阿克拉霉素对骨髓增生异常综合征细胞株（RAEB型）细胞作用的体外实验研究
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摘要
众所周知，骨髓增生异常综合征(Myelodysplastic syndrome，MDS)难治，尤其是高危MDS。原因可能与MDS恶性克隆对药物作用的敏感性降低有关。目前，MDS的治疗目的是消除或减少瘤细胞克隆，部分或完全重建正常造血，或诱导异常细胞向正常细胞分化成熟。为研究MDS细胞对药物治疗的反应，我们引进了对各种常用的分化诱导剂均无反应的MDS-RAEB型细胞株MUTZ-1。已有研究表明，以拓扑异构酶Ⅱ为作用靶点的第二代蒽环类抗肿瘤药物——阿克拉霉素(aclacinomycin，ACM)治疗某些MDS患者有效。体外实验研究表明，与其它的抗肿瘤剂相比，更不容易产生耐药性，其机理尚不明确。本文旨在研究ACM对MDS-RAEB型细胞株MUTZ-1细胞的体外治疗效应及其新的作用机理。试图证明ACM可诱导MUTZ-1细胞凋亡及(或)向成熟分化，以减少MDS恶性克隆；并试图阐明ACM抗MDS细胞的又一分子机制，从而为克服MDS细胞对化疗药物的耐受寻找新的治疗靶点，为设计新的治疗策略拓宽思路。
采用MTT比色法检测结果显示，0.05μmol/L ACM处理48h，即明显抑制MUTZ-1细胞的增殖，与空白对照组相比，差别有统计学意义(p＜0.05)。ACM作用48h的半数抑制浓度(IC_(50))为0.46μmol/L。为探讨这种抑制作用的机制，我们从细胞凋亡及细胞分化两个层面作了进一步的研究。
在体外，本研究采用瑞特-姬姆萨染色，光学显微镜观察ACM作用前后MUTZ-1细胞形态，结果显示，0.5μmol/L ACM处理后，MUTZ-1细胞出现凋亡细胞典型的形态学特征，
浙江大学博士学位论文
如胞浆空泡化，细胞体积缩小，核染色质固缩，核边聚，染色质断裂，出芽，凋亡小体形
成等（图I－2）。
为了对细胞凋亡现象进行准确地定性或定量分析，我们采用多种经典、特异而敏感的
实验指标，并利用近年来发展起来的流式细胞分析技术研究了细胞DNA含量、亚二倍体
及磷脂酞丝氨酸（PS）转位。结果显示，0．25Pmol／L－0．SPmol／L ACM作用 48h，可见典型
的 DNA梯形条带：采用 DNA片断原位末端（TLINEL法）标记方法证明，在一定的作用
时间点，随ACM浓度的增加，TUNEL阳性细胞也增加；一定的药物浓度范围内，随ACM
作用时间延长，TUNEL阳性细胞增加。从生物化学角度进一步证明，在体外，ACM能诱导
MUTZ—l细胞凋亡，且呈剂量一，时间一效应依赖关系。
采用流式细胞仪分析技术探讨了ACM作用前后MUTZ－1细胞DNA含量及亚二倍体的
改变。结果显示，0．in mol／L l．2卜mol／L ACM处理后，MUTZ－1细胞周期 G。／M期细胞增
多，GllG。期细胞减少，细胞被阻滞于GZ／M期。同时亚二倍体细胞增多，MUTZ－l细胞亚
GI期峰更明显（图1．小表明，ACM可能通过阻滞MUTZ－l细胞于细胞周期的GZ／M期，最
终导致MUTZ－l细胞凋亡。进一步采用AnnexinV－PI双标记法流式细胞仪分析，结果显示，
o．印（、！几＊＊M作用36h，早期凋亡细胞百分数（2！．83叨高于空白对照组（3．n叨；晚期凋
亡细胞在处理组仅稍高于空白对照组（厂9％VS 2．65％）。表明，一定浓度 ACM作用一定时
间，主要是诱导MUTZI细胞发生早期凋亡（图1．8）。
为研究ACM诱导MUTZ－！细胞凋亡的可能机制及信号传导途径，本研究采用转染报
告基因载体pNF。B－SEAP后化学发光测定法分析ACM作用前、后核转录因子NF。B活性
水平，并用 RT－PCR法在转录水平检测其调控的靶基因 clAPI及 clAPZ mRNA表达的改变；
WCstern blot法检测 ACM作用前、后 NF。B抑制蛋白质 I。B a水平表达的改变，并探讨了
ACM较少产生耐药性的可能原因。
研究结果显示，一定浓度 ACM（0．5 u mol／L）处理 MUTZ－l细胞不同时间（3h、6h、12h、
24h），导致了抗凋亡基因 clAP－l、cIAPZ mRNA表达水平以时间依赖的方式降低，并与
MUTZ－l细胞TUNEL阳性细胞百分数呈负相关（rs－0．991，－0．975）；不同浓度ACM（0三卜mol／L
十0 u mol／L）处理 24h，可引起 MUTZ－l细胞 cIAPI、cIAPZ mRNA表达以浓度依赖的方式
下调，并与其TUNEL阳性细胞百分数呈负相关（，－0．984，－0．951）。说明ACM诱导MUTZ－l
细胞凋亡与抗凋亡基因 clAPI、cIAPZ mRNA表达下调有关。
3
浙江大学博土学位论文
迢过瞬时转染报告基因载体pNF。B－SEAP后化学发光法检测培养上清液中报告基因
表达产物 SEAP活性，其活性代表相应转录因于 NF。B活性。结果显示，0．sapOUL ACM
作用 3h，MUTZ一！细胞 SEAP活性（8．72土 2．88）较同一时间点空白对照组（1184士 3．11）的为
低（cd．2 12，Pq）刀42）；0．SPmol／L ACM作用 6h时，MUTZ—l细胞 SEAP活性与在相应
时间点的空白对照间的差异非常显著（P＜0刀1），0．5卜m＊儿＊＊M作用至24卜时，细胞％AP
活性己降至相应空白对照组的二．9％。因此，0．5卜mol／L ACM作用 MUTZ—l细胞 3－24h，
以时间依赖方式抑制报告基因SEAP活性，当0门卜mol／L ACM处理24h，MUTZ—l细胞
SEAP活性（11．84士2．67?
