As_2O_3对急性淋巴细胞白血病Molt-4细胞系P15～（INK4b）基因的去甲基化实验研究
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摘要
P15~(INK4b)抑癌基因是近年来发现的位于染色体9P21上的新型抑癌基因，其表达产物P15蛋白是参与细胞周期调控的细胞周期蛋白依赖性激酶抑制因子(CKI)的重要成员。P15~(INK4b)抑癌基因的失活与白血病等多种肿瘤发生、发展密切相关。研究发现DNA的异常甲基化是引起P15~(INK4b)抑癌基因失活的重要方式之一，其中较常见的是P15~(INK4b)抑癌基因启动区5’—CpG岛的异常高甲基化，据报道高达86％的急性粒细胞白血病可存在DNA高甲基化。因此，针对P15~(INK4b)抑癌基因的去甲基化治疗成为恶性血液病防治的一条新思路。本课题为此进行该方向的实验研究。
目前国际上研究较多的去甲基化药物是5-杂氮-2-脱氧胞嘧啶(5—aza—CdR)。本课题选用国内最早报道原用于诱导治疗的化学物质(As_2O_3)作为去甲基实验药物，开展对P15~(INK4b)抑癌基因高甲基化的急性T淋巴母细胞白血病细胞系Molt-4细胞去甲基作用机制的实验研究，探讨As_2O_3对白血病细胞P15~(INK4B)抑癌基因的去甲基化效果及同基因表达的关系，旨在探讨As_2O_3与白血病DNA甲基化的关系及其去甲基化治疗的可能机制，并为As_2O_3作为去甲基化药物提供实验依据。
本课题研究主要包括2个部分：
1、As_2O_3对Molt-4细胞P15~(INK4b)抑癌基因DNA甲基化的影响：不同浓度(0μM、2.5μM、5.0μM和10.0μM)的As_2O_3与Molt-4细胞作用一定时间后，采用甲基敏感酶限制性酶切PCR(REP-PCR)比较加药前后P15~(INK4b)抑癌基因
DNA甲基化的改变;利用半定量RT一PcR法检测P 15’NK4b抑癌基因、DNA
转移酶(DNMT一l)和DNA去甲基化酶(MBD一2)的mRNA表达的变化;
甲基
通过
流式细胞仪和免疫细胞化学染色观察加药前后P15’NK4b蛋白表达的变化。以上分
别从基因组DNA水平、mRNA转录水平和蛋白翻译水平反映As203对P15’NK4b
抑癌基因甲基化和该基因表达的影响。结果表明:用药48小时后，P15基因甲
基化明显减低至消失，其mRNA部分或完全恢复表达。P巧’NK4”蛋白表达阳性率
及阳性强度均明显增强，2.5拼M、5，0林M和10.0协M AsZO3处理组表达分别增加
33.49%、56.84%和69.21%(P<0.05)，加药组与未处理组差别有显著性。此外，
采用半定量RT一PCR法检测加药前后DNA甲基转移酶(DNMT一1)和DNA去甲
基化酶(MBD一2)的mRNA表达情况，分析PI slNK4b抑癌基因甲基化与DNA甲
基化模式的关键酶表达的相关性，以探讨AsZO3的去甲基化机制。结果显示:伴
随P15’NK4b抑癌基因DNA甲基化的去除，DNMT一l表达下降，MBD一2表达增加。
2、AsZO3对Molt一4细胞增殖、活力和细胞周期的影响:不同浓度的AsZO3
与Molt一4细胞作用一定时间后，通过克隆形成率、细胞计数和MTT法观察AsZO3
对细胞增殖、活力的影响;通过流式细胞仪检测用药后Molt一4细胞周期的改变，
以探讨AsZO3去甲基化对细胞的影响。结果表明:AsZO3使Molt一4细胞周期阻滞
于GO、Gl期，且该增殖抑制作用具有时间和浓度依赖性。
结论:l、AsZO:可有效去除Molt一4细胞P15’NK4b抑癌基因甲基化，恢复该
基因表达，阻滞细胞周期于GO、Gl期，抑制细胞增殖。2、AsZO3去甲基化机
制主要是抑制DNMT一1表达、诱导MBD一2表达。
P15INK4b tumor suppressor gene, a new tumor suppressor gene , is located on the chromosome 9P21. P15 is an important factor of the cyclin dependent kinase inhibitors (CKI) concerning the regulation of cell cycle. Inactivation of P15INK4b gene is closedly related to the generation,^development and prognosis of various tumors. Studies show that DNA abnormal methylation is one of the important ways to inactivate P15INK4b gene. And 5' -CpG islands in P15 gene promoter are usually found abnormal hypermethylation. It was reported that about 86% of DNA hypermethylation is detected in acute myelogenous leukemia (AMI). Thus, demethylation therapy aimed at Pl5INK4b tumor suppressor gene becomes a new therapeutic strategy for hematologic malignancies.
