27): 25234-25246.
[28] Mary C. Perianayagam, Orfeas Liangos, Alexey Y. Kolyada, et al. NADPH Oxidase p22phox and Catalase Gene Variants Are Associated with Biomarkers of Oxidative Stress and Adverse Outcomes in Acute Renal Failure [J]. Am Soc Nephrol 2007.18: 255-263,
[29] San Jose G, Fortuno A, Beloqui O, et al. NADPH oxidase CYBA polymorphisms, oxidative stress and cardiovascular diseases. Clin Sci (Lond). 2008 Feb; 114(3): 173-82.
[30]K.G. Santos 1, L.H. Cananil, J.L. Grossl, et al. Relationship of p22phox C242T polymorphism with nephropathy in type 2 diabetic patients [J]. NEPHROL, 2005; 18:733-738
[31]Krause KH. Tissue distribution and putative physiological function of NOX family NADPH oxidase[ J]. Infact Dis, 2004 ; 57( 5): 28-29.
[32] Lassegue B,Clempus RE.Vascular. NAD(P)H oxidase: specific features, expression and regulation [J] . Physiol Regul Integr Comp Physiol, 2003 ; 285( 2): R277-297.
[33] Cui XL, Brockman D, Campos B, et al. Expression of NADPH oxidase isoform 1 (NOX1) in human placenta: involvement in preeclampsia [J]. Placenta, 2006 ; 27(4-5):422-431.
[34] Arbiser JL, Petros J, Klafter R, et al. Reactive oxygen generated by NOX1 triggers angiogenic switch[ J]. PNAS, 2002 ; 99( 2): 715-720
[35] Griendling KK, Sorescu D, Ushio-Fukai. NAD(P)H oxidase: role in cardiovascular biology and disease [ J]. Circ Res, 2000,86( 5): 494-501
[36]Vaquero EC,Edderkaoui M,Pandol SJ,et al.Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosis in pancreatic cancer cells[J].Biol Chem 2004,279:34643-34654
[37]Shimo-Nakanishi Y,Hasebe T,Suzuki A,et al.Functional effects of NAD(P)H oxidase p22(phox)C242T mutation in human leukocytes and association with thrombotic cerebral infarction[J].Atherosclerosis,2004;175:109-115
[38]Pedruzzi E,Guichard C,Ollivier V,et al.NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells[J].Mol Cell Biol 2004,24:10703-10717.
[39]Martyn KD,Frederick LM,von Loehneysen K,et al.Functional analysis of Nox4 reveals unique characteristics compared with other NADPH oxidases[J].Cell Signal 2006,18:69-82.
[40]Edderkaoui M,Hong P,Vaquero EC,et al.Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase[J].Physiol 2005,289:G1137-G1147.
[41]Shimo-Nakanishi Y,Hasebe T,Suzuki A,et al.Functional effects of NAD(P)H oxidase p22(phox)C242T mutation in human leukocytes and association with thrombotic cerebral infarction.Atherosclerosis.2004;175:109-115.
[42]Robert J,Mc Kallip,Wentao Jia,et al.Cannabidiol-Induced Apoptosis in Human Leukemia Cells:A Novel Role of Cannabidiol in the Regulation of p22phox and Nox4 Expression[J].The American Society for Pharmacology and Experimental Therapeutics.2006(3);70,897-908.
[43]Shimo-Nakanishi Y,Hasebe T,Suzuki A,et al.Functional effects of NAD(P)H oxidase p22(phox)C242T mutation in human leukocytes and association with thrombotic cerebral infarction.Atherosclerosis[J].2004;175:109-115.
[44]Leszek Wojnowski,Bettina Kulle,Markus Schirmer,et al.NAD(P)H Oxidase and Multidrug Resistance Protein Genetic Polymorphisms Are Associated With Doxorubicin-Induced Cardiotoxicity[J].Circulation.2005;112:3754-3762.)