It's well known that patients with MDS, especially with high-risk MDS, have poor prognosis, which may be related to decreased sensibility of MDS malignant clone to drugs. The aim of current therapy of MDS is to reduce, or even erase the malignant clone, reconstruct normal hematopoiesis partly or completely, and induce the abnormal cells differentiation into normal hematopoietic cells. In order to investigate the responses of MDS cells to drugs, a cell line MUTZ-1, derived from a patient with MDS-RAEB. has been introduced to our series, which has no response to traditional differentiation-inducing drugs. It's reported that aclacinomycin ( ACM ), the second-generation anthracyclines targeting at topoisomerase II, can be used to treat some MDS patients and acquire some
response. Compared with other anti-tumor drugs, ACM doesn't tend to cause drug resistance in experimental study in vitro, but the mechanisms remain unknown. The aim of this paper is to investigate the effect of ACM on MUTZ-1 cell line derived from MDS-RAEB and its new mechanism, try to prove that ACM can induce MUTZ-1 cells afoptosis and (or) differentiation and maturation, so as to reduce the malignant clone of MDS, and try to clarify another new molecular mechanism of ACM on MDS cells. Thereby it
would help to find a new treatment target and develop new strategy to overcome the drug resistance of MDS cells to chemotherapeutic agents.
Using MTT assay we investigated the effect of ACM on MUTZ-1 growth. The result showed that following treatment with ACM(0.05 μmol/L) for 48 h, the proliferation of MUTZ-1 cells was inhibited significantly, in comparison with the blank control group(P<0.05). IC50 was 0.46μ mol/L at 48h. To explore the possible mechanism of the inhibition of ACM on MUTZ-1 cells, further investigation was carried out from the two stratifications of inducing apoptosis and differentiation.
Using Wright-Giemsa stain, the morphology of MUTZ-1 cells was observed with or without ACM treatment under a light microscope. The result displayed that a series of typical morphological features of apoptosis were observed following treatment with ACM(0.5μmol/L) for 48h, such as vacuolization of cell plasma, shrinkage of cell and nucleus, condensation of nuclear chromatin, marginated against the nuclear membranes, karyorrhexis and convolution of
nuclear, and membrane-bound apoptotic bodies(Figi.2).
In order to exactly analyze the cell apoptosis quantitatively and qualitatively, in addition to
some classic and specific and sensitive methods, the flow cytometry was also used to evaluate the DNA content, percentage of hypodiploid and translocation of phosphatidyl serine( PS). It showed that following treatment with 0.25umol/L-0.5nmol/L ACM for 48h, a typical DNA ladder was observed. With TdT mediated-dUTP nick end labeling (TUNEL), it appeared that with the increasing concentration of ACM at a certain time-point, the TUNEL positive cells increased. Meanwhile, with the increasing treatment time at a certain concentration of ACM, the TUNEL positive cells increased. From biochemistry, it was proved that ACM could induce MUTZ-1 cells apoptosis with dose- and time- dependent manners in vitro.
