依地福新抑制粟酒裂殖酵母细胞胞质分裂机制的研究
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摘要
第一章依地福新对粟酒裂殖酵母细胞胞质分裂的影响
【背景与目的】：依地福新为人工合成的抗肿瘤醚酯类药物，它能抑制肿瘤细胞分裂，但并不抑制核的裂解，导致细胞聚集在G_2／M期，形成多核细胞，随后通过凋亡促使细胞死亡。也有报告指出，依地福新处理过的细胞能经历完全的细胞周期，但不能进行分裂，使细胞聚集在G_0／G_1期，形成四倍体或八倍体细胞。然而，依地福新抑制肿瘤细胞的胞质分裂的机制仍然不清楚。人体细胞与酵母细胞在生长周期存在着相似性。许多有关人体细胞的研究成果都是首先通过研究酵母细胞发现的。癌症细胞本质上来源于机体正常细胞。由于在细胞分裂的不同阶段受各种因素的影响，导致其出现分化和增殖的异常，从而使人们在利用癌细胞进行有关研究时带来许多困难。而以粟酒裂殖酵母作为研究对象，易于进行细胞学操作、具有明显的细胞周期，且生长迅速、易于培养。因此本研究以粟酒裂殖酵母作为实验材料，探讨依地福新(edelfosine，ET-18-OCH_3，1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine)抑制粟酒裂殖酵母(Schizosaccharomycespombe，S．pombe)细胞生长的作用剂量，及在此作用剂量下，依地福新对粟酒裂殖酵母细胞胞质分裂和胞核分裂的影响。
【方法】：(1)、应用粟酒裂殖酵母细胞和Jurkat细胞的生长抑制试验，确定依地福新抑制粟酒裂殖酵母细胞生长的作用剂量。
(2)、利用粟酒裂殖酵母细胞胞质分裂抑制试验，观察依地福新对粟酒裂殖酵母细胞胞质分裂的影响。
(3)、利用粟酒裂殖酵母细胞胞核分裂试验和粟酒裂殖酵母DNA含量检测试验，观察依地福新对粟酒裂殖酵母细胞胞核分裂的影响。
【结果】：(1)、在粟酒裂殖酵母细胞和Jurkat细胞的生长抑制试验中可见，野生型粟酒裂殖酵母细胞和Jurkat细胞培养6h后，其生长能被5.0μM、10.0μM和20.0μM的依地福新所抑制，与平行生长的0μM依地福新处理的细胞比较差异有统计学意义；培养8h后，其生长除能被5.0μM、10.0μM和20.0／aM的依地福新所抑制，还能被1.0μM的依地福新所抑制，与平行生长的0μM依地福新处理的细胞比较差异亦有统计学意义。
(2)、在粟酒裂殖酵母细胞胞质分裂抑制试验中可见，对照组(edelfosine 0μM)粟酒裂殖酵母细胞形态正常，隔膜位于细胞中央，厚度正常，两侧对称。低剂量观察组(edelfosine 1.0μM)粟酒裂殖酵母细胞出现多个隔膜或出现超长而没有隔膜的细胞；细胞隔膜增厚；细胞大小不一，透光性下降，且出现少量粒状细胞。高剂量观察组(edelfosine 5.0μM)粟酒裂殖酵母细胞形态不规则，出现较多奇异形细胞，细胞透光性差；细胞隔膜增厚；出现较多粒状细胞。粟酒裂殖酵母细胞隔膜计数结果显示，对照组(edelfosine 0μM)和低剂量观察组(edelfosine 1.0μM)比较，两组之间差异有统计学意义(X~2=16.089，P＜0.01)。
(3)、在粟酒裂殖酵母细胞胞核分裂试验中可见，对照组(edelfosine0μM组)细胞核大小和数目均正常，在裂殖细胞可见双核，未见异常核型；处理组(edelfosine 1.5μM组)粟酒裂殖酵母核染色较弱，可见子细胞尚未与母细胞分离，但母细胞已开始进入到下一个有丝分裂M期的细胞。
(4)、在粟酒裂殖酵母DNA含量检测试验中可见，对照组(edelfosine 0μM组)大部分细胞DNA含量为1C，只有极少量细胞DNA含量为2C；而edelfosine 0.5μM组、edelfosine 1.0μM组和edelfosine 1.5μM组细胞DNA含量为2 C。
【结论】：(1)、1.0μM-5.0μM浓度的依地福新能抑制粟酒裂殖酵母和Jurkat细胞生长，对粟酒裂殖酵母具有与其抗肿瘤细胞相似作用。(2)、0.5μM-1.5μM浓度的依地福新依地福新抑制粟酒裂殖酵母细胞的胞质分裂，但不影响细胞核DNA的合成和胞核的分离。
第二章依地福新对粟酒裂殖酵母胞质分裂突变体mid2Δ、spmlΔ和pmplΔ生长的影响
【背景与目的】：有报道指出，依地福新在其凋亡浓度(5-25μM)时，可抑制MAPK／ERK促有丝分裂途径和Akt／PKB(蛋白激酶B)生存途径。在粟酒裂殖酵母细胞人们已经发现了多条MAPK级联，其中Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径与细胞形态发生有关。Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径通过细胞膜上的Mid2接受细胞外信号后，经小GTP酶Rho4激活PKC_1-MPK_1细胞完整性通路，使Spm1磷酸化程度增加，最终激活Spm1；而Pmp1能抑制Spm1的磷酸化，使Spm1的磷酸化程度降低。spm1基因，也称之为pmk1，它的系统命名为SPBC119.08，编码生成的蛋白为MAP激酶Spm1／Pmk1(MAPkinase Spm1／Pmk1，MAPK~(Spm1/Pmk1)；pmp1基因的系统命名为SPBC1685.01，它编码生成的蛋白为双重特异的MAPK磷酸酶Pmp1(dual-specificity MAP kinase phosphatase Pmp1)，Pmp1参与MAPKKK级联反应(MAPKKK cascade)，与细胞裂殖时的信息传递有关，具有使蛋白氨基酸脱磷酸化作用，能影响Spin1蛋白磷酸化；mid2基因的系统命名为SPAPYUG7.03c，它编码生成的蛋白为香兰素同系物Mid2(anillin homologue Mid2)，Mid2影响裂殖酵母细胞隔膜的组装(organization)和解聚(disassembly)，它参与胞质分裂时的细胞分离过程。
为了验证依地福新对Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)信号级联的影响，需要判断出依地福新抑制胞质分裂是否与mid2、spm1和pmp1基因有关。其方法是利用这些细胞的突变体。这些突变体由于基因突变，而被封阻在细胞周期的某一特定阶段，从而使我们得知此突变的基因是与该特定阶段的调控有关的基因。本实验以胞质分裂突变体mid2Δ、spm1Δ和pmp1Δ为实验材料，探讨依地福新对MAPK级联信号相关基因的影响，从而阐明依地福新抑制胞质分裂的机制。
【方法】：应用粟酒裂殖酵母细胞突变体生长抑制试验，观察依地福新对野生型粟酒裂殖酵母细胞和mid2突变体(mid2Δ)、spm1突变体(spm1Δ)、pmp1突变体(pmp1A)生长率的影响。
【结果】：应用0μM、0.156μM、0.312μM、0.625μM、1.25μM、2.5μM、5μM、10μM依地福新处理野生型粟酒裂殖酵母及mid2突变体、spm1突变体、pmp1突变体，在培养20h后，spm1突变体在5μM和10μM浓度依地福新处理时的生长率均高于同样浓度处理的野生型粟酒裂殖酵母的生长率(5μM依地福新处理时，spm1A组vsWT组为88.3±7.6 vs 15.9±1.7；10μM依地福新处理时，spm1Δ组vsWT组为85.3±6.7 vs 15.7±1.6；t值分别为16.10，18.25；P＜0.01)。mid2A和pmp1A在5μM浓度时，它们的生长率均高于野生型粟酒裂殖酵母的生长率(mid2Δvs WT组为76.4±6.2 vs 15.9±1.7；pmp1ΔvsWT组为76.9±5.8 vs 15.9±1.7；t值分别为16.29，17.48；P＜0.01)；结果说明，培养20h后，spm1Δ对5μM和10μM依地福新都具有抵抗性，而mid2Δ和pmp1Δ则对5μM依地福新具有抵抗性。
【结论】：spm1、mid2和pmp1基因突变后对依地福新的抗性增强。因此，依地福新抑制粟酒裂殖酵母胞质分裂可能与MAPK级联相关的mid2、pmp1、spm1基因有关。
第三章spm1、pmp1和mid2基因在相应粟酒裂殖酵母突变体中的再表达
【背景与目的】：利用酵母突变体及其同源基因再表达的功能互补实验，是目前遗传学和分子生物学研究的一种重要实验方法，并使酵母成为筛查药物作用机制的工具。通过使用药物对特定的酵母基因突变株进行干预，筛选出对药物敏感或抵抗的突变株，然后利用酵母基因重组技术，使突变株再表达同源的基因，从而获得再表达同一基因的克隆。如果构建的同源克隆酵母可以挽救此突变株对药物的敏感性或抵抗性，那么就说明药物可能影响此基因。这将成为一种筛选抗癌和抗病毒药物的分析系统。因此，将spm1、pmp1和mid2基因在相应粟酒裂殖酵母突变株中进行再表达，观察其是否能恢复对依地福新的敏感性，可确定依地福新对这些基因是否产生影响。本实验的目的是，将粟酒裂殖酵母细胞胞质分裂相关基因spm1、pmp1和mid2在相应突变株中进行再表达，并筛选出有表达活性的重组子。
