小分子化合物As2S2对白血病干细胞的杀伤效应及其作用机制的初步研究
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摘要
第一部分
小分子化合物As2S2对白血病细胞的杀伤效应及其作用机制的研究
目的观察As2S2对白血病细胞的杀伤效应，揭示其杀伤机制，探索联用PI3K抑制剂PI-103时二者对白血病细胞的治疗获益。方法以不同浓度的As2S2作用白血病细胞不同时间后，应用MTT试验检测As2S2对白血病细胞的增殖抑制；流式细胞术及MGG染色检测As2S2及PI-103单用或联用时对白血病细胞、脐血单个核细胞及白血病患者骨髓单个核细胞的分化及凋亡诱导作用；克隆形成实验检测As2S2对白血病细胞克隆的清除能力；细胞免疫荧光、Western blot、Real-time PCR及Real-timePCR array检测As2S2对PML和PI3K信号通路中多个信号分子的蛋白及转录水平的调控。结果As2S2以时间、剂量依赖性方式抑制白血病细胞增殖、诱导其凋亡，抑制白血病细胞的克隆形成能力。As2S2作用后，PML蛋白的表达被显著下调，但转录水平无明显变化。PI3K信号通路中关键蛋白磷酸化水平短时间内被激活，随着作用时间的延长则转为下调，该通路基因变化的总体趋势是被下调。As2S2和PI-103联用对白血病细胞的凋亡诱导起到了协同增强的效应，但对正常的单个核细胞无明显的毒副作用。结论As2S2可能通过降调PML蛋白表达、长时间作用后抑制PI3K信号通路对白血病细胞显示出的时间、剂量依赖性杀伤效应，PI-103可协同增强上述效应，但对正常的单个核细胞无明显的毒副作用，具有较高的治疗指数。
第二部分
小分子化合物As2S2对白血病干细胞的杀伤效应及其作用机制的初步研究
目的检测小分子化合物As2S2单用或联用PI-103时对白血病干细胞的杀伤效应，初步研究其作用机制。方法应用流式细胞术分选白血病干细胞；经As2S2及PI-103单独或联合处理后，流式细胞术检测各组白血病干细胞的凋亡和分化；克隆形成实验检测药物干预后白血病干细胞及正常造血干细胞克隆形成能力的变化；多色流式细胞仪检测As2S2作用后白血病干细胞内p-AKT473表达的动态变化。结果流式细胞术分选的CD34+CD38-细胞占患者骨髓细胞总数的75．4％，分选纯度为96．4％。anti-ki67-Hoechst染色分析表明G0期细胞占83．9％，G1细胞期占7．93％。500 nmol／L的As2S2和1μmol／L PI-103分别单独作用时，细胞凋亡诱导效应并不明显，而两者联用时，凋亡率则从17．26％跃升至31．02％。As2S2和PI-103均有一定程度的白血病细胞克隆清除能力，二者联用时对白血病细胞克隆的清除能力显著增强。在有效清除白血病克隆的药物浓度范围内，二者对正常造血干细胞的克隆形成能力无明显影响。流式细胞术检测显示，As2S2处理组细胞CD11b表达升高，PI-103处理组CD11b表达无明显变化，As2S2与PI-103联用组较As2S2处理组CD11b表达升高更明显。6例白血病标本中，CD34+CD38-Lin-的细胞群体内p-AKT473的表达在As2S2作用细胞1 b时上调，在24h时下调。结论As2S2能有效诱导白血病干细胞的凋亡，促进其分化，抑制其自我更新及增殖能力，PI-103可协同增强上述效应。
Objective: To observe the killing effect of As2S2 on leukemic cells, explore the mechanism of the effect, and probe the therapy benefits from treatment combining As2S2 with a PI3K inhibitor, PI-103. Methods: After leukemia cells were treated with different concentrations of As2S2 for different times, MTT assay was used to test the effect of As2S2 on proliferation inhibition of treated cells; flow cytometry and MGG staining were used to examine the differentiation and apoptosis of leukemia cells, cord blood mononuclear mononuclear cells and bone marrow mononuclear cells of leukemia patients after being treated with As2S2, PI-103 or both. Colony-forming assay was used to asses the effect of cloning scavenging of As2S2 on leukemic cells. Cell immunofluorescence, western blot, real-time PCR and real-time PCR array were used to determine the regulation of PI3K signal pathway by As2S2 at the levels of transcription and protein. Results: As2S2 inhibited the proliferation and induced apoptosis of leukemia cells in a time- and dose-dependent manner, and supressed clone formation of leukemia cells significantly. After treatment with As2S2, PML protein expression was reduced significantly in leukemia cells, however no significant changes were seen in the level of transcription. The phosphorylation levels of key proteins in PI3K signaling pathway were activated in a short period of time, but they were downregulated in general with the extension of treatment time. As2S2 and PI-103 showed a synergistic enhanced effect on inducing apoptosis of leukemia cells, while showed no apparent toxicity on normal mononuclear cells. Conclusions: As2S2 shows a significant dose-dependent killing effect on leukemic cells through depletion of PML protein and downregulation of PI3K signaling pathway, but no obvious toxity on ormal mononuclear cells were seen, PI-103 can be coordinated to enhance the above effect. Therefore, As2S2 should hve a high therapeutic index.