With the help of flow cytometry, the DNA content and hypodiploid changes of MUTZ-1 cells were observed with or without ACM treatment. The results showed that" the percentage of MUTZ-1 cells in the G2/M phase of the cell cycle increased, while in the G1/G0 phase decreased
following treatment with 0.1μ mol/L 0.2μmoI/L ACM, indicating that MUTZ-1 cells were arrested in the Ga/M phase. At the same time, hypodiploid cells increased, and the sub-G| peak appeared (Fig1-7). This manifested that ACM at a certain concentration could arrest MUTZ-1 cells in the G2/M phase of cell cycle, and then induced the cells apoptosis. With the help of flow cytometry following annexinV-PI staining, exposure to 0.5nmol/L ACM for 36 h caused 21.83% early apoptotic cells, which was more than that of the untreated group (3.47%), while late apoptovic cells increased only sligh
采用MTT比色法检测结果显示，0.05μmol/L ACM处理48h，即明显抑制MUTZ-1细胞的增殖，与空白对照组相比，差别有统计学意义(p＜0.05)。ACM作用48h的半数抑制浓度(IC_(50))为0.46μmol/L。为探讨这种抑制作用的机制，我们从细胞凋亡及细胞分化两个层面作了进一步的研究。
在体外，本研究采用瑞特-姬姆萨染色，光学显微镜观察ACM作用前后MUTZ-1细胞形态，结果显示，0.5μmol/L ACM处理后，MUTZ-1细胞出现凋亡细胞典型的形态学特征，
浙江大学博士学位论文
如胞浆空泡化，细胞体积缩小，核染色质固缩，核边聚，染色质断裂，出芽，凋亡小体形
成等（图I－2）。
为了对细胞凋亡现象进行准确地定性或定量分析，我们采用多种经典、特异而敏感的
实验指标，并利用近年来发展起来的流式细胞分析技术研究了细胞DNA含量、亚二倍体
及磷脂酞丝氨酸（PS）转位。结果显示，0．25Pmol／L－0．SPmol／L ACM作用 48h，可见典型
的 DNA梯形条带：采用 DNA片断原位末端（TLINEL法）标记方法证明，在一定的作用
时间点，随ACM浓度的增加，TUNEL阳性细胞也增加；一定的药物浓度范围内，随ACM
作用时间延长，TUNEL阳性细胞增加。从生物化学角度进一步证明，在体外，ACM能诱导
MUTZ—l细胞凋亡，且呈剂量一，时间一效应依赖关系。
采用流式细胞仪分析技术探讨了ACM作用前后MUTZ－1细胞DNA含量及亚二倍体的
改变。结果显示，0．in mol／L l．2卜mol／L ACM处理后，MUTZ－1细胞周期 G。／M期细胞增
多，GllG。期细胞减少，细胞被阻滞于GZ／M期。同时亚二倍体细胞增多，MUTZ－l细胞亚
GI期峰更明显（图1．小表明，ACM可能通过阻滞MUTZ－l细胞于细胞周期的GZ／M期，最
终导致MUTZ－l细胞凋亡。进一步采用AnnexinV－PI双标记法流式细胞仪分析，结果显示，
o．印（、！几＊＊M作用36h，早期凋亡细胞百分数（2！．83叨高于空白对照组（3．n叨；晚期凋
亡细胞在处理组仅稍高于空白对照组（厂9％VS 2．65％）。表明，一定浓度 ACM作用一定时
间，主要是诱导MUTZI细胞发生早期凋亡（图1．8）。
为研究ACM诱导MUTZ－！细胞凋亡的可能机制及信号传导途径，本研究采用转染报
告基因载体pNF。B－SEAP后化学发光测定法分析ACM作用前、后核转录因子NF。B活性
水平，并用 RT－PCR法在转录水平检测其调控的靶基因 clAPI及 clAPZ mRNA表达的改变；
WCstern blot法检测 ACM作用前、后 NF。B抑制蛋白质 I。B a水平表达的改变，并探讨了
ACM较少产生耐药性的可能原因。
研究结果显示，一定浓度 ACM（0．5 u mol／L）处理 MUTZ－l细胞不同时间（3h、6h、12h、
24h），导致了抗凋亡基因 clAP－l、cIAPZ mRNA表达水平以时间依赖的方式降低，并与
MUTZ－l细胞TUNEL阳性细胞百分数呈负相关（rs－0．991，－0．975）；不同浓度ACM（0三卜mol／L
十0 u mol／L）处理 24h，可引起 MUTZ－l细胞 cIAPI、cIAPZ mRNA表达以浓度依赖的方式
下调，并与其TUNEL阳性细胞百分数呈负相关（，－0．984，－0．951）。说明ACM诱导MUTZ－l
细胞凋亡与抗凋亡基因 clAPI、cIAPZ mRNA表达下调有关。
3
浙江大学博土学位论文
迢过瞬时转染报告基因载体pNF。B－SEAP后化学发光法检测培养上清液中报告基因
表达产物 SEAP活性，其活性代表相应转录因于 NF。B活性。结果显示，0．sapOUL ACM
作用 3h，MUTZ一！细胞 SEAP活性（8．72土 2．88）较同一时间点空白对照组（1184士 3．11）的为
低（cd．2 12，Pq）刀42）；0．SPmol／L ACM作用 6h时，MUTZ—l细胞 SEAP活性与在相应
时间点的空白对照间的差异非常显著（P＜0刀1），0．5卜m＊儿＊＊M作用至24卜时，细胞％AP
活性己降至相应空白对照组的二．9％。因此，0．5卜mol／L ACM作用 MUTZ—l细胞 3－24h，
以时间依赖方式抑制报告基因SEAP活性，当0门卜mol／L ACM处理24h，MUTZ—l细胞
SEAP活性（11．84士2．67?
