骨髓增生异常综合征P15～（INK4B）基因甲基化及药物去甲基化作用的研究
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摘要
目的 检测骨髓增生异常综合征(MDS)p15~(INK4B)基因甲基化状态;探讨5-氮杂-2’-脱氧胞嘧啶(decitabine)和三氧化二砷(arsenic trioxide,As_2O_3)对MDS患者体外培养细胞的去甲基化作用。
方法 采用限制性内切酶联合PCR方法检测MDS患者骨髓单个核细胞p15~(INK4B)基因甲基化状态,并以β-globin(β珠蛋白)基因扩增产物作为试验内参照,分析其甲基化率。体外培养经检测p15~(INK4B)基因高度甲基化的MDS患者外周血单个核细胞,加入decitabine和As_2O_3进行处理,分析其甲基化率变化情况。
结果 低危组MDS患者均未发现p15~(INK4B)基因甲基化,80%高危组MDS患者和66.6%MDS转化为急性白血病的患者发现p15~(INK4B)基因高度甲基化。经decitabine和As_2O_3处理后,培养细胞的甲基化率由药物处理前的19.73%降至处理后的8.82～10.06%,明显降低至原来的50%左右,两种药物间去甲基化效应无明显差别。
结论 骨髓增生异常综合征的发生与p15~(INK4B)基因高度甲基化密切相关,且随病情进展,p15~(INK4B)基因高度甲基化出现越频繁。decitabine和As_2O_3对MDS患者来源的体外培养细胞高度甲基化的p15~(INK4B)基因具明显去甲基化作用,两种药物间去甲基化效应无明显差别。
Objective To detect the methylation status of p15~(INK4B) gene in the patients with myelodysplastic syndromes (MDS), and investigate the effects of decitabine and arsenic trioxide (As_2O_3) to demethylate the cultured cells from MDS patient.Methods The methylation status of p15 ~(INK4B) gene of the mononuclear cells obtained from the MDS patient's bone marrow aspiration were analyzed with restrictive endonucleases combined with PCR technique. β-globin gene was set as an internal reference of the specific amplified band to confirm the degree of methylation. The cultured peripheral blood mononuclear cells from a MDS patient, in whom the hypermethylation status of p15 ~(INK4B) gene had been identified, was chosen as the experimental target cells. The MDS cells was cultured with decitabine and As_2O_3 in vitro to explore the change of methylation degree.Results There was no p15 ~(INK4B)gene methylation detected in the low-risk group MDS patients, but this gene hypermethylation were detected in 80% patients of high-risk group and 66.6% patients in MDS progressing to AML group. After exposed to decitabine and AS2O3. the methylation degree of the
cultured cells fell down from 19.73% to 8.82-10.06%. No significant difference in demethylating effect was found between these two agents. Conclusion Our study has showed the results that pi 5 INK4B gene hypermethylation is closely associated with MDS pathogenesis, and the status of gene hypermethylation occurs more frequently with the progression of MDS. Decitabine and AS2O3 have obvious demethylating activity to the cultured cells from a MDS patient, and no significant difference in demethylating effect was found between the two agents.
方法 采用限制性内切酶联合PCR方法检测MDS患者骨髓单个核细胞p15~(INK4B)基因甲基化状态,并以β-globin(β珠蛋白)基因扩增产物作为试验内参照,分析其甲基化率。体外培养经检测p15~(INK4B)基因高度甲基化的MDS患者外周血单个核细胞,加入decitabine和As_2O_3进行处理,分析其甲基化率变化情况。
结果 低危组MDS患者均未发现p15~(INK4B)基因甲基化,80%高危组MDS患者和66.6%MDS转化为急性白血病的患者发现p15~(INK4B)基因高度甲基化。经decitabine和As_2O_3处理后,培养细胞的甲基化率由药物处理前的19.73%降至处理后的8.82～10.06%,明显降低至原来的50%左右,两种药物间去甲基化效应无明显差别。
结论 骨髓增生异常综合征的发生与p15~(INK4B)基因高度甲基化密切相关,且随病情进展,p15~(INK4B)基因高度甲基化出现越频繁。decitabine和As_2O_3对MDS患者来源的体外培养细胞高度甲基化的p15~(INK4B)基因具明显去甲基化作用,两种药物间去甲基化效应无明显差别。
Objective To detect the methylation status of p15~(INK4B) gene in the patients with myelodysplastic syndromes (MDS), and investigate the effects of decitabine and arsenic trioxide (As_2O_3) to demethylate the cultured cells from MDS patient.Methods The methylation status of p15 ~(INK4B) gene of the mononuclear cells obtained from the MDS patient's bone marrow aspiration were analyzed with restrictive endonucleases combined with PCR technique. β-globin gene was set as an internal reference of the specific amplified band to confirm the degree of methylation. The cultured peripheral blood mononuclear cells from a MDS patient, in whom the hypermethylation status of p15 ~(INK4B) gene had been identified, was chosen as the experimental target cells. The MDS cells was cultured with decitabine and As_2O_3 in vitro to explore the change of methylation degree.Results There was no p15 ~(INK4B)gene methylation detected in the low-risk group MDS patients, but this gene hypermethylation were detected in 80% patients of high-risk group and 66.6% patients in MDS progressing to AML group. After exposed to decitabine and AS2O3. the methylation degree of the
cultured cells fell down from 19.73% to 8.82-10.06%. No significant difference in demethylating effect was found between these two agents. Conclusion Our study has showed the results that pi 5 INK4B gene hypermethylation is closely associated with MDS pathogenesis, and the status of gene hypermethylation occurs more frequently with the progression of MDS. Decitabine and AS2O3 have obvious demethylating activity to the cultured cells from a MDS patient, and no significant difference in demethylating effect was found between the two agents.