【方法】：(1)、应用TRIZOL试剂法抽提粟酒裂殖酵母细胞的RNA；(2)、反转录PCR(RT-PCR)制备粟酒裂殖酵母细胞的cDNA；(3)、应用PCR扩增spm1、pmp1和mid2基因；(4)、将spm1、pmp1和mid2基因克隆到pREP3X-HA质粒中；(5)、将pREP3X-HA-spm1、pREP3X-HA-pmp1和pREP3X-HA-mid2电转化到粟酒裂殖酵母突变体spm1Δ、pmp1Δ和mid2Δ细胞中；(6)、应用硫胺素抑制试验筛选活性的pREP3X-HA-spm1、pREP3X-HA-pmp1和pREP3X-HA-mid2重组酵母。
【结果】：在35个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-spm1重组子。在43个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-pmp1重组子。在41个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-mid2重组子。
【结论】：pREP3X-HA-spm1穿梭载体、pREP3X-HA-pmp1穿梭载体和pREP3X-HA-mid2穿梭载体成功转入到相应的突变体细胞中，且转化子具有表达活性。
第四章依地福新影响粟酒裂殖酵母MAP激酶Spm1的磷酸化
【背景与目的】：蛋白激酶级联调节胞质和胞核对细胞外刺激的应答。MAPK(mitogen-activated protein kinase，丝裂原激活的蛋白激酶)级联是在单细胞和多细胞真核生物中发现的古老而保守的蛋白激酶级联。在粟酒裂殖酵母(S．pombe)细胞人们目前也已经发现了多条MAPK级联，其中Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径与细胞形态发生有关。在粟酒裂殖酵母细胞中，细胞胞膜上的Mid2接受细胞外信号后，经GTPase Rho4激活PKC_1-MPK_1细胞完整性通路，信号经Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)途径传递，最终影响细胞形态的发生。而Pmp1在体外能直接去除Spm1／Pmk1酪氨酸残基上的磷酸，导致Spm1／Pmk1去磷酸化；Pmp1在体内也能影响Spm1／Pmk1中酪氨酸的磷酸化。因此，本实验的目的是探讨依地福新对经Mid2介导的，Pmp1抑制的MAPK Spm1信号通路的影响，以阐明依地福新抑制粟酒裂殖酵母细胞胞质分裂的作用机制。
【方法】：(1)、通过野生型粟酒裂殖酵母和胞质分裂突变体spm1Δ、pmp1Δ、mid2Δ及其再转化株对依地福新的平行生长抵抗试验，进一步确定依地福新对spm1、pmp1、mid2基因的影响；(2)、应用依地福新对Spm1磷酸化的影响试验，阐明依地福新是否通过影响Mid2蛋白和Pmp1蛋白的表达而影响Spm1蛋白的磷酸化。
【结果】：(1)、在spm1、pmp1和mid2突变株及其再转染株对依地福新的平行生长抵抗试验中，spm1Δ组细胞培养24h后，在含有终浓度为5.0μM和10.0μM依地福新的培养液中仍生长良好；其生长率与在含有终浓度为5.0μM和10.0μM依地福新的培养液中培养的WT组细胞比较，差异有统计学意义(5.0μM依地福新处理时，spm1Δ组vs WT组细胞生长率为101.1±6.6 vs 5.3±0.7；10.0μM依地福新处理时，spm1Δ组vs WT组细胞生长率为105.5±9.8 vs 5.6±0.9；t值分别为25.00，17.58；P＜0.01)。依地福新对spm1Δ组细胞的IC_(50)(半数抑制浓度)为(50.12±4.31)μM，而对WT细胞的IC_(50)为(3.75±0.34)μM，两者比较有统计学意义(t值为18.58，P＜0.01)。mid2Δ组细胞和pmp1Δ组细胞培养24h后，在含有终浓度为5.0μM依地福新的培养液中生长良好；其生长率与在含有终浓度为5.0μM依地福新的培养液中培养的WT组细胞比较，其差异有统计学意义(mid2A组vs WT组细胞生长率为77.3±4.5 vs 5.3±0.7，t=27.38，P＜0.01，pmp1Δ组vsWT组细胞生长率为81.3±4.7 vs 5.3±0.7，t=27.70，P＜0.01)。依地福新对mid2Δ组细胞和pmp1Δ组细胞的IC_(50)分别为(7.12±0.63)μM和(7.25±0.65)μM，与WT细胞的IC_(50)[(3.75±0.34)μM]比较有统计学意义(t值分别为8.15和8.26，P＜0.01)。
(2)、在依地福新对Spm1磷酸化的影响试验中，依地福新处理组只有pREP3X-HA-mid2重组酵母、pREP3X-HA-pmp1重组酵母和pREP3X-HA-spm1重组酵母能检测到磷酸化的活性MAPK，而WT细胞、mid2Δ细胞、pmp1Δ细胞和spm1Δ细胞未检测到磷酸化的活性MAPK；而在依地福新未处理组，所有细胞均未检测到磷酸化的活性MAPK；结果说明，磷酸化活性MAPK Spm1的产生是由于依地福新诱导了Mid2的表达和／或依地福新抑制了Pmp1活性的缘故。
【结论】：(1)、依地福新通过影响粟酒裂殖酵母细胞mid2、spm1和pmp1基因而抑制粟酒裂殖酵母细胞的胞质分裂。(2)、细胞外依地福新诱导Mid2蛋白的表达，从而促进的Spm1磷酸化；细胞内依地福新通过抑制Pmp1蛋白的表达，取消Pmp1蛋白对Spm1的抑制作用，使Spm1的磷酸化程度增加，最终导致粟酒裂殖酵母细胞胞质分裂障碍。
Chapter 1 The Effect of Edelfosine on Cytokinesis of S. pombe
【Background and Objects】: Edelfosine is a synthetical alkyl-lysophospholipid analog, also known as antitumor ether lipids. It can inhibit cell division without concurrent inhibition of nuclear division, leading to accumulation of cells in G2/M, multinucleate cell formation, and subsequent cell death through apoptosis. It also reported that cells treated with edelfosine came through the whole cell cycle without nuclei cleavage, and cells were blocked in G_0/G_1 and subsequent formation of quadplex or octploid nuclei. However, the mechanism still is unknown that edelfosine inhibits cytokinesis. Cells in human are similar to yeast in cell cycle, and a lot of studies of human cells base on yeast research. Cancer cells derive from normal human cells affected by various factor. Differentiation and proliferation are abnormal in cancer cells, leading to a lot of difficulty when people to study it. However, the fission yeast S. pombe has become a powerful model organism with which to study the process of cytokinesis. Some of its key attributes and advantages in this regard include the ease with which cytological manipulations can be performed, a well-characterized mitotic cell cycle, and fast growth and culture easily. In this study, we utilized the S. pombe to explore the effect of dosage-dependent of edelfosine inhibiting the growth, cell division and nucleus division of S. pombe.
