AS_2O_3诱导人骨髓瘤U266细胞系p16基因去甲基化转录调节作用的实验研究
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摘要
目的利用巢式甲基特异性PCR法(nested methylation specific PCR)+ DNA克隆测序及限制性内切酶PCR法(REP-PCR)检测人骨髓瘤(MM) U266细胞系p16基因的甲基化状态;探讨三氧化二砷(As_2O_3)诱导p16基因去甲基化作用转录调节作用及其可能的机制;比较摸索一种高灵敏度的DNA甲基化检测方法。方法采用巢式甲基特异性PCR法(nested methylation specific PCR)+ DNA克隆测序及限制性内切酶PCR法(REP-PCR)检测U266细胞株P16基因甲基化状态,比较摸索高灵敏度的DNA甲基化检测方法;RT-PCR检测p16、甲基转移酶(DNMT)1、DNMT3A、DNMT3B基因mRNA的表达,生长曲线、MTT法检测As_2O_3对细胞的生长和增殖抑制。利用流式细胞仪DNA 含量分析法探讨As_2O_3对骨髓瘤细胞系U266周期的影响。结果(1)未处理组U266细胞基因组DNA 的胞嘧啶保持不变,而经As_2O_3作用的U266细胞基因组DNA 的胞嘧啶均已变为胸腺嘧啶,说明U266细胞存在p16基因甲基化,As_2O_3作用72h后p16 基因甲基化程度明显减弱至消失,p16 基因异常甲基化的现象被逆转; (2)未处理组细胞p16基因不表达,As_2O_3作用72h后p16基因表达增强,0.5umol/L组、1.0umol/L组和2.0umol/L组p16基因表达阳性条带灰度值与β-actin 比值分别为(0.22±0.10)、(0.59±0.11)、(0.68±0.09),阳性对照灰度比值为(0.77±0.13),其差异有统计学意义(P<0.01);(3)与未处理组相比,As_2O_3作用72h后甲基转移酶(DNMT)1、DNMT3A、DNMT3B 的表达下降并呈浓度依赖性。(4)与对照组相比,3 组不同浓度As_2O_3均能明显抑制肿瘤细胞生长,G0-G1期细胞增加。结论1、As2O3可能通过抑制甲基转移酶甲基转移酶(DNMT)
Objective To detect the methylated status of the multiple myeloma cell line U266by the nested-methylation specific PCR + DNA sequence analysis and restrictedendonucleases PCR(REP-PCR). To investigate the mechanism of ArsenicTrioxide(As_2O_3) induced p16 gene demethylation and to introduce a sensitive andspecific method PCR-based for rapid analysis of the promoter methylation status .Method Methylation of the U266 cell line was detected by the nested-methylationspecific PCR (n-MSP) + DNA sequence analysis and restricted endonucleasesPCR(REP-PCR), the expression of p16 gene、DNA methyltransferase 1(DNMT 1)、DNMT3A and 3B gene mRNA were determined by RT-PCR, the induced growthinhibition of U266 cell by growth curve and MTT, the DNA content of U266 cell wasanalyzed by flow cytometry after exposed by As_2O_3. Results (1) All cytosines inCpG dinucleotides in untreated U266 cell with As_2O_3 as C, while all cytosines intreated U266 cells have been converted to thymidine. (2) p16 gene fail to express inU266 cell line after methylation. As compared with the β-actin , The expression ofU266 cell p16 gene mRNA was increased to ( 0.22±0.10)、(0.59±0.11)、(0.68±0.09) after exposed to 0.5umol/L、1.0umol/L and 2.0umol/L As_2O_3 for 72h. (3)As_2O_3 could significantly down-regulate DNA methyltransferase 1(DNMT 1)、DNMT3A and 3B gene on mRNA level in a dose dependent manner.(4) U266 cells
line grew slowly and arrested at G0-G1 phrase after treatment with three differentconcentration As2O3.Conclusion As2O3 could activate the expression of p16gene by demethylation or/and by inhibiting DNMT 1、DNMT3A and 3B gene; n-MSP+ DNA sequence analysis is a simple、sensitive and specific method for rapid analysisof the promoter methylation status.
