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Experimental Studies on Possible Pharmacological Mechanism of Edelfosine Inhibiting Cytokinesis of S.pombe

Author: ZhangHui
Tutor: FangYunXiang;Faustino Mollinedo
School: Central South University
Course: Pharmacology
Keywords: According to Fu new Schizosaccharomyces pombe Cytokinesis Jurkat fine fat mid2 spm1 pmp1 Spm1 Gene expression Mid2
CLC: R96
Type: PhD thesis
Year: 2006
Downloads: 125
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Abstract


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 G0/G1 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 (χ2=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 PKC1-MPK1 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 PKC1-MPK1 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 IC50 of spm1 mutants and wild-type cells treated with edelfosine was (50.12±4.31)μM and (3.75±0.34)μM, respectively. The IC50 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 IC50 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 IC50 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.

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