骨髓增生异常综合征（MDS）细胞甲基化模式异常与三氧化二砷对其作用及机理研究
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摘要
骨髓增生异常综合征(myelodysplastic syndromes,MDS)是一组获得性造血干细胞克隆性疾病，根据临床预后分为低危MDS(难治性贫血／难治性贫血伴环铁粒幼红细胞增多，RA／RAS)和高危MDS(难治性贫血伴原始细胞增多／转变中的RAEB，RAEB／RAEB-t)。近年来，国内外研究报道，抑癌基因p151NK4B启动子甲基化导致其功能失活与MDS的疾病进展有关。p15INK4B蛋白质是细胞周期依赖性激酶(cyclin-dependent kinase,CDK)_4和CDK_6的抑制剂，通过抑制它们的丝氨酸／苏氨酸激酶效应，阻断分裂细胞由G_1期向S期过渡，从而抑制细胞增殖。p151NK4B基因启动子及转录起始区域内的CpG岛高度甲基化是导致该基因失活的重要方式。基因甲基化由DNA C5胞嘧啶甲基转移酶(DNA methyltransferases,DNMTs)催化，DNMTs以S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)为甲基供体，将甲基转移到胞嘧啶的第5位碳原子上。DNMTs在调节基因甲基化中起重要作用，迄今发现的人类DNA C5胞嘧啶甲基转移酶有三种，DMNT_1、DNMT_(3A)和DNMT_(3B)。国外有少数报道，急性髓细胞性白血病(AML)患者p151NK4B基因高度甲基化与DNMTs表达异常增高有关，DNMTs活性异常增高导致抑癌基因启动子CpG岛高度甲基化可能是AML发病机制之一，但在MDS细胞中DNMTs表达状况，迄今未见报道。
三氧化二砷(AS_2O_3)系我国民间验方癌灵1号的主要成分。我国学者临床观察发现，
浙江大学博士学位论文
静脉滴注防；0可有效地治疗急性早幼粒细胞性白血病（SCllte promyelOCytiC leUk6mi。，
APL），尤其是治疗复发和耐药的 APL患者。体外研究表明，AS203对某些肿瘤细胞具有诱导
调亡和分化、下调端粒酶活性、抗血管新生等作用。由于AS。03代谢过程中需要通过甲基化
被解毒，且甲基的来源是S一腺苦甲硫氨酸，使其有可能竞争甲基来干扰基因组DNA甲基化
模式。国外已有报道，AS。03可改变P53基因甲基化模式，国内仅见一篇报道，AS。0。可使人
急性淋巴细胞白血病系M。it4细胞P15INK4B基因去甲基化。但该方面的研究甚少。
有关AS。0。对hos的作用及其机理，国内外尚未见文献报道。本课题试图研究p15INK4B
基因甲基化模式改变与MDS发病的关系，及其可能的机制；阐明AS；0。治疗皿DS可能的作用
机理，为该药的临床应用提供实验和理论依据，为设计新的抗肿瘤策略拓宽思路。
本课题采用甲基化特异nR（methylation－speCific PCR，MSP）、RT－PCR和Western杂
交等方法研究了20例MDS患者，其中低危组10例，高危组10例。20例AL患者为阳性对
照组，其中急性髓细胞性白血病（AML）10例，急性淋巴细胞性白血病（ALL）10例。10
名正常骨髓志愿捐献者为阴性对照组。结果显示，正常对照组骨髓单个核细胞（MNCS）几
乎表达P15INK4B基因（90％，9／10）和P15INK4B蛋白质（90％，9／10），且均未检测到P15INK4B
基因甲基化。MDS患者骨髓MNCs s15INK4B基因 mRNA水平和蛋白质水平表达降低与
P15INK4B基因高度甲基化相关，表明MDS患者P15INK4B基因主要通过甲基化方式失活。
MDS低危组较正常组仅DNMT；表达增高，提示低危组骨髓单个核细胞P15INK4B基因甲基化
可能与DWT；高表达有关。高危组骨髓单个核细胞P15INK4B基因甲基化阳性率明显高于低
危组（60％ VS 10％），并伴有DNMT。＾、DNMT。mRNA表达异常增高。DNMT；在MDS低危组、高
危组患者均明显地增高，两组差异无统计学意义，表明，重新甲基转移酶DNMT。＾和DNMT。。mRNA
表达增高可能与血S高危有关。对照M组骨髓MNCS也有P15INK4B基因甲基化（45％，9／20）
和三种甲基转移酶 mRNA表达异常增高。与之比较，MI）S高危组患者骨髓 MNCs DNMT；、DNMT。B
mRNA表达差异无显著性，而DNMT。＾mRNA表达低于AL组。上述结果表明hos患者骨髓MNCs
甲基转移酶异常高度表达可能与MDS向AL演进有关。国内外未见类似文献报道。
本课题以人MDS－RAEB细胞株MUTZI细胞为主要研究对象，应用MTT比色法观察了
AS。0。对 MUTZ－l细胞及对照组 HL－60细胞、Raj i细胞的生长抑制作用。结果显示，AS众对
2
浙江大学博士学位论文
一
以上细胞都有不同程度的生长抑制作用，其对MUTZl细胞的抑制作用明显地高于HL乃0细
胞，而明显的低于 R幻i细胞。AS0。对 MUTZ＊细胞、HL乃0细胞与 R幻i细胞作用 24h的半
数抑制浓度（IC。）值分别为 14．94umol／L、21．06umol／L和4．48umol／L。AS。0。对MUTZ－l
细胞的生长抑制作用具有时间、剂量依赖关系。AS。0。处理MUTZ－1细胞24h、48h、72h的
IC。值分别为 14．94 u mol