【Methods】: (1)、We performed the experiment that edelfosine inhibited the growth of S. pombe and Jurkat cell, and confirmed the best effective dosage of edelfosine inhibiting the growth of the S. pombe.
(2)、We performed the experiment that edelfosine inhibiting the cytokinesis of S. pombe, and analyzed the effect of edelfosine on the cytokinesis of S. pombe.
(3)、We carried out the experiment that edelfosine acted on the nucleus division and detected the DNA content of S. pombe, and analyzed the effect of edelfosine on nucleus division of S. pombe.
【Results】: (1)、Treated with 5.0μM, 10.0μM and 20.0μM edelfosine for 6 h, the growth of S. pombe wild-type and Jurkat cell had been inhibited by edelfosine in the S. pombe's growth inhibition experiment. The difference has statistic significance (P＜0.01) between these cell treated with 5.0μM, 10.0μM and 20.0μM edelfosine for 6 h and those cell treated with 0μM edelfosine for 6 h. Treated with 1.0μM, 5.0μM, 10.0μM and 20.0μM edelfosine for 8 h, the growth of S. pombe wild-type and Jurkat cell had been inhibited not only by 5.0μM, 10.0μM and 20.0μM edelfosine, but also by 1.0μM edelfosine. The difference has statistic significance (P＜0.01) between these cell treated with 1.0μM, 5.0μM, 10.0μM and 20.0μM edelfosine for 8 h and those cell treated with 0μM edelfosine for 8 h.
(2)、In the inhibition experiment of cytokinesis of S. pombe, the S. pombe cells presented normal symmetrical shape with medium septum if they had not been treated by edelfosine (0μM); Meanwhile, the cells possessed cell wall with natural thickness. For the cells treated with 1.0μM edelfosine, they showed multiply septum or without septum, and also showed different size with decreased refraction.; In addition, the cells' septa were much thicker than the cells' untreated with edelfosine. A few granular cells were found. The number of the abnormal shape cells with decreased refraction become more and more, their septa become thicker and their shape become more asymmetrical, and the granule cell become more and more when the cells were treated with 5.0μM edelfosine for 6 h. The counted results of S. pombe septum showed a statistic significance between the cells treated with 0μM and 1.0μM edelfosine (χ~2=16.089, P＜0.01).
(3)、In the inhibition experiment of nucleus division of S. pombe, the size and number of the cells nucleus were in nature status, the cells with two nucleuses can be found, and the cells with abnormal nucleus had not existed in the cells if it had not been treated with edelfosine (0μM edelfosine); The fluorescence was generally weaker in the nucleus of the cells treated by 1.5μM edelfosine than untreated. A fissiparous cell shown that the mother cell had already started next mitosis while the young cell still had not split from the mother cell in the cells treated by 1.5μM edelfosine.
(4)、DNA amount detected by FACS showed that most cells has 1C DNA content and a few cells has 2C DNA content in the cells untreated with edelfosine (0μM); However, most cells has 2C DNA content in the cells treated by 0.5μM、1.0μM、1.5μM edelfosine.
【Conclusions】: (1)、1.0μM-5.0μM edelfosine has a similar effect on S. pombe and cancer cell to inhibit cell growth. (2)、0.5μM-1.5μM edelfosine inhibit cell division without inhibiting nucleus division.
Chapter 2 The Growth Effect of Edelfosine on mid2 Mutants, spm1 Mutants and pmp1 Mutants of S. pombe
【Background and Objects】: The concentration of apoptosis of 5-25μM edelfosine inhibits the MAPK/ERK and Akt/PKB pathway. Several MAPK cascades have been found in S. pombe. The Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) pathway has a relation with morphogenesis in S. pombe. Mid2 activates the PKC_1-MPK_1 cell integrity pathway via the small GTPase Rho4 resulting from exposure to extracellular signals and activate Spm1 finally, meanwhile, Pmp1 affect the photophosphorylation of Spm1. The system name of spm1 gene is SPBC119.08, and the coding protein of spm1 gene is MAP kinase Spm1/Pmk1. The system name of pmp1 gene is SPBC1685.01, and the coding protein of pmp1 gene is dual-specificity MAP kinase phosphatase Pmp1. Pmp1 is concerned with the MAPKKK cascade, and transfer information in cell division by affecting the photophosphorylation of Spm1. The system name of mid2 gene is SPAPYUG7.03c, and the coding protein of mid2 gene is anillin homologue Mid2. Mid2 affect the organization and disassembly of septum, and it is concerned with cell separation in cell division.
In order to confirm the effect of edelfosine on Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) signaling cascade, we need to judge whether the inhibition of cytokinesis have a correlation with mid2, spm1 and pmp1 genes. We designed the mutants of mid2, spm1 and pmp1, which have been blocked in a given stage of cell cycle. In this study, we explored the effect of edelfosine on mid2、spm1 and pmp1 mutants relating to MAPK cascade and elucidate the inhibition mechanism of cytokinesis.
【Methods】: The effect of edelfosine on the growth ratio of S. pombe wild-type, mid2 mutants, spm1 mutants and pmp1 mutants were observed via the inhibition experiment of growth of S. pombe mutants.
