HL-60细胞IRP_2mRNA、TfR mRNA、Fn mRNA的表达及其与肿瘤相关基因WT_1mRNA的关系研究
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摘要
铁是机体细胞生命活动所必需的微量元素之一。细胞内铁稳态(cellular iron
homeostasis)的维持对细胞的生长增殖及正常的机能活动来说都是非常重要的。研究发现，转铁蛋白受体(Transferrin Receptor，TfR)、铁蛋白(Ferritin，Fn)和铁调节蛋白(Iron Regulatory Protein，IRP)参与了细胞内铁稳态(cellular iron
homeostasis)的维持活动，其中TfR和Fn的表达都受IRP在转录或转录后水平上的调节。目前已知的铁调节蛋白形式有两种即IRP_1、IRP_2。
白血病是儿童时期较为常见的一种恶性肿瘤疾病，其发病机制目前尚不十分清楚。已有研究证实铁与白血病的发生、发展具有密切的关系。尽管IRP与IRE结合在转录后水平上调节相关基因的表达是当前研究的热点，但对白血病细胞铁代谢及其稳态调控的研究相对较少，尤其是对IRP_2在不同铁状态下调控基因转录作用的研究则更少。
本研究以人白血病细胞株HL-60细胞为实验对象，通过加铁和去铁干预试验，探讨白血病细胞铁代谢及其调节机制以及IRP_2在HL-60细胞铁代谢中的
郑州大学2004届研究生毕业论文
HL·60红11胞IRpZ mRNA、TI’RmRNA、FnmRNA的
表达及其，，肿瘤相关基眯1 WT，mRNA的关系‘JI究
作用。同时，探讨HL一60细胞中IRPZ、Tfl丈和Fn与肿瘤基因WTI的关系;
以揭示白血病细胞铁代谢机制以及铁在白血病发生发展中的作用。
方法
按照实验要求，将FeCI:或去铁胺(Desferrioxamine，DFO)加入含有10%
胎牛血清的RPMI一1 640培养液中，使HL-60细胞处于缺铁或富铁的不同状态
下进行培养。实验分为五组，即:FeC13一20组和FeC13一40组(培养液中FeC13
终浓度分别为20、40林mol/L);oFo一50组和nFo一1 00组(培养液中DFo
终浓度分别为50、100林mol/L);对照组(培养液中不加入FeC13和DFO)。
细胞培养后，分别于第12小时、24小时和48小时收集各组培养细胞，细胞
总RNA的提取参照组织/细胞总RNA提取试剂盒要求进行，提取后液氮冻存。
采用RT·PCR半定量法测定IRPZ mRNA、Fn;nRNA、T琅mRNA和wTI:nRNA
等指标的相对表达量
结果
1.IRP:mRNA:各实验组之间IRP:mRNA的表达量变化不大，经统计学
处理，组间差异无统计学意义(F娜庐1.199，P>0.05)。细胞培养时间对IRPZ
mRNA的表达有影响，经统计学处理，组内差异有统计学意义(F，。二43 .418，
尸<0.05)，表现为随时间的延长，IRP:mRNA的表达降低。
2.T仅mRNA:各实验组之间的差异有统计学意义(厂w一s二7.184，F。:。.，“
113.926，尸<0.01)。在加入去铁胺的DFO一50组和DFO一100组中TfRmRNA的
表达升高，并随培养时间的延长和DFO剂量的增加，其表达升高。在加铁的
FeC13一20组和FeC13一40组中，与对照组相比较，在12h时TfRmRNA的表达量
上升，24h时达到高峰，之后迅速下降。而在48h时，FeCI3一40组的表达量与
对照组相比较，约下降2倍，两组的差异具有统计学意义(尸<0.01)
3.Fn mRNA:细胞培养时间对Fn mRNA的表达无明显影响，经统计学处
理，组内差异无统计学意义(F，、内月514，尸>仓05)而各实验组之间的差异有
统计学意义(F。、。、=209、056，P<0.01)。FeC13一20组和FeC13一40组Fn mRNA
的表达量较高，大约是对照组的2倍，它们与对照组比较，差异具有统计学
郑州大学2004届研究生毕业论文
HL·60幻一l胞IRPZ mRNA、TfRmRNA、FnmRNA的
表达及其一与肿瘤相关基l州WT:mRNA的关系研究
意义(P<0.05);而DFO一50组、DFO一100组FnmRNA的表达与对照组相比
较，FnmRNA的表达无明显变化(尸>0.05)。
4.IRP:mRNA与T仅mRNA和Fn mRNA的相关性:相关分析结果显示:
IRPZ mRNA与T仅mRNA及Fn mRNA的表达均不相关(:=一0.005;r=0.074;P
>0 .05)。
5.WT，mRNA:各实验组之间T仅mRNA的表达量无明显变化，
异无统计学意义(厂，:。.，一l一07，P>0.05)。细胞培养时l’oJ又寸Fn mRNA
明显影响，组内差异无统计学意义(F。内=0.5“，尸>0.05)。
组间差
的表达无
6.WTI mRNA与IRP:n飞RNA、Fn mRNA以及TfR mRNA的相关性:相
关分析结果显示，WT;mRNA的表达与IRPZ mRNA、Fn mRNA以及TfR mRNA
的表达均不相关(p>0.05)。
结论
1.铁剂和去铁胺是通过IRP:或IRP:mRNA对HL一60细胞的铁代谢产生
作用。
2.在本实验所用铁剂和去铁胺剂量范围内，铁剂和去铁胺对IRPZ mRNA
表达的总量无影响。
3.铁剂可能通过影响IRPZ的表达，调控TfR mRNA、Fn mRNA的合成，
进而影响HL一60细胞TfR蛋白和Fn蛋白的表达量。
4.DFO可能通过影响IRPZ mRNA不同片段的合成，调控TfR mRNA的表
达，但对Fn mRNA的表达影响不大。
5.IRPZ mRNA与TfR mRNA、Fn mRNA无相关关系。
6.铁剂和去铁胺可能不影响HL一60细胞WT，mRNA的表达。
7.在
Fll mRNA
HL一60细胞中，肿瘤基因WT一mRNA与IRPZ mRNA、TfR mRNA、
无相关关系。
Background and Objection
Iron is an essential factor in many important cell functions, including growth, immunological response and energy production. It is important to maintain cellular iron homeostasis for cell proliferation and normal function. The iron regulatory proteins (IRP1 and IRP1) and the ferritin (Fn) and the transferrin receptor (TfR) involved in the maintenance of cellular iron homeostasis. Fn and TfR are used by cells to adjust intracellular iron concentration to levels in order to be adequate for their metabolic needs. IRP1 and IRP2 might bind to transcripts of ferritin, transferrin receptor to control the expression of iron metabolism proteins at the post-transcriptional level.