Myelodysplastic syndromes (MDS) are clonal stem cell disorders and they can be divided into two groups based on the clinical prognosis: low-risk MDS [refractory anemia (RA) and RA with ring sideroblasts (RAS)] and high-risk MDS [RA with excess of blasts (RAEB) and RAEB in transformation (RAEB-t)]. Recently, there are a few reports abroad and at home about that the p15INK4B gene inactivation may play an important role in the pathogenesis of MDS and during disease progression. The p15INK4B protein is a cell cycle regulator involved in the inhibitation of GI phase progression. It associates with cyclin-dependent kinase (CDK) 4 and CDK 6, and inhibits their kinase activities. Hypermethylation in CpG islands of the p15INK4B promoter region seems to be an important way in resulting gene silencing. The DNA methylation is catalyzed by C5 DNA methyltransferases (DNMTS), including DNMT,, DNMT3A, and DNMT3B, which transfer methyl groups proved by S-adenosylmethionine (SAM) to the carbon-5 position of cytosine. Hypermet
hylation of genes has a tendency to show significantly high expressive
levels of DNMTS. There are a few reports abroad about that the up-regulated DNMTS might contribute to the pathogenesis of acute myeloid leukemia (AML) by inducing aberrant regional hypermethylation of suppressor genes, which has yet not been reported in MDS.
Arsenic trioxide (AS2O3) is the chief compound of "Ailing 1', which is a kind of traditional Chinese agent. The drug has been found to be effective in the treatment of acute promyelocytic leukemia (APL), especially in the treatment of relapsed and refractory APL. It has been reported that AS2O3 can inhibit the growth of some tumor cells in vitro by inducing apoptosis and differentiation, down-regulating of telomerase activity and inhibiting of angiogenesis. Since AS2O3 is detoxified via methylation and it consumes methyl groups that deplete the SAM methyl donor pool. The lack of methyl groups would result in inability to properly maintain methylate cytosine in DNA. It has been reported that AS2O3 can alter methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells.
There is no report about AS2O3's antitumor effects on myelodysplasia cells. In this study, we tried to explode the p15TNK4B gene methylation status in myelodysplasia cells to verify the role of methylation as a gene-silencing mechanism involved in the pathogenesis of MDS, and to elucidate the antitumor mechanisms of AS2O3 in myelodysplasia cells in order to expend the train of thought of the clinical application of AS2O3 in MDS and to some extent, to provide a theoretical and experimental basis for designing new antitumor strategies.