【Results】: The S. pombe wild-type cells, mid2 mutants, spm1 mutants and pmp1 mutants were treated with a series of edelfosine (0μM、0.15μM、0.312μM、0.625μM、1.25μM、2.5μM、5μM、10μM) for 20 h. The growth ratios of spm1 mutants were higher than of wild-type cells treated with the same concentration of edelfosine (treated with 5μM edelfosine, the growth ratio of spm1 mutants vs of WT was 88.3±7.6 vs 15.9±1.7; treated with 10μM edelfosine, the growth ratio of spm1 mutants vs of WT was 85.3±6.7 vs 15.7±1.6; t value was 16.10, 18.25, respectively; P＜0.01). The growth ratio of mid2 mutants and pmp1 mutants treated with 5μM edelfosine were higher than of S. pombe wild-type cells (the growth ratio of mid2 mutants vs of WT was 76.4±6.2 vs 15.9±1.7; the growth ratio of pmp1 mutants vs of WT was 76.9±5.8 vs 15.9±1.7; the t value was 16.29, 17.48, respectively; P＜0.01). The results showed that the spm1 mutants were hyper resistant to 5μM and 10μM edelfosine; Meanwhile, the mid2 and pmp1 mutants were hyper resistant to 5μM edelfosine.
【Conclusions】: Edelfosine maybe have an effect on the mid2, pmp1 and spm1 gene relating to MAPK cascade in S. pombe cells. Chapter 3 The Re-Expression of spm1, pmp1 and mid2 Genes in Relevant Mutants of S. pombe
【Background and Objects】: Yeast is a model organism for studying eukaryote, especially in the research of humam genomics. Yeast also is an important research material and provides a detectable experimental system in genetics and molecular biology. The functional complementation assay of homeotic gene re-expression in yeast mutant has become a screen tool in mechanism research of drug. To screen the sensitive and resistant yeast mutant when they treated with drug, the recombine technology of yeast gene should be applied to obtain the clone expressing the homeotic gene. If the yeast with homeotic gene can retrieve the sensitivity or resistance, it could show that the drug affect the gene. The method is useful as an analysis system in screening anti-cancer and anti-virus drug research. Thus, if we re-express the spm1, pmp1 and mid2 genes in relevant yeast mutants, and observe whether they retrieve the sensitivity, we will judge whether edelfosine have an effect on these genes. In this study, we observed the retransfected mutants whether retrieved the sensitive to edelfosine, and confirmed the effect of edelfosine on the S. pombe cells that re-expressed the spm1, pmp1 and mid2 genes in relevant mutants.
【Methods】: (1)、Total RNA of S. pombe cells was extracted by TRIZOL reagent; (2)、First-strand cDNA of S. pombe cells was synthesized by RT-PCR; (3)、he spm1, pmp1 and mid2 genes were amplified by PCR; (4)、The spm1, pmp1 and mid2 genes were cloned into plasmid and formed a relevant shuttle carrier; (5)、The pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3X-HA-mid2 shuttle carriers were transformed into spm1, pmp1 and mid2 mutants by Electroporation, respectively; (6)、The active recombination was identified by the inhibition experiment of thiamine.
【Results】: An active recombination of pREP3X-HA-spm1 was identified from 35 single clones of relevant transformed S. pombe. An active recombination of pREP3X-HA-pmp1 was identified from 43 single clones of relevant transformed S. pombe. An active recombination of pREP3X-HA-mid2 was identified from 41 single clones of relevant transformed S. pombe.
【Conclusion】: The pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3X-HA-mid2 shuttle carriers have successfully been transformed into relevant S. pombe mutants; and the recombination of pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3 X-HA-mid2 possesses an expressed activity. Chapter 4 Edelfosine Affect the Phosphatization of MAPK Spm1 of S. pombe
【Background and Objects】: Protein kinase cascade regulates the response of extracellular stimulation in cytoplast and cell nuclei. The MAPK signal cascade is an ancestral and conservative protein kinase in eukaryote. In present, several MAPK cascades have been found in S. pombe. The Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) pathway has a relation with morphogenesis in S. pombe. Mid2p activates the PKC_1-MPK_1 cell integrity pathway via the small GTPase Rho4 resulting from exposure to extracellular signals and activate Spm1 finally, meanwhile, Pmp1 affect the photophosphorylation of Spm1/Pmk1 in vivo and in vitro. In this study, we explored the function mechanism of edelfosine inhibiting the cytokinesis of S. pombe and the effect of edelfosine on MAPK Spm1 pathway induced by Mid2 and inhibited by Pmp1.
【Methods】: Firstly, the parallel growth inhibition experiment of S. pombe wild-type cells, spm1△, pmp1△, mid2△and relevant retransform strains were carried out in order to farther confirmed the effect of edelfosine on spm1, pmp1 and mid2 genes. Then, the experiment that edelfosine inhibited the phosphatization of Spm1 was carried out in order to elucidate whether edelfosine affect on the phosphatization of Spm1 induced via Mid2 and inhibited via Pmp1.
【Results】: (1)、On the inhibition experiment of parallel growth of S. pombe wild-type cells, spm1 mutants, pmp1 mutants, mid2 mutants and relevant retransform strains, the spm1 mutants grew well treated with 5.0μM and 10.0μM edelfosine for 24 h; the growth ratio of spm1 mutants had a statistic significance in between the cells previously mentioned and the S. pombe wild-type cells treated with 5.0μM and 10.0μM edelfosine (treated with 5.0μM edelfosine, the growth ratio of spm1 mutants vs wild-type cells was 101.1±6.6 vs 5.34±0.7; treated with 10.0μM edelfosine, the growth ratio of spm1 mutants vs wild-type cells was 105.5±9.8 vs 5.6±0.9; the t values were 25.00, 17.58, respectively; P＜0.01). The IC_(50) of spm1 mutants and wild-type cells treated with edelfosine was (50.12±4.31)μM and (3.75±0.34)μM, respectively. The IC_(50) difference between spm1 mutants and wild-type cells had statistic significance (t=18.58, P＜0.01). The mid2 mutants and pmp1 mutants also grew well treated with 5.0μM edelfosine for 24 h. The growth ratio of mid2 mutants and pmp1 mutants had a statistic significance in between the cells previously mentioned and the S. pombe wild-type cells treated with 5.0μM edelfosine (treated with 5.0μM edelfosine, the growth ratio of mid2 mutants vs wild-type cells was 77.3±4.5 vs 5.3±0.7, t=27.38, P＜0.01; the growth ratio of pmp1 mutants vs wild-type cells was 81.3±4.7 vs 5.3±0.7, t=27.70, P＜0.01). The IC_(50) of mid2 and pmp1 mutants treated with edelfosine were (7.12±0.63)μM and (7.25±0.65)μM, respectively. Compared with wild-type cells treated with edelfosine, the difference of IC_(50) had statistic significance (the t value was 8.15 and 8.26, respectively, P＜0.01).
(2)、On the experiment of edelfosine inhibiting the phosphatization of Spm1, the active MAPK were detected in the cells contained pREP3 X-HA-mid2, pREP3 X-HA-pmp1 and pREP3 X-HA-spm1 plasmid when they treated with edelfosine, but the active MAPK were not detected in S. pombe wild-type cells, mid2 mutants, pmp1 mutants and spm1 mutants when they treated with edelfosine. Meanwhile, the active MAPK were not detected in all tested cells when they untreated with edelfosine. The results showed that the active MAPK Spm1 was produced via edelfosine inducing the expression of Mid2 and/or edelfosine inhibiting the expression of Pmp1.
【Conclusions】: (1)、Edelfosine inhibited cytokinesis of S. pombe cells via acting on the mid2, spm1 and pmp1 genes. (2)、The extracellular edelfosine promoted the phosphatization of Spmlvia inducing the expression of Mid2; The intracellular edelfosine increased the phosphatization of Spmlvia inhibiting the expression of Pmp1, and leading the holdback of cytokinesis.
