尿多酸肽抗骨髓增生异常综合征（MDS）作用及其机制的实验研究
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摘要
骨髓增生异常综合征(myelodysplastic syndrome,MDS)是一组以不同程度的外周血细胞减少伴随骨髓无效造血为特征的恶性克隆性疾病。目前仍被认为是不可治愈的。1997年世界卫生组织制定了MDS国际预后评估系统(IPSS),根据骨髓中原始细胞比例,染色体核型和外周血三系减少程度将MDS分为低危组(0分),中危组(0.5—2.0分)和高危组(2.5分以上)。中危和高危MDS患者虽然骨髓肿瘤细胞过度增殖,但是往往合并外周血三系减少,骨髓代偿能力差,治疗非常棘手。多数患者必须依靠输血才能维持生存,并最终转化为急性髓系白血病(acute myeloid leukemia,AWL)后死亡,预后极差。大部分MDS患者年龄超过70岁,无法接受强烈的化疗和骨髓移植治疗。虽然胞苷衍生物5—氮杂胞苷(5—aza—cytidine,Aza C)和5—氮杂—2‘一脱氧胞苷(5—aza-2'-deoxycitidine,Decitabine,DAC)已被美国FDA批准用于MDS的治疗,但是明显的骨髓抑制的副作用限制了其临床应用。因此寻找效果好且骨髓抑制轻的新的治疗方法以改善中危和高危MDS患者的预后,仍是目前MDS治疗研究的焦点。尿多酸肽(Uroacitides),又名CDA-Ⅱ(cell differentiation agentⅡ)是从健康人尿中经酸化、吸附、洗脱、真空干燥等过程制成的含有多种有机酸和多肽成分的非细胞毒性药物。CDA-Ⅱ的这些成分共同作用来发挥其抗肿瘤作用。前期研究表明,CDA-Ⅱ具有诱导肿瘤细胞分化的作用,在实体瘤的治疗中显示出一定的疗效。我们设想,CDA-Ⅱ可以与三氧化二砷一样,既具有促分化作用又具有诱导凋亡作用。如果这一假设成立,那么它的作用靶点与作用机理呢?此外,由于CDA-Ⅱ是从健康人尿中提取的有效成分,其毒副作用一般不会太大,比较适合老年病人,而MDS多见于老年患者。基于上述考虑,我们选择MDS-RAEB细胞株MUTZ-1进行体外及动物实验研究以观察CDA-Ⅱ对中危及高危MDS的作用及作用机制,以期为临床MDS治疗提供新的方法和思路。
本研究分三部分进行:
第一部分:尿多酸肽(CDA-Ⅱ)通过影响细胞生存信号通路诱导骨髓增生异常综合征细胞凋亡
第二部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞PTEN和p15INK4B基因异常甲基化模式的影响
第三部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞端粒酶活性及人端粒酶逆转录酶(hTERT)表达的影响及机制
第一部分:尿多酸肽(CDA-Ⅱ)通过影响细胞生存信号通路诱导骨髓增生异常综合征细胞凋亡
目的:探讨CDA-Ⅱ对MDS细胞的作用和作用机制以及对MDS细胞PI3K/Akt及NF-κB生存信号途径的影响,同时研究CDA-Ⅱ对人MDS荷瘤小鼠瘤体生长的抑制作用,以期从细胞水平和实验动物水平明确CDA-Ⅱ对MDS的作用及作用机制,为高危MDS的靶向治疗提供实验基础。
方法:采用MTT比色法研究CDA-Ⅱ对MDS,白血病和淋巴瘤细胞株以及MDS原代细胞和正常人骨髓单个核细胞的体外生长抑制作用:采用Wright-Giemsa染色、流式细胞仪PI染色检测亚G1峰和Annexin V/PI双染色检测细胞凋亡;采用流式细胞仪PI染色检测细胞周期、JC-1染色检测细胞线粒体膜电位的改变:用Western-blot方法检测CDA-Ⅱ作用后MUTZ-1细胞Caspase家族蛋白,细胞周期蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族和FLIP蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;提取核浆蛋白以观察CDA-Ⅱ作用对NF-κB信号通路的影响,并进一步加用TNF-α来证实CDA-Ⅱ对NF-κB核转位的影响;Western-blot方法检测CDA-Ⅱ对MUTZ-1细胞PI3K/Akt和NF-κB信号通路相关生存蛋白的影响,并进一步加用PI3K抑制剂LY294002和NF-κB抑制剂PDTC来明确两条信号通路的相关性及机制:用RT-PCR方法检测CDA-Ⅱ对表达PI3Kp110α蛋白的PIK3CA基因的影响,以明确CDA-Ⅱ治疗的靶点:用人MDS荷瘤小鼠模型观察CDA-Ⅱ对小鼠瘤体生长和平均生存期的影响以及比较用药前后瘤组织细胞形态和p-Akt的原位表达,从而明确CDA-Ⅱ在实验动物水平对MDS细胞的生长抑制作用,并评估实验治疗剂量的安全性。
结果:①CDA-Ⅱ体外可以抑制MDS,白血病和淋巴瘤细胞株生长,其中髓系白血病细胞株较淋系血液肿瘤细胞株更敏感,最敏感的为MUTZ-1细胞株:CDA-Ⅱ体外可以抑制MDS细胞株MUTZ-1及MDS原代细胞的生长,并呈浓度和时间依赖性,但不影响正常人骨髓单个核细胞的生长。②CDA-Ⅱ体外可以诱导MUTZ-1细胞株和MDS原代细胞凋亡,并呈浓度依赖性。③CDA-Ⅱ在体外可以诱导MUTZ-1细胞株出现G1期细胞周期阻滞,并下调细胞周期相关蛋白Cyclin D1和CDK4表达,呈剂量依赖性。④不同浓度CDA-Ⅱ作用可以激活Caspase-9,3,呈浓度依赖性,并伴有明显的线粒体膜电位下降:当CDA-Ⅱ浓度超过4mg/mL时激活Caspase-8,呈浓度依赖性:Caspase-3抑制剂可以抑制CDA-Ⅱ诱导的MUTZ-1细胞凋亡。⑤不同浓度CDA-Ⅱ作用可以下调Bcl-2家族抗凋亡蛋白Bcl-2,Mcl-1,p-Bad和上调促凋亡蛋白Bax的表达,呈浓度依赖性;CDA-Ⅱ在浓度超过4mg/mL时可以激活Bid,并呈浓度依赖性。⑥CDA-Ⅱ可以抑制MUTZ-1细胞IAPs家族中的CIAP1,XIAP和Survivin的表达,呈浓度依赖性,但不影响CLAP2的表达。⑦MUTZ-1细胞高表达FLIP_L:且CDA-Ⅱ在浓度超过4mg/mL时,可以明显抑制FLIP_L表达,呈浓度依赖性。⑧不同浓度CDA-Ⅱ作用可以抑制IκBα磷酸化和NF-κB核转位,也可以抑制TNF-α诱导的IκBα磷酸化和NF-κB核转位;CDA-Ⅱ与NF-κB抑制剂PDTC联用可以明显下调NF-κB信号通路下游信号分子IAPs家族,FLIP_L,Bcl-2,Cyclin D1的表达。⑨不同浓度和不同时间CDA-Ⅱ作用可以下调PIK3CA mRNA表达:PI3K抑制剂LY294002与CDA-Ⅱ联用可以明显抑制PI3K/Akt信号通路,并进一步影响其下游的信号蛋白,包括NF-κB信号通路及其下游的信号分子,p-Bad,Bax,Mcl-1和Caspase-9而诱导MUTZ-1细胞凋亡。⑩CDA-Ⅱ可以明显抑制人MDS荷瘤小鼠瘤体生长并延长小鼠平均生存期:CDA-Ⅱ的实验治疗剂量对小鼠体重无明显影响,且未发现有不明原因死亡的小鼠;HE染色和免疫组化检测显示CDA-Ⅱ在实验治疗剂量下可以明显抑制人MDS荷瘤小鼠瘤组织细胞的生长和p-Akt的原位表达。
小结:①CDA-Ⅱ体外可以抑制MDS,白血病和淋巴瘤细胞株生长,其中髓系白血病细胞株较淋系血液肿瘤细胞株更敏感,最敏感的为MUTZ-1细胞株:CDA-Ⅱ体外可以抑制MDS原代细胞的生长,但不影响正常人骨髓单个核细胞的生长,对正常骨髓无抑制作用。②CDA-Ⅱ体外可以诱导MUTZ-1细胞株和MDS原代细胞凋亡,并伴有G1期细胞周期阻滞,和细胞周期相关蛋白Cyclin D1和CDK4表达抑制。③CDA-Ⅱ通过启动内源性和外源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并以内源性(线粒体)途径为主,伴有明显的线粒体膜电位下降。④CDA-Ⅱ通过影响Bcl-2家族蛋白Bel-2,Mel-1,p-Bad,Bax和Bid的表达以及抑制IAPs家族中CIAP1,XIAP和Survivin的表达导致线粒体损伤和Caspase-9,3激活诱导细胞凋亡。⑤MUTZ-1细胞高表达FLIP_L;且CDA-Ⅱ在浓度超过4mg/mL时,可以明显抑制FLIP_L表达,从而解除对Caspase-8的抑制作用诱导凋亡。⑥CDA-Ⅱ可以通过抑制IκBα磷酸化,从而抑制NF-κB核转位来下调MUTZ-1细胞内源性及TNF-α诱导的NF-κB信号通路的活性;再通过作用于NF-κB信号通路下游的信号分子,包括IAPs家族,FLIP_L,Bcl-2,Cycl in D1导致MUTZ-1细胞凋亡和细胞周期阻滞:CDA-Ⅱ对MUTZ-1细胞NF-κB信号通路的影响主要通过对PI3K/Akt信号通路的调控来实现。⑦CDA-Ⅱ主要通过影响PIK3CA基因的表达而抑制PI3K/Akt信号通路,并进一步通过影响其下游的信号蛋白,包括NF-κB信号通路及其下游的信号分子,p-Bad,Bax,Mcl-1和Caspase-9而诱导MUTZ-1细胞凋亡。PIK3CA基因为CDA-Ⅱ作用的靶点。⑧CDA-Ⅱ在动物实验中可以明显抑制人MDS荷瘤小鼠瘤体生长并延长小鼠平均生存期;CDA-Ⅱ的实验治疗剂量对小鼠无明显毒副作用;CDA-Ⅱ在实验治疗剂量下可以明显抑制人MDS荷瘤小鼠瘤组织细胞的生长和p-Akt的原位表达。
第二部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞PTEN和p15INK4B基因异常甲基化模式的影响
目的:检测抑癌基因PTEN在MDS和MDS转化的AML中的表达和甲基化状态,从而明确PTEN基因与MDS发病和MDS转化为AML的相关性:探讨CDA-Ⅱ对MDS细胞中异常高表达的甲基转移酶DNMT1、3A和3B的作用以及对抑癌基因PTEN和p15INK4B的作用,并以去甲基化药物Decitabine作为阳性对照,为MDS的靶向治疗提供新的方法。
方法:用Western-blot方法检测MUTZ-1细胞株、低危MDS、高危MDS、MDS转化的AML患者和正常人骨髓单个核细胞中甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p15INK4B蛋白表达情况:用Western-blot方法检测CDA-Ⅱ作用对MUTZ-1细胞甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p15INK4B蛋白表达的影响:用RT-PCR方法检测CDA-Ⅱ作用对MUTZ-1细胞甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p151NK4B mRNA表达的影响,并以去甲基化药物Decitabine作为阳性对照:用甲基化PCR(MSP)方法检测MUTZ-1和AML细胞株、低危MDS、高危MDS、MDS转化的AML患者以及正常人骨髓单个核细胞中PTEN和p15INK4B的甲基化状态:用MSP方法检测CDA-Ⅱ作用对MUTZ-1细胞PTEN和p15INK4B甲基化状态的影响,同时与去甲基化药物Decitabine作对照,以评价CDA-Ⅱ的有效性。
结果:①MUTZ-1细胞株、高危WDS和MDS转化的AML患者高表达甲基转移酶DNMT1、3A和3B,并以DNMT3B为明显;低危组MDS和正常人弱表达DNMT1、3A和3B蛋白。②CDA-Ⅱ可以明显下调MUTZ-1细胞甲基转移酶DNMT1、3A和3B表达,呈时间和剂量依赖性,以对DNMT1的作用最明显。③发现了抑癌基因PTEN在MUTZ-1细胞株、高危MDS和MDS转化的AML患者细胞中均表达缺失,而在低危MDS和正常人细胞中均有表达。④用MSP方法检测并发现了MUTZ-1细胞株和高危MDS以及MDS转化的AML患者PTEN基因完全甲基化:AML细胞株Kasumi-1PTEN基因部分甲基化:AML细胞株KG1,CML细胞株K562,低危组MDS及正常人PTEN基因完全非甲基化,提示PTEN基因在高危MDS和MDS转化的AML中存在高度甲基化,很可能是MDS向AML转变的一个重要的发病机制,可以作为CDA-Ⅱ去甲基化治疗的靶基因。国内外均未见报道。⑤发现了CDA-Ⅱ可以通过去甲基化作用使PTEN启动子区CpG岛甲基化水平降低,导致PTEN mRNA水平和蛋白表达水平均上调,从而发挥对PI3K/Akt信号通路的抑制作用进而诱导MUTZ-1细胞凋亡。国内外均未见报道。⑥发现了CDA-Ⅱ可以通过去甲基化作用使p15INK4B甲基化水平降低,导致p15INK4B mRNA水平和蛋白表达水平均上调,从而发挥对CDK4的抑制作用进而造成MUTZ-1细胞G1期阻滞。
小结:PTEN基因在高危MDS和MDS转化的AML中存在高度甲基化,很可能是MDS向AML转变的一个重要的发病机制,靶向PTEN基因的去甲基化治疗可以作为高危MDS和MDS转化的AML患者的治疗策略:CDA-Ⅱ可以通过抑制甲基转移酶DNMT1、3A和3B导致抑癌基因PTEN和p15INK4B因去甲基化而被激活,激活的PTEN可以进一步加强CDA-Ⅱ对PI3K/Akt信号通路的抑制作用而诱导细胞凋亡;p15INK4B可以调控CDA-Ⅱ对CDK4的抑制作用而诱导细胞G1期阻滞。
第三部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞端粒酶活性及人端粒酶逆转录酶(hTERT)表达的影响及机制
目的:研究CDA-Ⅱ对MDS细胞端粒酶活性以及人端粒酶逆转录酶(hTERT)表达的影响,并探讨WT1基因和PI3K/Akt/NF-κB信号通路在CDA-Ⅱ影响MUTZ-1细胞hTERT表达中的作用,以期为临床上MDS的靶向治疗提供一个新思路。
方法:用TRAP法检测不同浓度和不同时间CDA-Ⅱ作用对MUTZ-1细胞端粒酶活性的影响:用荧光实时定量RT-PCR方法和Western-blot方法分别检测CDA-Ⅱ作用对MUTZ-1细胞hTERT mRNA和蛋白表达的影响:用RT-PCR方法检测CDA-Ⅱ作用对MUTZ-1细胞WT1基因表达的影响:用Western-blot方法检测CDA-Ⅱ与PI3K抑制剂LY294002和NF-κB抑制剂PDTC联用对MUTZ-1细胞hTERT蛋白表达的影响,以明确在CDA-Ⅱ诱导MUTZ-1细胞凋亡过程中PI3K/Akt/NF-κB信号通路对hTERT表达的调控作用。
结果:①阐明了CDA-Ⅱ对MDS细胞端粒酶和人端粒酶逆转录酶(hTERT)具有抑制作用,具有首创性。②阐明了CDA-Ⅱ可以通过抑制MUTZ-1细胞端粒酶活性来诱导细胞凋亡,并呈时间和剂量依赖性,这很可能是CDA-Ⅱ诱导MUTZ-1细胞凋亡的Caspase-3非依赖途径。③阐明了CDA-Ⅱ可以抑制MUTZ-1细胞hTERT mRNA和蛋白表达,并呈浓度和时间依赖性,这种抑制作用与CDA-Ⅱ对端粒酶的作用相一致,并与凋亡相关。因此,hTERT是CDA-Ⅱ治疗MDS的新的靶点。④CDA-Ⅱ可以下调MUTZ-1细胞WT1基因的表达,并呈浓度和时间依赖性,由于WT1基因是hTERT启动子的调控基因,因此我们的结果提示CDA-Ⅱ可以通过下调WT1基因表达来抑制hTERT表达从而抑制端粒酶活性。⑤CDA-Ⅱ主要通过抑制PI3K/Akt/NF-κB信号通路来抑制MUTZ-1细胞hTERT蛋白表达,而导致细胞端粒酶活性丧失并诱导凋亡。
小结:首次阐明了CDA-Ⅱ对肿瘤细胞端粒酶和人端粒酶逆转录酶(hTERT)具有抑制作用,为CDA-Ⅱ的临床应用提供了一个新的靶点,国内外均未见报道,具有首创性;CDA-Ⅱ可以通过抑制PI3K/Akt/NF-κB信号通路和WT1基因的表达来下调MUTZ-1细胞hTERT基因表达,进而抑制细胞端粒酶活性,并诱导Caspase-3非依赖型细胞凋亡。
结论:CDA-Ⅱ在细胞水平和动物水平均能抑制MDS细胞生长,其机理主要为诱导细胞凋亡;CDA-Ⅱ对正常人骨髓没有抑制作用,对实验动物也无明显毒副作用:CDA-Ⅱ是一个多靶向性药物,通过多种成分的共同作用来诱导MDS细胞凋亡,主要通过直接影响PI3K/Akt/NF-κB生存信号通路、通过去申基化作用激活抑癌基因PTEN和p15INK4B以及通过影响hTERT的表达来抑制细胞端粒酶活性三种机制发挥作用(见图3-4)。本研究为MDS治疗药物的选择及探讨药物作用机制提供了体外细胞水平和体内动物水平的实验基础,为临床上MDS的靶向治疗提供了新的思路和方法。
Myelodysplastic syndrome(MDS)is a heterogeneous group of clonal bone marrow disorders characterized by varying degrees of cytopenias with ineffective hematopoiesis.It is now still considered to be uncurable disease.The WHO instituted the IPSS for MDS according to the percentage of the blast cells,the karyotype of chromosome and the extent of cytopenias.The MDS patients are devided into three groups according to the IPSS,including low-risk group(score 0),middle-risk group (score 0.5-2.0)and high-risk group(score more than 2.5).The blast cells of the middle-risk and high-risk patients are hyperplasia,with severe cytopenias.Because the bone marrow compensation is poor in these patients,it is very difficult to treat them.Most patients need frequently for blood transfusion.Over time,there is worsening bone marrow fail or progression to acute myeloid leukemia(AML).The advanced age of the majority of MDS patients limits the therapeutic strategies often to supportive care.The patients can' t suffer the standard chemotherapy and bone marrow transplantation.A number of cytidine analogs such as 5-azacytidine(Aza C) and 5-Aza-2' -deoxycitidine(decitabine,DAC)have been approved by the FDA in the United States for the treatment of MDS.The drugs have intensive bone marrow suppression,which limits their clinical use.So there is a tremendous need for novel approaches which have better effect and no bone marrow toxicity in the management of middle-risk and high-risk MDS patients.