[45]康睿,曹励之,俞燕等。WAVE1基因在K562/A02白血病细胞多药耐药中的作用。中华血液学杂志,2007,28(6):379-382
[46] Li A, Prasad A, Mincemoyer F. Relationship of the C242T p22phox gene polymorphism to angiographic coronary artery disease and endothelial function [J ]. Med Genet ,1999 ,86 :57261.
[47] Saha N ,Sanghera DK, Kamboh MI. The p22phox polymorphism C242T is not associated with CHD risk in Asian Indians and Chinese [J]. Eur J Clin Invest , 1999,29:999-1002.
[48] Ito D, Murata M, Watanabe K. C242T Polymorphism of NADPH oxidase p22phox gene and ischemic cerebrovascular disease in the Japanese population[J]. Stroke, 2000 ,31 :9362939.
[49] Ungvari Z, Csiszar A, Edwards JG, et al. Increased superoxide production in coronary arteries in hyperhomocysteinemia: role of tumor necrosis factor-α, NAD(P)H oxidase, and inducible nitric oxide aynthase [J]. Arterioscler Thromb Vasc Biol, 2003,23( 3): 418-424.
[50] Lund AK, Peterson SL, Timmins GS, et al. Endothelin-1 -mediated increase in reactive oxygen species and NADPH oxidase activity in hearts of aryl hydrocarbon receptor(AhR) null mice [J]. Toxicol Sci, 2005,88(1): 265-273.
[51] Anderson KM , Ou D , Wu YB , et al . Induction of type 1 programmed cell death in U937 cells by the antioxidant , butylated hydroxytoluene or the free radical spin trap , NTBN [J]. Leuk Res , 1999 ;23 :665 - 673
[52] Banki K, Hutter E, Colombo E, et al . Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression [J]. Biol Chem , 1996; 271 :32994 - 33001
[53] Rabbi S, Hadi F, Rulio T, et al. Nitric oxide regulation of superoxide and peroxynitrite dependent lipid peroxidation [J]. Biol Chem ,1994 ; 269 :26066 -26075
[54] Ozcan ME, Gulec M, Ozerol E, et al. Antioxidant enzyme activities and oxidative stress in affective disorders [J]. Int Clin Psychopharmacol, 2004; 19: 89 - 95
[55] Tsuboi H, Shimoi K, Kinae N, et al. Depressive symptoms are independently correlated with lipid peroxidation in a female population comparison with vitamins and carotenoids [J]. Psychosom Res, 2004;56: 53 - 58
[56] Rubbo H, Radi R , Trujillo M , et al . Nitric oxide regulation of superoxide and peroxynitrite dependent lipid peroxidation. Formation of novel nitrogen containing oxidized lipid derivatives [J]. Biol Chem , 1994 ; 269 :26066 - 26075
[57] Gorbunov NV , Yalowich JC , Gaddam A, et al. Nitric oxide prevents oxidative damage produced by terbuty hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron. Electron paramagnetic resonance evidence [J]. Biol Chem, 1997 ; 272 :12328 - 12341
[58] Kelley EE , Wagner BA, Buettner GR , et al. Nitric oxide inhibits iron induced lipid peroxidation in HL260 cells [J]. Arch Biochem Biophys,1999; 370 :97 - 104
[59] Brett A. Wagner, Garry R. Buettner, Larry W. Oberley, et al. Myeloperoxidase Is Involved in NO-induced Apoptosis of HL-60 Human Leukemia Cells [J]. Biol Chem. 2000,275(29), 22461-22469
[60] Geng YJ, Hellstrand K, Wennmalm A, et al. Original Manuscript Nitric oxide induces the apoptosis of human BCR-ABL-positive myeloid leukemia cells: evidence for the chelation of intracellular iron [J] .Nature leukemia, 2002, 16(4): 708-715
[61] G Magrinat, SN Mason, PJ Shami ,et al. Nitric oxide modulation of human leukemia cell differentiation and gene expression [J].The American Society of Hematology,2002,80(8):1880-1884