It's well known that patients with MDS, especially with high-risk MDS, have poor prognosis, which may be related to decreased sensibility of MDS malignant clone to drugs. The aim of current therapy of MDS is to reduce, or even erase the malignant clone, reconstruct normal hematopoiesis partly or completely, and induce the abnormal cells differentiation into normal hematopoietic cells. In order to investigate the responses of MDS cells to drugs, a cell line MUTZ-1, derived from a patient with MDS-RAEB. has been introduced to our series, which has no response to traditional differentiation-inducing drugs. It's reported that aclacinomycin ( ACM ), the second-generation anthracyclines targeting at topoisomerase II, can be used to treat some MDS patients and acquire some
response. Compared with other anti-tumor drugs, ACM doesn't tend to cause drug resistance in experimental study in vitro, but the mechanisms remain unknown. The aim of this paper is to investigate the effect of ACM on MUTZ-1 cell line derived from MDS-RAEB and its new mechanism, try to prove that ACM can induce MUTZ-1 cells afoptosis and (or) differentiation and maturation, so as to reduce the malignant clone of MDS, and try to clarify another new molecular mechanism of ACM on MDS cells. Thereby it
would help to find a new treatment target and develop new strategy to overcome the drug resistance of MDS cells to chemotherapeutic agents.
Using MTT assay we investigated the effect of ACM on MUTZ-1 growth. The result showed that following treatment with ACM(0.05 μmol/L) for 48 h, the proliferation of MUTZ-1 cells was inhibited significantly, in comparison with the blank control group(P<0.05). IC50 was 0.46μ mol/L at 48h. To explore the possible mechanism of the inhibition of ACM on MUTZ-1 cells, further investigation was carried out from the two stratifications of inducing apoptosis and differentiation.
Using Wright-Giemsa stain, the morphology of MUTZ-1 cells was observed with or without ACM treatment under a light microscope. The result displayed that a series of typical morphological features of apoptosis were observed following treatment with ACM(0.5μmol/L) for 48h, such as vacuolization of cell plasma, shrinkage of cell and nucleus, condensation of nuclear chromatin, marginated against the nuclear membranes, karyorrhexis and convolution of
nuclear, and membrane-bound apoptotic bodies(Figi.2).
In order to exactly analyze the cell apoptosis quantitatively and qualitatively, in addition to
some classic and specific and sensitive methods, the flow cytometry was also used to evaluate the DNA content, percentage of hypodiploid and translocation of phosphatidyl serine( PS). It showed that following treatment with 0.25umol/L-0.5nmol/L ACM for 48h, a typical DNA ladder was observed. With TdT mediated-dUTP nick end labeling (TUNEL), it appeared that with the increasing concentration of ACM at a certain time-point, the TUNEL positive cells increased. Meanwhile, with the increasing treatment time at a certain concentration of ACM, the TUNEL positive cells increased. From biochemistry, it was proved that ACM could induce MUTZ-1 cells apoptosis with dose- and time- dependent manners in vitro.
With the help of flow cytometry, the DNA content and hypodiploid changes of MUTZ-1 cells were observed with or without ACM treatment. The results showed that" the percentage of MUTZ-1 cells in the G2/M phase of the cell cycle increased, while in the G1/G0 phase decreased
following treatment with 0.1μ mol/L 0.2μmoI/L ACM, indicating that MUTZ-1 cells were arrested in the Ga/M phase. At the same time, hypodiploid cells increased, and the sub-G| peak appeared (Fig1-7). This manifested that ACM at a certain concentration could arrest MUTZ-1 cells in the G2/M phase of cell cycle, and then induced the cells apoptosis. With the help of flow cytometry following annexinV-PI staining, exposure to 0.5nmol/L ACM for 36 h caused 21.83% early apoptotic cells, which was more than that of the untreated group (3.47%), while late apoptovic cells increased only sligh
引文
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22.Dive C, Evans CA, Whetton AD. Induction of apoptosis----new targets for cancer chemotherapy. Semin Cancer Biol, 1992, 3(6): 417-427
23.Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell, 1994,78(4): 539-542
24.Nagata T, Higasigawa M, Shimono Y, et al. Aclarubicine inhibits etoposide induced apoptosis through inhibition of RNA syntheisi in P388 mutine leukemic cells. J Exp Clin Cancer Res, 1998, 17(4): 435-442
25.Dong J, Naito M, Tatsuta T, et al. Difference between the resistance mechanisms of aclacinomycin-and adriamycin-resistant P388 cell line. Oncol Res, 1995, 7(5): 245-452
26.Fukushima T, Takemura H, Yamashita T, et al. Multidrug resistance due to impaired DNA cleavage in a VP-16 resistant human leukemia cell line. Anticancer Res, 1999, 19(68):ss
5111-5115