Tumor cells have a close relation with iron, as other cells do. And leukemia is one of common malignancies. The studies showed iron play a role in the development of leukemia. Although post-transcriptional gene regulation by the interaction of IRPs and IRE on select mRNAs is a very active research area, there is relatively little data on gene transcription which may be modified by IRP2 under
different iron status. In order to reveal the mechanism of iron metabolism and the roles of iron playing in leukemia ,we investigated the expression levels and the relationship of IRP2mRNA, TfRmRNA , Fn mRNA and WT1 mRNA under different iron status of HL-60 cells, and probed into the roles of IRP2 playing in HL-60 cells.
Methods
HL-60 cells (in 24-well plates) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 C in 5% CO2-95% air with 100 U/ml penicillin G and 100 U/ml streptomycin. To examine effects of iron, HL-60 cells were divided into five groups which treated with ferric chloride(FeCL3) or deferoxamine(DFO). HL-60 cells in the control group cultured in control media, in FeCL3-20 group cultured in control media plus of 20 M/L FeCL3, in FeCL3-40 group cultured in control media plus of 40 M/L FeCL3, in DFO-50 group cultured in control media plus of 50 M DFO and in DFO-100 group cultured in control media plus of 100 M DFO. The cells were harvested at 12, 24 and 48h proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative amounts of IRP2 mRNA , Fn mRNA and TfR mRNA were determined by RT-PCR.
Results
1. The levels of IRP2mRNA remained constant in all cells, whether or not treated with DFO or FeCL3.However, they were decreased when the cells prolonged in the media. There was not significant difference among the between-subjects (F=1.199,P > 0.05),but there was significant difference among the within-subjects (F=43.418, P<0.01).
2. The levels of TfRmRNA increased in the cells treated with DFO and they are dose-time dependant. In contrast to the control cells which treated neither with DFO nor ferric chloride ,there was significant difference (Fw-s = 7.184, FB-S =
113.926; P<0.01). Surprisingly, when cells grown with FeCLs, there wasn't a decline in expression of TfR mRNA, but it increased lightly at 12 hours and
peaked at 24 hours. This was followed by a drastically decline in its expression so that by 48 hours the levels of TfR mRNA were approximately two times fewer in the cells grown in the presence of 40 uM/L FeCLs than in the control cells. There was significant difference (P <0.01).
3. There was not significant difference among the within-subjects (F= 1.514.P > 0.05). It suggested that the levels of Fn mRNA didn't dependent on time.
However, there was significant difference among the between-subjects (F=209.056, P<0.01).The levels of Fn mRNA in the cells treated with FeCLswere approximately two times in the control cells. In contrast with the control cells, there was significant difference (P <0.05). The levels of Fn mRNA of the cells grown in DFO had little change. In contrast to the control cells, there was not significant difference (P > 0.05).