Objective To detect the methylated status of the multiple myeloma cell line U266by the nested-methylation specific PCR + DNA sequence analysis and restrictedendonucleases PCR(REP-PCR). To investigate the mechanism of ArsenicTrioxide(As_2O_3) induced p16 gene demethylation and to introduce a sensitive andspecific method PCR-based for rapid analysis of the promoter methylation status .Method Methylation of the U266 cell line was detected by the nested-methylationspecific PCR (n-MSP) + DNA sequence analysis and restricted endonucleasesPCR(REP-PCR), the expression of p16 gene、DNA methyltransferase 1(DNMT 1)、DNMT3A and 3B gene mRNA were determined by RT-PCR, the induced growthinhibition of U266 cell by growth curve and MTT, the DNA content of U266 cell wasanalyzed by flow cytometry after exposed by As_2O_3. Results (1) All cytosines inCpG dinucleotides in untreated U266 cell with As_2O_3 as C, while all cytosines intreated U266 cells have been converted to thymidine. (2) p16 gene fail to express inU266 cell line after methylation. As compared with the β-actin , The expression ofU266 cell p16 gene mRNA was increased to ( 0.22±0.10)、(0.59±0.11)、(0.68±0.09) after exposed to 0.5umol/L、1.0umol/L and 2.0umol/L As_2O_3 for 72h. (3)As_2O_3 could significantly down-regulate DNA methyltransferase 1(DNMT 1)、DNMT3A and 3B gene on mRNA level in a dose dependent manner.(4) U266 cells
line grew slowly and arrested at G0-G1 phrase after treatment with three differentconcentration As2O3.Conclusion As2O3 could activate the expression of p16gene by demethylation or/and by inhibiting DNMT 1、DNMT3A and 3B gene; n-MSP+ DNA sequence analysis is a simple、sensitive and specific method for rapid analysisof the promoter methylation status.
引文
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3. Faienza MF,Rajione FD,Basso G, et al. P16INK4a gene homozygous deletionsin human acute leukaemias with alterations of chromosome 9. Haematol,1996 Jun; 93:632-636.
4. Minamisawa M, Dicciami MB, Batora A, et al. Inactivation of P16 cellcycleregulatory pathmay by specific forgethy of P16 but not the downstreamcompoiunts T-cell acute Lyimph lashz Leukemia. Blood, 1997, 90:492-496.
5. Arshad Amannllah, Barbara trffman. Deregulated E-2F-1 blocks terminaldeffermticition and loss of leukemogencity of M1 myeloblastic Leukemiacell without abrogation induction of P15 and P16.Blood, 2000, 96:2731-2734.
6. Melki Jr, Wamecke P,Vincent PC,et al. Increased DNA ferase expressionleukemia,1998,12:311-316.
7. Baylin SB, Herman JG, Graff JG,et al. Alteration in DNA methylation:afundometal aspect of neoplasia. Cancer Res, 1998,72(8):141-146.
8. Huang L. Goodrow TL, Zhang SY, et al. Deletion and mutationanalysis of the P16/MTS1 tumor suppressor gene in humanductal pancreatic cancer keveals a higher frequency ofabnormalities in tumor-derived cell lines than in primaryductal adeno carcinomas, Cancer Res, 1996; 56(5): 1137.
9. Margeret HL, Ivyh Nwang, Cibert J et al.Aberrant P15 promotermethylation in adult and childhood acute Leukemias of nearly allmorphologic sultypes: potemtial prognostic implication.Blood, 2000,96(6):2306-2310.
10. Hegen Graf, Einsiedel T, Tillmam Tanbe, et al. Delection analysis of P~(16INK4A) and P~(15ZNKB) in relapsed childhood acute lymphoblash leukemin. Blood,
2002, 99(12):4629-4631.
11. Sopie Kusy, Marrieliidinetal, Kistnmial T.et al.P~(14ARF), P~(16INK4A) and P~(15ZNK4B)methylation states in chronic myelogenous leukemia.Blood, 2003,101(4):374-377.
12. Tu Dung T, Nguyeu, Aun F Quantitatire measure of C-ab_1 and P~(15)methylation in chronic myelogenons Leukemia: biological mplicationsBlood, 2000,95(9):2990-2992.
13. Rakesh singal, Goden D, Girde D. DNA methylation. Blood, 1999, 93(12):4009-4010.
14. Porosnicu M, Nimmanapalli R, Nguyen D, et al. Co-treatment with As2O3enhances selective cytotoxic effects of STI-571 against Brc-Abl-positiveacute leukemia cells. Leukemia 2001 May:15(5):772-778.