27.Koopman G, Reutelingsperger CP, Kuijem GA, et al. Annexin V for flow cytometric detection of phosphatidylmerine expression of B cells undergoing apoptosis. Blood, 1994, 84(5): 1415-1420
28.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(7): 2207-2216
29.Kroemer G, Petitpx, Zamzami N, et al. The biochemistry of apoptosis. FASEB J, 1995, 9(13): 1277-1282
30.O'Brien IE, Reutelingaperger CP, Holdaway KM. The use of annexin-V and TUNEL to monitor the progression of apoptosis in plants. Cytometry, 1997, 29(1): 28-33
31.Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flowcytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Meth, 1995, 184(1): 39-51
32.Andteson H, Roberge M. Toposomerase Ⅱ imhibitors affect entry into mitosis and chromosome condensation in BHK cells. Cell Growth Differ, 1996, 7(1): 83-90
33.Sugimoto K, Sasaki M, Tamayose K, et al. Inhibition of p34~(cdc2) dephosphorylation in DNA damage-and topoisomerase Ⅱ inactivation-induced G_2 arrests in HL-60 cells. Br J Haematol, 1999, 105(3): 720-729
34.Teillaud JL, Gruel N, Moncuit J, et al. Structurally different ahthracyclines provlke different effects on ceH cycle and tumor B cell differentiation. Biomed Pharmacother, 1998, 52(6): 282-290
35.Baeuerie PA, Baltimore D. NF-kappa B: ten years after. Cell, 1996, 87(I): 13-20
36.Cullen BR, Malim MH. Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol, 1992, 216:362-368
37.Berger J, Hauber J, Hauber R, et al. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene, 1988, 66(1): 1-10
38.Gr(?)ndker C, Schulz K, G(?)thert AR et al. Luteinizing hormone-releasing hormone induces nuclear factor κ B-activation and inhibits apoptosis in ovarian cancer cells. J Clin Endo Metab, 2000, 85(10): 3815-3820
39.Gehrt A, Erkel G, Anke T, et al. Cycloepoxydon, 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene and 1-hydrosy-2-hydrosymethyl-3-pent-1,3-dienylbenzene,new inhibitors of
eukaryotic signal transduction. J Antibiotics, 1998, 51(5): 455-463
40.Shah N, Thomas T, Shirahata A, et al. Activation of nuclear factor κ B by polyamines in breast cancer cells. Biochemistry, 1999, 38(45): 14763-14774
41.Hannun YA. Apoptosis and dilemma of cancer chemotherapy. Blood, 1997, 89(6): 1845-1853
42.Wang CY, Mayo MW, Komeluk RG, et al. NF-kappa B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1998, 281 (5383): 1680-1683
43.Sun C, Cai M, Gunasekera AH, et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature, 1999, 401(6755): 818-822
44.Fang G, kim CN, Perkins CL, et al. CGP57148B(ST1571)induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs. Blood, 2000, 96(6): 2246-2253
45.Ruemmele FM, Beaulieu JF, O'Connell J, et al. The susceptibility to Fas-induced apoptosis in normal enterocytes is regulated on the level of cIAP-1 and cIAP-2. Biochem Biophys Res Commun, 2002, 290(4): 1308-1314
46.Wu M Lee H, Bellas RE, et al. Inhibition fo NF-kappa B/Rel induces apoptosis of murine B cells. EMBO J, 1996, 15(17): 4682-4690
47.Wang CY, Mayo MW, Baldwin Jr AS. TNF-and cancer therapy-induced apoptosis: potentiation by imhibition of NF-kappa B. Science, 1996, 274(5288): 784-787
48.Shishodia S, Aggarwal BB. Nuclear factor-kappa B activation: a question of life and death. J Biochem Mol Biol, 2002, 35(1): 28-40
49.Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-kappa B in cell growth regulation. Am J Pathol, 2001, 159(2): 387-397
50.Pahl HL. Activators and target genes of Rel/NF-kappa B transcription factors. Oncogene, 1999, 18(49): 6853-6866
51.Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappa B. J Clin Invest, 2001, 107(3): 241-246
52.Guzman ML, Neering SJ, Upchurch D, et al. Nuclear factor kappa B is constitutively activated in primitive human acute myelogenous leukemia cells. Blood, 2001, 98(8): 2301-2307
53.Prasad AV, Mohan N, Chandrasekar B, et al. Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation. Radiat Res, 1994, 138(3): 367-372
54.Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer: its role in prevention and therapy. Biochem Pharmacol, 2002, 64(5-6): 883-888
55.Van Antwerp DJ, Martin SJ, Kafri T et al. Suppression of TNF-alpha-induced apoptosis by NF-kappa B. Science 1996, 274(5288): 787-789
56.Beg AA, Baltimore D. An essential role for NF-kappa B in preventing TNF-alpha-induced cell death. Science, 1996, 274(5288): 782-784
57.Sumitomo M, Tachibana M, Nakashima J, et al. An essential role for nuclear factor kappa B in preventing,TNF-alpha-induced cell death in prostate cancer cells. J Urol, 1999, 161(2): 674-679
58.Pahl HL, Baeuerle PA: A novel signal transduction pathway from the edoplasmatic reticulum to the nucleus is mediated by transcription factor NF-κB. EMBO J, 1995, 14(11): 2580-2588
59.Traenckner EB, Wilk S and Baeuerle PA. A proteasome inhibitor prevents activation of NF-κB and stabilizes a newly phosphorylated form of I κB-αthat is still bound to NF-κB. EMBO J, 1994, 13(22): 5433-5441.