引文
1. Uchida T, Kinoshita T, Nagai H, et al. Hypermethylation of the p15~(INK4B) gene in myelodysplastic syndromes. Blood, 1997; 90 (4) : 1403-1409.
2. Preisler HD, Li B, Chen H, et al. p15~(INK4B) gene methylation and expression in normal, myelodysplastic, and acute myelogenous leukemia cells and in the marrow cells of cured lymphoma patients. Leukemia, 2001; 15 (10) : 1589-1595.
3. Quesnel B, Guillerm G, Vereecque R, et al. Methylation of the p15~(INK4B) gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood, 1998; 91 : 2985-2990.
4. Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000; 21 (3) : 461—467.
5. Mace JM, Wang LJ. Arsenic alters cytosine methylation patterns of the promoter of the tumor supressor gene P53 in human lung cells: a model for a mechanism of carcinogenesis. Murat Res, 1997; 386: 263-277.
6.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p15~(INK4B)基因表达的研究.中华血液学杂志,2002;23(12):638-641.
7.任立敏,杜红玲,朱强,等.体外试验诱导白血病细胞p15~(INK4B)基因重新表达.中华内科杂志,2002;41 (11):762-765.
8. Aoki E, Uchida T, Ohashi H, et al. Methylation status of the p15~(INK4B) gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes. Leukemia, 2000; 14 (4) : 586-593.
9. Tien HF, Tang JH, Tsay W, et al. Methylation of the p1 5 ~INK4B gene in myelodysplastic syndrome: it can be detected early at diagnosis or during disease progression and is highly associated with leukaemic transformation. Br J Haematol, 2001; 112 (1): 148-154.
10. Silverman LR, Holland JF, Weinberg RS, et al. Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes. Leukemia , 1993;7 (suppl 1): 21-29.
11. Silverman LR, Holland JF, Demakos EP, et al. Azacytidine (aza C) in myelodysplastic syndromes (MDS): CALGb studies 8421 and 8921. Ann Hematol , 1994;68: A12.
12. Silverman LR. Targeting hypomethylation of DNA to achieve cellular differentiation in myelodysplastic syndromes (MDS) . Oncologist, 2001 ;6 (Suppl 5): 8-14.
13. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated p15 ~INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment. Blood, 2002; 100 (8): 2957-2964.
2. Preisler HD, Li B, Chen H, et al. p15~(INK4B) gene methylation and expression in normal, myelodysplastic, and acute myelogenous leukemia cells and in the marrow cells of cured lymphoma patients. Leukemia, 2001; 15 (10) : 1589-1595.
3. Quesnel B, Guillerm G, Vereecque R, et al. Methylation of the p15~(INK4B) gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood, 1998; 91 : 2985-2990.
4. Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis, 2000; 21 (3) : 461—467.
5. Mace JM, Wang LJ. Arsenic alters cytosine methylation patterns of the promoter of the tumor supressor gene P53 in human lung cells: a model for a mechanism of carcinogenesis. Murat Res, 1997; 386: 263-277.
6.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p15~(INK4B)基因表达的研究.中华血液学杂志,2002;23(12):638-641.
7.任立敏,杜红玲,朱强,等.体外试验诱导白血病细胞p15~(INK4B)基因重新表达.中华内科杂志,2002;41 (11):762-765.
8. Aoki E, Uchida T, Ohashi H, et al. Methylation status of the p15~(INK4B) gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes. Leukemia, 2000; 14 (4) : 586-593.
9. Tien HF, Tang JH, Tsay W, et al. Methylation of the p1 5 ~INK4B gene in myelodysplastic syndrome: it can be detected early at diagnosis or during disease progression and is highly associated with leukaemic transformation. Br J Haematol, 2001; 112 (1): 148-154.
10. Silverman LR, Holland JF, Weinberg RS, et al. Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes. Leukemia , 1993;7 (suppl 1): 21-29.
11. Silverman LR, Holland JF, Demakos EP, et al. Azacytidine (aza C) in myelodysplastic syndromes (MDS): CALGb studies 8421 and 8921. Ann Hematol , 1994;68: A12.
12. Silverman LR. Targeting hypomethylation of DNA to achieve cellular differentiation in myelodysplastic syndromes (MDS) . Oncologist, 2001 ;6 (Suppl 5): 8-14.
13. Daskalakis M, Nguyen TT, Nguyen C, et al. Demethylation of a hypermethylated p15 ~INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment. Blood, 2002; 100 (8): 2957-2964.