4. There wasn't significant correlation between IR?2 mRNA and TfR mRNA,Fn mRNA in HL-60 cells (P > 0.05 ).
5. The levels of WT| mRNA remained constant in all cells whether or not
homeostasis)的维持对细胞的生长增殖及正常的机能活动来说都是非常重要的。研究发现，转铁蛋白受体(Transferrin Receptor，TfR)、铁蛋白(Ferritin，Fn)和铁调节蛋白(Iron Regulatory Protein，IRP)参与了细胞内铁稳态(cellular iron
homeostasis)的维持活动，其中TfR和Fn的表达都受IRP在转录或转录后水平上的调节。目前已知的铁调节蛋白形式有两种即IRP_1、IRP_2。
白血病是儿童时期较为常见的一种恶性肿瘤疾病，其发病机制目前尚不十分清楚。已有研究证实铁与白血病的发生、发展具有密切的关系。尽管IRP与IRE结合在转录后水平上调节相关基因的表达是当前研究的热点，但对白血病细胞铁代谢及其稳态调控的研究相对较少，尤其是对IRP_2在不同铁状态下调控基因转录作用的研究则更少。
本研究以人白血病细胞株HL-60细胞为实验对象，通过加铁和去铁干预试验，探讨白血病细胞铁代谢及其调节机制以及IRP_2在HL-60细胞铁代谢中的
郑州大学2004届研究生毕业论文
HL·60红11胞IRpZ mRNA、TI’RmRNA、FnmRNA的
表达及其，，肿瘤相关基眯1 WT，mRNA的关系‘JI究
作用。同时，探讨HL一60细胞中IRPZ、Tfl丈和Fn与肿瘤基因WTI的关系;
以揭示白血病细胞铁代谢机制以及铁在白血病发生发展中的作用。
方法
按照实验要求，将FeCI:或去铁胺(Desferrioxamine，DFO)加入含有10%
胎牛血清的RPMI一1 640培养液中，使HL-60细胞处于缺铁或富铁的不同状态
下进行培养。实验分为五组，即:FeC13一20组和FeC13一40组(培养液中FeC13
终浓度分别为20、40林mol/L);oFo一50组和nFo一1 00组(培养液中DFo
终浓度分别为50、100林mol/L);对照组(培养液中不加入FeC13和DFO)。
细胞培养后，分别于第12小时、24小时和48小时收集各组培养细胞，细胞
总RNA的提取参照组织/细胞总RNA提取试剂盒要求进行，提取后液氮冻存。
采用RT·PCR半定量法测定IRPZ mRNA、Fn;nRNA、T琅mRNA和wTI:nRNA
等指标的相对表达量
结果
1.IRP:mRNA:各实验组之间IRP:mRNA的表达量变化不大，经统计学
处理，组间差异无统计学意义(F娜庐1.199，P>0.05)。细胞培养时间对IRPZ
mRNA的表达有影响，经统计学处理，组内差异有统计学意义(F，。二43 .418，
尸<0.05)，表现为随时间的延长，IRP:mRNA的表达降低。
2.T仅mRNA:各实验组之间的差异有统计学意义(厂w一s二7.184，F。:。.，“
113.926，尸<0.01)。在加入去铁胺的DFO一50组和DFO一100组中TfRmRNA的
表达升高，并随培养时间的延长和DFO剂量的增加，其表达升高。在加铁的
FeC13一20组和FeC13一40组中，与对照组相比较，在12h时TfRmRNA的表达量
上升，24h时达到高峰，之后迅速下降。而在48h时，FeCI3一40组的表达量与
对照组相比较，约下降2倍，两组的差异具有统计学意义(尸<0.01)
3.Fn mRNA:细胞培养时间对Fn mRNA的表达无明显影响，经统计学处
理，组内差异无统计学意义(F，、内月514，尸>仓05)而各实验组之间的差异有
统计学意义(F。、。、=209、056，P<0.01)。FeC13一20组和FeC13一40组Fn mRNA
的表达量较高，大约是对照组的2倍，它们与对照组比较，差异具有统计学
郑州大学2004届研究生毕业论文
HL·60幻一l胞IRPZ mRNA、TfRmRNA、FnmRNA的
表达及其一与肿瘤相关基l州WT:mRNA的关系研究
意义(P<0.05);而DFO一50组、DFO一100组FnmRNA的表达与对照组相比
较，FnmRNA的表达无明显变化(尸>0.05)。
4.IRP:mRNA与T仅mRNA和Fn mRNA的相关性:相关分析结果显示:
IRPZ mRNA与T仅mRNA及Fn mRNA的表达均不相关(:=一0.005;r=0.074;P
>0 .05)。
5.WT，mRNA:各实验组之间T仅mRNA的表达量无明显变化，
异无统计学意义(厂，:。.，一l一07，P>0.05)。细胞培养时l’oJ又寸Fn mRNA
明显影响，组内差异无统计学意义(F。内=0.5“，尸>0.05)。
组间差
的表达无
6.WTI mRNA与IRP:n飞RNA、Fn mRNA以及TfR mRNA的相关性:相
关分析结果显示，WT;mRNA的表达与IRPZ mRNA、Fn mRNA以及TfR mRNA
的表达均不相关(p>0.05)。
结论
1.铁剂和去铁胺是通过IRP:或IRP:mRNA对HL一60细胞的铁代谢产生
作用。
2.在本实验所用铁剂和去铁胺剂量范围内，铁剂和去铁胺对IRPZ mRNA
表达的总量无影响。
3.铁剂可能通过影响IRPZ的表达，调控TfR mRNA、Fn mRNA的合成，
进而影响HL一60细胞TfR蛋白和Fn蛋白的表达量。
4.DFO可能通过影响IRPZ mRNA不同片段的合成，调控TfR mRNA的表
达，但对Fn mRNA的表达影响不大。
5.IRPZ mRNA与TfR mRNA、Fn mRNA无相关关系。
6.铁剂和去铁胺可能不影响HL一60细胞WT，mRNA的表达。
7.在
Fll mRNA
HL一60细胞中，肿瘤基因WT一mRNA与IRPZ mRNA、TfR mRNA、
无相关关系。
Background and Objection
Iron is an essential factor in many important cell functions, including growth, immunological response and energy production. It is important to maintain cellular iron homeostasis for cell proliferation and normal function. The iron regulatory proteins (IRP1 and IRP1) and the ferritin (Fn) and the transferrin receptor (TfR) involved in the maintenance of cellular iron homeostasis. Fn and TfR are used by cells to adjust intracellular iron concentration to levels in order to be adequate for their metabolic needs. IRP1 and IRP2 might bind to transcripts of ferritin, transferrin receptor to control the expression of iron metabolism proteins at the post-transcriptional level.