60.Ray S, Bulloc G, Nunez G, et al. Enforced expression of Bcl-xs induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis mediated differentiation and apoptosis. Cell Growth Differ, 1996, 7(12): 1617-1623
61.Shibuya T, Teshima, Harada M, et al. Treatment of myelodysplastic syndrome and atypical leukemia with low-dose aclarubicin. Leuk Res, 1990, 14(2): 161-167
62.WANG Zhen-yi, CHEN Zhu. Treatment of inducing differentiation and apoptosis on tumor. Shanghai: Shanghai scientific technology publishing house, 1998, 40(in Chinese).
63.LIN Mao-fang, XIONG Hong, CAI Zhen,et al(林茂芳，熊红，蔡真，等). Preliminary investgation on aclacinomycin affecting myeiodysplastic syndrome cell line proliferation and its machanism. Journal of Practical Oncology, 2002,17(2): 88-92.(in Chinese)
64.Civin CI, Strauss LC, Brovall C, et al. Antigenic analysis of hematopoiesis. Ⅲ. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-la ceils. J Immunol, 1984, 133(1): 157-165.
65.Nakahata T, Okumura N. Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers. Leuk Lymphoma, 1994, 13(5-6): 401-409.
66.Loken MR, Shah VO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow. Ⅱ. Normal B lymphocyte development. Blood, 1987,70(5): 1316-1324
2.McNally RJ, Roman E, Cartwright RA. Leukemias and lymphomas:time trends in the UK, 1984-93. Cancr Causes Control, 1999, 10(1):35-42
3.Dartsch DC, Schaefer A, Boldt S, et al. Comparison of anthracycline-induced death of human leukemia cells: Programmed cell death versus necrosis. Apoplosis, 2002, 7(6): 537-48
4.Deveraux QL, N.Roy HR, Stennicke T, et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J, 1998, 17(8): 2215-2223
5.Hida A, Kawakami A, Nakashima T. Nuclear factor-kappa B and caspases co-operatively regulate the activation and apoptosis of human macrophages. Immunology, 2000, 99(4): 553-560
6.Richard D, Hollender P, Chenais B. Involvement of reactive oxygen species in aclarubicin-induced differentiation and invasiveness of HL-60 leukemia cells, Int J Oncol, 2002, 21(2): 393-399
7.Chenais B, Andriollo M, Guiraud P, et al. Oxidative stress involvement in chemically induced differentiation of K562 cells. Free Radic Biol Med, 2000, 28(1): 18-27
8.Giilet R, Devemy E, Dupont C, et al. Evidence of the role of protein kinase C during aclacinomycin induction of erythroid differentiation in K562 cells. FEBS Lett, 1999, 454(3): 331-334
9.Gillet R, Bobichon H, Trentesaux C, et al. Nuclear transcription factor GATA-1 is activated during aclacinomycin-induced erythroid differentiation. Biol Cell, 2002, 94(4-5): 267-273
10.Akashi K, Eto T, Shibuya T, et al. Aclarubicin induces differentiation of leukemic progenitors in myelodysplastic syndrome cooperating with granulocyte colony-stimulating factor. Leuk Res, 2000, 24(3): 243-248.