【背景与目的】：依地福新为人工合成的抗肿瘤醚酯类药物，它能抑制肿瘤细胞分裂，但并不抑制核的裂解，导致细胞聚集在G_2／M期，形成多核细胞，随后通过凋亡促使细胞死亡。也有报告指出，依地福新处理过的细胞能经历完全的细胞周期，但不能进行分裂，使细胞聚集在G_0／G_1期，形成四倍体或八倍体细胞。然而，依地福新抑制肿瘤细胞的胞质分裂的机制仍然不清楚。人体细胞与酵母细胞在生长周期存在着相似性。许多有关人体细胞的研究成果都是首先通过研究酵母细胞发现的。癌症细胞本质上来源于机体正常细胞。由于在细胞分裂的不同阶段受各种因素的影响，导致其出现分化和增殖的异常，从而使人们在利用癌细胞进行有关研究时带来许多困难。而以粟酒裂殖酵母作为研究对象，易于进行细胞学操作、具有明显的细胞周期，且生长迅速、易于培养。因此本研究以粟酒裂殖酵母作为实验材料，探讨依地福新(edelfosine，ET-18-OCH_3，1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine)抑制粟酒裂殖酵母(Schizosaccharomycespombe，S．pombe)细胞生长的作用剂量，及在此作用剂量下，依地福新对粟酒裂殖酵母细胞胞质分裂和胞核分裂的影响。
【方法】：(1)、应用粟酒裂殖酵母细胞和Jurkat细胞的生长抑制试验，确定依地福新抑制粟酒裂殖酵母细胞生长的作用剂量。
(2)、利用粟酒裂殖酵母细胞胞质分裂抑制试验，观察依地福新对粟酒裂殖酵母细胞胞质分裂的影响。
(3)、利用粟酒裂殖酵母细胞胞核分裂试验和粟酒裂殖酵母DNA含量检测试验，观察依地福新对粟酒裂殖酵母细胞胞核分裂的影响。
【结果】：(1)、在粟酒裂殖酵母细胞和Jurkat细胞的生长抑制试验中可见，野生型粟酒裂殖酵母细胞和Jurkat细胞培养6h后，其生长能被5.0μM、10.0μM和20.0μM的依地福新所抑制，与平行生长的0μM依地福新处理的细胞比较差异有统计学意义；培养8h后，其生长除能被5.0μM、10.0μM和20.0／aM的依地福新所抑制，还能被1.0μM的依地福新所抑制，与平行生长的0μM依地福新处理的细胞比较差异亦有统计学意义。
(2)、在粟酒裂殖酵母细胞胞质分裂抑制试验中可见，对照组(edelfosine 0μM)粟酒裂殖酵母细胞形态正常，隔膜位于细胞中央，厚度正常，两侧对称。低剂量观察组(edelfosine 1.0μM)粟酒裂殖酵母细胞出现多个隔膜或出现超长而没有隔膜的细胞；细胞隔膜增厚；细胞大小不一，透光性下降，且出现少量粒状细胞。高剂量观察组(edelfosine 5.0μM)粟酒裂殖酵母细胞形态不规则，出现较多奇异形细胞，细胞透光性差；细胞隔膜增厚；出现较多粒状细胞。粟酒裂殖酵母细胞隔膜计数结果显示，对照组(edelfosine 0μM)和低剂量观察组(edelfosine 1.0μM)比较，两组之间差异有统计学意义(X~2=16.089，P＜0.01)。
(3)、在粟酒裂殖酵母细胞胞核分裂试验中可见，对照组(edelfosine0μM组)细胞核大小和数目均正常，在裂殖细胞可见双核，未见异常核型；处理组(edelfosine 1.5μM组)粟酒裂殖酵母核染色较弱，可见子细胞尚未与母细胞分离，但母细胞已开始进入到下一个有丝分裂M期的细胞。
(4)、在粟酒裂殖酵母DNA含量检测试验中可见，对照组(edelfosine 0μM组)大部分细胞DNA含量为1C，只有极少量细胞DNA含量为2C；而edelfosine 0.5μM组、edelfosine 1.0μM组和edelfosine 1.5μM组细胞DNA含量为2 C。
【结论】：(1)、1.0μM-5.0μM浓度的依地福新能抑制粟酒裂殖酵母和Jurkat细胞生长，对粟酒裂殖酵母具有与其抗肿瘤细胞相似作用。(2)、0.5μM-1.5μM浓度的依地福新依地福新抑制粟酒裂殖酵母细胞的胞质分裂，但不影响细胞核DNA的合成和胞核的分离。
第二章依地福新对粟酒裂殖酵母胞质分裂突变体mid2Δ、spmlΔ和pmplΔ生长的影响
【背景与目的】：有报道指出，依地福新在其凋亡浓度(5-25μM)时，可抑制MAPK／ERK促有丝分裂途径和Akt／PKB(蛋白激酶B)生存途径。在粟酒裂殖酵母细胞人们已经发现了多条MAPK级联，其中Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径与细胞形态发生有关。Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径通过细胞膜上的Mid2接受细胞外信号后，经小GTP酶Rho4激活PKC_1-MPK_1细胞完整性通路，使Spm1磷酸化程度增加，最终激活Spm1；而Pmp1能抑制Spm1的磷酸化，使Spm1的磷酸化程度降低。spm1基因，也称之为pmk1，它的系统命名为SPBC119.08，编码生成的蛋白为MAP激酶Spm1／Pmk1(MAPkinase Spm1／Pmk1，MAPK~(Spm1/Pmk1)；pmp1基因的系统命名为SPBC1685.01，它编码生成的蛋白为双重特异的MAPK磷酸酶Pmp1(dual-specificity MAP kinase phosphatase Pmp1)，Pmp1参与MAPKKK级联反应(MAPKKK cascade)，与细胞裂殖时的信息传递有关，具有使蛋白氨基酸脱磷酸化作用，能影响Spin1蛋白磷酸化；mid2基因的系统命名为SPAPYUG7.03c，它编码生成的蛋白为香兰素同系物Mid2(anillin homologue Mid2)，Mid2影响裂殖酵母细胞隔膜的组装(organization)和解聚(disassembly)，它参与胞质分裂时的细胞分离过程。
为了验证依地福新对Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)信号级联的影响，需要判断出依地福新抑制胞质分裂是否与mid2、spm1和pmp1基因有关。其方法是利用这些细胞的突变体。这些突变体由于基因突变，而被封阻在细胞周期的某一特定阶段，从而使我们得知此突变的基因是与该特定阶段的调控有关的基因。本实验以胞质分裂突变体mid2Δ、spm1Δ和pmp1Δ为实验材料，探讨依地福新对MAPK级联信号相关基因的影响，从而阐明依地福新抑制胞质分裂的机制。
【方法】：应用粟酒裂殖酵母细胞突变体生长抑制试验，观察依地福新对野生型粟酒裂殖酵母细胞和mid2突变体(mid2Δ)、spm1突变体(spm1Δ)、pmp1突变体(pmp1A)生长率的影响。
【结果】：应用0μM、0.156μM、0.312μM、0.625μM、1.25μM、2.5μM、5μM、10μM依地福新处理野生型粟酒裂殖酵母及mid2突变体、spm1突变体、pmp1突变体，在培养20h后，spm1突变体在5μM和10μM浓度依地福新处理时的生长率均高于同样浓度处理的野生型粟酒裂殖酵母的生长率(5μM依地福新处理时，spm1A组vsWT组为88.3±7.6 vs 15.9±1.7；10μM依地福新处理时，spm1Δ组vsWT组为85.3±6.7 vs 15.7±1.6；t值分别为16.10，18.25；P＜0.01)。mid2A和pmp1A在5μM浓度时，它们的生长率均高于野生型粟酒裂殖酵母的生长率(mid2Δvs WT组为76.4±6.2 vs 15.9±1.7；pmp1ΔvsWT组为76.9±5.8 vs 15.9±1.7；t值分别为16.29，17.48；P＜0.01)；结果说明，培养20h后，spm1Δ对5μM和10μM依地福新都具有抵抗性，而mid2Δ和pmp1Δ则对5μM依地福新具有抵抗性。
【结论】：spm1、mid2和pmp1基因突变后对依地福新的抗性增强。因此，依地福新抑制粟酒裂殖酵母胞质分裂可能与MAPK级联相关的mid2、pmp1、spm1基因有关。
第三章spm1、pmp1和mid2基因在相应粟酒裂殖酵母突变体中的再表达
【背景与目的】：利用酵母突变体及其同源基因再表达的功能互补实验，是目前遗传学和分子生物学研究的一种重要实验方法，并使酵母成为筛查药物作用机制的工具。通过使用药物对特定的酵母基因突变株进行干预，筛选出对药物敏感或抵抗的突变株，然后利用酵母基因重组技术，使突变株再表达同源的基因，从而获得再表达同一基因的克隆。如果构建的同源克隆酵母可以挽救此突变株对药物的敏感性或抵抗性，那么就说明药物可能影响此基因。这将成为一种筛选抗癌和抗病毒药物的分析系统。因此，将spm1、pmp1和mid2基因在相应粟酒裂殖酵母突变株中进行再表达，观察其是否能恢复对依地福新的敏感性，可确定依地福新对这些基因是否产生影响。本实验的目的是，将粟酒裂殖酵母细胞胞质分裂相关基因spm1、pmp1和mid2在相应突变株中进行再表达，并筛选出有表达活性的重组子。