Tumor cells have a close relation with iron, as other cells do. And leukemia is one of common malignancies. The studies showed iron play a role in the development of leukemia. Although post-transcriptional gene regulation by the interaction of IRPs and IRE on select mRNAs is a very active research area, there is relatively little data on gene transcription which may be modified by IRP2 under
different iron status. In order to reveal the mechanism of iron metabolism and the roles of iron playing in leukemia ,we investigated the expression levels and the relationship of IRP2mRNA, TfRmRNA , Fn mRNA and WT1 mRNA under different iron status of HL-60 cells, and probed into the roles of IRP2 playing in HL-60 cells.
Methods
HL-60 cells (in 24-well plates) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 C in 5% CO2-95% air with 100 U/ml penicillin G and 100 U/ml streptomycin. To examine effects of iron, HL-60 cells were divided into five groups which treated with ferric chloride(FeCL3) or deferoxamine(DFO). HL-60 cells in the control group cultured in control media, in FeCL3-20 group cultured in control media plus of 20 M/L FeCL3, in FeCL3-40 group cultured in control media plus of 40 M/L FeCL3, in DFO-50 group cultured in control media plus of 50 M DFO and in DFO-100 group cultured in control media plus of 100 M DFO. The cells were harvested at 12, 24 and 48h proliferation, and total RNA was isolated; cDNA was synthesized by reverse transcription (RT), and relative amounts of IRP2 mRNA , Fn mRNA and TfR mRNA were determined by RT-PCR.
Results
1. The levels of IRP2mRNA remained constant in all cells, whether or not treated with DFO or FeCL3.However, they were decreased when the cells prolonged in the media. There was not significant difference among the between-subjects (F=1.199,P > 0.05),but there was significant difference among the within-subjects (F=43.418, P<0.01).
2. The levels of TfRmRNA increased in the cells treated with DFO and they are dose-time dependant. In contrast to the control cells which treated neither with DFO nor ferric chloride ,there was significant difference (Fw-s = 7.184, FB-S =
113.926; P<0.01). Surprisingly, when cells grown with FeCLs, there wasn't a decline in expression of TfR mRNA, but it increased lightly at 12 hours and
peaked at 24 hours. This was followed by a drastically decline in its expression so that by 48 hours the levels of TfR mRNA were approximately two times fewer in the cells grown in the presence of 40 uM/L FeCLs than in the control cells. There was significant difference (P <0.01).
3. There was not significant difference among the within-subjects (F= 1.514.P > 0.05). It suggested that the levels of Fn mRNA didn't dependent on time.
However, there was significant difference among the between-subjects (F=209.056, P<0.01).The levels of Fn mRNA in the cells treated with FeCLswere approximately two times in the control cells. In contrast with the control cells, there was significant difference (P <0.05). The levels of Fn mRNA of the cells grown in DFO had little change. In contrast to the control cells, there was not significant difference (P > 0.05).
4. There wasn't significant correlation between IR?2 mRNA and TfR mRNA,Fn mRNA in HL-60 cells (P > 0.05 ).
5. The levels of WT| mRNA remained constant in all cells whether or not
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19.Hugh A.Barton,Richard S.Eisenstein, et al.Determinants of the interaction between the iron-responsive element binding protein and its binding site in rat L-Ferritin mRNA.J Biol Ch, 1990,265(12): 7000-7008
20.Kuhn LC,Hentze MW.Coordination of cellular iron metabolism by posttranscriptional gene regulation.J Inorg Biochem, 1992,47:183
21.John D.Phillips,Daniel V.Kinikini,Yang Yu, et al Differential Regulation of IRP1 and IRP2 by Nitric Oxide in rat hepatoma cells.The American Society of Hematology, 1996,87(7):2983-2992
22.Guo B, Brown FM, Phillips JD, et al.Characterization and expression of iron regulatory protein 2 (IRP2).Presence of multiple IRP2 transcripts regulated by intracellular iron levels.J Biol Chem, 1995, 270(28):16529-16535
23.Dae-Kyung Kang, Jinsook Jeong, Steven K, et al.Iron Regulatory Protein 2 as Iron Sensor.J Biol Chem, 2003, 278(17): 14857-14864
24.Kaptain S, Downey WE, Tang C, et al.A regulated RNA binding protein also possesses aconitase activity.Proc Natl Acad Sci USA, 1991,88(22):10109- 10113
25.Kennedy MC,Mende-mueller L,Blondin GA,et al.Purification and characteriza- tion of cytosolic aconitase from liver and its relationship to the IRE binding protein.Proc Natl Acad Sci USA, 1992,89:11730