Uroacitides,another name CDA-Ⅱ(cell differentiation agentⅡ),is a mixture product and isolated from healthy human urine in China.It is characterized as a novel molecular targeting agent for cancer therapy.The human urine is acidified during collection and pass through an ultrafiltration process to remove molecules with molecular weight larger than 10,000 daltons.Multiple active components,such as peptides(MW 400-2800),organic acids,pigments and phenylacetylglutamine,with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-Ⅱ.Because CDA-Ⅱis healthy human urine extracts,it has no medullary and extramedullary toxicities,which makes it a suitable candidate for the treatment of MDS patients.However,there has been no report that clearly describes the mechanisms of CDA-Ⅱin MDS cells.In the present study,we evaluate both in-vitro and in-vivo anticancer activities and the mechanisms of action of CDA-Ⅱon MDS model, which might provide the experimental therapeutic data to expand its indications in clinical trials.There are three sections in our research.
Section 1:Uroacitides(CDA-Ⅱ)induces MDS cells into apoptosis via cell survival signaling pathway.
Objective:The aim of this study was to investigate the antitumor activity of CDA-Ⅱagainst MDS cells in vitro and in vivo and to determine if CDA-Ⅱ-induced apoptosis of MDS cells was associated with PI3K/Akt and NF-κB survival signaling pathways.
Methods:We used MTT assay to investigate the effects of CDA-Ⅱon the MDS,AML and lymphoma cell lines and normal bone marrow mononuclear cells(BMMCs)growth. MDS-RAEB cell line MUTZ-1 was used to examine the effects of CDA-Ⅱon the induction of growth arrest and apoptosis.Apoptosis was measured by flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer' s instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,8,3,Bcl-2 family,IAPs family and FLIP were studied by western-blot. Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NFκB signaling pathways.TNF-αwas used to determine the inhibition of CDA-Ⅱon the translocation of NF-κB(p65).The PIK3CA gene,which expressed the PI3Kp110 a protein,was examined by using RT-PCR to identify the target of CDA-Ⅱ- induced apoptosis.MUTZ-1 cell xenografted SCID mice were used for in-vivo study.
Results:①CDA-Ⅱcould inhibit growth of MUTZ-1 cells and human leukemia cell lines.The sensitivity of malignant lymphoid cell lines to CDA-Ⅱwas lower than those of MUTZ-1 cells and myeloid leukemia cells.MUTZ-1 cell line was most sensitive to CDA-Ⅱand cell growth was inhibited in a time- and dose-dependent manner, corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from three normal volunteers(p＜0.01).②CDA-Ⅱinduced apoptosis of MUTZ-1 cells in a dose-dependent manner.③CDA-Ⅱinduced G1 arrest, accompanied with a concomitant reduction of Cyclin D1 and CDK4 expression in a dose-dependent manner.④Treatment with CDA-Ⅱinduced marked activation of caspase-9,3 in a dose-dependent manner,however,caspase-8 was activated only at CDA-Ⅱconcentrations greater than 4 mg/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.⑤Bcl-2,Mcl-1 and p-Bad proteins were decreased,however,Bax and Smac proteins were increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h;Bid protein was cleavaged only at CDA-Ⅱconcentrations greater than 4 mg/mL.⑥The XIAP,CIAP1 and Survivine proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment,and not of CIAP2 protein.⑦MUTZ-1 cell line was high expression of FLIP_L and it was inhibited at CDA-Ⅱconcentrations greater than 4mg/mL.⑧CDA-Ⅱinhibited both intrinsic and TNF-α-induced IκBαphosphorylation and prevented NFκB (p65)nuclear translocation in a dose-dependent manner;CDA-Ⅱcombined with PDTC significantly inhibited the expression of XIAP,CIAP1,Survivine,FLIP_L,BCl-2 as well as Cyclin D1 proteins.⑨CDA-2 significantly reduced expression of PI3Kp110αas well as p-Akt in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time-and dose-dependent manner;CDA-Ⅱcombined with LY294002 significantly inhibited the expression of p-IκBα,p-Bad,Mcl-1 proteins and up-regulated the expression of Bax protein as well as activated caspase-9.⑩CDA-Ⅱinhibited tumor growth in MUTZ-1 xenografted SCID mice in a dose-dependent manner and improved the survival time of the animal.There was no significant body weight change in SCID mice during the experiment.
Conclusions:CDA-Ⅱcould induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.The main mechanisms were related to the inhibition of PI3Kp110αexpression at transcriptional level,which inactivated the phosphorylation of Akt involving prevention NF-κB nuclear translocation,downregulation of IAP family and FLIP_L protein as well as involvement of Bcl-2 family and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis.
Section 2:The effects of Uroacitides(CDA-Ⅱ)on the abnormal hypermethylation patterns of PTEN and p151NK4B genes in MDS cells.
Objective:The aim of this study was to investigate the methylation patterns of PTEN gene in MDS and MDS transformed AML in order to determine the relationship between the PTEN gene and the development of MDS.Another purpose of this study was to identify the effects of CDA-Ⅱon the abnormal high expressions of DNMT1,3A and 3B as well as on the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MDS cells.
Methods:We used western-blot to investigate the expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B proteins on the MUTZ-1 cell line,low-risk MDS, high-risk MDS and MDS transformed AML cells as well as normal BMMCs.RT-PCR and western-blot were used to determine the effects of CDA-Ⅱon the mRNA and protein expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B.The methylation patterns of PTEN and p15INK4B genes in MUTZ-1 and AML cell lines,low-risk MDS,high-risk MDS and MDS transformed AML cells as well as normal BMMCs were examined using methylation specific PCR(MSP).MSP was also used to identify the effects of CDA-Ⅱon the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MUTZ-1 cells. The demethylating agent decitabine was used as positive control.
Results:①The high expressions of DNMT1,3A and 3B proteins were found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells;The expression level of DNMT3B was higher than that of DNMT1 and 3A.The low-risk MDS and nomal BMMCs expressed DNMT1,3A and 3B proteins only at low levels.②Treatment with CDA-Ⅱinduced marked inhibition of DNMT1,3A and 3B in a time- and dose-dependent manner. Among them,DNMT1 was the most effective in CDA-Ⅱmanagement.③The absence of PTEN gene expression in protein level was found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells,however,the low-risk MDS and normal BMMCs cells all expressed the PTEN protein.④We used MSP to determine if the absence of PTEN gene expression was due to the abnormal hypermethylation pattern of it.We found that MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN completely methylation,the AML cell line Kasumi-1 was PTEN partly methylation,however,the KG1 and K562 cell lines,low-risk MDS and normal BMMCs were PTEN completely unmethylation,which indicated that the tumor suppressor gene PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy.⑤CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of PTEN gene in a time- and dose-dependent manner,resulting in the upregulation of PTEN mRNA and protein expressions and triggering inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.⑥CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of p15INK4B gene in a timeand dose-dependent manner,resulting in the upregulation of p15INK4B mRNA and protein expressions and triggering inhibition of CDK4 expression,which induced MUTZ-1 cells G1 arrest.
Conclusions:That MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN hypermethylation,however,low-risk MDS and normal BMMCs were PTEN completely unmethylation indicated that PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy. CDA-Ⅱcould downreglate the abnormal hypermethylation patterns of PTEN and p15INK4B genes in a time- and dose-dependent manner,resulting in the upregulation of PTEN and p15INK4B expressions.The high expression of PTEN triggered inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.The high expression of p15INK4B triggered inhibition of CDK4 protein,which induced MUTZ-1 cells G1 arrest.
Section 3:The study of Uroacitides(CDA-Ⅱ)on the telomerase activity and hTERT expression in MDS cells and its mechanism.
Objective:The aim of this study was to investigate the effects of CDA-Ⅱon the telomerase activity and hTERT expression in MDS cells and its mechanism.We wanted to identify if the WT1 gene and PI3K/Akt/NF-κB survival signaling pathway were involved in the effect of CDA-Ⅱon the expression of hTERT in MDS cells.
Methods:TRAP method was used to investigate the effect of CDA-Ⅱon the telomerase activity in MDS cells.We used real-time RT-PCR and western-blot to determine the effects of CDA-Ⅱon the mRNA and protein expressions of hTERT.RT-PCR was used to study the effect of CDA-Ⅱon the expression of WT1 gene on transcriptional level.The PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC were used to identify the involvement of PI3K/Akt/NF-κB survival signaling pathway in the effect of CDA-Ⅱon the expression of hTERT in MUTZ-1 cells.
Results:①CDA-Ⅱcould significantly inhibited the telomerase activity in a time- and dose-dependent manner,which was accordance with the CDA-Ⅱ-induced apoptosis.It suggested that the inhibition of telomerase activity might be the caspase-3 independent mechanism involved in CDA-Ⅱ-induced apoptosis.②CDA-Ⅱ exerted substantial inhibition of hTERT on transcriptional and protein levels in a time- and dose-dependent manner,which was concordance with that of telomerase activity in MDS cells.Therefore hTERT might be a new target for CDA-Ⅱtreatment.③Treatment with CDA-Ⅱinduced marked inhibition of WT1 gene expression in a timeand dose-dependent manner,suggesting that CDA-Ⅱcould inhibit hTERT through downregulation of WT1 gene expression.④CDA-Ⅱcombined with LY294002 and PDTC could significantly inhibit the expression of hTERT protein,which indicated that CDA-Ⅱmight inhibit hTERT expression and triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway in MDS cells.
Conclusions:CDA-Ⅱcould significantly inhibit the telomerase activity as well as the expressions of hTERT on transcriptional and protein levels,which might be a new target for CDA-Ⅱtreatment.CDA-Ⅱcould inhibit hTERT expression and telomerase activity as well as triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway and inhibition of WT1 gene in MDS cells.
Summary:CDA-Ⅱcan induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.CDA-Ⅱdoes not induce cytotoxicity in normal BMMCs,nor does it show any toxicity on the SCID mice.CDA-Ⅱis an effective medicine with multiple targets, including involvement of PI3K/Akt/NF-κB survival signaling pathway directly, downreglation of the abnormal hypermethylation patterns of PTEN and p15INK4B genes and inhibition of hTERT expression and telomerase activity.Different mechanisms of action acted concurrently to contribute to the CDA-Ⅱ-induced MDS cell apoptosis (See Figure 3-4).This research work may provide new insights for the treatment of high-risk MDS.
本研究分三部分进行:
第一部分:尿多酸肽(CDA-Ⅱ)通过影响细胞生存信号通路诱导骨髓增生异常综合征细胞凋亡
第二部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞PTEN和p15INK4B基因异常甲基化模式的影响
第三部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞端粒酶活性及人端粒酶逆转录酶(hTERT)表达的影响及机制
第一部分:尿多酸肽(CDA-Ⅱ)通过影响细胞生存信号通路诱导骨髓增生异常综合征细胞凋亡
目的:探讨CDA-Ⅱ对MDS细胞的作用和作用机制以及对MDS细胞PI3K/Akt及NF-κB生存信号途径的影响,同时研究CDA-Ⅱ对人MDS荷瘤小鼠瘤体生长的抑制作用,以期从细胞水平和实验动物水平明确CDA-Ⅱ对MDS的作用及作用机制,为高危MDS的靶向治疗提供实验基础。
方法:采用MTT比色法研究CDA-Ⅱ对MDS,白血病和淋巴瘤细胞株以及MDS原代细胞和正常人骨髓单个核细胞的体外生长抑制作用:采用Wright-Giemsa染色、流式细胞仪PI染色检测亚G1峰和Annexin V/PI双染色检测细胞凋亡;采用流式细胞仪PI染色检测细胞周期、JC-1染色检测细胞线粒体膜电位的改变:用Western-blot方法检测CDA-Ⅱ作用后MUTZ-1细胞Caspase家族蛋白,细胞周期蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族和FLIP蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;提取核浆蛋白以观察CDA-Ⅱ作用对NF-κB信号通路的影响,并进一步加用TNF-α来证实CDA-Ⅱ对NF-κB核转位的影响;Western-blot方法检测CDA-Ⅱ对MUTZ-1细胞PI3K/Akt和NF-κB信号通路相关生存蛋白的影响,并进一步加用PI3K抑制剂LY294002和NF-κB抑制剂PDTC来明确两条信号通路的相关性及机制:用RT-PCR方法检测CDA-Ⅱ对表达PI3Kp110α蛋白的PIK3CA基因的影响,以明确CDA-Ⅱ治疗的靶点:用人MDS荷瘤小鼠模型观察CDA-Ⅱ对小鼠瘤体生长和平均生存期的影响以及比较用药前后瘤组织细胞形态和p-Akt的原位表达,从而明确CDA-Ⅱ在实验动物水平对MDS细胞的生长抑制作用,并评估实验治疗剂量的安全性。
结果:①CDA-Ⅱ体外可以抑制MDS,白血病和淋巴瘤细胞株生长,其中髓系白血病细胞株较淋系血液肿瘤细胞株更敏感,最敏感的为MUTZ-1细胞株:CDA-Ⅱ体外可以抑制MDS细胞株MUTZ-1及MDS原代细胞的生长,并呈浓度和时间依赖性,但不影响正常人骨髓单个核细胞的生长。②CDA-Ⅱ体外可以诱导MUTZ-1细胞株和MDS原代细胞凋亡,并呈浓度依赖性。③CDA-Ⅱ在体外可以诱导MUTZ-1细胞株出现G1期细胞周期阻滞,并下调细胞周期相关蛋白Cyclin D1和CDK4表达,呈剂量依赖性。④不同浓度CDA-Ⅱ作用可以激活Caspase-9,3,呈浓度依赖性,并伴有明显的线粒体膜电位下降:当CDA-Ⅱ浓度超过4mg/mL时激活Caspase-8,呈浓度依赖性:Caspase-3抑制剂可以抑制CDA-Ⅱ诱导的MUTZ-1细胞凋亡。⑤不同浓度CDA-Ⅱ作用可以下调Bcl-2家族抗凋亡蛋白Bcl-2,Mcl-1,p-Bad和上调促凋亡蛋白Bax的表达,呈浓度依赖性;CDA-Ⅱ在浓度超过4mg/mL时可以激活Bid,并呈浓度依赖性。⑥CDA-Ⅱ可以抑制MUTZ-1细胞IAPs家族中的CIAP1,XIAP和Survivin的表达,呈浓度依赖性,但不影响CLAP2的表达。⑦MUTZ-1细胞高表达FLIP_L:且CDA-Ⅱ在浓度超过4mg/mL时,可以明显抑制FLIP_L表达,呈浓度依赖性。⑧不同浓度CDA-Ⅱ作用可以抑制IκBα磷酸化和NF-κB核转位,也可以抑制TNF-α诱导的IκBα磷酸化和NF-κB核转位;CDA-Ⅱ与NF-κB抑制剂PDTC联用可以明显下调NF-κB信号通路下游信号分子IAPs家族,FLIP_L,Bcl-2,Cyclin D1的表达。⑨不同浓度和不同时间CDA-Ⅱ作用可以下调PIK3CA mRNA表达:PI3K抑制剂LY294002与CDA-Ⅱ联用可以明显抑制PI3K/Akt信号通路,并进一步影响其下游的信号蛋白,包括NF-κB信号通路及其下游的信号分子,p-Bad,Bax,Mcl-1和Caspase-9而诱导MUTZ-1细胞凋亡。⑩CDA-Ⅱ可以明显抑制人MDS荷瘤小鼠瘤体生长并延长小鼠平均生存期:CDA-Ⅱ的实验治疗剂量对小鼠体重无明显影响,且未发现有不明原因死亡的小鼠;HE染色和免疫组化检测显示CDA-Ⅱ在实验治疗剂量下可以明显抑制人MDS荷瘤小鼠瘤组织细胞的生长和p-Akt的原位表达。
小结:①CDA-Ⅱ体外可以抑制MDS,白血病和淋巴瘤细胞株生长,其中髓系白血病细胞株较淋系血液肿瘤细胞株更敏感,最敏感的为MUTZ-1细胞株:CDA-Ⅱ体外可以抑制MDS原代细胞的生长,但不影响正常人骨髓单个核细胞的生长,对正常骨髓无抑制作用。②CDA-Ⅱ体外可以诱导MUTZ-1细胞株和MDS原代细胞凋亡,并伴有G1期细胞周期阻滞,和细胞周期相关蛋白Cyclin D1和CDK4表达抑制。③CDA-Ⅱ通过启动内源性和外源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并以内源性(线粒体)途径为主,伴有明显的线粒体膜电位下降。④CDA-Ⅱ通过影响Bcl-2家族蛋白Bel-2,Mel-1,p-Bad,Bax和Bid的表达以及抑制IAPs家族中CIAP1,XIAP和Survivin的表达导致线粒体损伤和Caspase-9,3激活诱导细胞凋亡。⑤MUTZ-1细胞高表达FLIP_L;且CDA-Ⅱ在浓度超过4mg/mL时,可以明显抑制FLIP_L表达,从而解除对Caspase-8的抑制作用诱导凋亡。⑥CDA-Ⅱ可以通过抑制IκBα磷酸化,从而抑制NF-κB核转位来下调MUTZ-1细胞内源性及TNF-α诱导的NF-κB信号通路的活性;再通过作用于NF-κB信号通路下游的信号分子,包括IAPs家族,FLIP_L,Bcl-2,Cycl in D1导致MUTZ-1细胞凋亡和细胞周期阻滞:CDA-Ⅱ对MUTZ-1细胞NF-κB信号通路的影响主要通过对PI3K/Akt信号通路的调控来实现。⑦CDA-Ⅱ主要通过影响PIK3CA基因的表达而抑制PI3K/Akt信号通路,并进一步通过影响其下游的信号蛋白,包括NF-κB信号通路及其下游的信号分子,p-Bad,Bax,Mcl-1和Caspase-9而诱导MUTZ-1细胞凋亡。PIK3CA基因为CDA-Ⅱ作用的靶点。⑧CDA-Ⅱ在动物实验中可以明显抑制人MDS荷瘤小鼠瘤体生长并延长小鼠平均生存期;CDA-Ⅱ的实验治疗剂量对小鼠无明显毒副作用;CDA-Ⅱ在实验治疗剂量下可以明显抑制人MDS荷瘤小鼠瘤组织细胞的生长和p-Akt的原位表达。
第二部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞PTEN和p15INK4B基因异常甲基化模式的影响
目的:检测抑癌基因PTEN在MDS和MDS转化的AML中的表达和甲基化状态,从而明确PTEN基因与MDS发病和MDS转化为AML的相关性:探讨CDA-Ⅱ对MDS细胞中异常高表达的甲基转移酶DNMT1、3A和3B的作用以及对抑癌基因PTEN和p15INK4B的作用,并以去甲基化药物Decitabine作为阳性对照,为MDS的靶向治疗提供新的方法。
方法:用Western-blot方法检测MUTZ-1细胞株、低危MDS、高危MDS、MDS转化的AML患者和正常人骨髓单个核细胞中甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p15INK4B蛋白表达情况:用Western-blot方法检测CDA-Ⅱ作用对MUTZ-1细胞甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p15INK4B蛋白表达的影响:用RT-PCR方法检测CDA-Ⅱ作用对MUTZ-1细胞甲基转移酶DNMT1、3A和3B以及抑癌基因PTEN和p151NK4B mRNA表达的影响,并以去甲基化药物Decitabine作为阳性对照:用甲基化PCR(MSP)方法检测MUTZ-1和AML细胞株、低危MDS、高危MDS、MDS转化的AML患者以及正常人骨髓单个核细胞中PTEN和p15INK4B的甲基化状态:用MSP方法检测CDA-Ⅱ作用对MUTZ-1细胞PTEN和p15INK4B甲基化状态的影响,同时与去甲基化药物Decitabine作对照,以评价CDA-Ⅱ的有效性。