11.Backx B, Broeders L, Touw I, et al. Blast colony-forming cells in myelodysplastic syndrome: Decreased potential to generate erythroid precursors. Leukemia, 1993, 7(1): 75-79
12.Hellstrom Lindberg E, Ahlgren T, Beguin Y, et al. Treatment of anemia in myelodysplastic
syndromes with granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase Ⅱ study and long-term follow-up of 71 patients. Blood, 1998, 92(1): 68-75
13.Rose EH, Abels RI, Nelson RA, et al. The use of r-HuEpo in the treatment of anaemia related to myelodysplasia(MDS). BrJ Haematol, 1995, 89(4):831-837
14.Steube KG, Gignac SM, Hu ZB, et al. In vitro culture studies of childhood meulodysplastic syndrome: Establishment of the cell line MUTZ-1. Leukemia and Lymphoma, 1997, 25(3-4): 345-363
15.Carter BZ, Milella M, Altieri DC, et al. Cytokine-regulated expression of survivin in myeloid leukemia. Blood, 2001,97(9): 2784-2790
16.Yoshida Y, Anzai N, Kawabata H, et al. Apoptosis in myelodysplasia: a paradox or paradigm. Leuk Res, 1995,19(12): 887-891
17.Bouscary D, Ves JD, Guesnu M, et al. Fas/Apo-1(CD95)expression and apoptosis in patients with myelodysplastic syndromes. Leukemia, 1997, 11(6): 839-845
18.Parker JE, Mufti GJ. Ineffective haemapoiesis and apoptosis in myelodysplastic syndromes. Br J Haematol, 1998, 101(2): 220-230
19.Mundle S, Shetty VT, Raza A. Caspases and apoptosis in myelodysplastic syndromes. Exp Hematol, 2000, 28(12): 1310-1313
20.Parker JE, Mufti GJ. The role of apoptosis in the pathogenesis of the myelodysplastic syndrome. Int J Hematol 2001, 73(4): 416-428
21.Parker JE, Fishlock KL, Mnovic A, et al. Low-risk myelodysplastic syndrome is associated associated with excessive apoptosis and an increated ratio of pro-versus anti-apoptotic bcl-2 related proteins. Br J Haematol, 1998, 103(4): 1075-1083
22.Dive C, Evans CA, Whetton AD. Induction of apoptosis----new targets for cancer chemotherapy. Semin Cancer Biol, 1992, 3(6): 417-427
23.Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell, 1994,78(4): 539-542
24.Nagata T, Higasigawa M, Shimono Y, et al. Aclarubicine inhibits etoposide induced apoptosis through inhibition of RNA syntheisi in P388 mutine leukemic cells. J Exp Clin Cancer Res, 1998, 17(4): 435-442
25.Dong J, Naito M, Tatsuta T, et al. Difference between the resistance mechanisms of aclacinomycin-and adriamycin-resistant P388 cell line. Oncol Res, 1995, 7(5): 245-452
26.Fukushima T, Takemura H, Yamashita T, et al. Multidrug resistance due to impaired DNA cleavage in a VP-16 resistant human leukemia cell line. Anticancer Res, 1999, 19(68):ss
5111-5115
27.Koopman G, Reutelingsperger CP, Kuijem GA, et al. Annexin V for flow cytometric detection of phosphatidylmerine expression of B cells undergoing apoptosis. Blood, 1994, 84(5): 1415-1420
28.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(7): 2207-2216
29.Kroemer G, Petitpx, Zamzami N, et al. The biochemistry of apoptosis. FASEB J, 1995, 9(13): 1277-1282
30.O'Brien IE, Reutelingaperger CP, Holdaway KM. The use of annexin-V and TUNEL to monitor the progression of apoptosis in plants. Cytometry, 1997, 29(1): 28-33
31.Vermes I, Haanen C, Steffens-Nakken H, et al. A novel assay for apoptosis. Flowcytometric detection of phosphatidylserine expression on early apoptosis cells using fluorescein labeled Annexin V. J Immunol Meth, 1995, 184(1): 39-51
32.Andteson H, Roberge M. Toposomerase Ⅱ imhibitors affect entry into mitosis and chromosome condensation in BHK cells. Cell Growth Differ, 1996, 7(1): 83-90
33.Sugimoto K, Sasaki M, Tamayose K, et al. Inhibition of p34~(cdc2) dephosphorylation in DNA damage-and topoisomerase Ⅱ inactivation-induced G_2 arrests in HL-60 cells. Br J Haematol, 1999, 105(3): 720-729
34.Teillaud JL, Gruel N, Moncuit J, et al. Structurally different ahthracyclines provlke different effects on ceH cycle and tumor B cell differentiation. Biomed Pharmacother, 1998, 52(6): 282-290
35.Baeuerie PA, Baltimore D. NF-kappa B: ten years after. Cell, 1996, 87(I): 13-20
36.Cullen BR, Malim MH. Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol, 1992, 216:362-368
37.Berger J, Hauber J, Hauber R, et al. Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells. Gene, 1988, 66(1): 1-10
38.Gr(?)ndker C, Schulz K, G(?)thert AR et al. Luteinizing hormone-releasing hormone induces nuclear factor κ B-activation and inhibits apoptosis in ovarian cancer cells. J Clin Endo Metab, 2000, 85(10): 3815-3820