26.Guo B, Yu Y, Leibold EA.Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity.J Biol Chem, 1994,269(39):24252-24260
27.Guo B, Phillips J D, Yu Y, et al.Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome.J Biol Chem, 1995,270:21645-21651
28.Iwai K, Klausner RD, Rouault TA.Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2.EMBO J, 1995, 14:5350-5357
29.Wang J, Pantopoulos K.Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP 1 mutant.Mol Cell Biol, 2002,22(13):4638-4651
30.Nunez MT, Nunez-Millacura C, Tapia V, et al.Iron-activated iron uptake: a positive feedback loop mediated by iron regulatory protein 1.Biometals, 2003, 16(1):83-90
31.G?nter Weiss, Ivo Graziadei, Martina Urbaneket, al.Divergent effects of 1-antitrypsin on the regulation of iron metabolism in human erythroleukaemic (K562) and myelomonocytic (THP-1) cells.Biochem J, 1996, 319:897-902
32.G?nter Weiss, Tracey Houston, Stefan Kastner, et al.Regulation of Cellular Iron Metabolism by Erythropoietin: Activation of Iron-Regulatory Protein and Upregulation of Transferrin Receptor Expression in Erythroid Cells.Blood, 1997, 89(2): 680-687
33．周密，廖清奎，李丰益，等．甲状腺激素T3影响K562细胞转铁蛋白受体和铁蛋白表达的分子机制研究．中华血液学杂志，2003，24(4)：181～184
34.Iwai K, Drake SK, Wehr NB, et al.Iron-dependent oxidation, ubiquitination and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins.Proc Natl Acad Sci USA, 1998, 95:4924-4928
35.Jing S Q,Trowbridge I S.Identification of intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.EMBO J, 1987,6:327-331
36.Enns CA, Clinton EM, Reckhow CL, et al.Acquisition of the functional properties of the transferrin receptor during its biosynthsis.J Biol Chem,1991, 256:13272-13277
37.Davis RJ,Johnson GL,Kelleher DJ, et al.Identification of serine 24 as unique residue on the transferrin receptor phosphotylated by kinase C.J Biol Chem, 1986, 261:9034-9041
38.Roberts MR, Miskimins WK, Ruddle FH.Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferring receptor gene and appear to be associated as a heterodimer.Cell Regul, 1989,1(1):151-164
39.Roberts MR, Han Y, Fienberg A, et al.A DNA-binding activity, TRAC,specific for the TRA element of the transferrin receptor gene copurifies with the Ku autoantigen.Proc Natl Acad Sci USA, 1994, 91(14):6354-6358
40.Vaismasn B, Fibach E, Koniji AM.Utilization of intracellular ferritin iron hemoglobin synthesis in developing human erythroid precursoes.Blood, 1997, 90:831-883
41.Chitambar CR, Massey EJ, Seligman PA.Regulation of transferrin receptor exp- ression on human leukemic cells during proliferation and induction of differenti- ation.J Clin Invest, 1983,72:11314-1325
42．赵晓晴，刘玉峰．急性白血病患儿血清转铁蛋白受体与铁参数变化的研究．中华血液学杂志，2002，23(7)：354—355
43.Higgy NA, Salicioni AM, Russo IH, et al.Differential expression of human
ferritin H chain gene in immortal human breast epithelial MCF-10F cells.Mol Carcinog, 1997, 20:332-339
44.Testa U.Transferrin receptor: structure and function.Curr Top Hematol, 1985, 5:127-161
45.Huebers HA, Finch CA.The physiology of transferrin and transferrin receprors.Physiol Rev, 1987,67(2):520-582
46．王伟良，李蓉生，陈嘉林，李琦．MEL细胞系中铁对转铁蛋白受体mRNA和铁螯合酶mRNA表达的调节．中华血液学杂志，1999,20(1)：17-20
47.Iwai K.An ubiquitin ligase recognizing a protein oxidized by iron: implications for the turnover of oxidatively damaged proteins.J Biochem, 2003, 134(2):175-182
48.Taetle R, Ralph S, Smedsrud S, et al.Regulation of transferrin receptor expression in myeloid leukemia cells.Blood, 1987,70(3):852-859
49．钱忠明主编．铁代谢—基础与临床，第一版．北京，科学出版社，2000．
50.Topham R, Goger M, Pearce K, et al.The mobilization of ferritin iron by liver cytosol.Biolchem J, 1989,261:137
51.Munro HN, Linder MC.Ferritin: structure, biosynthesis and role in iron metabolism.Physiol Rev, 1978, 58:317-396
52.Higgy NA, Salicioni AM, Russo IH, et al.Differential expression of human ferritin H chain gene in immortal human breast epithelial MCF-10F cells.Mol Carcinog, 1997,20:332-339
53.Broxmeyer HE, Cooper S, Levi S, et al.Mutated recombinant human heavy- chain ferritins and myelosuppression in vitro and in vivo: a link between ferritin ferroxidase activity and biological, function.Proc Natl Acad Sci USA, 1991, 88:770-774
54.Broxmeyer HE, Bognack J, Dorner MH, et al.Identification of leukemia- associated inhibitory activity as acidic isoferritins.J Exp Med, 1981, 153:1426-1444
55.Kikyo N, Hagiwara K, Fujisawa M, et al.Purification of a cell growth factor from a human lung cancer cell line: its relationship with ferritin.J Cell Physiol,1994,161:106-110
56.Kikyo N, Suda M, Kikyo N, et al.Purification and characterization of a cell growth factor from a human leukemia cell line: immunological identity with ferritin.Cancer Res, 1994,54:268-271