Objectives TO determine the effect of small molecule compounds As2S2 alone or in combination with PI-103 on the leukemia stem cells and conduct. Methods Leukemia stem cells were sorted by flow cytometry, and treated with As2S2 or / and PI-103. Then the apoptosis and differentiation of treated cells was detected by flow cytometry. The effect of As2S2 or / and PI-103 on the colony-forming ability of leukemia stem cells and normal hematopoietic stem cell cells was assessed by colony forming assay. The dynamic changes of p-AKT473 expression in leukemia stem cells treated with As2S2 was determined by multi-color flow cytometry. Results CD34 + CD38-cells sorted from the bone marrow cells of patients accounted for 75.4 percent of the total number, and sorting purity was 96.4%. Anti-ki67-Hoechst staining analysis showed that the cells in GO phase accounted for 83.9%, that in G1 phase accounted for 7.93%., respectively, the cell apoptosis-inducing effect of 1μmol / L of As2S2 or PI-103 was not apparent, but when used together, the apoptosis rate of leukemia stem cells jumped to 31.02% from 17.26%. As2S2 or PI-103 had a mild effect on inhibiting colony formation of leukemia cell when used alone, but the effect was significantly enhanced when used together. They had no significant effect on the colony-forming capacity of normal hematopoietic stem cells when used at concentrations above. The number of cells expressing CD11b was significantly increased in As2S2 treatment group, but the number of cells expressing CD11b in PI-103 treatment group was not changed significantly. The number of cells expressing CD11b was increased more significantly in group treated with As2S2 and PI-103 than with As2S2 alone. The expression of p-AKT473 in CD34+CD38-Lin- cells from specimens of six leukemia patients was increased after being treated with As2S2 for 1 h, but after 24 h, it was downregulated. Conclusion As2S2 could effectively induce apoptosis of leukemia stem cells, promote differentiation, inhibit their ability of self-renewal and proliferation, PI-103 can be coordinated to enhance the above effect.
小分子化合物As2S2对白血病细胞的杀伤效应及其作用机制的研究
目的观察As2S2对白血病细胞的杀伤效应，揭示其杀伤机制，探索联用PI3K抑制剂PI-103时二者对白血病细胞的治疗获益。方法以不同浓度的As2S2作用白血病细胞不同时间后，应用MTT试验检测As2S2对白血病细胞的增殖抑制；流式细胞术及MGG染色检测As2S2及PI-103单用或联用时对白血病细胞、脐血单个核细胞及白血病患者骨髓单个核细胞的分化及凋亡诱导作用；克隆形成实验检测As2S2对白血病细胞克隆的清除能力；细胞免疫荧光、Western blot、Real-time PCR及Real-timePCR array检测As2S2对PML和PI3K信号通路中多个信号分子的蛋白及转录水平的调控。结果As2S2以时间、剂量依赖性方式抑制白血病细胞增殖、诱导其凋亡，抑制白血病细胞的克隆形成能力。As2S2作用后，PML蛋白的表达被显著下调，但转录水平无明显变化。PI3K信号通路中关键蛋白磷酸化水平短时间内被激活，随着作用时间的延长则转为下调，该通路基因变化的总体趋势是被下调。As2S2和PI-103联用对白血病细胞的凋亡诱导起到了协同增强的效应，但对正常的单个核细胞无明显的毒副作用。结论As2S2可能通过降调PML蛋白表达、长时间作用后抑制PI3K信号通路对白血病细胞显示出的时间、剂量依赖性杀伤效应，PI-103可协同增强上述效应，但对正常的单个核细胞无明显的毒副作用，具有较高的治疗指数。