We studied the mononuclear cells (MNCs) from bone marrow of 20 cases of MDS by RT-PCR, Western blot, and methylation specific PCR (MSP). Twenty cases of acute leukemia (AL) were studied as positive controls. Of the 40 patients, 10 cases were low-risk MDS, 10 cases were high-risk MDS, 10 cases were acute myeloid leukemia (AML), and 10 cases were acute lymphocytic leukemia (ALL). Ten normal persons were studied as nective controls. The results showed that the incidence of p!5INK4B methylation in cells of high-risk MDS was higher than that in low-risk MDS (60% VS 10%), and the p15INK4B methylation was found to be associated with the down-regulation of the expressions of p15INK4B gene on both mRNA and protein levels, which indicated that the silencing of p15INK4B gene was in conjunction with
hypermethylation in MDS. The expressions of p15INK4B on mRNA level and protein levels were almost detected in the MNCs from bone marrow of normal persons without the p15INK4B methylation. We also found high-risk myelodysplasia cells with methylated p15INK4B tended to express high levels of DNA methyltransferases DNMT3A and DNMT3B. The expression level of DNMT] was higher in both low-risk and high-risk MDS, which indicated up-regulated DNMTS might contribute to the hypermethylation of p15INK4B, and the higher expressions of de novo methyltransferases DNMT3A and
三氧化二砷(AS_2O_3)系我国民间验方癌灵1号的主要成分。我国学者临床观察发现，
浙江大学博士学位论文
静脉滴注防；0可有效地治疗急性早幼粒细胞性白血病（SCllte promyelOCytiC leUk6mi。，
APL），尤其是治疗复发和耐药的 APL患者。体外研究表明，AS203对某些肿瘤细胞具有诱导
调亡和分化、下调端粒酶活性、抗血管新生等作用。由于AS。03代谢过程中需要通过甲基化
被解毒，且甲基的来源是S一腺苦甲硫氨酸，使其有可能竞争甲基来干扰基因组DNA甲基化
模式。国外已有报道，AS。03可改变P53基因甲基化模式，国内仅见一篇报道，AS。0。可使人
急性淋巴细胞白血病系M。it4细胞P15INK4B基因去甲基化。但该方面的研究甚少。
有关AS。0。对hos的作用及其机理，国内外尚未见文献报道。本课题试图研究p15INK4B
基因甲基化模式改变与MDS发病的关系，及其可能的机制；阐明AS；0。治疗皿DS可能的作用
机理，为该药的临床应用提供实验和理论依据，为设计新的抗肿瘤策略拓宽思路。
本课题采用甲基化特异nR（methylation－speCific PCR，MSP）、RT－PCR和Western杂
交等方法研究了20例MDS患者，其中低危组10例，高危组10例。20例AL患者为阳性对
照组，其中急性髓细胞性白血病（AML）10例，急性淋巴细胞性白血病（ALL）10例。10
名正常骨髓志愿捐献者为阴性对照组。结果显示，正常对照组骨髓单个核细胞（MNCS）几
乎表达P15INK4B基因（90％，9／10）和P15INK4B蛋白质（90％，9／10），且均未检测到P15INK4B
基因甲基化。MDS患者骨髓MNCs s15INK4B基因 mRNA水平和蛋白质水平表达降低与
P15INK4B基因高度甲基化相关，表明MDS患者P15INK4B基因主要通过甲基化方式失活。
MDS低危组较正常组仅DNMT；表达增高，提示低危组骨髓单个核细胞P15INK4B基因甲基化
可能与DWT；高表达有关。高危组骨髓单个核细胞P15INK4B基因甲基化阳性率明显高于低
危组（60％ VS 10％），并伴有DNMT。＾、DNMT。mRNA表达异常增高。DNMT；在MDS低危组、高
危组患者均明显地增高，两组差异无统计学意义，表明，重新甲基转移酶DNMT。＾和DNMT。。mRNA
表达增高可能与血S高危有关。对照M组骨髓MNCS也有P15INK4B基因甲基化（45％，9／20）
和三种甲基转移酶 mRNA表达异常增高。与之比较，MI）S高危组患者骨髓 MNCs DNMT；、DNMT。B
mRNA表达差异无显著性，而DNMT。＾mRNA表达低于AL组。上述结果表明hos患者骨髓MNCs
甲基转移酶异常高度表达可能与MDS向AL演进有关。国内外未见类似文献报道。
本课题以人MDS－RAEB细胞株MUTZI细胞为主要研究对象，应用MTT比色法观察了
AS。0。对 MUTZ－l细胞及对照组 HL－60细胞、Raj i细胞的生长抑制作用。结果显示，AS众对
2
浙江大学博士学位论文
一
以上细胞都有不同程度的生长抑制作用，其对MUTZl细胞的抑制作用明显地高于HL乃0细
胞，而明显的低于 R幻i细胞。AS0。对 MUTZ＊细胞、HL乃0细胞与 R幻i细胞作用 24h的半
数抑制浓度（IC。）值分别为 14．94umol／L、21．06umol／L和4．48umol／L。AS。0。对MUTZ－l
细胞的生长抑制作用具有时间、剂量依赖关系。AS。0。处理MUTZ－1细胞24h、48h、72h的
IC。值分别为 14．94 u mol
Myelodysplastic syndromes (MDS) are clonal stem cell disorders and they can be divided into two groups based on the clinical prognosis: low-risk MDS [refractory anemia (RA) and RA with ring sideroblasts (RAS)] and high-risk MDS [RA with excess of blasts (RAEB) and RAEB in transformation (RAEB-t)]. Recently, there are a few reports abroad and at home about that the p15INK4B gene inactivation may play an important role in the pathogenesis of MDS and during disease progression. The p15INK4B protein is a cell cycle regulator involved in the inhibitation of GI phase progression. It associates with cyclin-dependent kinase (CDK) 4 and CDK 6, and inhibits their kinase activities. Hypermethylation in CpG islands of the p15INK4B promoter region seems to be an important way in resulting gene silencing. The DNA methylation is catalyzed by C5 DNA methyltransferases (DNMTS), including DNMT,, DNMT3A, and DNMT3B, which transfer methyl groups proved by S-adenosylmethionine (SAM) to the carbon-5 position of cytosine. Hypermet