结果:①MUTZ-1细胞株、高危WDS和MDS转化的AML患者高表达甲基转移酶DNMT1、3A和3B,并以DNMT3B为明显;低危组MDS和正常人弱表达DNMT1、3A和3B蛋白。②CDA-Ⅱ可以明显下调MUTZ-1细胞甲基转移酶DNMT1、3A和3B表达,呈时间和剂量依赖性,以对DNMT1的作用最明显。③发现了抑癌基因PTEN在MUTZ-1细胞株、高危MDS和MDS转化的AML患者细胞中均表达缺失,而在低危MDS和正常人细胞中均有表达。④用MSP方法检测并发现了MUTZ-1细胞株和高危MDS以及MDS转化的AML患者PTEN基因完全甲基化:AML细胞株Kasumi-1PTEN基因部分甲基化:AML细胞株KG1,CML细胞株K562,低危组MDS及正常人PTEN基因完全非甲基化,提示PTEN基因在高危MDS和MDS转化的AML中存在高度甲基化,很可能是MDS向AML转变的一个重要的发病机制,可以作为CDA-Ⅱ去甲基化治疗的靶基因。国内外均未见报道。⑤发现了CDA-Ⅱ可以通过去甲基化作用使PTEN启动子区CpG岛甲基化水平降低,导致PTEN mRNA水平和蛋白表达水平均上调,从而发挥对PI3K/Akt信号通路的抑制作用进而诱导MUTZ-1细胞凋亡。国内外均未见报道。⑥发现了CDA-Ⅱ可以通过去甲基化作用使p15INK4B甲基化水平降低,导致p15INK4B mRNA水平和蛋白表达水平均上调,从而发挥对CDK4的抑制作用进而造成MUTZ-1细胞G1期阻滞。
小结:PTEN基因在高危MDS和MDS转化的AML中存在高度甲基化,很可能是MDS向AML转变的一个重要的发病机制,靶向PTEN基因的去甲基化治疗可以作为高危MDS和MDS转化的AML患者的治疗策略:CDA-Ⅱ可以通过抑制甲基转移酶DNMT1、3A和3B导致抑癌基因PTEN和p15INK4B因去甲基化而被激活,激活的PTEN可以进一步加强CDA-Ⅱ对PI3K/Akt信号通路的抑制作用而诱导细胞凋亡;p15INK4B可以调控CDA-Ⅱ对CDK4的抑制作用而诱导细胞G1期阻滞。
第三部分:尿多酸肽(CDA-Ⅱ)对骨髓增生异常综合征细胞端粒酶活性及人端粒酶逆转录酶(hTERT)表达的影响及机制
目的:研究CDA-Ⅱ对MDS细胞端粒酶活性以及人端粒酶逆转录酶(hTERT)表达的影响,并探讨WT1基因和PI3K/Akt/NF-κB信号通路在CDA-Ⅱ影响MUTZ-1细胞hTERT表达中的作用,以期为临床上MDS的靶向治疗提供一个新思路。
方法:用TRAP法检测不同浓度和不同时间CDA-Ⅱ作用对MUTZ-1细胞端粒酶活性的影响:用荧光实时定量RT-PCR方法和Western-blot方法分别检测CDA-Ⅱ作用对MUTZ-1细胞hTERT mRNA和蛋白表达的影响:用RT-PCR方法检测CDA-Ⅱ作用对MUTZ-1细胞WT1基因表达的影响:用Western-blot方法检测CDA-Ⅱ与PI3K抑制剂LY294002和NF-κB抑制剂PDTC联用对MUTZ-1细胞hTERT蛋白表达的影响,以明确在CDA-Ⅱ诱导MUTZ-1细胞凋亡过程中PI3K/Akt/NF-κB信号通路对hTERT表达的调控作用。
结果:①阐明了CDA-Ⅱ对MDS细胞端粒酶和人端粒酶逆转录酶(hTERT)具有抑制作用,具有首创性。②阐明了CDA-Ⅱ可以通过抑制MUTZ-1细胞端粒酶活性来诱导细胞凋亡,并呈时间和剂量依赖性,这很可能是CDA-Ⅱ诱导MUTZ-1细胞凋亡的Caspase-3非依赖途径。③阐明了CDA-Ⅱ可以抑制MUTZ-1细胞hTERT mRNA和蛋白表达,并呈浓度和时间依赖性,这种抑制作用与CDA-Ⅱ对端粒酶的作用相一致,并与凋亡相关。因此,hTERT是CDA-Ⅱ治疗MDS的新的靶点。④CDA-Ⅱ可以下调MUTZ-1细胞WT1基因的表达,并呈浓度和时间依赖性,由于WT1基因是hTERT启动子的调控基因,因此我们的结果提示CDA-Ⅱ可以通过下调WT1基因表达来抑制hTERT表达从而抑制端粒酶活性。⑤CDA-Ⅱ主要通过抑制PI3K/Akt/NF-κB信号通路来抑制MUTZ-1细胞hTERT蛋白表达,而导致细胞端粒酶活性丧失并诱导凋亡。
小结:首次阐明了CDA-Ⅱ对肿瘤细胞端粒酶和人端粒酶逆转录酶(hTERT)具有抑制作用,为CDA-Ⅱ的临床应用提供了一个新的靶点,国内外均未见报道,具有首创性;CDA-Ⅱ可以通过抑制PI3K/Akt/NF-κB信号通路和WT1基因的表达来下调MUTZ-1细胞hTERT基因表达,进而抑制细胞端粒酶活性,并诱导Caspase-3非依赖型细胞凋亡。
结论:CDA-Ⅱ在细胞水平和动物水平均能抑制MDS细胞生长,其机理主要为诱导细胞凋亡;CDA-Ⅱ对正常人骨髓没有抑制作用,对实验动物也无明显毒副作用:CDA-Ⅱ是一个多靶向性药物,通过多种成分的共同作用来诱导MDS细胞凋亡,主要通过直接影响PI3K/Akt/NF-κB生存信号通路、通过去申基化作用激活抑癌基因PTEN和p15INK4B以及通过影响hTERT的表达来抑制细胞端粒酶活性三种机制发挥作用(见图3-4)。本研究为MDS治疗药物的选择及探讨药物作用机制提供了体外细胞水平和体内动物水平的实验基础,为临床上MDS的靶向治疗提供了新的思路和方法。
Myelodysplastic syndrome(MDS)is a heterogeneous group of clonal bone marrow disorders characterized by varying degrees of cytopenias with ineffective hematopoiesis.It is now still considered to be uncurable disease.The WHO instituted the IPSS for MDS according to the percentage of the blast cells,the karyotype of chromosome and the extent of cytopenias.The MDS patients are devided into three groups according to the IPSS,including low-risk group(score 0),middle-risk group (score 0.5-2.0)and high-risk group(score more than 2.5).The blast cells of the middle-risk and high-risk patients are hyperplasia,with severe cytopenias.Because the bone marrow compensation is poor in these patients,it is very difficult to treat them.Most patients need frequently for blood transfusion.Over time,there is worsening bone marrow fail or progression to acute myeloid leukemia(AML).The advanced age of the majority of MDS patients limits the therapeutic strategies often to supportive care.The patients can' t suffer the standard chemotherapy and bone marrow transplantation.A number of cytidine analogs such as 5-azacytidine(Aza C) and 5-Aza-2' -deoxycitidine(decitabine,DAC)have been approved by the FDA in the United States for the treatment of MDS.The drugs have intensive bone marrow suppression,which limits their clinical use.So there is a tremendous need for novel approaches which have better effect and no bone marrow toxicity in the management of middle-risk and high-risk MDS patients.
Uroacitides,another name CDA-Ⅱ(cell differentiation agentⅡ),is a mixture product and isolated from healthy human urine in China.It is characterized as a novel molecular targeting agent for cancer therapy.The human urine is acidified during collection and pass through an ultrafiltration process to remove molecules with molecular weight larger than 10,000 daltons.Multiple active components,such as peptides(MW 400-2800),organic acids,pigments and phenylacetylglutamine,with different mechanisms of action act concurrently to contribute to the anticancer effect of CDA-Ⅱ.Because CDA-Ⅱis healthy human urine extracts,it has no medullary and extramedullary toxicities,which makes it a suitable candidate for the treatment of MDS patients.However,there has been no report that clearly describes the mechanisms of CDA-Ⅱin MDS cells.In the present study,we evaluate both in-vitro and in-vivo anticancer activities and the mechanisms of action of CDA-Ⅱon MDS model, which might provide the experimental therapeutic data to expand its indications in clinical trials.There are three sections in our research.
Section 1:Uroacitides(CDA-Ⅱ)induces MDS cells into apoptosis via cell survival signaling pathway.
Objective:The aim of this study was to investigate the antitumor activity of CDA-Ⅱagainst MDS cells in vitro and in vivo and to determine if CDA-Ⅱ-induced apoptosis of MDS cells was associated with PI3K/Akt and NF-κB survival signaling pathways.
Methods:We used MTT assay to investigate the effects of CDA-Ⅱon the MDS,AML and lymphoma cell lines and normal bone marrow mononuclear cells(BMMCs)growth. MDS-RAEB cell line MUTZ-1 was used to examine the effects of CDA-Ⅱon the induction of growth arrest and apoptosis.Apoptosis was measured by flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer' s instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,8,3,Bcl-2 family,IAPs family and FLIP were studied by western-blot. Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NFκB signaling pathways.TNF-αwas used to determine the inhibition of CDA-Ⅱon the translocation of NF-κB(p65).The PIK3CA gene,which expressed the PI3Kp110 a protein,was examined by using RT-PCR to identify the target of CDA-Ⅱ- induced apoptosis.MUTZ-1 cell xenografted SCID mice were used for in-vivo study.
Results:①CDA-Ⅱcould inhibit growth of MUTZ-1 cells and human leukemia cell lines.The sensitivity of malignant lymphoid cell lines to CDA-Ⅱwas lower than those of MUTZ-1 cells and myeloid leukemia cells.MUTZ-1 cell line was most sensitive to CDA-Ⅱand cell growth was inhibited in a time- and dose-dependent manner, corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from three normal volunteers(p＜0.01).②CDA-Ⅱinduced apoptosis of MUTZ-1 cells in a dose-dependent manner.③CDA-Ⅱinduced G1 arrest, accompanied with a concomitant reduction of Cyclin D1 and CDK4 expression in a dose-dependent manner.④Treatment with CDA-Ⅱinduced marked activation of caspase-9,3 in a dose-dependent manner,however,caspase-8 was activated only at CDA-Ⅱconcentrations greater than 4 mg/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.⑤Bcl-2,Mcl-1 and p-Bad proteins were decreased,however,Bax and Smac proteins were increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h;Bid protein was cleavaged only at CDA-Ⅱconcentrations greater than 4 mg/mL.⑥The XIAP,CIAP1 and Survivine proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment,and not of CIAP2 protein.⑦MUTZ-1 cell line was high expression of FLIP_L and it was inhibited at CDA-Ⅱconcentrations greater than 4mg/mL.⑧CDA-Ⅱinhibited both intrinsic and TNF-α-induced IκBαphosphorylation and prevented NFκB (p65)nuclear translocation in a dose-dependent manner;CDA-Ⅱcombined with PDTC significantly inhibited the expression of XIAP,CIAP1,Survivine,FLIP_L,BCl-2 as well as Cyclin D1 proteins.⑨CDA-2 significantly reduced expression of PI3Kp110αas well as p-Akt in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time-and dose-dependent manner;CDA-Ⅱcombined with LY294002 significantly inhibited the expression of p-IκBα,p-Bad,Mcl-1 proteins and up-regulated the expression of Bax protein as well as activated caspase-9.⑩CDA-Ⅱinhibited tumor growth in MUTZ-1 xenografted SCID mice in a dose-dependent manner and improved the survival time of the animal.There was no significant body weight change in SCID mice during the experiment.
Conclusions:CDA-Ⅱcould induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.The main mechanisms were related to the inhibition of PI3Kp110αexpression at transcriptional level,which inactivated the phosphorylation of Akt involving prevention NF-κB nuclear translocation,downregulation of IAP family and FLIP_L protein as well as involvement of Bcl-2 family and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis.
Section 2:The effects of Uroacitides(CDA-Ⅱ)on the abnormal hypermethylation patterns of PTEN and p151NK4B genes in MDS cells.
Objective:The aim of this study was to investigate the methylation patterns of PTEN gene in MDS and MDS transformed AML in order to determine the relationship between the PTEN gene and the development of MDS.Another purpose of this study was to identify the effects of CDA-Ⅱon the abnormal high expressions of DNMT1,3A and 3B as well as on the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MDS cells.
Methods:We used western-blot to investigate the expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B proteins on the MUTZ-1 cell line,low-risk MDS, high-risk MDS and MDS transformed AML cells as well as normal BMMCs.RT-PCR and western-blot were used to determine the effects of CDA-Ⅱon the mRNA and protein expressions of DNMT1,3A and 3B as well as PTEN and p15INK4B.The methylation patterns of PTEN and p15INK4B genes in MUTZ-1 and AML cell lines,low-risk MDS,high-risk MDS and MDS transformed AML cells as well as normal BMMCs were examined using methylation specific PCR(MSP).MSP was also used to identify the effects of CDA-Ⅱon the abnormal hypermethylation patterns of PTEN and p15INK4B genes in MUTZ-1 cells. The demethylating agent decitabine was used as positive control.
Results:①The high expressions of DNMT1,3A and 3B proteins were found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells;The expression level of DNMT3B was higher than that of DNMT1 and 3A.The low-risk MDS and nomal BMMCs expressed DNMT1,3A and 3B proteins only at low levels.②Treatment with CDA-Ⅱinduced marked inhibition of DNMT1,3A and 3B in a time- and dose-dependent manner. Among them,DNMT1 was the most effective in CDA-Ⅱmanagement.③The absence of PTEN gene expression in protein level was found in MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells,however,the low-risk MDS and normal BMMCs cells all expressed the PTEN protein.④We used MSP to determine if the absence of PTEN gene expression was due to the abnormal hypermethylation pattern of it.We found that MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN completely methylation,the AML cell line Kasumi-1 was PTEN partly methylation,however,the KG1 and K562 cell lines,low-risk MDS and normal BMMCs were PTEN completely unmethylation,which indicated that the tumor suppressor gene PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy.⑤CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of PTEN gene in a time- and dose-dependent manner,resulting in the upregulation of PTEN mRNA and protein expressions and triggering inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.⑥CDA-Ⅱcould downreglate the abnormal hypermethylation pattern of p15INK4B gene in a timeand dose-dependent manner,resulting in the upregulation of p15INK4B mRNA and protein expressions and triggering inhibition of CDK4 expression,which induced MUTZ-1 cells G1 arrest.
Conclusions:That MUTZ-1 cell line,high-risk MDS and MDS transformed AML cells were PTEN hypermethylation,however,low-risk MDS and normal BMMCs were PTEN completely unmethylation indicated that PTEN abnormal hypermethylation was associated with the development of MDS and it might be a new target in MDS therapy. CDA-Ⅱcould downreglate the abnormal hypermethylation patterns of PTEN and p15INK4B genes in a time- and dose-dependent manner,resulting in the upregulation of PTEN and p15INK4B expressions.The high expression of PTEN triggered inhibition of PI3K/Akt survival signaling pathway,which induced MUTZ-1 cells apoptosis.The high expression of p15INK4B triggered inhibition of CDK4 protein,which induced MUTZ-1 cells G1 arrest.
Section 3:The study of Uroacitides(CDA-Ⅱ)on the telomerase activity and hTERT expression in MDS cells and its mechanism.
Objective:The aim of this study was to investigate the effects of CDA-Ⅱon the telomerase activity and hTERT expression in MDS cells and its mechanism.We wanted to identify if the WT1 gene and PI3K/Akt/NF-κB survival signaling pathway were involved in the effect of CDA-Ⅱon the expression of hTERT in MDS cells.
Methods:TRAP method was used to investigate the effect of CDA-Ⅱon the telomerase activity in MDS cells.We used real-time RT-PCR and western-blot to determine the effects of CDA-Ⅱon the mRNA and protein expressions of hTERT.RT-PCR was used to study the effect of CDA-Ⅱon the expression of WT1 gene on transcriptional level.The PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC were used to identify the involvement of PI3K/Akt/NF-κB survival signaling pathway in the effect of CDA-Ⅱon the expression of hTERT in MUTZ-1 cells.
Results:①CDA-Ⅱcould significantly inhibited the telomerase activity in a time- and dose-dependent manner,which was accordance with the CDA-Ⅱ-induced apoptosis.It suggested that the inhibition of telomerase activity might be the caspase-3 independent mechanism involved in CDA-Ⅱ-induced apoptosis.②CDA-Ⅱ exerted substantial inhibition of hTERT on transcriptional and protein levels in a time- and dose-dependent manner,which was concordance with that of telomerase activity in MDS cells.Therefore hTERT might be a new target for CDA-Ⅱtreatment.③Treatment with CDA-Ⅱinduced marked inhibition of WT1 gene expression in a timeand dose-dependent manner,suggesting that CDA-Ⅱcould inhibit hTERT through downregulation of WT1 gene expression.④CDA-Ⅱcombined with LY294002 and PDTC could significantly inhibit the expression of hTERT protein,which indicated that CDA-Ⅱmight inhibit hTERT expression and triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway in MDS cells.
Conclusions:CDA-Ⅱcould significantly inhibit the telomerase activity as well as the expressions of hTERT on transcriptional and protein levels,which might be a new target for CDA-Ⅱtreatment.CDA-Ⅱcould inhibit hTERT expression and telomerase activity as well as triggered caspase-3 independent apoptosis through PI3K/Akt/NF-κB survival signaling pathway and inhibition of WT1 gene in MDS cells.
Summary:CDA-Ⅱcan induce growth arrest and apoptosis of MUTZ-1 cells in vitro and in vivo.CDA-Ⅱdoes not induce cytotoxicity in normal BMMCs,nor does it show any toxicity on the SCID mice.CDA-Ⅱis an effective medicine with multiple targets, including involvement of PI3K/Akt/NF-κB survival signaling pathway directly, downreglation of the abnormal hypermethylation patterns of PTEN and p15INK4B genes and inhibition of hTERT expression and telomerase activity.Different mechanisms of action acted concurrently to contribute to the CDA-Ⅱ-induced MDS cell apoptosis (See Figure 3-4).This research work may provide new insights for the treatment of high-risk MDS.