第二部分
小分子化合物As2S2对白血病干细胞的杀伤效应及其作用机制的初步研究
目的检测小分子化合物As2S2单用或联用PI-103时对白血病干细胞的杀伤效应，初步研究其作用机制。方法应用流式细胞术分选白血病干细胞；经As2S2及PI-103单独或联合处理后，流式细胞术检测各组白血病干细胞的凋亡和分化；克隆形成实验检测药物干预后白血病干细胞及正常造血干细胞克隆形成能力的变化；多色流式细胞仪检测As2S2作用后白血病干细胞内p-AKT473表达的动态变化。结果流式细胞术分选的CD34+CD38-细胞占患者骨髓细胞总数的75．4％，分选纯度为96．4％。anti-ki67-Hoechst染色分析表明G0期细胞占83．9％，G1细胞期占7．93％。500 nmol／L的As2S2和1μmol／L PI-103分别单独作用时，细胞凋亡诱导效应并不明显，而两者联用时，凋亡率则从17．26％跃升至31．02％。As2S2和PI-103均有一定程度的白血病细胞克隆清除能力，二者联用时对白血病细胞克隆的清除能力显著增强。在有效清除白血病克隆的药物浓度范围内，二者对正常造血干细胞的克隆形成能力无明显影响。流式细胞术检测显示，As2S2处理组细胞CD11b表达升高，PI-103处理组CD11b表达无明显变化，As2S2与PI-103联用组较As2S2处理组CD11b表达升高更明显。6例白血病标本中，CD34+CD38-Lin-的细胞群体内p-AKT473的表达在As2S2作用细胞1 b时上调，在24h时下调。结论As2S2能有效诱导白血病干细胞的凋亡，促进其分化，抑制其自我更新及增殖能力，PI-103可协同增强上述效应。
Objective: To observe the killing effect of As2S2 on leukemic cells, explore the mechanism of the effect, and probe the therapy benefits from treatment combining As2S2 with a PI3K inhibitor, PI-103. Methods: After leukemia cells were treated with different concentrations of As2S2 for different times, MTT assay was used to test the effect of As2S2 on proliferation inhibition of treated cells; flow cytometry and MGG staining were used to examine the differentiation and apoptosis of leukemia cells, cord blood mononuclear mononuclear cells and bone marrow mononuclear cells of leukemia patients after being treated with As2S2, PI-103 or both. Colony-forming assay was used to asses the effect of cloning scavenging of As2S2 on leukemic cells. Cell immunofluorescence, western blot, real-time PCR and real-time PCR array were used to determine the regulation of PI3K signal pathway by As2S2 at the levels of transcription and protein. Results: As2S2 inhibited the proliferation and induced apoptosis of leukemia cells in a time- and dose-dependent manner, and supressed clone formation of leukemia cells significantly. After treatment with As2S2, PML protein expression was reduced significantly in leukemia cells, however no significant changes were seen in the level of transcription. The phosphorylation levels of key proteins in PI3K signaling pathway were activated in a short period of time, but they were downregulated in general with the extension of treatment time. As2S2 and PI-103 showed a synergistic enhanced effect on inducing apoptosis of leukemia cells, while showed no apparent toxicity on normal mononuclear cells. Conclusions: As2S2 shows a significant dose-dependent killing effect on leukemic cells through depletion of PML protein and downregulation of PI3K signaling pathway, but no obvious toxity on ormal mononuclear cells were seen, PI-103 can be coordinated to enhance the above effect. Therefore, As2S2 should hve a high therapeutic index.