Current researches about methylation inhibitors mainly focus on 5-aza-2-deoxycyitidine(5-aza-CdR), but the potential mechanisms of demethylation and the possible approach to regulate cell cycle are still not illuminated clearly. In the other hand, that arsenic trioxide is used to treat leukemia as methylation inhibitor is rarely reported. Accordingly in our study acute T-cell lymphoblastic leukemia cell line
Molt-4 was treated by arsenic trioxide in which P15INK4b gene expression was suppressed due to DNA hypermethylation. The purpose is to investigate the demethylating effect on P15INK4b gene in leukemia cell and the relations between demethylation and gene expression, to explore the correlation between arsenic trioxide and DNA methylaion and clarify the possible mechanisms of demethylating treatment, also to provide experimental basis for arsenic trioxide as methylation inhibitor.
The subject of the thesis were focused on the following two aspects :
1. The impacts on P15INK4b genes in Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentrations (0μM, 2.5 μM,5.0μM and 10.0μM)of arsenic trioxide for some time. Then restricted endonucleases PCR (REP-PCR) was used to detect the methylation of P15INK4b gene. The expression of P15 gene , DNA methyltransferase-1 (DNMT-1) and DNA demethylases (MBD-2) gene mRNA were determined by RT-PCR . Immunocytochemistry and flow cytometry were used to investigate the expression of Pl5INK4b proteins. Those foregoing means can reflect the connection between DNA methylation and P15INK4b gene expression from genome DNA replication, mRNA transcription and protein translation levels. The major results indicated that after 48 hours treatment, the methylation of P15 gene decreased markedly to disappear. Compared with the untreated group, p15INK4b gene mRNA expressions were recovered partly or completely. And P15 protein positive ratios and intensities went up obviously. As results showed , P15INK4b pro
tein expressions in arsenic trioxide (2. 5μM, 5. 0μM and 10. 0μM) -treated groups increased 33.49%, 56.84% and 69.21% respectively (P<0.05). Moreover we can analyse the correlation between P15INK4b gene methylation and expression of key enzyme in methylation pattern. The results indicated that with P15INK4b gene methylaation decreasing , expression of DNMT-1 went down
while MBD-2 went up.
2. The effects on proliferation energy and cell cycle of Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentration of arsenic trioxide for a certain time. Then, we observed the clone formation, do cell-counting and MTT assay to analyse the impact of arsenic trioxide on the cell proliferation and energy. Flow cytometry was used to detect the change of cell cycle for Molt-4 cells in order to explore the demethylation effect of arsenic trioxide. Results showed that arsenic trioxide displayed concentration-dependent and time-dependent inhibition of cell proliferation and cell cycle arrest.