引文
1. Steensma DP, Bennett JM: The myelodysplastic syndromes: diagnosis and treatment. Mayo Clin Proc 2006; 81: 104-130.
2. Greenberg P, Cox C, Le Beau, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89:2079-2088.
3. Jaffe ES, Harris NL, Stein H, et al. World Health Organization classification of tumors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon (France): IARC Press; 2001.
4. Mufti G, List AF, Gore SD, et al. Myelodysplastic syndrome. Hematology Am Soc Hematol Educ Program 2003; 176-199.
5. Look AT. Molecular pathogenesis of MDS. Hematology Am Soc Hematol Educ Program 2005; 156-160.
6. Parker JE, Mufti GJ. The myelodysplastic syndromes: a matter of life or death. Acta Haematol 2004; 111: 78-99.
7. Richard K, Shadduck JM, Latsko JM, et al. Recent advances in myelodysplastic syndromes. Exp Hematol 2007; 35: 137-143.
8. Kaminskas E, Farrell A, Abraham S, et al. FDA Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 2005; 11:3604 - 3608.
9. Saba H, Rosenfeld C, Issa J-P, et al. First report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS). Blood 2004; 104:23a.
10. Burzynski SR. Antineoplastons: history of the research (I). Drugs Exp Clin Res 1986; 12 (suppl 1):1-9.
11. Badria F, Mabed M, Khafagy W, et al. Potential utility of antineoplaston A-10 levels in breast cancer. Cancer Lett 2000; 155: 67-70.
12. Lin WC, Wu YW, Lai TY, et al. Effect of CDA-II, urinary preparation, on lipofuscin, lipid peroxidation and antioxidant systems in young and middle-aged rat brain. Am J Chin Med 2001; 29: 91-99.
13. Lin WC, Liao YC, Liau MC, et al. Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes. Food Chem Toxicol 2006;44:546-551.
14. Steelman LS, Pohnert SC, Shelton JG, et al. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 2004; 18: 189-218.
15. Woodgett JR. Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol 2005; 17: 150-157.
16. Thoompson JE, Thompson CB. Putting the Rap on Akt. J Clin Oncol 2004; 1522: 4217-4226.
17. Zhou HL, Li XM, Meinkoth J, et al. Akt Regulates cell Survival and Apoptosis at a Postmitochondrial Level. J Cell Biol 2000; 151: 483-494.
18. Feng J, Tamaskovie R, Yang Z, et al. Stabilization of Mdm-2 via decreased ubiquitination is mediated by protein kinase B / Akt-dependent phosphorylation . J Biol Chem 2004; 279: 35510-35517.
19. Chang F, Lee JT, Navolanie PM, et al. Involvement of P13-K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003; 17: 590-603.
20. Romashkova JA, Makarov SS. NF-κB is a target of Akt in anti-apoptotic PDGF signaling. Nature 1999; 401: 86-90.
21. Harrey RD, Lonial S. PI3 kinase/Akt pathway as a therapeutic target in multiple myeloma. Future Oncol 2007; 316: 639-647.
22. Takada Y, Kobayashi Y, Aggarwal BB. Evodiamine abolishes constitutive and inducible NF—kappa B activation by inhibiting I kappa B alpha kinase activation, thereby suppressing NF-kappa B—regulated antiapoptotic and metastatic gene expression, upregulating apoptosis, and inhibiting invasion. J Biol Chem 2005; 280: 17203-17212.
23. Micheau O, Lens S, Gaide O, et al. NF-kappaB signals induce the expression of c-FLIP. Mol Cell Biol 2001; 21:5299-5305.
24. Nyakern M, Tazzari PL, Finelli C, et al. Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients. Leukemia 2006; 20:230-238.
25. Braun T, Carvalho G, Coquelle A, et al. NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome. Blood 2006; 107:1156-1165.
26. Sanz C, Richard C, Prosper F, et al. Nuclear factor k B is activated in myelodysplastic bone marrow cells. Haematologica 2002; 87:1005-1006.
27. Esteller M. DNA methylation and cancer therapy: new developments and expectations. Curr Opin Oncol 2005;17: 55-60.
28. Das PM, Singal R. DNA methylation and cancer. J Clin Oncol 2004; 22: 4632-4642.
29. Davis CD, Uthus EO. DNA methylation, cancer susceptibility, and nutrient interactions. Exp Biol Med(Maywood) 2004;229:988-995.
30. Brown R, Strathdee G. Epigenomics and epigenetic therapy of cancer. Trends Mol Med 2002;8 Suppl: 43-48.
31. Schmutte C, Yang AS, Nguyen TT, et al. Mechanisms for the involvement of DNA methylation in the colon carcinogenesis. Cancer Res 1996; 56:2375-2381.
32. Wu JC, Santi DV. Kinetic and catalytic mechanism of Hha I methyltransferase. J Biol Chem 1987; 262: 4778-4786.
33. Xie S, Wang Z, Okano M, et al. Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 1999; 236: 87-95.
34. Langer F, Dingemann J, Kreipe H, et al. Up-regulation of DNA methyl transferases DNMT1, 3A, and 3B in myelodysplastic syndrome. Leukemia Res 2005; 29: 325-329.
35. Herman JG, Jen J, Merlo A, et al. Hypermerhylation-associated inactivation indicates a tumor suppressor role for pl5INK4B. Cancer Res 1996; 56: 722-727.
36. Quesnel B, Guillerm G, Vereecque R, et al. Methylation of the p15INK4B gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood 1998; 91: 2985-2990.
37. Steck PA, Pershouse MA, Jasser SA, et al. Identifcation of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23. 3 that is mutated in multiple advanced cancers. Nat Genet 1997; 15: 356-362.
38. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943-1947.
39. Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res 1997: 57: 2124-2129.
40. Zhang J, Grindley JC, Yin T, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 2006;441:518-522.
41. Ohshima K, Karube K, Shimazaki K, et al. Imbalance between apoptosis and telomerase activity in myelodysplastic syndromes: possible role in ineffective hemopoiesis. Leuk Lymphoma 2003; 44:1339-1346.
42. Bock O, Serinsoz E, Schlue J, et al. Different expression levels of the telomerase catalytic subunit hTERT in myeloproliferative and myelodysplastic diseases. Leuk Res 2004;28:457-460.
43. Huang J, Jin J, Xu WL. Studies of WT1 and hTERT genes expression in myelodysplastic syndrome patients. Chinese Journal of Medical Genetics. 2005; 22:350-352. (in Chinese).
44. Wang ZY, Qiu QQ, Deuel TF. The Wilms' tumor gene product WT1 activates or suppresses transcription through separate functional domains. J Biol Chem 1993; 268: 9172-9175.
45. Huang J, Jin J, Xu WL. WT1 gene expression in myelodysplastic syndrome and its clinical implication. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2006; 35:132-135. (in Chinese).
46. Kyo S, Inoue M. Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene 2002; 21:688-97.
47. Steube KG, Gignac SM, Hu ZB, et al. In vitro culture studies of childhood myelodysplastic syndrome: establishment of the cell line MUTZ-1. Leuk Lymphoma 1997; 25:345-63.
48. Drexler HG. Malignant hematopoietic cell lines: in vitro models for the study of myelodysplastic syndromes. Leuk Res 2000; 24:109-115.
49. Zhang ZN, Shen T. Criteria of diagnosis and therapeutic reponse in hematologic diseases. 2nd ed. Beijing: Scientific Publication, 1998. 258-267. (in Chinese)
50. Brunet A, Datta SR, Greenberg ME. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 2001; 11: 297-305.
51. Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl-2 interacting protein, mediates cytochrome C release from mitochondria in response to activation of cell surface death receptors. Cell 1998; 94: 481-490.
52. Gross A, Yin XM, Wang K, et al. Caspase cleaved BID targets mitochondrial and is required for cytochrome c release, while Bcl-x1 prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 1999; 93: 3044-3052.
53. Kerbauy DB, Joachim DH. Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome. Exp Hematol 2007; 35: 1739-1746.
54. Kerbauy DM, Lesnikov V, Abbasi N, et al. NF-kappaB and FLIP in arsenic trioxide (ATO)-induced apoptosis in myelodysplastic syndromes (MDSs). Blood 2005; 106:3917-3925.
55. Fadok VA, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophage. J Immunol 1992;148: 2207-2216.
56. Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Meth 1995; 184: 39-51.
57. Kroemer G, Petit PX, Zamzami N, et al. The biochemistry of apoptosis. FASEB J 1995; 9: 1277-1287.
58. Nicoletti I, er al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139: 271-279.
59. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770-776.
60. Green D, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309-1312.
61. Krammer PH. CD95' s deadly mission in the immune system. Nature 2000; 407: 789-795.
62. Woo M, Hakem R, Soengas MS, et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 1998;12: 806-819.
63. Burkle A. Physiology and pathophysiology of poly(ADP-ribosyl)ation. Bioessays 2001; 23: 795-806.
64. Tong WM., Cortes U, Wang ZQ. Poly (ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis. Biochim Biophys Acta 2001; 1552: 27-37.
65. Bharti AC, Takada Y, Aggarwal BB. PARP cleavage and caspase activity to assess chemosensitivity. Methods Mol Med 2005; 11:69-78.
66. Sherr CJ. Cancer cell cycle. Science 1996; 274: 1672 -1677.
67. Dynlacht BD. Regulation of transcription by proteins that control the cell cycle. Nature 1997; 389: 149-152.
68. Peter M, Herskowitz I. Joining the complex: cycl in-dependent kinase inhibitory proteins and the cell cycle. Cell 1994; 79: 181 -184.
69. Erba E. Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine. Br J Cancer 2002; 86: 1510-1517.
70. Decker RH, Dai Y, Grant S. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway. Cell Death Differ 2001; 8: 715-724.
71. Di Bacco A. Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: 405-415.
72. Yang LJ, Si XH. Expression and significance of Cyclin D1 in humanhepatocellular carcinoma. Chin J Cancer Res 2001; 13: 144-146.
73. Si X, Liu Z. Expression and significance of cell cycle-related proteins cyclin D1, CDK4, p27, E2F 1 and cts-1 in chondrosarcoma of the jaws. Oral Oncol 2001; 37: 431-436.
74. Aladjem MI, Pasa S, Parodi S, et al. Molecular interaction maps-a diagrammatic graphical language for bioregulatory networks. Sci STKE 2004; 2004:8.
75. Al-Aynati MM, Radulovical N, Ho J, et al. Overexpression of G1-S cyclins and cyclin dependent kinases during multistage human pancreatic duct cell carcinogenesis. Clin Cancer Res 2004; 10: 6598-6605.
76. Armstrong JS. Mitochondrial medicine: pharmacological targeting of mitochondria in disease. Br J Pharmacol 2007; 151:1154-1165.
77. BoudardD, VasselonC, BertheasMF, et al. Expression and prognostic significance of Bcl-2 family proteins in myelodysplastic syndromes. Am J Hematol 2002;70:115-125.
78. HockenberyD, Nunez G, MillimanC, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990; 348: 334-336.
79. Krajewski S, Tanaka S, Takayama S, et al. Investigation of the subcellular distribution of bcl-2 oncoprotein: Residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 1993; 53: 4701-4714.
80. de Jong D, Prins FA, Mason DY, et al. Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res 1994; 54:256-260.
81. Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-X( L) during apoptosis. Proc Natl Acad Sci U S A. 1997; 94: 3668-3672.
82. Gross A, Jockel J, Wei MC, et al. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998 ; 17:3878-3885.
83. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74: 609-619.
84. Del Poeta G, Venditti A, Del Principe MI, et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003; 101: 2125-2131.
85. Dlugosz PJ, Billen LP, Annis MG, et al. Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J 2006; 25: 2287-2296.
86. Sedletska Y, Giraud-Panis MJ, Malinge JM. Cisplatin is a DNA-damaging antitumour compound triggering multifactorial biochemical responses in cancer cells: importance of apoptotic pathways. Curr Med Chem Anticancer Agents 2005; 5:251-265.
87. Ryningen A, Ersvaer E, Oyan AM, et al. Stress-induced in vitro apoptosis of native human acute myelogenous leukemia (AML) cells shows a wide variation between patients and is associated with low BCL-2: Bax ratio and low levels of heat shock protein 70 and 90. Leuk Res 2006; 30: 1531-1540.
88. Kelekar A, Chang BS, Harlan JE, et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997; 17:7040-7046.
89. Rahmani M, Davis EM, Bauer C, et al. Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation. J Biol Chem 2005; 280:35217-35227.
90. Moulding DA, Giles RV, Spiller DG, et al. Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells. Blood 2000; 96: 1756 -1763.
91. Zhou P, Qian L, Bieszczad CK, et al. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998; 92:3226-3239.
92. Derenne S, Monia B, Dean N M, et al. Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-X_1. is an essential survival protein of human nyeloma cells. Blood 2002; 100: 194-199.
93. JiaL, Patwari Y, Kelsey SM, et al. Role of Smac in human leukaemic cell apoptosis and proliferation. Oncogene 2003; 22: 1589-1599.
94. Van Eden ME, Byrd MP, Sherrill KW, et al. Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA 2004; 10: 469-481.
95. de Graaf AO, de Witte T, Jansen JH. Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer? Leukemia 2004; 18:1751-1759.
96. Rajcan-Separovic E, Liston P, Lefebvre C, et al. Assignment of human inhibitor of apoptosis protein (IAP) genes xiap, hiap-1, and hiap-2 to chromosomes Xq25 and 11q22-q23 by fluorescence in situ hybridization. Genomics 1996; 37:404-406.
97. Carter BZ, Mak DH, Schober WD, et al. Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells. Blood 2006; 108: 630-637.
98. Carter BZ, Milella M, Tsao T, et al. Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia. Leukemia 2003; 17: 2081-2089.
99. Yamamoto K, Abe S, Nakagawa Y, et al. Expression of IAP family proteins in myelodysplastic syndromes transforming to overt leukemia. Leuk Res 2004; 28:1203-1211.
100. Rasper DM, Vaillancourt JP, Hadana S,et al. Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD95(Fas, APO-1)receptor complex. Cell Death Differ 1998; 5: 271—288.
101. Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997; 388: 190-195.
102. Micheau O, Lens S, Gaide O, et al. NF-κB signals induce the expression of c-FLIP. Mol Cell Biol 2001; 21: 5299-5305.
103. Wachter T, Spriek M, Hausmann D, et al. cFLIPL inhibits tumor necrosis factor-related apoptosis-inducing ligand-mediated NF-kappaB activation at the death-inducing signaling complex in human keratinoeytes. J Biol Chem 2004; 279: 52824—52834.
104. Herman MA, Ch'ng Q, Hettenbach SM, et al. EGL227 is similar to a metastasis2-associated factor an d controls cell polarity and cell migration in C elegans. Development 1999; 126: 1055-1064.
105. Toh Y, Kuninaka S, Endo K, et al. Molecular analysis of a candidate metastasis2associated gene, MTA1: possible interaction with histone descetylase 1. J Exp Clin Cancer Res 2000; 19: 105-111.
106. Benesch M, Platzbecker U, Ward J, et al. Expression of FLIP_(long) and FLIP_(short) in bone marrow mononuclear and CD34+ cells in patients with myelodysplastic syndrome: correlation with apoptosis. Leukemia 2003; 17: 2460-2466.
107. Philipp V, Carsten B, Bernd N, et al. Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase. J Biol Chem 2005; 280: 5121-5127.
108. Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarilv conserved mediators of immune responses. Annu Rev Immunol 1998; 16: 225-260.
109. Huxford T, Malek S, Ghosh G. Structure and mechanism in NF-kappa B / I kappa B signaling. Cold Spring Harb Symp Quant Biol 1999; 64: 533-540.
110. Hatada EN, Nieters A, Wulczyn FG, et al. The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding. Proc Natl Acad Sci USA 1992; 89: 2489-2493.
111. Malek S, Huang DB, Huxford T, et al. X-ray crystal structure of an IkappaBbe ta x NF-kappaB p65 homodimer complex. J Biol Chem 2003; 278: 23094—23100.
112. Ghosh S, Karin M . Missing pieces in the NF-kappaB puzzle. Cell 2002; 109 Suppl: S81-96.
113. Rothwarf DM, Karin M . The NF-kappa B activation pathway: a paradigm in information transfer from membran e to nucleus. Sci STKE 1999; 1999: RE1.
114. May MJ, Marienfeld RB, Ghosh S. Characterization of the Ikappa B-kinase NEM 0 binding domain. J Biol Chem 2002: 277: 45992-46000.
115. Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatorv signalling. Semin Immunol 2000; 12: 85-98.
116. Luo JL, Kamata H, Karin M. IKK / NF-kappaB signal ing: balancing life and death-a new approach to cancer therapy. J Clin Invest 2005; 115: 2625-2632.
117. Li ZW ,Chu W, Hu Y, et al.The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. J Exp Med 1999; 189: 1839-1845.
118. Rothe M, Sarma V, Dixit VM, et al. TRAF2-mediated activation of NF-kappa B by TNF-receptor 2 and CD40. Science 1995; 269: 1424-1429.
119. Kucharczak J, Simmons MJ, Fan Y, et al. To be, or not to be : NF-kappaB is the answer-role of Rel / NF-kappaB in the regulation of apoptosis. Oncogene 2003; 22: 8961-8982.
120. Mattson MP, Meffert MK. Roles for NF-kappaB in nerve cell survival, plasticity, and disease. Cell Death Differ 2006; 13: 852-860.
121. Varfolomeev E, Wayson SM, Dixit VM , et al. The inhibitor of apoptosis protein fusion C-IAP2. MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2. J Biol Chem 2006; 281: 29022-29029.
122. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998; 281: 1322-1326.
123. Karin M, Cao Y, Greten FR, et al. NF-kappa B in cancer. Nat Rev Ancer 2002; 2: 301-310.
124. Derouet M, Thomas L, Cross A, et al. Granulocyte Macrophage Colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem 2004; 279: 26915-26921.
125. Cowburn AS, Cadwallader KA, Reed BJ, et al. Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 2002; 100: 2607—2616.
126. Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood 2001; 97: 1172-1179.
127. Chen BG, Zhu M, Luo WD, et al. The correlation study of PTEN gene expression and Akt phosphorylation in myelodysplastic syndrome. Chin J Hematol 2007;28:470-473. (in Chinese)
128. Toyota M, Kopecky KJ, YoyotaMO, et al. Methylation profiling in acute myeloid leukemia. Blood 2001; 97: 2824-2829.
129. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: A novel PCR assay for methylation status of CpG island. Proc. Natl. Acad. Sci. USA. 1996; 93: 9821-9826.
130. Salvesen HB, MacDonald N, Ryan A, et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. Int J Cancer 2001; 91: 22-26.
131. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer 2005; 5:223-231.
132. Goffin J, Eisenhauer E. DNA methyhransferase inhibitors-state of the art. Ann Oncol 2002; 13:1699—1716.
133. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349: 2042—2054.
134. Okano M, Xie S, Li E, et al. Dnmt 2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res 1998; 26: 2536-2540.
135. Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5)methyltransferases. Nat Genet 1998; 19: 219-220.
136. Xie S, Wang Z, Okano M, et al. Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 1999; 236: 87-95.
137. VertinoPM, Sekowski JA, Coil JM, et al. DNMT1 is a component of a multiprotein DNA replication complex. Cell Cycle 2002; 1: 416-423.