57.Zheng Ye, James R,Connor.Identification of iorn responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions, Nucleic Acids Research,2000,28(8):1802-1807
58.Call KM, Glaser T, Ito CY, et al.Isolation and characterization of a zinc finger poly- peptide gene at the human chromosome 11 wilms' tumor locus.Cell, 1990, 60:509-518
59.Haber DA, Parks, Maheswaran S, et al.WT1-mediated growth suppression of Wilms' tumor cell sex pressing a WT1 splicing variant.Science, 1993, 262: 2057
60.Werner H, Shnorr Z, Rauscher FJ, et al.Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor Ⅰ receptor gene expression.Mol Cell Biol, 1995, 15:3516
61.Englert C, Hou X, Maheswaran S, et al.WT1 suppresses synthesis of the epidermal growth factor receptor and iduces apoptosis.EMBOJ, 1995, 14: 4662-4675
62.Armstrong JF, Pritchard-Jones K, Bickmore WA, et al.The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo.Mech Dev, 1993,40(1-2):85-97
63.Taetle R, Ralph S, Smedsrud S, et al.Regulation of transferrin receptor expression in myeloid leukemia cells.Blood, 1987,70(3):852-859
64.Miwa H, Beran M, Saunders GF.Expression of the Wilms' tumor gene (WT1) in human leukemias.Leukemia, 1992, 6(5):405-409
65.Inoue K, Ogawa H, Yamagami T, et al.Long-termfol-low-up of minimal residual disease in leukemia patients by monitoring WT1 expression levels.Blood, 1996, 88:2267-2282
66.Bergmann L,Cornelius M,Maurer U et al.High levels of wilms' tumor gene mRNA inacute myeloid leukemia sare associated with long-term outcome.Blood, 1997,90:12175-12191
67.King-Underwood L, Renshaw J, Pritchard-Jones K et al.Mutations in the wilms' tumor gene WT1 inleukemias.Blood, 1996, 87(2):2171-2185
68.Magyarosy E, Varga N, Timar J, et al.Follow-up of minimal residual disease in acute childhood lymphoblastic leukemia by WT1 gene expression in the peripheral blood: the Hungarian experience.Pediatr Hematol Oncol, 2003,20(1):65-74
69.Ogawa H, Tamaki H, Ikegame K, et al.The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia.Blood, 2003,101(5):1698-1704.
70.Kletzel M, Olzewski M, Huang W, et al.Utility of WT1 as a reliable tool for the detection of minimal residual disease in children with leukemia.Pediatr Dev Pathol, 2002,5(3):269-275
71.Le NT, Richardson DR.The role of iron in cell cycle progression and the proliferation of neoplastic cells Biochim Biophys Acta, 2002,1603(1):31-46.
72.Wu KJ, Polack A, Dalla-Favera R.Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC.Science, 1999,283(5402):676-679.
73.Ashizuka M, Fukuda T, Nakamura T, et al.Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2.Mol Cell Biol,2002,22(18):6375-6383
2.Richardson DR, Ponka P.The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells.Biochem Biophys Acta, 1997, 1331(1):1-40
3.Chitambar CR, Wereley JP.Resistance to the antitumor agent gallium nitrate in human leukemic cells is associated with decreased gallium/iron uptake, increased activity of iron regulatory protein- 1, and decreased ferritin production.J Biol Chem, 1997,272(18):112151-12157
4.Hu J, Connor JR.Demonstration and characterization of the iron regulatory protein in human brain.J Neurochem, 1996,67(2):838-844
5.Henderson BR, Menotti E, Kuhn LC.Iron regulatory proteins 1 and 2 bind distinct sets of RNA target sequences.J Biol Chem, 1996, 271 (9):4900-4908
6.Qian Z M,Tang P L,Wang.Ion crosses the endosomal membrane by a cari-ermediated process.Prog Biophys Molec Biol, 1997,67:1-15
7.kevin L,Schalinske, Kenneth P, et al.Iron regulatory protein 1 is not required for the modulation of ferritin and transferring receptor expression by iron in a murine pro-B lymphocyte cell line.Proc Natl Acad Sci USA, 1997, 94: 10681-10686
8．贾国存，刘玉峰．富铁对HL-60细胞增殖及化疗药物诱导其凋亡的影响．中华血液学杂志，2001，22(7)：381-382
9．刘玉峰，贾国存．铁剥夺诱导HL—60细胞凋亡及对化疗药物诱导HL—60细胞捌亡的影响．中华儿科杂志，2001，39(12)：735-738
10.Richardson DR.The use of iron chelators as therapeutic agents for the treatment of cancer.Crit Rev Oncol Hematol, 2002, 42(3):267-281
11.Trowbridge IS, Lopez F.Monoclonal antibody to transferrin receptor blocks transferrin binding and inhibits tumor cell growth in vitro.Proc Natl Acad Sci USA, 1982, 79:1175-1179
12.Bassett ML, Haliday JW, Powell LW.Genetic hemochromatosis.Semin Liver Dis, 1984, 4(3):217-227
13.Klausner R D, Rouault T A, Harford J B.Regulating the fate of mRNA: the control of cellular iron metabolism.Cell, 1993, 72:19-28
14.Hentze MW, K?hn L C.Molecular control of vertebrate iron metabolism: mRNA-based circuits operated by iron, nitric oxide, and oxidative stress.Proc Natl Acad Sci USA, 1996, 93:8175-8182
15.Hentze MW, Rouault TA, Harford JB, et al.Oxidation-reduction and the molecular mechanism of a regulatory RNA-protein interaction.Science, 1989, 244(4902): 357-359