Objectives TO determine the effect of small molecule compounds As2S2 alone or in combination with PI-103 on the leukemia stem cells and conduct. Methods Leukemia stem cells were sorted by flow cytometry, and treated with As2S2 or / and PI-103. Then the apoptosis and differentiation of treated cells was detected by flow cytometry. The effect of As2S2 or / and PI-103 on the colony-forming ability of leukemia stem cells and normal hematopoietic stem cell cells was assessed by colony forming assay. The dynamic changes of p-AKT473 expression in leukemia stem cells treated with As2S2 was determined by multi-color flow cytometry. Results CD34 + CD38-cells sorted from the bone marrow cells of patients accounted for 75.4 percent of the total number, and sorting purity was 96.4%. Anti-ki67-Hoechst staining analysis showed that the cells in GO phase accounted for 83.9%, that in G1 phase accounted for 7.93%., respectively, the cell apoptosis-inducing effect of 1μmol / L of As2S2 or PI-103 was not apparent, but when used together, the apoptosis rate of leukemia stem cells jumped to 31.02% from 17.26%. As2S2 or PI-103 had a mild effect on inhibiting colony formation of leukemia cell when used alone, but the effect was significantly enhanced when used together. They had no significant effect on the colony-forming capacity of normal hematopoietic stem cells when used at concentrations above. The number of cells expressing CD11b was significantly increased in As2S2 treatment group, but the number of cells expressing CD11b in PI-103 treatment group was not changed significantly. The number of cells expressing CD11b was increased more significantly in group treated with As2S2 and PI-103 than with As2S2 alone. The expression of p-AKT473 in CD34+CD38-Lin- cells from specimens of six leukemia patients was increased after being treated with As2S2 for 1 h, but after 24 h, it was downregulated. Conclusion As2S2 could effectively induce apoptosis of leukemia stem cells, promote differentiation, inhibit their ability of self-renewal and proliferation, PI-103 can be coordinated to enhance the above effect.
引文
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8. Yilmaz O" H., Valdez R, Theisen BK., et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006, 441:475-82.
9. Park S, Chapuis N, Bardet V, et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, hasantileukemic activity in AML. Leukemia. 2008,22:1698-706.
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1．Wicha MS， Liu SL and Dontu G．Cancer Stem Cells： An Old Idea-A Paradigm Shift．Cancer Res．2006， 66：1883-90．
2．Lan Wang， Guang-Biao Zhou， Ping Liu， et al．Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia．Proc Natl Acad Sci U S A．2008， 105： 4826-31．
3．Yin T， Wu YL， Sun HP， et al．Combined effects of As4S4 and imatinib on chronic myeloid leukemia cells and BCR-ABL oncoprotein．Blood， 2004， 104：4219-25．
4．Lu DP， Qiu JY， Jiang B， et al．Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia： a pilot report．Blood， 2002，99： 3136-43．
5．刘延方，江滨，陆道培．硫化砷诱导NB4细胞凋亡及细胞周期阻滞的研究．中华血液学杂志．2000，21 ：647-8．
6．Ito K， Bernardi R， Morotti A et al．PML targeting eradicates quiescent leukaemia-initiating cells．Nature．2008， 453：1072-8．
7．Tatham MH．， Geoffroy MC， Shen LN， et al．RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation．Nature cell biology．2008，10：538-46．
8．Bernardi R， Guernah I， Jin D， et al．PML inhibits HIF-1 a translation and neoangiogenesis through repression of mTOR．Nature．442：779-85．
9．Park S， Chapuis N， Bardet V， et al．PI-103， a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR， hasantileukemic activity in AML．Leukemia．2008，22：1698-706．
1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997, 3:730-7.
2. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature. 2004, 432:396-401.
3. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003,100:3983-8.
4. Jordan CT, Guzman ML, and Noble M. Cancer stem cells. N Engl J Med. 2006, 355:1253-61.
5. Jin LQ, Hope KJ, Zhai QL, et al. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006,12:1167-74.
6. Park S, Chapuis N, Bardet V, et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, hasantileukemic activity in AML. Leukemia. 2008,22:1698-706.
7. Yilmaz O" H., Valdez R, Theisen BK., et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006,441:475-82.
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32. Barnett SC, Robertson L, Graham D, et al. Oligodendrocytetype-2 astrocyte (O-2A) progenitor cells transformed with c-myc and H-ras form high-grade glioma after stereotactic injection into the rat brain. Carcinogenesis 1998; 19:1529-37.
33. Stingl J, Eirew P, Ricketson I, et al. Purification and unique properties of mammary epithelial stem cells. Nature 2006;439:993-7.
34. Shackleton M, Vaillant F, Simpson KJ, et al. Generation of a functional mammary gland from a single stem cell. Nature 2006;439:84-8.
35. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005;5:275-84.
36. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2:48-58.
37. Donnenberg VS, Donnenberg AD. Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis. J Clin Pharmacol 2005;45:872-7.