138. Okano M, Bell DW, Haber DA, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99: 247-257.
139. Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 2002; 416: 552-556.
140. Mizuno S, Chijiwa T, Okam ura T, et al. Expression of DNA methyltransferases DNMTI. 3A, an d 3B in normal hematopoiesis and in acute an d chronic myelogenous leukemia. Blood. 2001. 97: 1 172-1 179. 2823-2829.
141. Robertson KD,Uzvolgyi E, Liang G, et al. The human DNA methyltransferase (DNMTs) 1,3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 1999; 27: 2291-2298.
142. Robertson KD, Keyomarsi K, Gonzales FA, et al. Differential mRNA expression of the human DNA methyl transferases (DNMTs) 1, 3a and 3b during the G(0)G(1)to S phase transition in normal and tumor ceils. Nucleic Acids Res 2000; 28: 2108-2113.
143. Hedge DR, Xiao W, Clausen PA, et al. Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) geBe in human erythroleukemia cells. J Biol Chem 2001; 276: 39508-39511.
144. Shen H, Wang L, Spitz MR, et al. A novel polymorphism in human cytosine DNA-methyltransferase-3B promoter is associated with an increased risk of lung cancer. Cancer Res 2002; 62: 4992-4995.
145. Goel A, Arnold CN, Niedzwiecki D, et al. Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Res 2004 ; 64: 3014-3021
146. Soria JC, Lee HY, Lee JI, et al. Lack of PTEN expression in nonsmall cell lung cancer could be related to promoter methylation. Clin Cancer Res 2002; 8: 1178-1184.
147. Garcia JM, Silva J, Pena C, et al. Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes Cancer 2004; 41: 117-124.
148. Yu J, Zhang H, Gu J, et al. Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma . BMC Cancer 2004; 4: 65.
149. Schondorf T, Ebert MP, Hoffmann J, et al. Hypermethylation of the PTEN gene in ovarian cancer cell lines. Cancer Lett 2004; 207: 215-220.
150. Baeza N, Weller M , Yonekawa Y, et al. PTEN methylation and expression in glioblastomas. Acta Neuropathol 2003; 106: 479-485.
151. Salvesen HB, Stefansson I, Kretzschmar El, et al. Significance of PTEN alterations in endometrial carcinoma: a population—based study of mutations, promoter methylation an PTEN protein expression. Int J Oncol 2004; 25: 1615-1623.
152. Kusy S, Cividin M , Sorel N, et al. pI4ARF,p15INK4b, and p16INK4a methylation status in chronic myelogenous leukemia. Blood 2003;101: 374-375.
153. Wong IH, Ng MH, Huang DP, et al. Aberrant pl5 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications. Blood 2000;95:1942-1949.
154. Chim CS, Kwong YL, Kwong YL. et al. Methylation profiling in multiple myeloma. Leuk Res 2004;28:379-385.
155. Garcia MJ, Martinez-Delgado B, Cebrian A, et al. Different Incidence and Pattern of p15INK4b and pl6INK4a Promoter Region Hypermethylation in Hodgkin's and CD30-Positive Non-Hodgkin's Lymphomas. Am J Pathol 2002;161: 1007-1013.
156. Chen H, Wu S. Hypermethylation of the p15(INK4B)gene in acute leukemia and myelodysplastic syndromes. Chin Med J(Engl) 2002; 115: 987—990.
157. Sandhu C, Garbe GJ, Bllattacharya N, et al. Transforming growth factor β stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclinD1-cdk4 association in human mammary epithelial cells. Molecular and Cellular Biology 1997;17: 2458-2467.
158. Hackett JA, Gredier CW. Balancing instability; dual roles for telomerase and telomere dysfunction in tumorigensis. Oncogene, 2002;21:619-625.
159. Blackburn EH. Telomere states and cell fates. Nature 2000;408: 53-56.
160. Norgaard JM, Hokland P. Biology of multiple drug resistance in acute leukemia. Int J Hemat 2000;72:290-297.
161. Bearss DJ, Hurley LH, Von Hoff DD. Telomere maintenance mechanisms as a target for drug development. Oncogene 2000;19:6632-6642.
162. Ramirez R, Carracedo J, Jimenez R, et al. Massive telomere loss is an early event of DNA damage-induced apoptosis. J Biol Chem 2003;278:836-842.
163. Akeshima R, Kigawa J, Takahashi M, et al. Telomerase activity and p53-dependent apoptosis in ovarian cancer cells. Br J Cancer 2001;84:1551-1555.
164. Kushner DM, Paranjape JM, Bandyopadhyay B, et al. 2-5A antisence directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol Oncol 2000; 76:183-192.
165. Counter CM, Meyerson M, Eaton EN, et al. Telomerase activity is restored in human cells byectopic expression of hTERT(hEST2), the catalytic subunit of telomerase. Oncogene 1998;16:1217.
166. Bryce LA, Morrison N, Hoare SF, et al. Mapping of the gene for the human telomerase reverse transcriptase, hTERT, to chromosome 5p15. 33 by fluorescence in situ hybridization. Neoplasia 2000;2:197-201.
167. Szutorisz H, Linguer J, Cuthbert AP, et al. A chromosome 3 encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin. Cancer Res 2003;63:689-695.
168. Oh S, Song YH, Yim J, et al. The films' tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene. J Biol Chem 1999;274:37473-37478.
169. Oh S, Song YH, Yim J, et al. Identification of Mad as a repressor of the human telomerase(hTERT) gene. Oncogene 2000;19:1485-1490.
170. Bergmann L, Miething C, Maurer U, et al. High levels of Wilms' tumor gene(wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood 1997; 90:1217-1225.
171. Ostergaard M, Olesen LH, Hasle H, et al. WT1 gene expression: an excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukaemia patients-results from a single-centre stugy. Br J Haematol 2004;125:590-600.
172. Galimberti S, Guerrini F, Carulli G, et al. Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis. Eur J Haematol 2004;72:45-51.
173. Sekiya M, Adachi M, Hinoda Y, et al. Downregulation of Wilms' tumor gene(wt1) during myelomonocytic differentiation in HL60 cells. Blood 1994;83:1876-1882.
174. Tamaki H, Ogawa H, Ohyashiki K, et al. The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia 1999;13:393-399.
175. Patmasiriwat P, Fraizer G, Kantarjian H, et al.WTl and GATA1 expression in myelodysplastic syndrome and acute leukemia. Leukemia 1999;3:891-900.
176. Yin L, Hubbard AK, Giardina C. NF-kappa B regulates transcription of the mouse telomerase catalytic subunit. J Biol Chem 2000;275:36671-36675.
177. Akiyama M, Hideshima T, Hayashi T, et al. Nuclear factor-kappaB p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein. Cancer Res 2003;63:18-21.
178. Sinha-Datta U, Horikawa I, Michishita E, et al. Transcriptional activation of hTERT through the NF-kappaB pathway in HTLV-I-transformed cells. Blood 2004;104:2523-2531.
179. Akiyama M , Yamada O, Hideshima T, et al. TNFalpha induces rapid activation and nuclear translocation of telomerase in human lymphocytes. Biochem Biophys Res Commun 2004;316: 528-532.
1.Steensma DP,Bennett JM:The myelodysplastic syndromes:diagnosis and treatment.Mayo C1in Proc 2006;81:104-130.
2.Cazzola M,Malcovati L.Myelodysplastic syndromes-coping with ineffective hematopoiesis.N Engl J Med.2005;352:536-538.
3.Richard Z.Shadduck,Joan M.et al.Recent advances in myelodysplastic syndromes.Experimental Hematology 2007;35:137-143.
4.Inokuchi K:Chronic myelogenous leukemia:from molecular biology to clinical aspects and novel targeted therapies.J Nippon Med Sch 2006;73:178-192.
5.Warner JK,Wang JC,Hope KJ,et al.Concepts of human leukemic development.Oncogene 2004;23:7164-7177.
6.Nilsson L,Astrand-Grundstrom I,Anderson K,et at.:Involvement and functional impairment of the CD34(+)CD38(-)Thy-1(+)hematopoietic stem cell pool in myelodysplastic Syndromes with trisomy 8.Blood 2002;100:259-267.
7.Thanopoulou E,Cashman J,Kakagianne T,et al.Engraftment of NOD_SCIDbeta2microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.Blood 2004;103:4285-4293.
8.Young NS,SloandEM,Barrett J.Myelodysplastic syndrome.In:Young NS,Gerson SL,High KS,eds.Clinical Hematology.New York:Elsevier Health Sciences;2005.p.203-230.
9.Sloand EM,Mainwaring L,Fuhrer M,et at.:Preferential suppression of trisomy 8 compared with normal hematopoietic cell growth by autologous lymphocytes in patients with trisomy 8 myelodysplastic syndrome.Blood 2005;106:841-851.
10.Sloand EM,Ramkissoon S,Mainwaring L,et al.Granulocyte colony stimulating factor preferentially causes proliferation of pre-existing monosomy 7 cells in aplastic anemia and myelodysplastic syndrome because of abnormal truncated GCSF-receptors on these cells [abstract]. Blood. 2004;104:Abstract 458.
11. Fenaux P: Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol 2001;73: 429-437.
12. Kaiser J: First pass at cancer genome reveals complex landscape. Science 2006; 313: 1370.
13. Esteller M: Epigenetics provides a new generation of oncogenes and tumour-suppressor genes. Br J Cancer 2006; 94: 179—183.
14. Navas TA, Mohindru M, Estes M, et al. : Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors. Blood 2006; 108:4170-4177.
15. Shih LY, Lin TL, Wang PN, et al. Internal tandem duplication of fms-like tyrosine kinase 3 is associated with poor outcome in patients with myelodysplastic syndrome. Cancer 2004; 101:989-998.
16. Speranza A, Pellizzaro C, Coradini D. Hyaluronic acid butyric esters in cancer therapy. Anticancer Drugs 2005; 16:373- 379.
17. Choudhary C, Schwable J, Brandts C, et al. AML-associated Flt3 kinase domain mutations show signal transduction differences in comparison to Flt3 ITD mutations. Blood 2005; 106:265-273.
18. Levis M, Murphy KM, Pham R, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood 2005; 106:673-680.
19. ChalandonY, Schwaller J. Targeting mutated proteinbtyrosine kinases and their signaling pathways in hematologic malignancies. Haematologica 2005; 90:949-968.
20. Giles FJ, Stopeck AT, Silverman LR, et al. SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplasticsyndromes. Blood 2003; 102:795-801.
21. Chen J, Lee BH, Williams IR, et al. FGFR3 as a therapeutic target of the small molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene 2005; 24: 8259-8267.
22. Gotlib J, Berube C, Growney JD, et al. Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation. Blood 2005; 106:2865-2870.
23. GrowneyJD, Clark JJ, Adelsperger J, et al. Activation mutations of human c-KIT resistant to imatinib are sensitive to the tyrosine kinase inhibitor PKC412. Blood 2005; 106:721-724.
24. Stone RM, DeAngelo DJ, Klimek V, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105:54-60.
25. Kurzrock R, Albitar M, Cortes JE, et al. Phase II study of R115777, a farnesyl transferase inhibitor, in myelodysplastic syndrome. J Clin Oncol 2004;22: 1287 - 1292.
26. Kurzrock R, Fenaux P, Raza A, et al. High-Risk Myelodysplastic Syndrome (MDS): first results of international phase 2 study with Oral Farnesyltransferase Inhibitor R115777 (ZARNESTRA). Blood 2004; 104:23a.
27. Ravoet C, Mineur P, Robin V, et al. Phase I—II study of a Farnesyl Transf erase Inhibitor (FTI), SCH66336, in patients with Myelodysplastic Syndrome (MDS) or Secondary Acute Myeloid Leukemia (sAML). Blood 2002; 100:794a.
28. Cortes J, Faderl S, Estey E, et al. Phase I study of BMS- 214662, a farnesyl transferase inhibitor in patients with acute leukemias and high-risk myelodysplastic syndromes. J Clin Oncol 2005; 23:2805-2812.
29. Alexandrakis MG, Passam FH, Pappa CA, et al. Serum evaluation of angiogenic cytokine basic fibroblast growth factor, hepatocyte growth factor and TNF - alpha in patients with myelodysplastic syndromes: correlation with bone marrow microvascular density. Int J Immunopathol Pharmacol 2005;18:287-295.
30. Alexandrakis MG, Passam FH, Pappa CA, et al. Relation between bone marrow angiogenesis and serum levels of angiogenin in patients with myelodysplastic syndromes. Leuk Res 2005;29:41 -46.
31. Hu Q, Dey AL, Yang Y, et al. Soluble vascular endothelial growth factor receptor 1, and not receptor 2, is an independent prognostic factor in acute myeloid leukemia and myelodysplastic syndromes. Cancer 2004;100:1884- 1891.
32. Ribatti D, Vacca A. Therapeutic renaissance of thalidomide in the treatment of haematological malignancies. Leukemia 2005; 19:1525-1531.
33. Bouscary D, Quarre MC, Vassilief D, et al. Thalidomide for the treatment of Low Risk Myelodysplasia. The Thal-SMD-200 Trial from the French Group of Myelodysplasia (GFM). Blood 2004; 104:403a.
34. Koh KR, Janz M, Mapara MY, et al. Immunomodulatory derivative of thalidomide (IMiD CC-4047) induces a shift in lineage commitment by suppressing erythropoiesis and promoting myelopoiesis. Blood 2005; 105:3833-3840.
35. List A, Kurtin S, Roe DJ, et al. Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 2005; 352:549-557.
36. Cazzola M, Malcovati L. Myelodysplastic syndromescoping with ineffective hematopoiesis. N Engl J Med 2005; 352:536-538.
37. Paz K, Zhu Z. Development of angiogenesis inhibitors to vascular endothelial growth factor receptor 2. Current status and future perspective. Front Biosci 2005; 10:1415-1439.
38. Ferrara N, Hillan KJ, Novotny W. Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 2005; 333:328-335.
39. .Giles FJ, Steinfeldt H, Bellamy WT, et al. Phase 2 study of the Anti-Angiogenesis Agent AG-013736 in patients with Poor Prognosis Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS). Blood 2004; 104:502a.
40. Roboz GJ, List AF, Giles F, et al. Phase I Trial of PTK787/ZK 222584, an inhibitor Of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases, In Acute Myeloid Leukemia and Myelodysplastic Syndrome. Blood 2002; 100:337a.
41. Ellis LM . Bevacizumab. Nat Rev Drug Discov (Suppl) 2005:S8-S9.
42. Hasegawa D, Manabe A, Kubota T, et al. Methylation status of the pl5 and pl6 genes in paediatric myelodysplastic syndrome and juvenile myelomonocytic leukaemia. Br J Haematol 2005; 128:805-812.
43. Johan MF, Bowen DT, Frew ME, et al .Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia. Br J Haematol 2005; 129:60-65.
44. Langer F, Dingemann J, Kreipe H, et al. Upregulation of DNA methyltransferases DNMT1, 3A, and 3B in myelodysplastic syndrome. Leuk Res 2005; 29:325-329.
45. Kaminskas E, Farrell A, Abraham S, et al. FDA Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 2005; 11: 3604 - 3608.
46. Oka M, Meacham AM, Hamazaki T, et al. De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'- deoxycytidine. Oncogene 2005; 24:3091-3099.
47. van den Bosch J, Lubbert M, Verhoef G, et al. The effects of 5-aza-2'-deoxycytidine (Decitabine) on the platelet count in patients with intermediate and high-risk myelodysplastic syndromes. Leuk Res 2004; 28:785 - 790.
48. Issa JP, Garcia-Manero G, Giles FJ, et al. Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2' -deoxycytidine (decitabine) in hematopoietic malignancies. Blood 2004; 103:1635-1640.
49. Saba H, Rosenfeld C, Issa J-P, et al. First report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS). Blood 2004; 104:23a.
50. Lubbert M, Wijermans, PW, Ruter, BH. Re-Treatment with Low-Dose 5-Aza-2' -Deoxycytidine (Decitabine) results in second remissions of previously responsive MDS patients. Blood 2004; 104:405a.
51. Ben-Kasus T, Ben-Zvi Z, Marquez VE, et al. Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Biochem Pharmacol 2005; 70:121-133.
52. Guo H, Rao N, Xu Q, et al. Origin of tight binding of a near-perfect transition-state analogue by cytidine deaminase: implications for enzyme catalysis. J Am Chem Soc 2005; 127:3191-3197.
53. Holleran JL, Parise RA, Joseph E, et al. Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine. Clin Cancer Res 2005; 11:3862-3868.
54. Drummond DC, Noble CO, Kirpotin DB, et al. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 2005; 45:495-528.
55. Kuendgen A, Strupp C, Aivado M, et al. Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-trans retinoic acid. Blood 2004; 104:1266-1269.
56. Garcia-Manero G, Kantarjian H, Sanchez-Gonzalez B, et al. Results of a Phase I/II study of the combination of 5-aza-2-deoxycytidine (DAC) and Valproic Acid (VPA) in patients (pts) with Leukemia. Blood 2004; 104:78a.
57. Giles FJ, Fischer T, Cortes J, et al. A Phase I/II Study of intravenous LBH589, a novel histone deacetylase (HDAC) inhibitor, in patients (pts) with advanced hematologic malignancies. Blood 2004; 104:499a.
58. Michael A. Morgan. Christoph W. M. Reuter. Molecularly targeted therapies in myelodysplastic syndromes and acute myeloid leukemias. Ann Hematol 2006; 85: 139 - 163.
1 Ravandi F,Talpaz M,Estrov Z.Modulation of cellular signaling pathways:prospects for targeted therapy in hematological malignancies.Clin Cancer Res 2003;9:535-550.
2 Krause DS,Van Etten RA.Tyrosine kinases as targets for cancer therapy.N Engl J Med 2005; 353: 172-187.
3 Mizuki M, Ueda S, Matsumura I et al. Oncogenic receptor tyrosine kinase in leukemia. Cell Mol Biol 2003; 49: 907-922.
4 Abe A, Emi N, Tanimoto M et al. Fusion of the platelet-derived growth factor receptor beta to a novel gene CEV14 in acute myelogenous leukemia after clonal evolution. Blood 1997; 90: 4271-4277.
5 Cools J, DeAngelo DJ, Gotlib J et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 2003; 48: 1201-1214.
6 Curtis CE, Grand FH, Musto P et al. Two novel imatinib-responsive PDGFRA fusion genes in chronic eosinophilic leukaemia. Br J Haematol 2007; 138: 77-81.
7 Score J, Curtis C, Waghorn K et al. Identification of a novel imatinib responsive KIF5B-PDGFRA fusion gene following screening for PDGFRA overexpression in patients with hypereosinophilia. Leukemia 2006; 20: 827-832.
8 Safley AM, Sebastian S, Collins TS et al. Molecular and cytogenetic characterization of a novel translocation t (4;22) involving the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with atypical chronic myeloid leukemia. Genes Chromosomes Cancer 2004; 40: 44-50.
9 Cross NC, Reiter A. Tyrosine kinase fusion genes in chronic myeloproliferative diseases. Leukemia 2002; 16: 1207-12.
10 Chesi E, Nardini LA, Brents E et al. Frequent translocation t (4;14) (p16. 3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 1997; 16: 260-264.