15. Sordet O, Wrebe C,Leroy I,et al. Mitochondria-targeting drugs arsenictrioxide and lonidamine bypass the resistance of TPA-differentiatedleukemic cells to apoptosis. Blood, 2001 Jun 15;97(12):3931-3940.
16. 朱新华,陈国强,陈竺,等. 砷剂对肿瘤的生物学作用. 国外医学输血及血液学分册,1998 21:314-316.
17. 刘延芳, 江滨, 陆道培. 硫化砷诱导NB4 细胞凋亡及细胞周期阻滞的研究.中华血液学杂志, 2000 , 21 :647 -648.
18. Chen F , Xu Gl , Li Y, et al . Regulation of telomerase activityin HL-60 and NB4 cells by arsenic trioxide . Hematol , 2000 , 20 : 366-369.
19.肖冬梅, 孙关林, 邬维礼, 等. As2O3 诱导K562 细胞凋亡过程中酪氨酸蛋白激酶活性的变化. 中华血液学杂志,1998 ,19 :234 -236.
20. Chen Z, Wang ZY, Chen SJ . Acute promyelocytie lerkemia: cellular andmolecular basis of differentiation and apoptosis. Pharmacol Ther, 1997,76: 141 -149.
21. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis inmyeloid leukemia cells. Cancer Res.,1999, 59 :776 -780.
22. Ishitsuka K, Hanada S, Uozumi K, et al. Arsenic trioxide and the growthof human T-cell leukemia virus type Ⅰinfected T-cell lines. LeukLymphoma, 2000, 37: 649 -655.
23. Hannon GL. P16 is a potential effector of TGF-β-inducedcell cycle arrest. Nature, 1994, 351-357.
24. Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by5-Aza-2′-deoxycytidine suppresses the growth of human tumor cell lines.Cancer Res, 1998, 58: 95-101
25. Momparler RL, Cote S, Eliopoulos N. Pharmacological approach foroptimization of the dose schedule of 5-Aza-2′-deoxycytidine (Decitabine)for the therapy of leukemia. Leukemia 1997 Mar, 11: 1-6.
26.傅卫军,侯健,王东星,等.多发性骨髓瘤P15、P16 基因启动子区甲基化的研究.肿瘤防治研究,2002,29:18-19.
27.卢圣栋主编,现代分子生物学实验技术。北京:高等教育出版社,1993,95-102。
28. Herman J G, Graff J R,Myohanen S et al.Methylation specific-PCR: anovel PCR assay of for methylation status of CpG islands.Proc Natl AcadSci USA,1996,93:9821.
29. Palmisano W A,Divine K K,Saccominno G,et al.Predition lung cancer bydetecting aberrant promoter methylation in spusum.Cancer Res, 2000, 60:5954-5956.
30. Latham KE. Mechanisms and control of embryonic genome activationin mammalian embryos. Int. Rev. Cytol. 1999, 193: 71-124.
31. Ehrlich M, Wang RYH, 5-Methylcytosine in eukaryotic DNA. Science.1981, 19: 212(4501):1350-1357.
32.Dodge JE, List AF, Futscher BW. Selective rariegated methylation ofthe P15CpG island in acute myeloid Leukemia.Cancer,1998,78:561-567.
33.Aggerholm A Guldberg P, Hokland M, et al. Extensive intra-andinter-individnal heterogencity of P15INK4B methylation in acute myeloidLeukemia.Cancer Res, 1999,59:436-441.
34.蒋俊煌,沈建箴,严俊等. MSP 检测P15 基因甲基化在儿童急性白血病微小残留病灶诊断中的价值. 中国小儿血液, 2002.02.17; 7(1): 1-3.
35. Patra SK, Patra A ,Zhao H,et al. DNA methyltransferase and demethylasein human prostate cancer.Mol Carcinog,2002 Mar;33(3);163-171.
36.Mizuno S, Chijina T, Okamura T, et al 。Expression of DNAmethyltransferases DNMT1,3A and 3B in normal hematopoiesis and in acuteand chronic myelogenous leukemia. Blood, 2001,97:1172.
37.Gonsebatt ME,Vega L,Salazar AM,et al. Cytogenetic effects in humanexposure to arsenic.Mutat Res,1997,368:219-228.