【方法】：(1)、应用TRIZOL试剂法抽提粟酒裂殖酵母细胞的RNA；(2)、反转录PCR(RT-PCR)制备粟酒裂殖酵母细胞的cDNA；(3)、应用PCR扩增spm1、pmp1和mid2基因；(4)、将spm1、pmp1和mid2基因克隆到pREP3X-HA质粒中；(5)、将pREP3X-HA-spm1、pREP3X-HA-pmp1和pREP3X-HA-mid2电转化到粟酒裂殖酵母突变体spm1Δ、pmp1Δ和mid2Δ细胞中；(6)、应用硫胺素抑制试验筛选活性的pREP3X-HA-spm1、pREP3X-HA-pmp1和pREP3X-HA-mid2重组酵母。
【结果】：在35个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-spm1重组子。在43个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-pmp1重组子。在41个酵母转化单菌落中，筛选到一个有表达活性的pREP3X-HA-mid2重组子。
【结论】：pREP3X-HA-spm1穿梭载体、pREP3X-HA-pmp1穿梭载体和pREP3X-HA-mid2穿梭载体成功转入到相应的突变体细胞中，且转化子具有表达活性。
第四章依地福新影响粟酒裂殖酵母MAP激酶Spm1的磷酸化
【背景与目的】：蛋白激酶级联调节胞质和胞核对细胞外刺激的应答。MAPK(mitogen-activated protein kinase，丝裂原激活的蛋白激酶)级联是在单细胞和多细胞真核生物中发现的古老而保守的蛋白激酶级联。在粟酒裂殖酵母(S．pombe)细胞人们目前也已经发现了多条MAPK级联，其中Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)细胞信号途径与细胞形态发生有关。在粟酒裂殖酵母细胞中，细胞胞膜上的Mid2接受细胞外信号后，经GTPase Rho4激活PKC_1-MPK_1细胞完整性通路，信号经Mkh1(MEKK)-Skh1／Pek1(MEK)-Spm1／Pmk1(MAPK)途径传递，最终影响细胞形态的发生。而Pmp1在体外能直接去除Spm1／Pmk1酪氨酸残基上的磷酸，导致Spm1／Pmk1去磷酸化；Pmp1在体内也能影响Spm1／Pmk1中酪氨酸的磷酸化。因此，本实验的目的是探讨依地福新对经Mid2介导的，Pmp1抑制的MAPK Spm1信号通路的影响，以阐明依地福新抑制粟酒裂殖酵母细胞胞质分裂的作用机制。
【方法】：(1)、通过野生型粟酒裂殖酵母和胞质分裂突变体spm1Δ、pmp1Δ、mid2Δ及其再转化株对依地福新的平行生长抵抗试验，进一步确定依地福新对spm1、pmp1、mid2基因的影响；(2)、应用依地福新对Spm1磷酸化的影响试验，阐明依地福新是否通过影响Mid2蛋白和Pmp1蛋白的表达而影响Spm1蛋白的磷酸化。
【结果】：(1)、在spm1、pmp1和mid2突变株及其再转染株对依地福新的平行生长抵抗试验中，spm1Δ组细胞培养24h后，在含有终浓度为5.0μM和10.0μM依地福新的培养液中仍生长良好；其生长率与在含有终浓度为5.0μM和10.0μM依地福新的培养液中培养的WT组细胞比较，差异有统计学意义(5.0μM依地福新处理时，spm1Δ组vs WT组细胞生长率为101.1±6.6 vs 5.3±0.7；10.0μM依地福新处理时，spm1Δ组vs WT组细胞生长率为105.5±9.8 vs 5.6±0.9；t值分别为25.00，17.58；P＜0.01)。依地福新对spm1Δ组细胞的IC_(50)(半数抑制浓度)为(50.12±4.31)μM，而对WT细胞的IC_(50)为(3.75±0.34)μM，两者比较有统计学意义(t值为18.58，P＜0.01)。mid2Δ组细胞和pmp1Δ组细胞培养24h后，在含有终浓度为5.0μM依地福新的培养液中生长良好；其生长率与在含有终浓度为5.0μM依地福新的培养液中培养的WT组细胞比较，其差异有统计学意义(mid2A组vs WT组细胞生长率为77.3±4.5 vs 5.3±0.7，t=27.38，P＜0.01，pmp1Δ组vsWT组细胞生长率为81.3±4.7 vs 5.3±0.7，t=27.70，P＜0.01)。依地福新对mid2Δ组细胞和pmp1Δ组细胞的IC_(50)分别为(7.12±0.63)μM和(7.25±0.65)μM，与WT细胞的IC_(50)[(3.75±0.34)μM]比较有统计学意义(t值分别为8.15和8.26，P＜0.01)。
(2)、在依地福新对Spm1磷酸化的影响试验中，依地福新处理组只有pREP3X-HA-mid2重组酵母、pREP3X-HA-pmp1重组酵母和pREP3X-HA-spm1重组酵母能检测到磷酸化的活性MAPK，而WT细胞、mid2Δ细胞、pmp1Δ细胞和spm1Δ细胞未检测到磷酸化的活性MAPK；而在依地福新未处理组，所有细胞均未检测到磷酸化的活性MAPK；结果说明，磷酸化活性MAPK Spm1的产生是由于依地福新诱导了Mid2的表达和／或依地福新抑制了Pmp1活性的缘故。
【结论】：(1)、依地福新通过影响粟酒裂殖酵母细胞mid2、spm1和pmp1基因而抑制粟酒裂殖酵母细胞的胞质分裂。(2)、细胞外依地福新诱导Mid2蛋白的表达，从而促进的Spm1磷酸化；细胞内依地福新通过抑制Pmp1蛋白的表达，取消Pmp1蛋白对Spm1的抑制作用，使Spm1的磷酸化程度增加，最终导致粟酒裂殖酵母细胞胞质分裂障碍。
Chapter 1 The Effect of Edelfosine on Cytokinesis of S. pombe
【Background and Objects】: Edelfosine is a synthetical alkyl-lysophospholipid analog, also known as antitumor ether lipids. It can inhibit cell division without concurrent inhibition of nuclear division, leading to accumulation of cells in G2/M, multinucleate cell formation, and subsequent cell death through apoptosis. It also reported that cells treated with edelfosine came through the whole cell cycle without nuclei cleavage, and cells were blocked in G_0/G_1 and subsequent formation of quadplex or octploid nuclei. However, the mechanism still is unknown that edelfosine inhibits cytokinesis. Cells in human are similar to yeast in cell cycle, and a lot of studies of human cells base on yeast research. Cancer cells derive from normal human cells affected by various factor. Differentiation and proliferation are abnormal in cancer cells, leading to a lot of difficulty when people to study it. However, the fission yeast S. pombe has become a powerful model organism with which to study the process of cytokinesis. Some of its key attributes and advantages in this regard include the ease with which cytological manipulations can be performed, a well-characterized mitotic cell cycle, and fast growth and culture easily. In this study, we utilized the S. pombe to explore the effect of dosage-dependent of edelfosine inhibiting the growth, cell division and nucleus division of S. pombe.