11 Yagasaki D, Wakao Y, Yokoyama Y et al. Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t (4; 12) (p16;p13) chromosomal translocation, Cancer Res 2001; 61: 8371-4.
12 Furitsu T, Tsujimura T, Tono T et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 1993; 92: 1736-44.
13 Tsujimura T, Morimoto M, Hashimoto K et al. Constitutive activation of ckit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain. Blood 1996; 87: 273-83.
14 London CA, Galli SJ, Yuuki T. et al. Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Exp Hematol 1999;27: 689-697.
15 Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998; 279: 577-580.
16 Nishida T, Hirota S, Taniguchi M. et al. Familial gastrointestinal stromal tumours with germline mutation of the KIT gene. Nat Genet 1998; 19: 323-324.
17 Heinrich MC, Corless CL, Duensing A et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708-710.
18 Nakao M, Yokota S, Iwai T et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911-1918.
19 Kiyoi H, Naoe T, Nakano Y et al. Prognostic implication of FLT3 and NRAS gene mutations in acute myeloid leukemia. Blood 1999; 93: 3074-3080.
20 Irusta PM, Luo Y, Bakht 0 et al. Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor. J Biol Chem 2002; 277: 38627-38634.
21 Ma Y, Cunningham ME, Wang X et al. Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region. J Biol Chem 1999; 274: 13399-133402.
22 Kitayama H, Kanakura Y, Furitsu T et al. Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood 1995; 85: 790 - 798.
23 Kiyoi H, Towatari M, Yokota S et al. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia 1998; 12: 1333-1337.
24 Longley BJ Jr, Metcalfe DD, Tharp M et al. Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci USA 1999; 96: 1609-1614.
25 Nagata H, Worobec AS, Oh CK. et al. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci USA 1995; 92: 10560-10564.
26 Worobec AS, Semere T, Nagata H et al. Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis. Cancer 1998; 83: 2120-2129.
27 Carre RS, Goodeve A, Abu-Duhier FM et al. Incidence and prognosis of ckit and Flt3 mutations in core binding factor (CBF) acute myeloid leukemia. Blood 2002: 746a.
28 Zwaan CM, Miller M, Goemans BF et al. Frequency and clinical significance of c-kit exon 17 mutations in childfood acute myeloid leukemia. Blood 2002: 746a.
29 Abu-Duhier FM, Goodeve AC, Wilson GA et al. Identification of novel FLT- 3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol 2001; 113: 983-988.
30 Yamamoto Y, Kiyoi H, Nakano Y et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97: 2434 - 2439.
31 Spiekermann K, Bagrintseva K, Schoch C et al. A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. Blood 2002; 100: 3423 - 3425.
32 Mead AJ, Linch DC, Hills RK et al. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood 2007; 110: 1262-1270.
33 Moriyama Y, Tsujimura T, Hashimoto K et al. Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase. J Biol Chem 1996; 271: 3347 - 3350.
34 Tsujimura T, Hashimoto K, Kitayama H et al. Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self association not at the ligand-induced dimerization site. Blood 1999; 93: 1319-1329.
35 Hashimoto K, Matsumura I, Tsujimura T et al. Necessity of tyrosine 719 and phosphatidylinositol 3' -kinase-mediated signal pathway in constitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the Asp814Val mutation. Blood 2003; 101: 1094-1102.
36 Gari M, Goodeve A, Wilson G et al. c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia. Br J Haematol 1999; 105: 894 - 900.
37 Care RS, Valk PJ, Goodeve AC et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol 2003; 121: 775-777.
38 Boissel N, Leroy H, Brethon B et al. Incidence and prognostic impact of c- Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia 2006; 20: 965-970.
39 Ridge SA, Worwood M, Oscier D et al. FMS mutations in myelodysplastic, leukemic, and normal subjects. Proc Natl Acad Sci USA 1990; 87: 1377-1380.
40 Tobal K, Pagliuca A, Bhatt B et al. Mutation of the human FMS gene (MCSF receptor) in myelodysplastic syndromes and acute myeloid leukemia. Leukemia 1990; 4: 486 - 489.
41 Roussel MF, Downing JR, Rettenmier CW et al. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell 1988; 55: 979-988.
42 Plotnikov AN, Schlessinger J, Hubbard SR et al. Structural basis for FGF receptor dimerization and activation. Cell 1999; 98: 641-650.
43 Lancet JE, Karp JE. Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy. Blood 2003; 102: 3880-3889.
44 Morgan MA, Ganser A, Reuter CW. Therapeutic efficacy of prenylation inhibitors in the treatment of myeloid leukemia. Leukemia 2003; 17: 1482-1498.
45 Hayakawa F, Towatari M, Kiyoi H. et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000; 19: 624-631.
46 MizukiM, Fenski R, Halfter H et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 2000; 96: 3907-3914.
47 Chian R, Young S, Danilkovitch-Miagkova A et al. Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant. Blood 2001; 98: 1365-1373.
48 Blume-Jensen P, Jiang G, Hyman R et al. Kit/stem cell factor receptorinduced activation of phosphatidylinositol 3' -kinase is essential for male fertility. Nat Genet 2000; 24: 157-162.
49 Kissel H, Timokhina I, Hardy MP et al. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 2000; 19: 1312-1326.
50 Rane SG, Reddy EP. JAKs, STATs and Src kinases in hematopoiesis. Oncogene 2002; 21: 3334-3358.
51 Darnell JE Jr. STATs and gene regulation. Science 1997; 277: 1630-1635.
52 Ning ZQ, Li J, Arceci RJ. Signal transducer and activator of transcription 3 activation is required for Asp (816) mutant c-Kit-mediated cytokineindependent survival and proliferation in human leukemia cells. Blood 2001; 97: 3559-3567.
53 Ning ZQ, Li J, McGuinness M et al. STAT3 activation is required for Asp (816) mutant c-Kit induced tumorigenicity. Oncogene 2001; 20: 4528-4536.
54 Sternberg DW, Tomasson MH, Carroll M et al. The TEL/PDGFbetaR fusion in chronic myelomonocytic leukemia signals through STAT5-dependent and STAT5-independent pathways. Blood 2001; 98: 3390-3397.
55 Wilbanks AM, Mahajan S, Frank DA et al. TEL/PDGFbetaR fusion protein activates STAT1 and STAT5: a common mechanism for transformation by tyrosine kinase fusion proteins. Exp Hematol 2000; 28: 584-593.
56 Mizuki M, Schwable J, Steur C et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations. Blood 2003; 101: 3164-3173.
57 Watanabe D, Ezoe S, Fujimoto M et al. Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol 2004; 126: 726-735.
58 Magnusson MK, Meade KE, Nakamura R et al. Activity of STI571 in chronic myelomonocytic leukemia with a platelet- derived growth factor β receptor fusion oncogene. Blood 2002; 100: 1088-1091.
59 Levis M, Tse KF, Smith BD et al. FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations. Blood 2001; 98, 885-887.
60 Tse KF, Novelli E, Civin CI et al. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 2001; 15: 1001-1010.
61 Kurzrock R, Sebti SM, Kantarjian HM et al. Phase I Study of a Farnesyl Transferase Inhibitor. R115777, in Patients with Myelodysplastic Syndrome. Blood 2001; 98: 623a.
62 Propper DJ, McDonald AC, Man A et al. Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C. J Clin Oncol 2001; 19: 1485- 1492.
63 Sebolt-Leopold JS. Development of anticancer drugs targeting the MAP kinase pathway. Oncogene 2000; 19: 6594-6599.
64 Witzig TE, Kaufmann SH. Inhibition of the phosphatidylinositol 3-kinase/ mammalian target of rapamycin pathway in hematologic malignancies. Curr Treat Options Oncol 2006; 7: 285-294.
65 Smolewski P. Recent developments in targeting the mammalian target of rapamycin (mTOR) kinase pathway. Anticancer Drugs 2006; 17: 487-494.
2. Greenberg P, Cox C, Le Beau, et al. International scoring system for evaluating prognosis in myelodysplastic syndromes. Blood 1997; 89:2079-2088.
3. Jaffe ES, Harris NL, Stein H, et al. World Health Organization classification of tumors. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. Lyon (France): IARC Press; 2001.
4. Mufti G, List AF, Gore SD, et al. Myelodysplastic syndrome. Hematology Am Soc Hematol Educ Program 2003; 176-199.
5. Look AT. Molecular pathogenesis of MDS. Hematology Am Soc Hematol Educ Program 2005; 156-160.
6. Parker JE, Mufti GJ. The myelodysplastic syndromes: a matter of life or death. Acta Haematol 2004; 111: 78-99.
7. Richard K, Shadduck JM, Latsko JM, et al. Recent advances in myelodysplastic syndromes. Exp Hematol 2007; 35: 137-143.
8. Kaminskas E, Farrell A, Abraham S, et al. FDA Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 2005; 11:3604 - 3608.
9. Saba H, Rosenfeld C, Issa J-P, et al. First report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS). Blood 2004; 104:23a.
10. Burzynski SR. Antineoplastons: history of the research (I). Drugs Exp Clin Res 1986; 12 (suppl 1):1-9.
11. Badria F, Mabed M, Khafagy W, et al. Potential utility of antineoplaston A-10 levels in breast cancer. Cancer Lett 2000; 155: 67-70.
12. Lin WC, Wu YW, Lai TY, et al. Effect of CDA-II, urinary preparation, on lipofuscin, lipid peroxidation and antioxidant systems in young and middle-aged rat brain. Am J Chin Med 2001; 29: 91-99.
13. Lin WC, Liao YC, Liau MC, et al. Inhibitory effect of CDA-II, a urinary preparation, on aflatoxin B(1)-induced oxidative stress and DNA damage in primary cultured rat hepatocytes. Food Chem Toxicol 2006;44:546-551.
14. Steelman LS, Pohnert SC, Shelton JG, et al. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis. Leukemia 2004; 18: 189-218.
15. Woodgett JR. Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol 2005; 17: 150-157.
16. Thoompson JE, Thompson CB. Putting the Rap on Akt. J Clin Oncol 2004; 1522: 4217-4226.
17. Zhou HL, Li XM, Meinkoth J, et al. Akt Regulates cell Survival and Apoptosis at a Postmitochondrial Level. J Cell Biol 2000; 151: 483-494.
18. Feng J, Tamaskovie R, Yang Z, et al. Stabilization of Mdm-2 via decreased ubiquitination is mediated by protein kinase B / Akt-dependent phosphorylation . J Biol Chem 2004; 279: 35510-35517.
19. Chang F, Lee JT, Navolanie PM, et al. Involvement of P13-K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia 2003; 17: 590-603.
20. Romashkova JA, Makarov SS. NF-κB is a target of Akt in anti-apoptotic PDGF signaling. Nature 1999; 401: 86-90.
21. Harrey RD, Lonial S. PI3 kinase/Akt pathway as a therapeutic target in multiple myeloma. Future Oncol 2007; 316: 639-647.
22. Takada Y, Kobayashi Y, Aggarwal BB. Evodiamine abolishes constitutive and inducible NF—kappa B activation by inhibiting I kappa B alpha kinase activation, thereby suppressing NF-kappa B—regulated antiapoptotic and metastatic gene expression, upregulating apoptosis, and inhibiting invasion. J Biol Chem 2005; 280: 17203-17212.
23. Micheau O, Lens S, Gaide O, et al. NF-kappaB signals induce the expression of c-FLIP. Mol Cell Biol 2001; 21:5299-5305.
24. Nyakern M, Tazzari PL, Finelli C, et al. Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients. Leukemia 2006; 20:230-238.
25. Braun T, Carvalho G, Coquelle A, et al. NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome. Blood 2006; 107:1156-1165.
26. Sanz C, Richard C, Prosper F, et al. Nuclear factor k B is activated in myelodysplastic bone marrow cells. Haematologica 2002; 87:1005-1006.
27. Esteller M. DNA methylation and cancer therapy: new developments and expectations. Curr Opin Oncol 2005;17: 55-60.
28. Das PM, Singal R. DNA methylation and cancer. J Clin Oncol 2004; 22: 4632-4642.
29. Davis CD, Uthus EO. DNA methylation, cancer susceptibility, and nutrient interactions. Exp Biol Med(Maywood) 2004;229:988-995.
30. Brown R, Strathdee G. Epigenomics and epigenetic therapy of cancer. Trends Mol Med 2002;8 Suppl: 43-48.
31. Schmutte C, Yang AS, Nguyen TT, et al. Mechanisms for the involvement of DNA methylation in the colon carcinogenesis. Cancer Res 1996; 56:2375-2381.
32. Wu JC, Santi DV. Kinetic and catalytic mechanism of Hha I methyltransferase. J Biol Chem 1987; 262: 4778-4786.
33. Xie S, Wang Z, Okano M, et al. Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 1999; 236: 87-95.
34. Langer F, Dingemann J, Kreipe H, et al. Up-regulation of DNA methyl transferases DNMT1, 3A, and 3B in myelodysplastic syndrome. Leukemia Res 2005; 29: 325-329.
35. Herman JG, Jen J, Merlo A, et al. Hypermerhylation-associated inactivation indicates a tumor suppressor role for pl5INK4B. Cancer Res 1996; 56: 722-727.
36. Quesnel B, Guillerm G, Vereecque R, et al. Methylation of the p15INK4B gene in myelodysplastic syndromes is frequent and acquired during disease progression. Blood 1998; 91: 2985-2990.
37. Steck PA, Pershouse MA, Jasser SA, et al. Identifcation of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23. 3 that is mutated in multiple advanced cancers. Nat Genet 1997; 15: 356-362.
38. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943-1947.
39. Li DM, Sun H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res 1997: 57: 2124-2129.
40. Zhang J, Grindley JC, Yin T, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 2006;441:518-522.
41. Ohshima K, Karube K, Shimazaki K, et al. Imbalance between apoptosis and telomerase activity in myelodysplastic syndromes: possible role in ineffective hemopoiesis. Leuk Lymphoma 2003; 44:1339-1346.
42. Bock O, Serinsoz E, Schlue J, et al. Different expression levels of the telomerase catalytic subunit hTERT in myeloproliferative and myelodysplastic diseases. Leuk Res 2004;28:457-460.
43. Huang J, Jin J, Xu WL. Studies of WT1 and hTERT genes expression in myelodysplastic syndrome patients. Chinese Journal of Medical Genetics. 2005; 22:350-352. (in Chinese).
44. Wang ZY, Qiu QQ, Deuel TF. The Wilms' tumor gene product WT1 activates or suppresses transcription through separate functional domains. J Biol Chem 1993; 268: 9172-9175.
45. Huang J, Jin J, Xu WL. WT1 gene expression in myelodysplastic syndrome and its clinical implication. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2006; 35:132-135. (in Chinese).
46. Kyo S, Inoue M. Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene 2002; 21:688-97.
47. Steube KG, Gignac SM, Hu ZB, et al. In vitro culture studies of childhood myelodysplastic syndrome: establishment of the cell line MUTZ-1. Leuk Lymphoma 1997; 25:345-63.
48. Drexler HG. Malignant hematopoietic cell lines: in vitro models for the study of myelodysplastic syndromes. Leuk Res 2000; 24:109-115.
49. Zhang ZN, Shen T. Criteria of diagnosis and therapeutic reponse in hematologic diseases. 2nd ed. Beijing: Scientific Publication, 1998. 258-267. (in Chinese)
50. Brunet A, Datta SR, Greenberg ME. Transcription-dependent and -independent control of neuronal survival by the PI3K-Akt signaling pathway. Curr Opin Neurobiol 2001; 11: 297-305.
51. Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl-2 interacting protein, mediates cytochrome C release from mitochondria in response to activation of cell surface death receptors. Cell 1998; 94: 481-490.
52. Gross A, Yin XM, Wang K, et al. Caspase cleaved BID targets mitochondrial and is required for cytochrome c release, while Bcl-x1 prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 1999; 93: 3044-3052.
53. Kerbauy DB, Joachim DH. Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome. Exp Hematol 2007; 35: 1739-1746.
54. Kerbauy DM, Lesnikov V, Abbasi N, et al. NF-kappaB and FLIP in arsenic trioxide (ATO)-induced apoptosis in myelodysplastic syndromes (MDSs). Blood 2005; 106:3917-3925.
55. Fadok VA, Voelker DR, Campbell PA, et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophage. J Immunol 1992;148: 2207-2216.
56. Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Meth 1995; 184: 39-51.
57. Kroemer G, Petit PX, Zamzami N, et al. The biochemistry of apoptosis. FASEB J 1995; 9: 1277-1287.
58. Nicoletti I, er al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139: 271-279.
59. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770-776.
60. Green D, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309-1312.
61. Krammer PH. CD95' s deadly mission in the immune system. Nature 2000; 407: 789-795.
62. Woo M, Hakem R, Soengas MS, et al. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev 1998;12: 806-819.
63. Burkle A. Physiology and pathophysiology of poly(ADP-ribosyl)ation. Bioessays 2001; 23: 795-806.
64. Tong WM., Cortes U, Wang ZQ. Poly (ADP-ribose) polymerase: a guardian angel protecting the genome and suppressing tumorigenesis. Biochim Biophys Acta 2001; 1552: 27-37.
65. Bharti AC, Takada Y, Aggarwal BB. PARP cleavage and caspase activity to assess chemosensitivity. Methods Mol Med 2005; 11:69-78.
66. Sherr CJ. Cancer cell cycle. Science 1996; 274: 1672 -1677.
67. Dynlacht BD. Regulation of transcription by proteins that control the cell cycle. Nature 1997; 389: 149-152.
68. Peter M, Herskowitz I. Joining the complex: cycl in-dependent kinase inhibitory proteins and the cell cycle. Cell 1994; 79: 181 -184.
69. Erba E. Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine. Br J Cancer 2002; 86: 1510-1517.
70. Decker RH, Dai Y, Grant S. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway. Cell Death Differ 2001; 8: 715-724.
71. Di Bacco A. Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis. Oncologist 2000; 5: 405-415.
72. Yang LJ, Si XH. Expression and significance of Cyclin D1 in humanhepatocellular carcinoma. Chin J Cancer Res 2001; 13: 144-146.
73. Si X, Liu Z. Expression and significance of cell cycle-related proteins cyclin D1, CDK4, p27, E2F 1 and cts-1 in chondrosarcoma of the jaws. Oral Oncol 2001; 37: 431-436.
74. Aladjem MI, Pasa S, Parodi S, et al. Molecular interaction maps-a diagrammatic graphical language for bioregulatory networks. Sci STKE 2004; 2004:8.
75. Al-Aynati MM, Radulovical N, Ho J, et al. Overexpression of G1-S cyclins and cyclin dependent kinases during multistage human pancreatic duct cell carcinogenesis. Clin Cancer Res 2004; 10: 6598-6605.
76. Armstrong JS. Mitochondrial medicine: pharmacological targeting of mitochondria in disease. Br J Pharmacol 2007; 151:1154-1165.
77. BoudardD, VasselonC, BertheasMF, et al. Expression and prognostic significance of Bcl-2 family proteins in myelodysplastic syndromes. Am J Hematol 2002;70:115-125.