38. Cairns J. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis.Proc Natl Acad Sci U S A 2002;99: 10567-70.
39. Wang Y, Schulte BA, Larue AC, et al. Total body irradiation selectively induces murine hematopoietic stem cell senescence. Blood 2006;107:358-66.
40. Meng A, Wang Y, Van Zant G, et al. Ionizing radiation and busulfan induce premature senescence in murine bone marrow hematopoietic cells. Cancer Res 2003;63:5414-9.
41. Narita M, Lowe SW. Senescence comes of age. Nat Med 2005; 11:920-2.
42. Lowe SW, Cepero E, Evan G. Intrinsic tumour suppression. Nature 2004;432:307-15.
43. van Rhenen A, Feller N, Kelder A, et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005;11:6520-7.
1. Sancak Y, Thoreen CC, Peterson TR, et al. PRAS40 Is an Insulin-Regulated Inhibitor of the mTORCl Protein Kinase. Mol. Cell 2007;25:903-15.
2. Guertin DA, and Sabatini DM. An expanding role for mTOR in cancer. Trends Mol. Med. 2005;11:353-61.
3. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent
4. pathways. Nat. Med. 2004;10:594-601.
5. Jaeschke A, Hartkamp J, Saitoh M, et al. Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J. Cell Biol. 2002;159:217-24.
6. Haar EV, Lee SI, Bandhakavi S, et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 2007;9:316-23.
7. Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101.
8. Hresko RC and Mueckler M. mTOR/RICTOR is the Ser473 kinase for Akt/PKB in 3T3-L1 adipocytes. J. Biol. Chem. 2005;280:40406-16.
9. Brognard J, Sierecki E, Gao T, et al. PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol. Cell 2007;25:917-931.
10. Lee S, Comer FI, Sasaki A, et al. TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium. Mol. Biol. Cell 2005;16:4572-83.
11. Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 2002;10:457-68.
12. Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101.
13. Manning, B.D., Tee, A.R., Logsdon, M.N., et al. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 2002; 10: 151-162.
14. Guertin, D.A., Stevens, D.M., Thoreen, CC, et al. Ablation in Mice of the mTORC Components raptor, rictor, or mLST8 Reveals that mT0RC2 Is Required for Signaling to Akt-FOXO and PKCalpha, but Not S6K1. Dev. Cell 2006; 11: 859-871.
15. Jacinto, E., Facchinetti, V., Liu, D., et al. SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity. Cell 2006; 127: 125-137
16. 15. Hietakangas, V., and Cohen, S.M. Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth. Genes Dev. 2007; 21: 632-637.
17. Chiang, G.G., and Abraham, R.T. Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase. J. Biol. Chem. 2005; 280: 25485-25490.
18. Holz, M.K., and Blenis, J. Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase. J. Biol. Chem. 2005; 280: 26089-26093.
19. Zhang, H.H., Lipovsky, A.I., Dibble, C.C., et al. S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. Mol. Cell 2006; 24: 185-197.
20. McManus, E.J., Collins, B.J., Ashby, P.R., et al. The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. EMBO J. 2004; 23: 2071-2082.
21. Dong, J., and Pan, D. Tsc2 is not a critical target of Akt during normal Drosophila development. Genes Dev. 2004; 18; 2479-2484.
22. Tavazoie, S.F., Alvarez, V.A., Ridenour, D.A., et al. Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat. Neurosci. 2005; 8: 1727-1734.
23. Haar, E.V., Lee, S.I., Bandhakavi, S., Griffin, T.J., et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 2007; 9: 316-323.
24. Sancak, Y., Thoreen, C.C., Peterson, T.R., et al. PRAS40 Is an Insulin-Regulated Inhibitor of the mTORCl Protein Kinase. Mol. Cell 2007; 25: 903-915.
25. Samuels, Y., Diaz, L.A., Jr., Schmidt-Kittler, O., et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005; 7: 561-573.
26. Greer, E.L., and Brunet, A. (). FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 2005; 24: 7410-7425.
27. Potente, M., Urbich, C, Sasaki, K., et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J. Clin. Invest. 2005; 115: 2382-2392.
28. Paik, J.H., Kollipara, R., Chu, G., et al. FoxOs Are Lineage-Restricted Redundant Tumor Suppressors and Regulate Endothelial Cell Homeostasis. Cell 2007; 128: 309-323.
29. Tothova, Z., Kollipara, R., Huntly, B.J., et al. FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress. Cell 2007; 128: 325-339.