Conclusions:(1) arsenic trioxide is able to effectively wipe off the methylation of P15INK4b tumor suppressor gene, recover the expression of P15 , block the cell cycle in Go/Gl and ultimately inhibit the growth and proliferation of Molt-4 cell. ?The possible mechanism of demethylation of arsenic trio
目前国际上研究较多的去甲基化药物是5-杂氮-2-脱氧胞嘧啶(5—aza—CdR)。本课题选用国内最早报道原用于诱导治疗的化学物质(As_2O_3)作为去甲基实验药物，开展对P15~(INK4b)抑癌基因高甲基化的急性T淋巴母细胞白血病细胞系Molt-4细胞去甲基作用机制的实验研究，探讨As_2O_3对白血病细胞P15~(INK4B)抑癌基因的去甲基化效果及同基因表达的关系，旨在探讨As_2O_3与白血病DNA甲基化的关系及其去甲基化治疗的可能机制，并为As_2O_3作为去甲基化药物提供实验依据。
本课题研究主要包括2个部分：
1、As_2O_3对Molt-4细胞P15~(INK4b)抑癌基因DNA甲基化的影响：不同浓度(0μM、2.5μM、5.0μM和10.0μM)的As_2O_3与Molt-4细胞作用一定时间后，采用甲基敏感酶限制性酶切PCR(REP-PCR)比较加药前后P15~(INK4b)抑癌基因
DNA甲基化的改变;利用半定量RT一PcR法检测P 15’NK4b抑癌基因、DNA
转移酶(DNMT一l)和DNA去甲基化酶(MBD一2)的mRNA表达的变化;
甲基
通过
流式细胞仪和免疫细胞化学染色观察加药前后P15’NK4b蛋白表达的变化。以上分
别从基因组DNA水平、mRNA转录水平和蛋白翻译水平反映As203对P15’NK4b
抑癌基因甲基化和该基因表达的影响。结果表明:用药48小时后，P15基因甲
基化明显减低至消失，其mRNA部分或完全恢复表达。P巧’NK4”蛋白表达阳性率
及阳性强度均明显增强，2.5拼M、5，0林M和10.0协M AsZO3处理组表达分别增加
33.49%、56.84%和69.21%(P<0.05)，加药组与未处理组差别有显著性。此外，
采用半定量RT一PCR法检测加药前后DNA甲基转移酶(DNMT一1)和DNA去甲
基化酶(MBD一2)的mRNA表达情况，分析PI slNK4b抑癌基因甲基化与DNA甲
基化模式的关键酶表达的相关性，以探讨AsZO3的去甲基化机制。结果显示:伴
随P15’NK4b抑癌基因DNA甲基化的去除，DNMT一l表达下降，MBD一2表达增加。
2、AsZO3对Molt一4细胞增殖、活力和细胞周期的影响:不同浓度的AsZO3
与Molt一4细胞作用一定时间后，通过克隆形成率、细胞计数和MTT法观察AsZO3
对细胞增殖、活力的影响;通过流式细胞仪检测用药后Molt一4细胞周期的改变，
以探讨AsZO3去甲基化对细胞的影响。结果表明:AsZO3使Molt一4细胞周期阻滞
于GO、Gl期，且该增殖抑制作用具有时间和浓度依赖性。
结论:l、AsZO:可有效去除Molt一4细胞P15’NK4b抑癌基因甲基化，恢复该
基因表达，阻滞细胞周期于GO、Gl期，抑制细胞增殖。2、AsZO3去甲基化机
制主要是抑制DNMT一1表达、诱导MBD一2表达。
P15INK4b tumor suppressor gene, a new tumor suppressor gene , is located on the chromosome 9P21. P15 is an important factor of the cyclin dependent kinase inhibitors (CKI) concerning the regulation of cell cycle. Inactivation of P15INK4b gene is closedly related to the generation,^development and prognosis of various tumors. Studies show that DNA abnormal methylation is one of the important ways to inactivate P15INK4b gene. And 5' -CpG islands in P15 gene promoter are usually found abnormal hypermethylation. It was reported that about 86% of DNA hypermethylation is detected in acute myelogenous leukemia (AMI). Thus, demethylation therapy aimed at Pl5INK4b tumor suppressor gene becomes a new therapeutic strategy for hematologic malignancies.
Current researches about methylation inhibitors mainly focus on 5-aza-2-deoxycyitidine(5-aza-CdR), but the potential mechanisms of demethylation and the possible approach to regulate cell cycle are still not illuminated clearly. In the other hand, that arsenic trioxide is used to treat leukemia as methylation inhibitor is rarely reported. Accordingly in our study acute T-cell lymphoblastic leukemia cell line
Molt-4 was treated by arsenic trioxide in which P15INK4b gene expression was suppressed due to DNA hypermethylation. The purpose is to investigate the demethylating effect on P15INK4b gene in leukemia cell and the relations between demethylation and gene expression, to explore the correlation between arsenic trioxide and DNA methylaion and clarify the possible mechanisms of demethylating treatment, also to provide experimental basis for arsenic trioxide as methylation inhibitor.