【Methods】: (1)、We performed the experiment that edelfosine inhibited the growth of S. pombe and Jurkat cell, and confirmed the best effective dosage of edelfosine inhibiting the growth of the S. pombe.
(2)、We performed the experiment that edelfosine inhibiting the cytokinesis of S. pombe, and analyzed the effect of edelfosine on the cytokinesis of S. pombe.
(3)、We carried out the experiment that edelfosine acted on the nucleus division and detected the DNA content of S. pombe, and analyzed the effect of edelfosine on nucleus division of S. pombe.
【Results】: (1)、Treated with 5.0μM, 10.0μM and 20.0μM edelfosine for 6 h, the growth of S. pombe wild-type and Jurkat cell had been inhibited by edelfosine in the S. pombe's growth inhibition experiment. The difference has statistic significance (P＜0.01) between these cell treated with 5.0μM, 10.0μM and 20.0μM edelfosine for 6 h and those cell treated with 0μM edelfosine for 6 h. Treated with 1.0μM, 5.0μM, 10.0μM and 20.0μM edelfosine for 8 h, the growth of S. pombe wild-type and Jurkat cell had been inhibited not only by 5.0μM, 10.0μM and 20.0μM edelfosine, but also by 1.0μM edelfosine. The difference has statistic significance (P＜0.01) between these cell treated with 1.0μM, 5.0μM, 10.0μM and 20.0μM edelfosine for 8 h and those cell treated with 0μM edelfosine for 8 h.
(2)、In the inhibition experiment of cytokinesis of S. pombe, the S. pombe cells presented normal symmetrical shape with medium septum if they had not been treated by edelfosine (0μM); Meanwhile, the cells possessed cell wall with natural thickness. For the cells treated with 1.0μM edelfosine, they showed multiply septum or without septum, and also showed different size with decreased refraction.; In addition, the cells' septa were much thicker than the cells' untreated with edelfosine. A few granular cells were found. The number of the abnormal shape cells with decreased refraction become more and more, their septa become thicker and their shape become more asymmetrical, and the granule cell become more and more when the cells were treated with 5.0μM edelfosine for 6 h. The counted results of S. pombe septum showed a statistic significance between the cells treated with 0μM and 1.0μM edelfosine (χ~2=16.089, P＜0.01).
(3)、In the inhibition experiment of nucleus division of S. pombe, the size and number of the cells nucleus were in nature status, the cells with two nucleuses can be found, and the cells with abnormal nucleus had not existed in the cells if it had not been treated with edelfosine (0μM edelfosine); The fluorescence was generally weaker in the nucleus of the cells treated by 1.5μM edelfosine than untreated. A fissiparous cell shown that the mother cell had already started next mitosis while the young cell still had not split from the mother cell in the cells treated by 1.5μM edelfosine.
(4)、DNA amount detected by FACS showed that most cells has 1C DNA content and a few cells has 2C DNA content in the cells untreated with edelfosine (0μM); However, most cells has 2C DNA content in the cells treated by 0.5μM、1.0μM、1.5μM edelfosine.
【Conclusions】: (1)、1.0μM-5.0μM edelfosine has a similar effect on S. pombe and cancer cell to inhibit cell growth. (2)、0.5μM-1.5μM edelfosine inhibit cell division without inhibiting nucleus division.
Chapter 2 The Growth Effect of Edelfosine on mid2 Mutants, spm1 Mutants and pmp1 Mutants of S. pombe
【Background and Objects】: The concentration of apoptosis of 5-25μM edelfosine inhibits the MAPK/ERK and Akt/PKB pathway. Several MAPK cascades have been found in S. pombe. The Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) pathway has a relation with morphogenesis in S. pombe. Mid2 activates the PKC_1-MPK_1 cell integrity pathway via the small GTPase Rho4 resulting from exposure to extracellular signals and activate Spm1 finally, meanwhile, Pmp1 affect the photophosphorylation of Spm1. The system name of spm1 gene is SPBC119.08, and the coding protein of spm1 gene is MAP kinase Spm1/Pmk1. The system name of pmp1 gene is SPBC1685.01, and the coding protein of pmp1 gene is dual-specificity MAP kinase phosphatase Pmp1. Pmp1 is concerned with the MAPKKK cascade, and transfer information in cell division by affecting the photophosphorylation of Spm1. The system name of mid2 gene is SPAPYUG7.03c, and the coding protein of mid2 gene is anillin homologue Mid2. Mid2 affect the organization and disassembly of septum, and it is concerned with cell separation in cell division.
In order to confirm the effect of edelfosine on Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) signaling cascade, we need to judge whether the inhibition of cytokinesis have a correlation with mid2, spm1 and pmp1 genes. We designed the mutants of mid2, spm1 and pmp1, which have been blocked in a given stage of cell cycle. In this study, we explored the effect of edelfosine on mid2、spm1 and pmp1 mutants relating to MAPK cascade and elucidate the inhibition mechanism of cytokinesis.
【Methods】: The effect of edelfosine on the growth ratio of S. pombe wild-type, mid2 mutants, spm1 mutants and pmp1 mutants were observed via the inhibition experiment of growth of S. pombe mutants.
【Results】: The S. pombe wild-type cells, mid2 mutants, spm1 mutants and pmp1 mutants were treated with a series of edelfosine (0μM、0.15μM、0.312μM、0.625μM、1.25μM、2.5μM、5μM、10μM) for 20 h. The growth ratios of spm1 mutants were higher than of wild-type cells treated with the same concentration of edelfosine (treated with 5μM edelfosine, the growth ratio of spm1 mutants vs of WT was 88.3±7.6 vs 15.9±1.7; treated with 10μM edelfosine, the growth ratio of spm1 mutants vs of WT was 85.3±6.7 vs 15.7±1.6; t value was 16.10, 18.25, respectively; P＜0.01). The growth ratio of mid2 mutants and pmp1 mutants treated with 5μM edelfosine were higher than of S. pombe wild-type cells (the growth ratio of mid2 mutants vs of WT was 76.4±6.2 vs 15.9±1.7; the growth ratio of pmp1 mutants vs of WT was 76.9±5.8 vs 15.9±1.7; the t value was 16.29, 17.48, respectively; P＜0.01). The results showed that the spm1 mutants were hyper resistant to 5μM and 10μM edelfosine; Meanwhile, the mid2 and pmp1 mutants were hyper resistant to 5μM edelfosine.