39.Gehrt A, Erkel G, Anke T, et al. Cycloepoxydon, 1-hydroxy-2-hydroxymethyl-3-pent-1-enylbenzene and 1-hydrosy-2-hydrosymethyl-3-pent-1,3-dienylbenzene,new inhibitors of
eukaryotic signal transduction. J Antibiotics, 1998, 51(5): 455-463
40.Shah N, Thomas T, Shirahata A, et al. Activation of nuclear factor κ B by polyamines in breast cancer cells. Biochemistry, 1999, 38(45): 14763-14774
41.Hannun YA. Apoptosis and dilemma of cancer chemotherapy. Blood, 1997, 89(6): 1845-1853
42.Wang CY, Mayo MW, Komeluk RG, et al. NF-kappa B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1998, 281 (5383): 1680-1683
43.Sun C, Cai M, Gunasekera AH, et al. NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature, 1999, 401(6755): 818-822
44.Fang G, kim CN, Perkins CL, et al. CGP57148B(ST1571)induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs. Blood, 2000, 96(6): 2246-2253
45.Ruemmele FM, Beaulieu JF, O'Connell J, et al. The susceptibility to Fas-induced apoptosis in normal enterocytes is regulated on the level of cIAP-1 and cIAP-2. Biochem Biophys Res Commun, 2002, 290(4): 1308-1314
46.Wu M Lee H, Bellas RE, et al. Inhibition fo NF-kappa B/Rel induces apoptosis of murine B cells. EMBO J, 1996, 15(17): 4682-4690
47.Wang CY, Mayo MW, Baldwin Jr AS. TNF-and cancer therapy-induced apoptosis: potentiation by imhibition of NF-kappa B. Science, 1996, 274(5288): 784-787
48.Shishodia S, Aggarwal BB. Nuclear factor-kappa B activation: a question of life and death. J Biochem Mol Biol, 2002, 35(1): 28-40
49.Chen F, Castranova V, Shi X. New insights into the role of nuclear factor-kappa B in cell growth regulation. Am J Pathol, 2001, 159(2): 387-397
50.Pahl HL. Activators and target genes of Rel/NF-kappa B transcription factors. Oncogene, 1999, 18(49): 6853-6866
51.Baldwin AS. Control of oncogenesis and cancer therapy resistance by the transcription factor NF-kappa B. J Clin Invest, 2001, 107(3): 241-246
52.Guzman ML, Neering SJ, Upchurch D, et al. Nuclear factor kappa B is constitutively activated in primitive human acute myelogenous leukemia cells. Blood, 2001, 98(8): 2301-2307
53.Prasad AV, Mohan N, Chandrasekar B, et al. Activation of nuclear factor kappa B in human lymphoblastoid cells by low-dose ionizing radiation. Radiat Res, 1994, 138(3): 367-372
54.Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer: its role in prevention and therapy. Biochem Pharmacol, 2002, 64(5-6): 883-888
55.Van Antwerp DJ, Martin SJ, Kafri T et al. Suppression of TNF-alpha-induced apoptosis by NF-kappa B. Science 1996, 274(5288): 787-789
56.Beg AA, Baltimore D. An essential role for NF-kappa B in preventing TNF-alpha-induced cell death. Science, 1996, 274(5288): 782-784
57.Sumitomo M, Tachibana M, Nakashima J, et al. An essential role for nuclear factor kappa B in preventing,TNF-alpha-induced cell death in prostate cancer cells. J Urol, 1999, 161(2): 674-679
58.Pahl HL, Baeuerle PA: A novel signal transduction pathway from the edoplasmatic reticulum to the nucleus is mediated by transcription factor NF-κB. EMBO J, 1995, 14(11): 2580-2588
59.Traenckner EB, Wilk S and Baeuerle PA. A proteasome inhibitor prevents activation of NF-κB and stabilizes a newly phosphorylated form of I κB-αthat is still bound to NF-κB. EMBO J, 1994, 13(22): 5433-5441.
60.Ray S, Bulloc G, Nunez G, et al. Enforced expression of Bcl-xs induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis mediated differentiation and apoptosis. Cell Growth Differ, 1996, 7(12): 1617-1623
61.Shibuya T, Teshima, Harada M, et al. Treatment of myelodysplastic syndrome and atypical leukemia with low-dose aclarubicin. Leuk Res, 1990, 14(2): 161-167
62.WANG Zhen-yi, CHEN Zhu. Treatment of inducing differentiation and apoptosis on tumor. Shanghai: Shanghai scientific technology publishing house, 1998, 40(in Chinese).
63.LIN Mao-fang, XIONG Hong, CAI Zhen,et al(林茂芳，熊红，蔡真，等). Preliminary investgation on aclacinomycin affecting myeiodysplastic syndrome cell line proliferation and its machanism. Journal of Practical Oncology, 2002,17(2): 88-92.(in Chinese)
64.Civin CI, Strauss LC, Brovall C, et al. Antigenic analysis of hematopoiesis. Ⅲ. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-la ceils. J Immunol, 1984, 133(1): 157-165.
65.Nakahata T, Okumura N. Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers. Leuk Lymphoma, 1994, 13(5-6): 401-409.
66.Loken MR, Shah VO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow. Ⅱ. Normal B lymphocyte development. Blood, 1987,70(5): 1316-1324