The subject of the thesis were focused on the following two aspects :
1. The impacts on P15INK4b genes in Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentrations (0μM, 2.5 μM,5.0μM and 10.0μM)of arsenic trioxide for some time. Then restricted endonucleases PCR (REP-PCR) was used to detect the methylation of P15INK4b gene. The expression of P15 gene , DNA methyltransferase-1 (DNMT-1) and DNA demethylases (MBD-2) gene mRNA were determined by RT-PCR . Immunocytochemistry and flow cytometry were used to investigate the expression of Pl5INK4b proteins. Those foregoing means can reflect the connection between DNA methylation and P15INK4b gene expression from genome DNA replication, mRNA transcription and protein translation levels. The major results indicated that after 48 hours treatment, the methylation of P15 gene decreased markedly to disappear. Compared with the untreated group, p15INK4b gene mRNA expressions were recovered partly or completely. And P15 protein positive ratios and intensities went up obviously. As results showed , P15INK4b pro
tein expressions in arsenic trioxide (2. 5μM, 5. 0μM and 10. 0μM) -treated groups increased 33.49%, 56.84% and 69.21% respectively (P<0.05). Moreover we can analyse the correlation between P15INK4b gene methylation and expression of key enzyme in methylation pattern. The results indicated that with P15INK4b gene methylaation decreasing , expression of DNMT-1 went down
while MBD-2 went up.
2. The effects on proliferation energy and cell cycle of Molt-4 cell by arsenic trioxide: Molt-4 cells were incubated with different concentration of arsenic trioxide for a certain time. Then, we observed the clone formation, do cell-counting and MTT assay to analyse the impact of arsenic trioxide on the cell proliferation and energy. Flow cytometry was used to detect the change of cell cycle for Molt-4 cells in order to explore the demethylation effect of arsenic trioxide. Results showed that arsenic trioxide displayed concentration-dependent and time-dependent inhibition of cell proliferation and cell cycle arrest.
Conclusions:(1) arsenic trioxide is able to effectively wipe off the methylation of P15INK4b tumor suppressor gene, recover the expression of P15 , block the cell cycle in Go/Gl and ultimately inhibit the growth and proliferation of Molt-4 cell. ?The possible mechanism of demethylation of arsenic trio
引文
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2. Kamb A, Gruis NA, Feldhaus JW, et al. A cell cycle regulator potentially involved in genesis of many twmor types. Science, 1994; 264: 436-440
3. Marx J. New tumor Suppressor may rival P53. Science, 1994, 264:344
4. Faienza MF, Rajione FD, Basso G, et al. P16~(INK4a) gene homozygous deletions in human acute leukaemias with alterations of chromosome 9. Br J Haematol. 1996 Jun;93(3):632-6.
5．付国，吕昌龙．P16和P15基因缺失与白血病．国外医学肿瘤学分册，1997，24(1)：52
6. Huang L. Goodrow TL, Zhang SY, et al. Deletion and mutation analysis of the P16/MTS1 tumor suppressor gene in human ductal pancreatic cancer keveals a higher frequency of abnormalities in tumor-derived cell lines than in primary ductal adeno carcinomas, Cancer Res, 1996; 56(5): 1137.
7. Graff JR, Herman JG, Lapidus RG, et al E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate cancer Cancer Res, 1995, 55:5195
8. Greger V, Passarge E,Hopping W, et al, Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet. 1989 Sep;83 (2): 155-8.
9. Fueyo J, Gomez-Manzano C, Bruner JM Hypermethylation of the CpG island of P16/CDKN2 correlates with gene inactivation in gliomas Oncogene, 1996,13:1615
10. Herman JG, Graff JR,Myohanen S, et al Methylation-specific PCR:a novol PCR assay for methylation status of CpG islands, Proc Natl Acad Sci USA,
1996,93:9821-9826
11. Szyf M DNA methylation machinery as a target for anticancer therapy Pharmacol Ther,1996,76(7):501
12. bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res, 1998,58:95-101
13. Momparler RL, Cote S, Eliopoulos N. Pharmacological approach for optimization of the dose schedule of 5-Aza-2'-deoxycytidine(Decitabine)for the therapy of leukemia Leukemia 1997 Mar, 11(Suppl 1):S1-6
14. Porosnicu M, Nimmanapalli R, Nguyen D,et al. Co-treatment with As2O3 enhances selective cytotoxic effects of STI-571 against Brc-Abl-positive acute leukemia cells. Leukemia 2001 May;15(5):772-8
15. Sordet O,Wrebe C,Leroy I,et al. Mitochondria-targeting drugs arsenic trioxide and lonidarnine bypass the resistance of TPA-differentiated leukemic cells to apoptosis. Blood. 2001 Jun 15;97(12):3931-40.
16．李霞，刘陕西．砷剂抗癌作用机制研究进展．国外医学输血及血液学分册．2002，25(2)：130-133
17. Attadia V. Effects of 5-aza-2'-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937. Leukemia, 1993;7(suppl 1): 9-16
18．姜泊，张亚历，周殿元主编．分子生物学常用实验方法．人民军医出版社．第一版 1996；2．231-235
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20. Monk M. Epigenetic programming of differential gene expression in development and evolution. Dev Genet. 1995; 17(3): 188-97.