30. Podsypanina, K., Ellenson, L.H., Nemes, A., et al. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. USA 1999; 96: 1563-1568.
31. Fumarola C, La Monica S, AlfieriRR, et al. Cell size reduction induced by inhibition of the mTOR/S6Ksignaling pathway protects Jurkat cells from apoptosis. Cell Death Differ. 2005;12:1344-57.
32. Ghosh S, Tergaonkar V, Rothlin CV, et al. Essential role of tuberous sclerosis 33. genes TSC1 and TSC2 in NF-kappaB activation and cell survival. Cancer Cell 2006;10:215-226.
34. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB. Molecular Cell 2006;22:159-68.
35. Granville CA, Warfel N, Tsurutani J, et al. Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen-Induced Murine Lung Tumors. Clin. Cancer Res. 2007;13:2281-9.
36. Faivre S, Kroemer G and Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat. Rev. Drug Discov. 2006;5:671-88.
37. Granville CA, Memmott RM, Gills JJ, et al. Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway. Clin. Cancer Res. 2006;12: 679-689.
2. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997, 3:730-7.
3. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature. 2004,432:396-401.
4. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003, 100:3983-8.
5. Jordan CT, Guzman ML, and Noble M. Cancer stem cells. N Engl J Med. 2006, 355:1253-61.
6. Jin LQ, Hope KJ, Zhai QL, et al. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006,12:1167-74.
7. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood 2003,102:972-80.
8. Yilmaz O" H., Valdez R, Theisen BK., et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006, 441:475-82.
9. Park S, Chapuis N, Bardet V, et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, hasantileukemic activity in AML. Leukemia. 2008,22:1698-706.
10. Bernardi R, Guernah I, Jin D, et al. PML inhibits HIF-1a translation and neoangiogenesis through repression of mTOR. Nature. 442:779-85.
11. Ito K, Bernardi R, Morotti A et al. PML targeting eradicates quiescent leukaemia-initiating cells. Nature. 2008, 453:1072-8.
12. Lan Wang, Guang-Biao Zhou, Ping Liu, et al. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia. Proc Natl Acad Sci U S A. 2008, 105: 4826-31.
13. Lu DP, Qiu JY, Jiang B, et al. Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia: a pilot report. Blood, 2002, 99: 3136-43.
14. Yin T, Wu YL, Sun HP, et al. Combined effects of As4S4 and imatinib on chronic myeloid leukemia cells and BCR-ABL oncoprotein. Blood, 2004, 104:4219-25.
1．Wicha MS， Liu SL and Dontu G．Cancer Stem Cells： An Old Idea-A Paradigm Shift．Cancer Res．2006， 66：1883-90．
2．Lan Wang， Guang-Biao Zhou， Ping Liu， et al．Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia．Proc Natl Acad Sci U S A．2008， 105： 4826-31．
3．Yin T， Wu YL， Sun HP， et al．Combined effects of As4S4 and imatinib on chronic myeloid leukemia cells and BCR-ABL oncoprotein．Blood， 2004， 104：4219-25．
4．Lu DP， Qiu JY， Jiang B， et al．Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia： a pilot report．Blood， 2002，99： 3136-43．
5．刘延方，江滨，陆道培．硫化砷诱导NB4细胞凋亡及细胞周期阻滞的研究．中华血液学杂志．2000，21 ：647-8．
6．Ito K， Bernardi R， Morotti A et al．PML targeting eradicates quiescent leukaemia-initiating cells．Nature．2008， 453：1072-8．
7．Tatham MH．， Geoffroy MC， Shen LN， et al．RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation．Nature cell biology．2008，10：538-46．
8．Bernardi R， Guernah I， Jin D， et al．PML inhibits HIF-1 a translation and neoangiogenesis through repression of mTOR．Nature．442：779-85．
9．Park S， Chapuis N， Bardet V， et al．PI-103， a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR， hasantileukemic activity in AML．Leukemia．2008，22：1698-706．
1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997, 3:730-7.
2. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature. 2004, 432:396-401.
3. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003,100:3983-8.
4. Jordan CT, Guzman ML, and Noble M. Cancer stem cells. N Engl J Med. 2006, 355:1253-61.
5. Jin LQ, Hope KJ, Zhai QL, et al. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat Med. 2006,12:1167-74.
6. Park S, Chapuis N, Bardet V, et al. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, hasantileukemic activity in AML. Leukemia. 2008,22:1698-706.