38.Marc JM,Wang LJ.Arsenic alters cytosine methylation pattems of thetumor suppressor gene p53 in human lung cells:a model for a mechanism ofcarcigenesis. Mutat Res, 1997,368:263-277.
39. 佟红艳,林茂芳.三氧化二砷诱导人骨髓增生异常综合征细胞株MUTZ-1细胞P15INK4B基因表达的研究.中华血液学杂志,2002, 23:638-640.
40.Rein T,Depamphilis M L,Zorbos H.Indentifying 5-methylcytosine andrelated modifications in DNA genomes. Nucleic Acids Res,1998,26: 2255.
41.Grunau C,Clark S J,Rosenthal A.Bisulfite genomic sequencing :systematic investigation of critical experimental parameters.NucleicAcids Res,2001,29: 65.
2.Kamb A, Gruis NA, Feldhaus JW, et al.A cell cycle regulator potentiallyinvolved in genesis of many tumor types. Science, 1994; 264: 436-440.
3. Faienza MF,Rajione FD,Basso G, et al. P16INK4a gene homozygous deletionsin human acute leukaemias with alterations of chromosome 9. Haematol,1996 Jun; 93:632-636.
4. Minamisawa M, Dicciami MB, Batora A, et al. Inactivation of P16 cellcycleregulatory pathmay by specific forgethy of P16 but not the downstreamcompoiunts T-cell acute Lyimph lashz Leukemia. Blood, 1997, 90:492-496.
5. Arshad Amannllah, Barbara trffman. Deregulated E-2F-1 blocks terminaldeffermticition and loss of leukemogencity of M1 myeloblastic Leukemiacell without abrogation induction of P15 and P16.Blood, 2000, 96:2731-2734.
6. Melki Jr, Wamecke P,Vincent PC,et al. Increased DNA ferase expressionleukemia,1998,12:311-316.
7. Baylin SB, Herman JG, Graff JG,et al. Alteration in DNA methylation:afundometal aspect of neoplasia. Cancer Res, 1998,72(8):141-146.
8. Huang L. Goodrow TL, Zhang SY, et al. Deletion and mutationanalysis of the P16/MTS1 tumor suppressor gene in humanductal pancreatic cancer keveals a higher frequency ofabnormalities in tumor-derived cell lines than in primaryductal adeno carcinomas, Cancer Res, 1996; 56(5): 1137.
9. Margeret HL, Ivyh Nwang, Cibert J et al.Aberrant P15 promotermethylation in adult and childhood acute Leukemias of nearly allmorphologic sultypes: potemtial prognostic implication.Blood, 2000,96(6):2306-2310.
10. Hegen Graf, Einsiedel T, Tillmam Tanbe, et al. Delection analysis of P~(16INK4A) and P~(15ZNKB) in relapsed childhood acute lymphoblash leukemin. Blood,
2002, 99(12):4629-4631.
11. Sopie Kusy, Marrieliidinetal, Kistnmial T.et al.P~(14ARF), P~(16INK4A) and P~(15ZNK4B)methylation states in chronic myelogenous leukemia.Blood, 2003,101(4):374-377.
12. Tu Dung T, Nguyeu, Aun F Quantitatire measure of C-ab_1 and P~(15)methylation in chronic myelogenons Leukemia: biological mplicationsBlood, 2000,95(9):2990-2992.
13. Rakesh singal, Goden D, Girde D. DNA methylation. Blood, 1999, 93(12):4009-4010.
14. Porosnicu M, Nimmanapalli R, Nguyen D, et al. Co-treatment with As2O3enhances selective cytotoxic effects of STI-571 against Brc-Abl-positiveacute leukemia cells. Leukemia 2001 May:15(5):772-778.
15. Sordet O, Wrebe C,Leroy I,et al. Mitochondria-targeting drugs arsenictrioxide and lonidamine bypass the resistance of TPA-differentiatedleukemic cells to apoptosis. Blood, 2001 Jun 15;97(12):3931-3940.
16. 朱新华,陈国强,陈竺,等. 砷剂对肿瘤的生物学作用. 国外医学输血及血液学分册,1998 21:314-316.
17. 刘延芳, 江滨, 陆道培. 硫化砷诱导NB4 细胞凋亡及细胞周期阻滞的研究.中华血液学杂志, 2000 , 21 :647 -648.
18. Chen F , Xu Gl , Li Y, et al . Regulation of telomerase activityin HL-60 and NB4 cells by arsenic trioxide . Hematol , 2000 , 20 : 366-369.