78. HockenberyD, Nunez G, MillimanC, et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990; 348: 334-336.
79. Krajewski S, Tanaka S, Takayama S, et al. Investigation of the subcellular distribution of bcl-2 oncoprotein: Residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 1993; 53: 4701-4714.
80. de Jong D, Prins FA, Mason DY, et al. Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res 1994; 54:256-260.
81. Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-X( L) during apoptosis. Proc Natl Acad Sci U S A. 1997; 94: 3668-3672.
82. Gross A, Jockel J, Wei MC, et al. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998 ; 17:3878-3885.
83. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993; 74: 609-619.
84. Del Poeta G, Venditti A, Del Principe MI, et al. Amount of spontaneous apoptosis detected by Bax/Bcl-2 ratio predicts outcome in acute myeloid leukemia (AML). Blood 2003; 101: 2125-2131.
85. Dlugosz PJ, Billen LP, Annis MG, et al. Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J 2006; 25: 2287-2296.
86. Sedletska Y, Giraud-Panis MJ, Malinge JM. Cisplatin is a DNA-damaging antitumour compound triggering multifactorial biochemical responses in cancer cells: importance of apoptotic pathways. Curr Med Chem Anticancer Agents 2005; 5:251-265.
87. Ryningen A, Ersvaer E, Oyan AM, et al. Stress-induced in vitro apoptosis of native human acute myelogenous leukemia (AML) cells shows a wide variation between patients and is associated with low BCL-2: Bax ratio and low levels of heat shock protein 70 and 90. Leuk Res 2006; 30: 1531-1540.
88. Kelekar A, Chang BS, Harlan JE, et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997; 17:7040-7046.
89. Rahmani M, Davis EM, Bauer C, et al. Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation. J Biol Chem 2005; 280:35217-35227.
90. Moulding DA, Giles RV, Spiller DG, et al. Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells. Blood 2000; 96: 1756 -1763.
91. Zhou P, Qian L, Bieszczad CK, et al. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood 1998; 92:3226-3239.
92. Derenne S, Monia B, Dean N M, et al. Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-X_1. is an essential survival protein of human nyeloma cells. Blood 2002; 100: 194-199.
93. JiaL, Patwari Y, Kelsey SM, et al. Role of Smac in human leukaemic cell apoptosis and proliferation. Oncogene 2003; 22: 1589-1599.
94. Van Eden ME, Byrd MP, Sherrill KW, et al. Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA 2004; 10: 469-481.
95. de Graaf AO, de Witte T, Jansen JH. Inhibitor of apoptosis proteins: new therapeutic targets in hematological cancer? Leukemia 2004; 18:1751-1759.
96. Rajcan-Separovic E, Liston P, Lefebvre C, et al. Assignment of human inhibitor of apoptosis protein (IAP) genes xiap, hiap-1, and hiap-2 to chromosomes Xq25 and 11q22-q23 by fluorescence in situ hybridization. Genomics 1996; 37:404-406.
97. Carter BZ, Mak DH, Schober WD, et al. Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells. Blood 2006; 108: 630-637.
98. Carter BZ, Milella M, Tsao T, et al. Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia. Leukemia 2003; 17: 2081-2089.
99. Yamamoto K, Abe S, Nakagawa Y, et al. Expression of IAP family proteins in myelodysplastic syndromes transforming to overt leukemia. Leuk Res 2004; 28:1203-1211.
100. Rasper DM, Vaillancourt JP, Hadana S,et al. Cell death attenuation by 'Usurpin', a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD95(Fas, APO-1)receptor complex. Cell Death Differ 1998; 5: 271—288.
101. Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP. Nature 1997; 388: 190-195.
102. Micheau O, Lens S, Gaide O, et al. NF-κB signals induce the expression of c-FLIP. Mol Cell Biol 2001; 21: 5299-5305.
103. Wachter T, Spriek M, Hausmann D, et al. cFLIPL inhibits tumor necrosis factor-related apoptosis-inducing ligand-mediated NF-kappaB activation at the death-inducing signaling complex in human keratinoeytes. J Biol Chem 2004; 279: 52824—52834.
104. Herman MA, Ch'ng Q, Hettenbach SM, et al. EGL227 is similar to a metastasis2-associated factor an d controls cell polarity and cell migration in C elegans. Development 1999; 126: 1055-1064.
105. Toh Y, Kuninaka S, Endo K, et al. Molecular analysis of a candidate metastasis2associated gene, MTA1: possible interaction with histone descetylase 1. J Exp Clin Cancer Res 2000; 19: 105-111.
106. Benesch M, Platzbecker U, Ward J, et al. Expression of FLIP_(long) and FLIP_(short) in bone marrow mononuclear and CD34+ cells in patients with myelodysplastic syndrome: correlation with apoptosis. Leukemia 2003; 17: 2460-2466.
107. Philipp V, Carsten B, Bernd N, et al. Assigning functional domains within the p101 regulatory subunit of phosphoinositide 3-kinase. J Biol Chem 2005; 280: 5121-5127.
108. Ghosh S, May MJ, Kopp EB. NF-kappa B and Rel proteins: evolutionarilv conserved mediators of immune responses. Annu Rev Immunol 1998; 16: 225-260.
109. Huxford T, Malek S, Ghosh G. Structure and mechanism in NF-kappa B / I kappa B signaling. Cold Spring Harb Symp Quant Biol 1999; 64: 533-540.
110. Hatada EN, Nieters A, Wulczyn FG, et al. The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding. Proc Natl Acad Sci USA 1992; 89: 2489-2493.
111. Malek S, Huang DB, Huxford T, et al. X-ray crystal structure of an IkappaBbe ta x NF-kappaB p65 homodimer complex. J Biol Chem 2003; 278: 23094—23100.
112. Ghosh S, Karin M . Missing pieces in the NF-kappaB puzzle. Cell 2002; 109 Suppl: S81-96.
113. Rothwarf DM, Karin M . The NF-kappa B activation pathway: a paradigm in information transfer from membran e to nucleus. Sci STKE 1999; 1999: RE1.
114. May MJ, Marienfeld RB, Ghosh S. Characterization of the Ikappa B-kinase NEM 0 binding domain. J Biol Chem 2002: 277: 45992-46000.
115. Karin M, Delhase M. The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatorv signalling. Semin Immunol 2000; 12: 85-98.
116. Luo JL, Kamata H, Karin M. IKK / NF-kappaB signal ing: balancing life and death-a new approach to cancer therapy. J Clin Invest 2005; 115: 2625-2632.
117. Li ZW ,Chu W, Hu Y, et al.The IKKbeta subunit of IkappaB kinase (IKK) is essential for nuclear factor kappaB activation and prevention of apoptosis. J Exp Med 1999; 189: 1839-1845.
118. Rothe M, Sarma V, Dixit VM, et al. TRAF2-mediated activation of NF-kappa B by TNF-receptor 2 and CD40. Science 1995; 269: 1424-1429.
119. Kucharczak J, Simmons MJ, Fan Y, et al. To be, or not to be : NF-kappaB is the answer-role of Rel / NF-kappaB in the regulation of apoptosis. Oncogene 2003; 22: 8961-8982.
120. Mattson MP, Meffert MK. Roles for NF-kappaB in nerve cell survival, plasticity, and disease. Cell Death Differ 2006; 13: 852-860.
121. Varfolomeev E, Wayson SM, Dixit VM , et al. The inhibitor of apoptosis protein fusion C-IAP2. MALT1 stimulates NF-kappaB activation independently of TRAF1 AND TRAF2. J Biol Chem 2006; 281: 29022-29029.
122. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998; 281: 1322-1326.
123. Karin M, Cao Y, Greten FR, et al. NF-kappa B in cancer. Nat Rev Ancer 2002; 2: 301-310.
124. Derouet M, Thomas L, Cross A, et al. Granulocyte Macrophage Colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1. J Biol Chem 2004; 279: 26915-26921.
125. Cowburn AS, Cadwallader KA, Reed BJ, et al. Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival. Blood 2002; 100: 2607—2616.
126. Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1, 3A and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood 2001; 97: 1172-1179.
127. Chen BG, Zhu M, Luo WD, et al. The correlation study of PTEN gene expression and Akt phosphorylation in myelodysplastic syndrome. Chin J Hematol 2007;28:470-473. (in Chinese)
128. Toyota M, Kopecky KJ, YoyotaMO, et al. Methylation profiling in acute myeloid leukemia. Blood 2001; 97: 2824-2829.
129. Herman JG, Graff JR, Myohanen S, et al. Methylation-specific PCR: A novel PCR assay for methylation status of CpG island. Proc. Natl. Acad. Sci. USA. 1996; 93: 9821-9826.
130. Salvesen HB, MacDonald N, Ryan A, et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. Int J Cancer 2001; 91: 22-26.
131. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer 2005; 5:223-231.
132. Goffin J, Eisenhauer E. DNA methyhransferase inhibitors-state of the art. Ann Oncol 2002; 13:1699—1716.
133. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349: 2042—2054.
134. Okano M, Xie S, Li E, et al. Dnmt 2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res 1998; 26: 2536-2540.
135. Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5)methyltransferases. Nat Genet 1998; 19: 219-220.
136. Xie S, Wang Z, Okano M, et al. Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 1999; 236: 87-95.
137. VertinoPM, Sekowski JA, Coil JM, et al. DNMT1 is a component of a multiprotein DNA replication complex. Cell Cycle 2002; 1: 416-423.
138. Okano M, Bell DW, Haber DA, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99: 247-257.
139. Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 2002; 416: 552-556.
140. Mizuno S, Chijiwa T, Okam ura T, et al. Expression of DNA methyltransferases DNMTI. 3A, an d 3B in normal hematopoiesis and in acute an d chronic myelogenous leukemia. Blood. 2001. 97: 1 172-1 179. 2823-2829.
141. Robertson KD,Uzvolgyi E, Liang G, et al. The human DNA methyltransferase (DNMTs) 1,3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 1999; 27: 2291-2298.
142. Robertson KD, Keyomarsi K, Gonzales FA, et al. Differential mRNA expression of the human DNA methyl transferases (DNMTs) 1, 3a and 3b during the G(0)G(1)to S phase transition in normal and tumor ceils. Nucleic Acids Res 2000; 28: 2108-2113.
143. Hedge DR, Xiao W, Clausen PA, et al. Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) geBe in human erythroleukemia cells. J Biol Chem 2001; 276: 39508-39511.
144. Shen H, Wang L, Spitz MR, et al. A novel polymorphism in human cytosine DNA-methyltransferase-3B promoter is associated with an increased risk of lung cancer. Cancer Res 2002; 62: 4992-4995.
145. Goel A, Arnold CN, Niedzwiecki D, et al. Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers. Cancer Res 2004 ; 64: 3014-3021
146. Soria JC, Lee HY, Lee JI, et al. Lack of PTEN expression in nonsmall cell lung cancer could be related to promoter methylation. Clin Cancer Res 2002; 8: 1178-1184.
147. Garcia JM, Silva J, Pena C, et al. Promoter methylation of the PTEN gene is a common molecular change in breast cancer. Genes Chromosomes Cancer 2004; 41: 117-124.
148. Yu J, Zhang H, Gu J, et al. Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma . BMC Cancer 2004; 4: 65.
149. Schondorf T, Ebert MP, Hoffmann J, et al. Hypermethylation of the PTEN gene in ovarian cancer cell lines. Cancer Lett 2004; 207: 215-220.
150. Baeza N, Weller M , Yonekawa Y, et al. PTEN methylation and expression in glioblastomas. Acta Neuropathol 2003; 106: 479-485.
151. Salvesen HB, Stefansson I, Kretzschmar El, et al. Significance of PTEN alterations in endometrial carcinoma: a population—based study of mutations, promoter methylation an PTEN protein expression. Int J Oncol 2004; 25: 1615-1623.
152. Kusy S, Cividin M , Sorel N, et al. pI4ARF,p15INK4b, and p16INK4a methylation status in chronic myelogenous leukemia. Blood 2003;101: 374-375.
153. Wong IH, Ng MH, Huang DP, et al. Aberrant pl5 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications. Blood 2000;95:1942-1949.
154. Chim CS, Kwong YL, Kwong YL. et al. Methylation profiling in multiple myeloma. Leuk Res 2004;28:379-385.
155. Garcia MJ, Martinez-Delgado B, Cebrian A, et al. Different Incidence and Pattern of p15INK4b and pl6INK4a Promoter Region Hypermethylation in Hodgkin's and CD30-Positive Non-Hodgkin's Lymphomas. Am J Pathol 2002;161: 1007-1013.
156. Chen H, Wu S. Hypermethylation of the p15(INK4B)gene in acute leukemia and myelodysplastic syndromes. Chin Med J(Engl) 2002; 115: 987—990.
157. Sandhu C, Garbe GJ, Bllattacharya N, et al. Transforming growth factor β stabilizes p15INK4B protein, increases p15INK4B-cdk4 complexes, and inhibits cyclinD1-cdk4 association in human mammary epithelial cells. Molecular and Cellular Biology 1997;17: 2458-2467.
158. Hackett JA, Gredier CW. Balancing instability; dual roles for telomerase and telomere dysfunction in tumorigensis. Oncogene, 2002;21:619-625.
159. Blackburn EH. Telomere states and cell fates. Nature 2000;408: 53-56.
160. Norgaard JM, Hokland P. Biology of multiple drug resistance in acute leukemia. Int J Hemat 2000;72:290-297.
161. Bearss DJ, Hurley LH, Von Hoff DD. Telomere maintenance mechanisms as a target for drug development. Oncogene 2000;19:6632-6642.
162. Ramirez R, Carracedo J, Jimenez R, et al. Massive telomere loss is an early event of DNA damage-induced apoptosis. J Biol Chem 2003;278:836-842.
163. Akeshima R, Kigawa J, Takahashi M, et al. Telomerase activity and p53-dependent apoptosis in ovarian cancer cells. Br J Cancer 2001;84:1551-1555.
164. Kushner DM, Paranjape JM, Bandyopadhyay B, et al. 2-5A antisence directed against telomerase RNA produces apoptosis in ovarian cancer cells. Gynecol Oncol 2000; 76:183-192.
165. Counter CM, Meyerson M, Eaton EN, et al. Telomerase activity is restored in human cells byectopic expression of hTERT(hEST2), the catalytic subunit of telomerase. Oncogene 1998;16:1217.
166. Bryce LA, Morrison N, Hoare SF, et al. Mapping of the gene for the human telomerase reverse transcriptase, hTERT, to chromosome 5p15. 33 by fluorescence in situ hybridization. Neoplasia 2000;2:197-201.
167. Szutorisz H, Linguer J, Cuthbert AP, et al. A chromosome 3 encoded repressor of the human telomerase reverse transcriptase (hTERT) gene controls the state of hTERT chromatin. Cancer Res 2003;63:689-695.
168. Oh S, Song YH, Yim J, et al. The films' tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene. J Biol Chem 1999;274:37473-37478.
169. Oh S, Song YH, Yim J, et al. Identification of Mad as a repressor of the human telomerase(hTERT) gene. Oncogene 2000;19:1485-1490.
170. Bergmann L, Miething C, Maurer U, et al. High levels of Wilms' tumor gene(wt1) mRNA in acute myeloid leukemias are associated with a worse long-term outcome. Blood 1997; 90:1217-1225.
171. Ostergaard M, Olesen LH, Hasle H, et al. WT1 gene expression: an excellent tool for monitoring minimal residual disease in 70% of acute myeloid leukaemia patients-results from a single-centre stugy. Br J Haematol 2004;125:590-600.
172. Galimberti S, Guerrini F, Carulli G, et al. Significant co-expression of WT1 and MDR1 genes in acute myeloid leukemia patients at diagnosis. Eur J Haematol 2004;72:45-51.
173. Sekiya M, Adachi M, Hinoda Y, et al. Downregulation of Wilms' tumor gene(wt1) during myelomonocytic differentiation in HL60 cells. Blood 1994;83:1876-1882.
174. Tamaki H, Ogawa H, Ohyashiki K, et al. The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes. Leukemia 1999;13:393-399.
175. Patmasiriwat P, Fraizer G, Kantarjian H, et al.WTl and GATA1 expression in myelodysplastic syndrome and acute leukemia. Leukemia 1999;3:891-900.
176. Yin L, Hubbard AK, Giardina C. NF-kappa B regulates transcription of the mouse telomerase catalytic subunit. J Biol Chem 2000;275:36671-36675.
177. Akiyama M, Hideshima T, Hayashi T, et al. Nuclear factor-kappaB p65 mediates tumor necrosis factor alpha-induced nuclear translocation of telomerase reverse transcriptase protein. Cancer Res 2003;63:18-21.
178. Sinha-Datta U, Horikawa I, Michishita E, et al. Transcriptional activation of hTERT through the NF-kappaB pathway in HTLV-I-transformed cells. Blood 2004;104:2523-2531.
179. Akiyama M , Yamada O, Hideshima T, et al. TNFalpha induces rapid activation and nuclear translocation of telomerase in human lymphocytes. Biochem Biophys Res Commun 2004;316: 528-532.
1.Steensma DP,Bennett JM:The myelodysplastic syndromes:diagnosis and treatment.Mayo C1in Proc 2006;81:104-130.
2.Cazzola M,Malcovati L.Myelodysplastic syndromes-coping with ineffective hematopoiesis.N Engl J Med.2005;352:536-538.
3.Richard Z.Shadduck,Joan M.et al.Recent advances in myelodysplastic syndromes.Experimental Hematology 2007;35:137-143.
4.Inokuchi K:Chronic myelogenous leukemia:from molecular biology to clinical aspects and novel targeted therapies.J Nippon Med Sch 2006;73:178-192.
5.Warner JK,Wang JC,Hope KJ,et al.Concepts of human leukemic development.Oncogene 2004;23:7164-7177.
6.Nilsson L,Astrand-Grundstrom I,Anderson K,et at.:Involvement and functional impairment of the CD34(+)CD38(-)Thy-1(+)hematopoietic stem cell pool in myelodysplastic Syndromes with trisomy 8.Blood 2002;100:259-267.
7.Thanopoulou E,Cashman J,Kakagianne T,et al.Engraftment of NOD_SCIDbeta2microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.Blood 2004;103:4285-4293.
8.Young NS,SloandEM,Barrett J.Myelodysplastic syndrome.In:Young NS,Gerson SL,High KS,eds.Clinical Hematology.New York:Elsevier Health Sciences;2005.p.203-230.
9.Sloand EM,Mainwaring L,Fuhrer M,et at.:Preferential suppression of trisomy 8 compared with normal hematopoietic cell growth by autologous lymphocytes in patients with trisomy 8 myelodysplastic syndrome.Blood 2005;106:841-851.
10.Sloand EM,Ramkissoon S,Mainwaring L,et al.Granulocyte colony stimulating factor preferentially causes proliferation of pre-existing monosomy 7 cells in aplastic anemia and myelodysplastic syndrome because of abnormal truncated GCSF-receptors on these cells [abstract]. Blood. 2004;104:Abstract 458.
11. Fenaux P: Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol 2001;73: 429-437.
12. Kaiser J: First pass at cancer genome reveals complex landscape. Science 2006; 313: 1370.
13. Esteller M: Epigenetics provides a new generation of oncogenes and tumour-suppressor genes. Br J Cancer 2006; 94: 179—183.