7. Yilmaz O" H., Valdez R, Theisen BK., et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006,441:475-82.
1. Wang JC, Dick JE. Cancer stem cells: lessons from leukemia. Trends Cell Biol 2005; 15:4 94-501.
2. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105-11.
3. Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 2003;3:895-902.
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6. Fialkow PJ, Gartler SM, Yoshida A. Clonal origin of chronic myelocytic leukemia in man. Proc Natl Acad Sci U S A 1967;58:1468-71.
7. Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science 1977; 197:461-3.
8. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3:730-7.
9. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature 2004;432:396-401.
10. Al-Hajj M, Wicha MS, Benito-Hernandez A, et al. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100:3983-8.
11. Jamieson CH, Ailles LE, Dylla SJ, et al. Granulocyte-macrophage progenitors as candidate leukemic stem cells in blastcrisis CML. N Engl J Med 2004;351:657-67.
12. Cozzio A, Passegue E, Ayton PM, et al. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors. Genes Dev 2003; 17: 3029-35.
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26. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood 2003; 102:972-80.
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28. Xu Q, Thompson JE, Carroll M. mTOR regulates cell survival after etoposide treatment in primary AML cells. Blood 2005;106:4261-8.
29. Yilmaz OH, Valdez R, Theisen BK, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 2006;441:475-82.
30. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707-10.
31. Uchida N, Buck DW, He D, et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2000;97:14720-5.
32. Barnett SC, Robertson L, Graham D, et al. Oligodendrocytetype-2 astrocyte (O-2A) progenitor cells transformed with c-myc and H-ras form high-grade glioma after stereotactic injection into the rat brain. Carcinogenesis 1998; 19:1529-37.
33. Stingl J, Eirew P, Ricketson I, et al. Purification and unique properties of mammary epithelial stem cells. Nature 2006;439:993-7.
34. Shackleton M, Vaillant F, Simpson KJ, et al. Generation of a functional mammary gland from a single stem cell. Nature 2006;439:84-8.
35. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005;5:275-84.
36. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002;2:48-58.
37. Donnenberg VS, Donnenberg AD. Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis. J Clin Pharmacol 2005;45:872-7.
38. Cairns J. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis.Proc Natl Acad Sci U S A 2002;99: 10567-70.
39. Wang Y, Schulte BA, Larue AC, et al. Total body irradiation selectively induces murine hematopoietic stem cell senescence. Blood 2006;107:358-66.
40. Meng A, Wang Y, Van Zant G, et al. Ionizing radiation and busulfan induce premature senescence in murine bone marrow hematopoietic cells. Cancer Res 2003;63:5414-9.
41. Narita M, Lowe SW. Senescence comes of age. Nat Med 2005; 11:920-2.
42. Lowe SW, Cepero E, Evan G. Intrinsic tumour suppression. Nature 2004;432:307-15.
43. van Rhenen A, Feller N, Kelder A, et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005;11:6520-7.
1. Sancak Y, Thoreen CC, Peterson TR, et al. PRAS40 Is an Insulin-Regulated Inhibitor of the mTORCl Protein Kinase. Mol. Cell 2007;25:903-15.
2. Guertin DA, and Sabatini DM. An expanding role for mTOR in cancer. Trends Mol. Med. 2005;11:353-61.
3. Majumder PK, Febbo PG, Bikoff R, et al. mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent
4. pathways. Nat. Med. 2004;10:594-601.
5. Jaeschke A, Hartkamp J, Saitoh M, et al. Tuberous sclerosis complex tumor suppressor-mediated S6 kinase inhibition by phosphatidylinositide-3-OH kinase is mTOR independent. J. Cell Biol. 2002;159:217-24.
6. Haar EV, Lee SI, Bandhakavi S, et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 2007;9:316-23.
7. Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101.
8. Hresko RC and Mueckler M. mTOR/RICTOR is the Ser473 kinase for Akt/PKB in 3T3-L1 adipocytes. J. Biol. Chem. 2005;280:40406-16.
9. Brognard J, Sierecki E, Gao T, et al. PHLPP and a second isoform, PHLPP2, differentially attenuate the amplitude of Akt signaling by regulating distinct Akt isoforms. Mol. Cell 2007;25:917-931.