16.Domachowske JB.The role of nitric oxide in the regulation of cellular iron metabolism.Biol Mol Mell,1997, 60:1-12
17.Gray NK, Pantopoulos K, DandekarT.Translation regulation of mammalianand drosophila citric acid cycle enzymes vias IREs.Proc Natl Aci Sci USA,1996,93:4925-4930
18.Richard SR, Kenneth PB.Iron regulatory protein,iron responsine element andiron homeostasis.J Nutr, 1998,128:22951-2298
19.Hugh A.Barton,Richard S.Eisenstein, et al.Determinants of the interaction between the iron-responsive element binding protein and its binding site in rat L-Ferritin mRNA.J Biol Ch, 1990,265(12): 7000-7008
20.Kuhn LC,Hentze MW.Coordination of cellular iron metabolism by posttranscriptional gene regulation.J Inorg Biochem, 1992,47:183
21.John D.Phillips,Daniel V.Kinikini,Yang Yu, et al Differential Regulation of IRP1 and IRP2 by Nitric Oxide in rat hepatoma cells.The American Society of Hematology, 1996,87(7):2983-2992
22.Guo B, Brown FM, Phillips JD, et al.Characterization and expression of iron regulatory protein 2 (IRP2).Presence of multiple IRP2 transcripts regulated by intracellular iron levels.J Biol Chem, 1995, 270(28):16529-16535
23.Dae-Kyung Kang, Jinsook Jeong, Steven K, et al.Iron Regulatory Protein 2 as Iron Sensor.J Biol Chem, 2003, 278(17): 14857-14864
24.Kaptain S, Downey WE, Tang C, et al.A regulated RNA binding protein also possesses aconitase activity.Proc Natl Acad Sci USA, 1991,88(22):10109- 10113
25.Kennedy MC,Mende-mueller L,Blondin GA,et al.Purification and characteriza- tion of cytosolic aconitase from liver and its relationship to the IRE binding protein.Proc Natl Acad Sci USA, 1992,89:11730
26.Guo B, Yu Y, Leibold EA.Iron regulates cytoplasmic levels of a novel iron-responsive element-binding protein without aconitase activity.J Biol Chem, 1994,269(39):24252-24260
27.Guo B, Phillips J D, Yu Y, et al.Iron regulates the intracellular degradation of iron regulatory protein 2 by the proteasome.J Biol Chem, 1995,270:21645-21651
28.Iwai K, Klausner RD, Rouault TA.Requirements for iron-regulated degradation of the RNA binding protein, iron regulatory protein 2.EMBO J, 1995, 14:5350-5357
29.Wang J, Pantopoulos K.Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP 1 mutant.Mol Cell Biol, 2002,22(13):4638-4651
30.Nunez MT, Nunez-Millacura C, Tapia V, et al.Iron-activated iron uptake: a positive feedback loop mediated by iron regulatory protein 1.Biometals, 2003, 16(1):83-90
31.G?nter Weiss, Ivo Graziadei, Martina Urbaneket, al.Divergent effects of 1-antitrypsin on the regulation of iron metabolism in human erythroleukaemic (K562) and myelomonocytic (THP-1) cells.Biochem J, 1996, 319:897-902
32.G?nter Weiss, Tracey Houston, Stefan Kastner, et al.Regulation of Cellular Iron Metabolism by Erythropoietin: Activation of Iron-Regulatory Protein and Upregulation of Transferrin Receptor Expression in Erythroid Cells.Blood, 1997, 89(2): 680-687
33．周密，廖清奎，李丰益，等．甲状腺激素T3影响K562细胞转铁蛋白受体和铁蛋白表达的分子机制研究．中华血液学杂志，2003，24(4)：181～184
34.Iwai K, Drake SK, Wehr NB, et al.Iron-dependent oxidation, ubiquitination and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins.Proc Natl Acad Sci USA, 1998, 95:4924-4928
35.Jing S Q,Trowbridge I S.Identification of intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site.EMBO J, 1987,6:327-331
36.Enns CA, Clinton EM, Reckhow CL, et al.Acquisition of the functional properties of the transferrin receptor during its biosynthsis.J Biol Chem,1991, 256:13272-13277
37.Davis RJ,Johnson GL,Kelleher DJ, et al.Identification of serine 24 as unique residue on the transferrin receptor phosphotylated by kinase C.J Biol Chem, 1986, 261:9034-9041
38.Roberts MR, Miskimins WK, Ruddle FH.Nuclear proteins TREF1 and TREF2 bind to the transcriptional control element of the transferring receptor gene and appear to be associated as a heterodimer.Cell Regul, 1989,1(1):151-164
39.Roberts MR, Han Y, Fienberg A, et al.A DNA-binding activity, TRAC,specific for the TRA element of the transferrin receptor gene copurifies with the Ku autoantigen.Proc Natl Acad Sci USA, 1994, 91(14):6354-6358
40.Vaismasn B, Fibach E, Koniji AM.Utilization of intracellular ferritin iron hemoglobin synthesis in developing human erythroid precursoes.Blood, 1997, 90:831-883
41.Chitambar CR, Massey EJ, Seligman PA.Regulation of transferrin receptor exp- ression on human leukemic cells during proliferation and induction of differenti- ation.J Clin Invest, 1983,72:11314-1325
42．赵晓晴，刘玉峰．急性白血病患儿血清转铁蛋白受体与铁参数变化的研究．中华血液学杂志，2002，23(7)：354—355
43.Higgy NA, Salicioni AM, Russo IH, et al.Differential expression of human