14. Navas TA, Mohindru M, Estes M, et al. : Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors. Blood 2006; 108:4170-4177.
15. Shih LY, Lin TL, Wang PN, et al. Internal tandem duplication of fms-like tyrosine kinase 3 is associated with poor outcome in patients with myelodysplastic syndrome. Cancer 2004; 101:989-998.
16. Speranza A, Pellizzaro C, Coradini D. Hyaluronic acid butyric esters in cancer therapy. Anticancer Drugs 2005; 16:373- 379.
17. Choudhary C, Schwable J, Brandts C, et al. AML-associated Flt3 kinase domain mutations show signal transduction differences in comparison to Flt3 ITD mutations. Blood 2005; 106:265-273.
18. Levis M, Murphy KM, Pham R, et al. Internal tandem duplications of the FLT3 gene are present in leukemia stem cells. Blood 2005; 106:673-680.
19. ChalandonY, Schwaller J. Targeting mutated proteinbtyrosine kinases and their signaling pathways in hematologic malignancies. Haematologica 2005; 90:949-968.
20. Giles FJ, Stopeck AT, Silverman LR, et al. SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplasticsyndromes. Blood 2003; 102:795-801.
21. Chen J, Lee BH, Williams IR, et al. FGFR3 as a therapeutic target of the small molecule inhibitor PKC412 in hematopoietic malignancies. Oncogene 2005; 24: 8259-8267.
22. Gotlib J, Berube C, Growney JD, et al. Activity of the tyrosine kinase inhibitor PKC412 in a patient with mast cell leukemia with the D816V KIT mutation. Blood 2005; 106:2865-2870.
23. GrowneyJD, Clark JJ, Adelsperger J, et al. Activation mutations of human c-KIT resistant to imatinib are sensitive to the tyrosine kinase inhibitor PKC412. Blood 2005; 106:721-724.
24. Stone RM, DeAngelo DJ, Klimek V, et al. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 2005; 105:54-60.
25. Kurzrock R, Albitar M, Cortes JE, et al. Phase II study of R115777, a farnesyl transferase inhibitor, in myelodysplastic syndrome. J Clin Oncol 2004;22: 1287 - 1292.
26. Kurzrock R, Fenaux P, Raza A, et al. High-Risk Myelodysplastic Syndrome (MDS): first results of international phase 2 study with Oral Farnesyltransferase Inhibitor R115777 (ZARNESTRA). Blood 2004; 104:23a.
27. Ravoet C, Mineur P, Robin V, et al. Phase I—II study of a Farnesyl Transf erase Inhibitor (FTI), SCH66336, in patients with Myelodysplastic Syndrome (MDS) or Secondary Acute Myeloid Leukemia (sAML). Blood 2002; 100:794a.
28. Cortes J, Faderl S, Estey E, et al. Phase I study of BMS- 214662, a farnesyl transferase inhibitor in patients with acute leukemias and high-risk myelodysplastic syndromes. J Clin Oncol 2005; 23:2805-2812.
29. Alexandrakis MG, Passam FH, Pappa CA, et al. Serum evaluation of angiogenic cytokine basic fibroblast growth factor, hepatocyte growth factor and TNF - alpha in patients with myelodysplastic syndromes: correlation with bone marrow microvascular density. Int J Immunopathol Pharmacol 2005;18:287-295.
30. Alexandrakis MG, Passam FH, Pappa CA, et al. Relation between bone marrow angiogenesis and serum levels of angiogenin in patients with myelodysplastic syndromes. Leuk Res 2005;29:41 -46.
31. Hu Q, Dey AL, Yang Y, et al. Soluble vascular endothelial growth factor receptor 1, and not receptor 2, is an independent prognostic factor in acute myeloid leukemia and myelodysplastic syndromes. Cancer 2004;100:1884- 1891.
32. Ribatti D, Vacca A. Therapeutic renaissance of thalidomide in the treatment of haematological malignancies. Leukemia 2005; 19:1525-1531.
33. Bouscary D, Quarre MC, Vassilief D, et al. Thalidomide for the treatment of Low Risk Myelodysplasia. The Thal-SMD-200 Trial from the French Group of Myelodysplasia (GFM). Blood 2004; 104:403a.
34. Koh KR, Janz M, Mapara MY, et al. Immunomodulatory derivative of thalidomide (IMiD CC-4047) induces a shift in lineage commitment by suppressing erythropoiesis and promoting myelopoiesis. Blood 2005; 105:3833-3840.
35. List A, Kurtin S, Roe DJ, et al. Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 2005; 352:549-557.
36. Cazzola M, Malcovati L. Myelodysplastic syndromescoping with ineffective hematopoiesis. N Engl J Med 2005; 352:536-538.
37. Paz K, Zhu Z. Development of angiogenesis inhibitors to vascular endothelial growth factor receptor 2. Current status and future perspective. Front Biosci 2005; 10:1415-1439.
38. Ferrara N, Hillan KJ, Novotny W. Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochem Biophys Res Commun 2005; 333:328-335.
39. .Giles FJ, Steinfeldt H, Bellamy WT, et al. Phase 2 study of the Anti-Angiogenesis Agent AG-013736 in patients with Poor Prognosis Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS). Blood 2004; 104:502a.
40. Roboz GJ, List AF, Giles F, et al. Phase I Trial of PTK787/ZK 222584, an inhibitor Of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases, In Acute Myeloid Leukemia and Myelodysplastic Syndrome. Blood 2002; 100:337a.
41. Ellis LM . Bevacizumab. Nat Rev Drug Discov (Suppl) 2005:S8-S9.
42. Hasegawa D, Manabe A, Kubota T, et al. Methylation status of the pl5 and pl6 genes in paediatric myelodysplastic syndrome and juvenile myelomonocytic leukaemia. Br J Haematol 2005; 128:805-812.
43. Johan MF, Bowen DT, Frew ME, et al .Aberrant methylation of the negative regulators RASSFIA, SHP-1 and SOCS-1 in myelodysplastic syndromes and acute myeloid leukaemia. Br J Haematol 2005; 129:60-65.
44. Langer F, Dingemann J, Kreipe H, et al. Upregulation of DNA methyltransferases DNMT1, 3A, and 3B in myelodysplastic syndrome. Leuk Res 2005; 29:325-329.
45. Kaminskas E, Farrell A, Abraham S, et al. FDA Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 2005; 11: 3604 - 3608.
46. Oka M, Meacham AM, Hamazaki T, et al. De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'- deoxycytidine. Oncogene 2005; 24:3091-3099.
47. van den Bosch J, Lubbert M, Verhoef G, et al. The effects of 5-aza-2'-deoxycytidine (Decitabine) on the platelet count in patients with intermediate and high-risk myelodysplastic syndromes. Leuk Res 2004; 28:785 - 790.
48. Issa JP, Garcia-Manero G, Giles FJ, et al. Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2' -deoxycytidine (decitabine) in hematopoietic malignancies. Blood 2004; 103:1635-1640.
49. Saba H, Rosenfeld C, Issa J-P, et al. First report of the Phase III North American Trial of Decitabine in Advanced Myelodysplastic Syndrome (MDS). Blood 2004; 104:23a.
50. Lubbert M, Wijermans, PW, Ruter, BH. Re-Treatment with Low-Dose 5-Aza-2' -Deoxycytidine (Decitabine) results in second remissions of previously responsive MDS patients. Blood 2004; 104:405a.
51. Ben-Kasus T, Ben-Zvi Z, Marquez VE, et al. Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Biochem Pharmacol 2005; 70:121-133.
52. Guo H, Rao N, Xu Q, et al. Origin of tight binding of a near-perfect transition-state analogue by cytidine deaminase: implications for enzyme catalysis. J Am Chem Soc 2005; 127:3191-3197.
53. Holleran JL, Parise RA, Joseph E, et al. Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine. Clin Cancer Res 2005; 11:3862-3868.
54. Drummond DC, Noble CO, Kirpotin DB, et al. Clinical development of histone deacetylase inhibitors as anticancer agents. Annu Rev Pharmacol Toxicol 2005; 45:495-528.
55. Kuendgen A, Strupp C, Aivado M, et al. Treatment of myelodysplastic syndromes with valproic acid alone or in combination with all-trans retinoic acid. Blood 2004; 104:1266-1269.
56. Garcia-Manero G, Kantarjian H, Sanchez-Gonzalez B, et al. Results of a Phase I/II study of the combination of 5-aza-2-deoxycytidine (DAC) and Valproic Acid (VPA) in patients (pts) with Leukemia. Blood 2004; 104:78a.
57. Giles FJ, Fischer T, Cortes J, et al. A Phase I/II Study of intravenous LBH589, a novel histone deacetylase (HDAC) inhibitor, in patients (pts) with advanced hematologic malignancies. Blood 2004; 104:499a.
58. Michael A. Morgan. Christoph W. M. Reuter. Molecularly targeted therapies in myelodysplastic syndromes and acute myeloid leukemias. Ann Hematol 2006; 85: 139 - 163.
1 Ravandi F,Talpaz M,Estrov Z.Modulation of cellular signaling pathways:prospects for targeted therapy in hematological malignancies.Clin Cancer Res 2003;9:535-550.
2 Krause DS,Van Etten RA.Tyrosine kinases as targets for cancer therapy.N Engl J Med 2005; 353: 172-187.
3 Mizuki M, Ueda S, Matsumura I et al. Oncogenic receptor tyrosine kinase in leukemia. Cell Mol Biol 2003; 49: 907-922.
4 Abe A, Emi N, Tanimoto M et al. Fusion of the platelet-derived growth factor receptor beta to a novel gene CEV14 in acute myelogenous leukemia after clonal evolution. Blood 1997; 90: 4271-4277.
5 Cools J, DeAngelo DJ, Gotlib J et al. A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome. N Engl J Med 2003; 48: 1201-1214.
6 Curtis CE, Grand FH, Musto P et al. Two novel imatinib-responsive PDGFRA fusion genes in chronic eosinophilic leukaemia. Br J Haematol 2007; 138: 77-81.
7 Score J, Curtis C, Waghorn K et al. Identification of a novel imatinib responsive KIF5B-PDGFRA fusion gene following screening for PDGFRA overexpression in patients with hypereosinophilia. Leukemia 2006; 20: 827-832.
8 Safley AM, Sebastian S, Collins TS et al. Molecular and cytogenetic characterization of a novel translocation t (4;22) involving the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with atypical chronic myeloid leukemia. Genes Chromosomes Cancer 2004; 40: 44-50.
9 Cross NC, Reiter A. Tyrosine kinase fusion genes in chronic myeloproliferative diseases. Leukemia 2002; 16: 1207-12.
10 Chesi E, Nardini LA, Brents E et al. Frequent translocation t (4;14) (p16. 3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 1997; 16: 260-264.
11 Yagasaki D, Wakao Y, Yokoyama Y et al. Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t (4; 12) (p16;p13) chromosomal translocation, Cancer Res 2001; 61: 8371-4.
12 Furitsu T, Tsujimura T, Tono T et al. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product. J Clin Invest 1993; 92: 1736-44.
13 Tsujimura T, Morimoto M, Hashimoto K et al. Constitutive activation of ckit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain. Blood 1996; 87: 273-83.
14 London CA, Galli SJ, Yuuki T. et al. Spontaneous canine mast cell tumors express tandem duplications in the proto-oncogene c-kit. Exp Hematol 1999;27: 689-697.
15 Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998; 279: 577-580.
16 Nishida T, Hirota S, Taniguchi M. et al. Familial gastrointestinal stromal tumours with germline mutation of the KIT gene. Nat Genet 1998; 19: 323-324.
17 Heinrich MC, Corless CL, Duensing A et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708-710.
18 Nakao M, Yokota S, Iwai T et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911-1918.
19 Kiyoi H, Naoe T, Nakano Y et al. Prognostic implication of FLT3 and NRAS gene mutations in acute myeloid leukemia. Blood 1999; 93: 3074-3080.
20 Irusta PM, Luo Y, Bakht 0 et al. Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor. J Biol Chem 2002; 277: 38627-38634.
21 Ma Y, Cunningham ME, Wang X et al. Inhibition of spontaneous receptor phosphorylation by residues in a putative alpha-helix in the KIT intracellular juxtamembrane region. J Biol Chem 1999; 274: 13399-133402.
22 Kitayama H, Kanakura Y, Furitsu T et al. Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood 1995; 85: 790 - 798.
23 Kiyoi H, Towatari M, Yokota S et al. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia 1998; 12: 1333-1337.
24 Longley BJ Jr, Metcalfe DD, Tharp M et al. Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci USA 1999; 96: 1609-1614.
25 Nagata H, Worobec AS, Oh CK. et al. Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci USA 1995; 92: 10560-10564.
26 Worobec AS, Semere T, Nagata H et al. Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis. Cancer 1998; 83: 2120-2129.
27 Carre RS, Goodeve A, Abu-Duhier FM et al. Incidence and prognosis of ckit and Flt3 mutations in core binding factor (CBF) acute myeloid leukemia. Blood 2002: 746a.
28 Zwaan CM, Miller M, Goemans BF et al. Frequency and clinical significance of c-kit exon 17 mutations in childfood acute myeloid leukemia. Blood 2002: 746a.
29 Abu-Duhier FM, Goodeve AC, Wilson GA et al. Identification of novel FLT- 3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol 2001; 113: 983-988.
30 Yamamoto Y, Kiyoi H, Nakano Y et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97: 2434 - 2439.
31 Spiekermann K, Bagrintseva K, Schoch C et al. A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. Blood 2002; 100: 3423 - 3425.
32 Mead AJ, Linch DC, Hills RK et al. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood 2007; 110: 1262-1270.
33 Moriyama Y, Tsujimura T, Hashimoto K et al. Role of aspartic acid 814 in the function and expression of c-kit receptor tyrosine kinase. J Biol Chem 1996; 271: 3347 - 3350.
34 Tsujimura T, Hashimoto K, Kitayama H et al. Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self association not at the ligand-induced dimerization site. Blood 1999; 93: 1319-1329.
35 Hashimoto K, Matsumura I, Tsujimura T et al. Necessity of tyrosine 719 and phosphatidylinositol 3' -kinase-mediated signal pathway in constitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the Asp814Val mutation. Blood 2003; 101: 1094-1102.
36 Gari M, Goodeve A, Wilson G et al. c-kit proto-oncogene exon 8 in-frame deletion plus insertion mutations in acute myeloid leukaemia. Br J Haematol 1999; 105: 894 - 900.
37 Care RS, Valk PJ, Goodeve AC et al. Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias. Br J Haematol 2003; 121: 775-777.
38 Boissel N, Leroy H, Brethon B et al. Incidence and prognostic impact of c- Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML). Leukemia 2006; 20: 965-970.
39 Ridge SA, Worwood M, Oscier D et al. FMS mutations in myelodysplastic, leukemic, and normal subjects. Proc Natl Acad Sci USA 1990; 87: 1377-1380.
40 Tobal K, Pagliuca A, Bhatt B et al. Mutation of the human FMS gene (MCSF receptor) in myelodysplastic syndromes and acute myeloid leukemia. Leukemia 1990; 4: 486 - 489.
41 Roussel MF, Downing JR, Rettenmier CW et al. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene product) activates its transforming potential. Cell 1988; 55: 979-988.
42 Plotnikov AN, Schlessinger J, Hubbard SR et al. Structural basis for FGF receptor dimerization and activation. Cell 1999; 98: 641-650.
43 Lancet JE, Karp JE. Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy. Blood 2003; 102: 3880-3889.
44 Morgan MA, Ganser A, Reuter CW. Therapeutic efficacy of prenylation inhibitors in the treatment of myeloid leukemia. Leukemia 2003; 17: 1482-1498.
45 Hayakawa F, Towatari M, Kiyoi H. et al. Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines. Oncogene 2000; 19: 624-631.
46 MizukiM, Fenski R, Halfter H et al. Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 2000; 96: 3907-3914.
47 Chian R, Young S, Danilkovitch-Miagkova A et al. Phosphatidylinositol 3 kinase contributes to the transformation of hematopoietic cells by the D816V c-Kit mutant. Blood 2001; 98: 1365-1373.
48 Blume-Jensen P, Jiang G, Hyman R et al. Kit/stem cell factor receptorinduced activation of phosphatidylinositol 3' -kinase is essential for male fertility. Nat Genet 2000; 24: 157-162.
49 Kissel H, Timokhina I, Hardy MP et al. Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses. EMBO J 2000; 19: 1312-1326.
50 Rane SG, Reddy EP. JAKs, STATs and Src kinases in hematopoiesis. Oncogene 2002; 21: 3334-3358.
51 Darnell JE Jr. STATs and gene regulation. Science 1997; 277: 1630-1635.
52 Ning ZQ, Li J, Arceci RJ. Signal transducer and activator of transcription 3 activation is required for Asp (816) mutant c-Kit-mediated cytokineindependent survival and proliferation in human leukemia cells. Blood 2001; 97: 3559-3567.
53 Ning ZQ, Li J, McGuinness M et al. STAT3 activation is required for Asp (816) mutant c-Kit induced tumorigenicity. Oncogene 2001; 20: 4528-4536.
54 Sternberg DW, Tomasson MH, Carroll M et al. The TEL/PDGFbetaR fusion in chronic myelomonocytic leukemia signals through STAT5-dependent and STAT5-independent pathways. Blood 2001; 98: 3390-3397.
55 Wilbanks AM, Mahajan S, Frank DA et al. TEL/PDGFbetaR fusion protein activates STAT1 and STAT5: a common mechanism for transformation by tyrosine kinase fusion proteins. Exp Hematol 2000; 28: 584-593.
56 Mizuki M, Schwable J, Steur C et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations. Blood 2003; 101: 3164-3173.
57 Watanabe D, Ezoe S, Fujimoto M et al. Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol 2004; 126: 726-735.
58 Magnusson MK, Meade KE, Nakamura R et al. Activity of STI571 in chronic myelomonocytic leukemia with a platelet- derived growth factor β receptor fusion oncogene. Blood 2002; 100: 1088-1091.
59 Levis M, Tse KF, Smith BD et al. FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations. Blood 2001; 98, 885-887.
60 Tse KF, Novelli E, Civin CI et al. Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia 2001; 15: 1001-1010.
61 Kurzrock R, Sebti SM, Kantarjian HM et al. Phase I Study of a Farnesyl Transferase Inhibitor. R115777, in Patients with Myelodysplastic Syndrome. Blood 2001; 98: 623a.
62 Propper DJ, McDonald AC, Man A et al. Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C. J Clin Oncol 2001; 19: 1485- 1492.
63 Sebolt-Leopold JS. Development of anticancer drugs targeting the MAP kinase pathway. Oncogene 2000; 19: 6594-6599.
64 Witzig TE, Kaufmann SH. Inhibition of the phosphatidylinositol 3-kinase/ mammalian target of rapamycin pathway in hematologic malignancies. Curr Treat Options Oncol 2006; 7: 285-294.
65 Smolewski P. Recent developments in targeting the mammalian target of rapamycin (mTOR) kinase pathway. Anticancer Drugs 2006; 17: 487-494.