10. Lee S, Comer FI, Sasaki A, et al. TOR Complex 2 Integrates Cell Movement during Chemotaxis and Signal Relay in Dictyostelium. Mol. Biol. Cell 2005;16:4572-83.
11. Loewith R, Jacinto E, Wullschleger S, et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 2002;10:457-68.
12. Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005;307:1098-101.
13. Manning, B.D., Tee, A.R., Logsdon, M.N., et al. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol. Cell 2002; 10: 151-162.
14. Guertin, D.A., Stevens, D.M., Thoreen, CC, et al. Ablation in Mice of the mTORC Components raptor, rictor, or mLST8 Reveals that mT0RC2 Is Required for Signaling to Akt-FOXO and PKCalpha, but Not S6K1. Dev. Cell 2006; 11: 859-871.
15. Jacinto, E., Facchinetti, V., Liu, D., et al. SIN1/MIP1 Maintains rictor-mTOR Complex Integrity and Regulates Akt Phosphorylation and Substrate Specificity. Cell 2006; 127: 125-137
16. 15. Hietakangas, V., and Cohen, S.M. Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth. Genes Dev. 2007; 21: 632-637.
17. Chiang, G.G., and Abraham, R.T. Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase. J. Biol. Chem. 2005; 280: 25485-25490.
18. Holz, M.K., and Blenis, J. Identification of S6 kinase 1 as a novel mammalian target of rapamycin (mTOR)-phosphorylating kinase. J. Biol. Chem. 2005; 280: 26089-26093.
19. Zhang, H.H., Lipovsky, A.I., Dibble, C.C., et al. S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. Mol. Cell 2006; 24: 185-197.
20. McManus, E.J., Collins, B.J., Ashby, P.R., et al. The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation. EMBO J. 2004; 23: 2071-2082.
21. Dong, J., and Pan, D. Tsc2 is not a critical target of Akt during normal Drosophila development. Genes Dev. 2004; 18; 2479-2484.
22. Tavazoie, S.F., Alvarez, V.A., Ridenour, D.A., et al. Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat. Neurosci. 2005; 8: 1727-1734.
23. Haar, E.V., Lee, S.I., Bandhakavi, S., Griffin, T.J., et al. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 2007; 9: 316-323.
24. Sancak, Y., Thoreen, C.C., Peterson, T.R., et al. PRAS40 Is an Insulin-Regulated Inhibitor of the mTORCl Protein Kinase. Mol. Cell 2007; 25: 903-915.
25. Samuels, Y., Diaz, L.A., Jr., Schmidt-Kittler, O., et al. Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 2005; 7: 561-573.
26. Greer, E.L., and Brunet, A. (). FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 2005; 24: 7410-7425.
27. Potente, M., Urbich, C, Sasaki, K., et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J. Clin. Invest. 2005; 115: 2382-2392.
28. Paik, J.H., Kollipara, R., Chu, G., et al. FoxOs Are Lineage-Restricted Redundant Tumor Suppressors and Regulate Endothelial Cell Homeostasis. Cell 2007; 128: 309-323.
29. Tothova, Z., Kollipara, R., Huntly, B.J., et al. FoxOs Are Critical Mediators of Hematopoietic Stem Cell Resistance to Physiologic Oxidative Stress. Cell 2007; 128: 325-339.
30. Podsypanina, K., Ellenson, L.H., Nemes, A., et al. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. USA 1999; 96: 1563-1568.
31. Fumarola C, La Monica S, AlfieriRR, et al. Cell size reduction induced by inhibition of the mTOR/S6Ksignaling pathway protects Jurkat cells from apoptosis. Cell Death Differ. 2005;12:1344-57.
32. Ghosh S, Tergaonkar V, Rothlin CV, et al. Essential role of tuberous sclerosis 33. genes TSC1 and TSC2 in NF-kappaB activation and cell survival. Cancer Cell 2006;10:215-226.
34. Sarbassov DD, Ali SM, Sengupta S, et al. Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB. Molecular Cell 2006;22:159-68.
35. Granville CA, Warfel N, Tsurutani J, et al. Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen-Induced Murine Lung Tumors. Clin. Cancer Res. 2007;13:2281-9.
36. Faivre S, Kroemer G and Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat. Rev. Drug Discov. 2006;5:671-88.
37. Granville CA, Memmott RM, Gills JJ, et al. Handicapping the race to develop inhibitors of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin pathway. Clin. Cancer Res. 2006;12: 679-689.