ferritin H chain gene in immortal human breast epithelial MCF-10F cells.Mol Carcinog, 1997, 20:332-339
44.Testa U.Transferrin receptor: structure and function.Curr Top Hematol, 1985, 5:127-161
45.Huebers HA, Finch CA.The physiology of transferrin and transferrin receprors.Physiol Rev, 1987,67(2):520-582
46．王伟良，李蓉生，陈嘉林，李琦．MEL细胞系中铁对转铁蛋白受体mRNA和铁螯合酶mRNA表达的调节．中华血液学杂志，1999,20(1)：17-20
47.Iwai K.An ubiquitin ligase recognizing a protein oxidized by iron: implications for the turnover of oxidatively damaged proteins.J Biochem, 2003, 134(2):175-182
48.Taetle R, Ralph S, Smedsrud S, et al.Regulation of transferrin receptor expression in myeloid leukemia cells.Blood, 1987,70(3):852-859
49．钱忠明主编．铁代谢—基础与临床，第一版．北京，科学出版社，2000．
50.Topham R, Goger M, Pearce K, et al.The mobilization of ferritin iron by liver cytosol.Biolchem J, 1989,261:137
51.Munro HN, Linder MC.Ferritin: structure, biosynthesis and role in iron metabolism.Physiol Rev, 1978, 58:317-396
52.Higgy NA, Salicioni AM, Russo IH, et al.Differential expression of human ferritin H chain gene in immortal human breast epithelial MCF-10F cells.Mol Carcinog, 1997,20:332-339
53.Broxmeyer HE, Cooper S, Levi S, et al.Mutated recombinant human heavy- chain ferritins and myelosuppression in vitro and in vivo: a link between ferritin ferroxidase activity and biological, function.Proc Natl Acad Sci USA, 1991, 88:770-774
54.Broxmeyer HE, Bognack J, Dorner MH, et al.Identification of leukemia- associated inhibitory activity as acidic isoferritins.J Exp Med, 1981, 153:1426-1444
55.Kikyo N, Hagiwara K, Fujisawa M, et al.Purification of a cell growth factor from a human lung cancer cell line: its relationship with ferritin.J Cell Physiol,1994,161:106-110
56.Kikyo N, Suda M, Kikyo N, et al.Purification and characterization of a cell growth factor from a human leukemia cell line: immunological identity with ferritin.Cancer Res, 1994,54:268-271
57.Zheng Ye, James R,Connor.Identification of iorn responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions, Nucleic Acids Research,2000,28(8):1802-1807
58.Call KM, Glaser T, Ito CY, et al.Isolation and characterization of a zinc finger poly- peptide gene at the human chromosome 11 wilms' tumor locus.Cell, 1990, 60:509-518
59.Haber DA, Parks, Maheswaran S, et al.WT1-mediated growth suppression of Wilms' tumor cell sex pressing a WT1 splicing variant.Science, 1993, 262: 2057
60.Werner H, Shnorr Z, Rauscher FJ, et al.Inhibition of cellular proliferation by the Wilms' tumor suppressor WT1 is associated with suppression of insulin-like growth factor Ⅰ receptor gene expression.Mol Cell Biol, 1995, 15:3516
61.Englert C, Hou X, Maheswaran S, et al.WT1 suppresses synthesis of the epidermal growth factor receptor and iduces apoptosis.EMBOJ, 1995, 14: 4662-4675
62.Armstrong JF, Pritchard-Jones K, Bickmore WA, et al.The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo.Mech Dev, 1993,40(1-2):85-97
63.Taetle R, Ralph S, Smedsrud S, et al.Regulation of transferrin receptor expression in myeloid leukemia cells.Blood, 1987,70(3):852-859
64.Miwa H, Beran M, Saunders GF.Expression of the Wilms' tumor gene (WT1) in human leukemias.Leukemia, 1992, 6(5):405-409
65.Inoue K, Ogawa H, Yamagami T, et al.Long-termfol-low-up of minimal residual disease in leukemia patients by monitoring WT1 expression levels.Blood, 1996, 88:2267-2282
66.Bergmann L,Cornelius M,Maurer U et al.High levels of wilms' tumor gene mRNA inacute myeloid leukemia sare associated with long-term outcome.Blood, 1997,90:12175-12191
67.King-Underwood L, Renshaw J, Pritchard-Jones K et al.Mutations in the wilms' tumor gene WT1 inleukemias.Blood, 1996, 87(2):2171-2185
68.Magyarosy E, Varga N, Timar J, et al.Follow-up of minimal residual disease in acute childhood lymphoblastic leukemia by WT1 gene expression in the peripheral blood: the Hungarian experience.Pediatr Hematol Oncol, 2003,20(1):65-74
69.Ogawa H, Tamaki H, Ikegame K, et al.The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia.Blood, 2003,101(5):1698-1704.
70.Kletzel M, Olzewski M, Huang W, et al.Utility of WT1 as a reliable tool for the detection of minimal residual disease in children with leukemia.Pediatr Dev Pathol, 2002,5(3):269-275
71.Le NT, Richardson DR.The role of iron in cell cycle progression and the proliferation of neoplastic cells Biochim Biophys Acta, 2002,1603(1):31-46.
72.Wu KJ, Polack A, Dalla-Favera R.Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC.Science, 1999,283(5402):676-679.
73.Ashizuka M, Fukuda T, Nakamura T, et al.Novel translational control through an iron-responsive element by interaction of multifunctional protein YB-1 and IRP2.Mol Cell Biol,2002,22(18):6375-6383