21. Ehrlich M, Wang RYH, 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350-7.
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31. Hannon GL, et al. P_(15)~(INK4B) is a potential effector of TGF-β-induced cell cycle arrest. Nature, 1994; 371: 357.
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33．金哈斯，楼方定，于力 As_2O_3诱导急性淋巴细胞白血病细胞系Molt4细胞P15基因表达的研究 中华血液学杂志 2001，22(1)：24-26
34. Shin-ichi Mizuno,Takahito Chijiwa,Takashi Okamura,et al, Expression of DNA methyltransferases DNMT1,3A and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia Blood, 2001 Mar,97(5): 1172-1179
35．白志勇，武淑兰，徐国宾．白血病细胞DNA甲基转移酶活性改变及其意义．中国实验血液学杂志，1999：7：221-224
36．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
2. Kamb A, Gruis NA, Feldhaus JW, et al. A cell cycle regulator potentially involved in genesis of many twmor types. Science, 1994; 264: 436-440
3. Marx J. New tumor Suppressor may rival P53. Science, 1994, 264:344
4. Faienza MF, Rajione FD, Basso G, et al. P16~(INK4a) gene homozygous deletions in human acute leukaemias with alterations of chromosome 9. Br J Haematol. 1996 Jun;93(3):632-6.
5．付国，吕昌龙．P16和P15基因缺失与白血病．国外医学肿瘤学分册，1997，24(1)：52
6. Huang L. Goodrow TL, Zhang SY, et al. Deletion and mutation analysis of the P16/MTS1 tumor suppressor gene in human ductal pancreatic cancer keveals a higher frequency of abnormalities in tumor-derived cell lines than in primary ductal adeno carcinomas, Cancer Res, 1996; 56(5): 1137.
7. Graff JR, Herman JG, Lapidus RG, et al E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate cancer Cancer Res, 1995, 55:5195
8. Greger V, Passarge E,Hopping W, et al, Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet. 1989 Sep;83 (2): 155-8.
9. Fueyo J, Gomez-Manzano C, Bruner JM Hypermethylation of the CpG island of P16/CDKN2 correlates with gene inactivation in gliomas Oncogene, 1996,13:1615
10. Herman JG, Graff JR,Myohanen S, et al Methylation-specific PCR:a novol PCR assay for methylation status of CpG islands, Proc Natl Acad Sci USA,
1996,93:9821-9826
11. Szyf M DNA methylation machinery as a target for anticancer therapy Pharmacol Ther,1996,76(7):501
12. bender CM, Pao MM, Jones PA. Inhibition of DNA methylation by 5-Aza-2'-deoxycytidine suppresses the growth of human tumor cell lines. Cancer Res, 1998,58:95-101
13. Momparler RL, Cote S, Eliopoulos N. Pharmacological approach for optimization of the dose schedule of 5-Aza-2'-deoxycytidine(Decitabine)for the therapy of leukemia Leukemia 1997 Mar, 11(Suppl 1):S1-6
14. Porosnicu M, Nimmanapalli R, Nguyen D,et al. Co-treatment with As2O3 enhances selective cytotoxic effects of STI-571 against Brc-Abl-positive acute leukemia cells. Leukemia 2001 May;15(5):772-8
15. Sordet O,Wrebe C,Leroy I,et al. Mitochondria-targeting drugs arsenic trioxide and lonidarnine bypass the resistance of TPA-differentiated leukemic cells to apoptosis. Blood. 2001 Jun 15;97(12):3931-40.
16．李霞，刘陕西．砷剂抗癌作用机制研究进展．国外医学输血及血液学分册．2002，25(2)：130-133
17. Attadia V. Effects of 5-aza-2'-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937. Leukemia, 1993;7(suppl 1): 9-16
18．姜泊，张亚历，周殿元主编．分子生物学常用实验方法．人民军医出版社．第一版 1996；2．231-235
19. Latham KE, Mechanisms and control of embryonic genome activation in mammalian embryos. Int Rev Cytol. 1999;193:71-124.
20. Monk M. Epigenetic programming of differential gene expression in development and evolution. Dev Genet. 1995; 17(3): 188-97.
21. Ehrlich M, Wang RYH, 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350-7.
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