hylation of genes has a tendency to show significantly high expressive
levels of DNMTS. There are a few reports abroad about that the up-regulated DNMTS might contribute to the pathogenesis of acute myeloid leukemia (AML) by inducing aberrant regional hypermethylation of suppressor genes, which has yet not been reported in MDS.
Arsenic trioxide (AS2O3) is the chief compound of "Ailing 1', which is a kind of traditional Chinese agent. The drug has been found to be effective in the treatment of acute promyelocytic leukemia (APL), especially in the treatment of relapsed and refractory APL. It has been reported that AS2O3 can inhibit the growth of some tumor cells in vitro by inducing apoptosis and differentiation, down-regulating of telomerase activity and inhibiting of angiogenesis. Since AS2O3 is detoxified via methylation and it consumes methyl groups that deplete the SAM methyl donor pool. The lack of methyl groups would result in inability to properly maintain methylate cytosine in DNA. It has been reported that AS2O3 can alter methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells.
There is no report about AS2O3's antitumor effects on myelodysplasia cells. In this study, we tried to explode the p15TNK4B gene methylation status in myelodysplasia cells to verify the role of methylation as a gene-silencing mechanism involved in the pathogenesis of MDS, and to elucidate the antitumor mechanisms of AS2O3 in myelodysplasia cells in order to expend the train of thought of the clinical application of AS2O3 in MDS and to some extent, to provide a theoretical and experimental basis for designing new antitumor strategies.
We studied the mononuclear cells (MNCs) from bone marrow of 20 cases of MDS by RT-PCR, Western blot, and methylation specific PCR (MSP). Twenty cases of acute leukemia (AL) were studied as positive controls. Of the 40 patients, 10 cases were low-risk MDS, 10 cases were high-risk MDS, 10 cases were acute myeloid leukemia (AML), and 10 cases were acute lymphocytic leukemia (ALL). Ten normal persons were studied as nective controls. The results showed that the incidence of p!5INK4B methylation in cells of high-risk MDS was higher than that in low-risk MDS (60% VS 10%), and the p15INK4B methylation was found to be associated with the down-regulation of the expressions of p15INK4B gene on both mRNA and protein levels, which indicated that the silencing of p15INK4B gene was in conjunction with
hypermethylation in MDS. The expressions of p15INK4B on mRNA level and protein levels were almost detected in the MNCs from bone marrow of normal persons without the p15INK4B methylation. We also found high-risk myelodysplasia cells with methylated p15INK4B tended to express high levels of DNA methyltransferases DNMT3A and DNMT3B. The expression level of DNMT] was higher in both low-risk and high-risk MDS, which indicated up-regulated DNMTS might contribute to the hypermethylation of p15INK4B, and the higher expressions of de novo methyltransferases DNMT3A and
引文
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