19.肖冬梅, 孙关林, 邬维礼, 等. As2O3 诱导K562 细胞凋亡过程中酪氨酸蛋白激酶活性的变化. 中华血液学杂志,1998 ,19 :234 -236.
20. Chen Z, Wang ZY, Chen SJ . Acute promyelocytie lerkemia: cellular andmolecular basis of differentiation and apoptosis. Pharmacol Ther, 1997,76: 141 -149.
21. Li YM, Broome JD. Arsenic targets tubulins to induce apoptosis inmyeloid leukemia cells. Cancer Res.,1999, 59 :776 -780.
22. Ishitsuka K, Hanada S, Uozumi K, et al. Arsenic trioxide and the growthof human T-cell leukemia virus type Ⅰinfected T-cell lines. LeukLymphoma, 2000, 37: 649 -655.
23. Hannon GL. P16 is a potential effector of TGF-β-inducedcell cycle arrest. Nature, 1994, 351-357.
24. Bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by5-Aza-2′-deoxycytidine suppresses the growth of human tumor cell lines.Cancer Res, 1998, 58: 95-101
25. Momparler RL, Cote S, Eliopoulos N. Pharmacological approach foroptimization of the dose schedule of 5-Aza-2′-deoxycytidine (Decitabine)for the therapy of leukemia. Leukemia 1997 Mar, 11: 1-6.
26.傅卫军,侯健,王东星,等.多发性骨髓瘤P15、P16 基因启动子区甲基化的研究.肿瘤防治研究,2002,29:18-19.
27.卢圣栋主编,现代分子生物学实验技术。北京:高等教育出版社,1993,95-102。
28. Herman J G, Graff J R,Myohanen S et al.Methylation specific-PCR: anovel PCR assay of for methylation status of CpG islands.Proc Natl AcadSci USA,1996,93:9821.
29. Palmisano W A,Divine K K,Saccominno G,et al.Predition lung cancer bydetecting aberrant promoter methylation in spusum.Cancer Res, 2000, 60:5954-5956.
30. Latham KE. Mechanisms and control of embryonic genome activationin mammalian embryos. Int. Rev. Cytol. 1999, 193: 71-124.
31. Ehrlich M, Wang RYH, 5-Methylcytosine in eukaryotic DNA. Science.1981, 19: 212(4501):1350-1357.
32.Dodge JE, List AF, Futscher BW. Selective rariegated methylation ofthe P15CpG island in acute myeloid Leukemia.Cancer,1998,78:561-567.
33.Aggerholm A Guldberg P, Hokland M, et al. Extensive intra-andinter-individnal heterogencity of P15INK4B methylation in acute myeloidLeukemia.Cancer Res, 1999,59:436-441.
34.蒋俊煌,沈建箴,严俊等. MSP 检测P15 基因甲基化在儿童急性白血病微小残留病灶诊断中的价值. 中国小儿血液, 2002.02.17; 7(1): 1-3.
35. Patra SK, Patra A ,Zhao H,et al. DNA methyltransferase and demethylasein human prostate cancer.Mol Carcinog,2002 Mar;33(3);163-171.
36.Mizuno S, Chijina T, Okamura T, et al 。Expression of DNAmethyltransferases DNMT1,3A and 3B in normal hematopoiesis and in acuteand chronic myelogenous leukemia. Blood, 2001,97:1172.
37.Gonsebatt ME,Vega L,Salazar AM,et al. Cytogenetic effects in humanexposure to arsenic.Mutat Res,1997,368:219-228.
38.Marc JM,Wang LJ.Arsenic alters cytosine methylation pattems of thetumor suppressor gene p53 in human lung cells:a model for a mechanism ofcarcigenesis. Mutat Res, 1997,368:263-277.
39. 佟红艳,林茂芳.三氧化二砷诱导人骨髓增生异常综合征细胞株MUTZ-1细胞P15INK4B基因表达的研究.中华血液学杂志,2002, 23:638-640.
40.Rein T,Depamphilis M L,Zorbos H.Indentifying 5-methylcytosine andrelated modifications in DNA genomes. Nucleic Acids Res,1998,26: 2255.
41.Grunau C,Clark S J,Rosenthal A.Bisulfite genomic sequencing :systematic investigation of critical experimental parameters.NucleicAcids Res,2001,29: 65.