【Conclusions】: Edelfosine maybe have an effect on the mid2, pmp1 and spm1 gene relating to MAPK cascade in S. pombe cells. Chapter 3 The Re-Expression of spm1, pmp1 and mid2 Genes in Relevant Mutants of S. pombe
【Background and Objects】: Yeast is a model organism for studying eukaryote, especially in the research of humam genomics. Yeast also is an important research material and provides a detectable experimental system in genetics and molecular biology. The functional complementation assay of homeotic gene re-expression in yeast mutant has become a screen tool in mechanism research of drug. To screen the sensitive and resistant yeast mutant when they treated with drug, the recombine technology of yeast gene should be applied to obtain the clone expressing the homeotic gene. If the yeast with homeotic gene can retrieve the sensitivity or resistance, it could show that the drug affect the gene. The method is useful as an analysis system in screening anti-cancer and anti-virus drug research. Thus, if we re-express the spm1, pmp1 and mid2 genes in relevant yeast mutants, and observe whether they retrieve the sensitivity, we will judge whether edelfosine have an effect on these genes. In this study, we observed the retransfected mutants whether retrieved the sensitive to edelfosine, and confirmed the effect of edelfosine on the S. pombe cells that re-expressed the spm1, pmp1 and mid2 genes in relevant mutants.
【Methods】: (1)、Total RNA of S. pombe cells was extracted by TRIZOL reagent; (2)、First-strand cDNA of S. pombe cells was synthesized by RT-PCR; (3)、he spm1, pmp1 and mid2 genes were amplified by PCR; (4)、The spm1, pmp1 and mid2 genes were cloned into plasmid and formed a relevant shuttle carrier; (5)、The pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3X-HA-mid2 shuttle carriers were transformed into spm1, pmp1 and mid2 mutants by Electroporation, respectively; (6)、The active recombination was identified by the inhibition experiment of thiamine.
【Results】: An active recombination of pREP3X-HA-spm1 was identified from 35 single clones of relevant transformed S. pombe. An active recombination of pREP3X-HA-pmp1 was identified from 43 single clones of relevant transformed S. pombe. An active recombination of pREP3X-HA-mid2 was identified from 41 single clones of relevant transformed S. pombe.
【Conclusion】: The pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3X-HA-mid2 shuttle carriers have successfully been transformed into relevant S. pombe mutants; and the recombination of pREP3X-HA-spm1, pREP3X-HA-pmp1 and pREP3 X-HA-mid2 possesses an expressed activity. Chapter 4 Edelfosine Affect the Phosphatization of MAPK Spm1 of S. pombe
【Background and Objects】: Protein kinase cascade regulates the response of extracellular stimulation in cytoplast and cell nuclei. The MAPK signal cascade is an ancestral and conservative protein kinase in eukaryote. In present, several MAPK cascades have been found in S. pombe. The Mkh1(MEKK)-Skh1/Pek1(MEK)-Spm1/Pmk1(MAPK) pathway has a relation with morphogenesis in S. pombe. Mid2p activates the PKC_1-MPK_1 cell integrity pathway via the small GTPase Rho4 resulting from exposure to extracellular signals and activate Spm1 finally, meanwhile, Pmp1 affect the photophosphorylation of Spm1/Pmk1 in vivo and in vitro. In this study, we explored the function mechanism of edelfosine inhibiting the cytokinesis of S. pombe and the effect of edelfosine on MAPK Spm1 pathway induced by Mid2 and inhibited by Pmp1.
【Methods】: Firstly, the parallel growth inhibition experiment of S. pombe wild-type cells, spm1△, pmp1△, mid2△and relevant retransform strains were carried out in order to farther confirmed the effect of edelfosine on spm1, pmp1 and mid2 genes. Then, the experiment that edelfosine inhibited the phosphatization of Spm1 was carried out in order to elucidate whether edelfosine affect on the phosphatization of Spm1 induced via Mid2 and inhibited via Pmp1.
【Results】: (1)、On the inhibition experiment of parallel growth of S. pombe wild-type cells, spm1 mutants, pmp1 mutants, mid2 mutants and relevant retransform strains, the spm1 mutants grew well treated with 5.0μM and 10.0μM edelfosine for 24 h; the growth ratio of spm1 mutants had a statistic significance in between the cells previously mentioned and the S. pombe wild-type cells treated with 5.0μM and 10.0μM edelfosine (treated with 5.0μM edelfosine, the growth ratio of spm1 mutants vs wild-type cells was 101.1±6.6 vs 5.34±0.7; treated with 10.0μM edelfosine, the growth ratio of spm1 mutants vs wild-type cells was 105.5±9.8 vs 5.6±0.9; the t values were 25.00, 17.58, respectively; P＜0.01). The IC_(50) of spm1 mutants and wild-type cells treated with edelfosine was (50.12±4.31)μM and (3.75±0.34)μM, respectively. The IC_(50) difference between spm1 mutants and wild-type cells had statistic significance (t=18.58, P＜0.01). The mid2 mutants and pmp1 mutants also grew well treated with 5.0μM edelfosine for 24 h. The growth ratio of mid2 mutants and pmp1 mutants had a statistic significance in between the cells previously mentioned and the S. pombe wild-type cells treated with 5.0μM edelfosine (treated with 5.0μM edelfosine, the growth ratio of mid2 mutants vs wild-type cells was 77.3±4.5 vs 5.3±0.7, t=27.38, P＜0.01; the growth ratio of pmp1 mutants vs wild-type cells was 81.3±4.7 vs 5.3±0.7, t=27.70, P＜0.01). The IC_(50) of mid2 and pmp1 mutants treated with edelfosine were (7.12±0.63)μM and (7.25±0.65)μM, respectively. Compared with wild-type cells treated with edelfosine, the difference of IC_(50) had statistic significance (the t value was 8.15 and 8.26, respectively, P＜0.01).
(2)、On the experiment of edelfosine inhibiting the phosphatization of Spm1, the active MAPK were detected in the cells contained pREP3 X-HA-mid2, pREP3 X-HA-pmp1 and pREP3 X-HA-spm1 plasmid when they treated with edelfosine, but the active MAPK were not detected in S. pombe wild-type cells, mid2 mutants, pmp1 mutants and spm1 mutants when they treated with edelfosine. Meanwhile, the active MAPK were not detected in all tested cells when they untreated with edelfosine. The results showed that the active MAPK Spm1 was produced via edelfosine inducing the expression of Mid2 and/or edelfosine inhibiting the expression of Pmp1.
【Conclusions】: (1)、Edelfosine inhibited cytokinesis of S. pombe cells via acting on the mid2, spm1 and pmp1 genes. (2)、The extracellular edelfosine promoted the phosphatization of Spmlvia inducing the expression of Mid2; The intracellular edelfosine increased the phosphatization of Spmlvia inhibiting the expression of Pmp1, and leading the holdback of cytokinesis.
引文
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