Trichosanthin suppresses the elevation of p38 MAPK, and Bcl-2 induced by HSV-1 infection in Vero cells

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) effective against HIV-1 and HSV-1 replication. The mechanism of its antiviral activity is not clear. Many believe that it is related to ribosome inactivation. Some RIPs and viral infectio

MAPK在天花粉蛋白抗HIV-1中的作用以及syncytin基因在白血病细胞中的表达

本学位论文主要包括两部分的内容： 一是关于MAPK信号转导在天花粉蛋白（trichosanthin, TCS）抗人类免疫缺陷病毒-1（HIV-1）中的作用的研究。TCS是I型核糖体失活蛋白（RIP），分子量27Kd，可从传统的中期流产和抗绒癌中药栝楼根块茎（天花粉）中提纯获得。该蛋白具有抗HIV-1活性，但其机制尚不清楚。本文用JNK抑制剂CEP-11004，预处理宿主细胞，检测其对TCS抗HIV-1的影响。用以下两种方法检测病毒的复制：一是用ELISA方法检测细胞培养上清中p24抗原的水平，二是检测上清中病毒粒子的逆转录酶（RT）活性。结果显示，TCS剂量依赖性地抑制HIV-1在C8166细胞中的复制。在TCS实验浓度下，HIV-1的复制水平平均为68 ± 4％（p24抗原检测）和52 ± 4％（RT活性检测）。如果用0.4μM CEP-11004对C8166细胞预处理2小时，TCS似乎失去了抗HIV-1活性，HIV-1的复制水平分别恢复为101 ± 4％和101 ± 7％。但无论是p24抗原检测还是RT活性检测，当不含TCS时，CEP-11004预处理病毒宿主细胞，本身并不影响病毒粒子的复制。这说明CEP-11004能够拮抗TCS的抗病毒活性，或者说CEP-11004抑制的信号转导途径的某些信号分子，与TCS的抗HIV-1活性相关。Western Blot方法检测的结果也证明，TCS能够以时间依赖和剂量依赖的方式激活JNK激酶，0.4μM的CEP-11004能有效抑制JNK的磷酸化。因此，TCS与它激活MAPK信号转导途径有关。 二是关于人类内源性病毒HERV-W家族囊膜蛋白基因syncytin在白血病细胞中的表达的研究。该基因在人的胎盘组织中特异性表达，可能与合胞滋养层的形成有关。另外也少量表达于睾丸组织。本论文采用实时定量RT-PCR的方法证明，syncytin能够在白血病细胞系中表达。进一步的检测还表明，syncytin的mRNA也表达于白血病/淋巴瘤患者的外周血细胞，而不表达于作为对照的10名健康志愿者的血细胞。在15名不同类型的白血病/淋巴瘤患者中，有11名有syncytin的表达。细胞系的表达相对稳定，与C8166细胞系的表达量相比较，介于0.5-2.0倍之间；而在白血病患者外周血细胞中的表达则介于0.8-21.7倍不等。上述结果提示，syncytin可能与白血病的形成有关。

MAPK途径与细胞的辐射应答

MAPK信号转导途径在细胞辐射应答中起着重要作用,ERK,JNK,P38 MAPK和大MAPK 4条不同的MAPK通路在受辐射细胞中扮演着不同角色,它们的协调控制作用决定着细胞受辐照后的命运。描述了辐射应答中的这4条不同的MAPK途径,并讨论了这些途径在辐射应答中的作用。

Funneled landscape leads to robustness of cellular networks: MAPK signal transduction

We uncover the underlying potential energy landscape for a cellular network. We find that the potential energy landscape of the mitogen-activated protein-kinase signal transduction network is funneled toward the global minimum. The funneled landscape is quite robust against random perturbations. This naturally explains robustness from a physical point of view. The ratio of slope versus roughness of the landscape becomes a quantitative measure of robustness of the network. Funneled landscape is a realization of the Darwinian principle of natural selection at the cellular network level. It provides an optimal criterion for network connections and design. Our approach is general and can be applied to other cellular networks.

Porównawcza analiza udziału pokrewnych kinaz MAPK (MKK1, MKK2, MKK4 i MKK5) Arabidopsis thaliana w odpowiedzi na stres suszy

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Novel insights into the expression and function of p38δ MAPK in oesophageal squamous cell carcinoma

Oesophageal cancer is an aggressive malignancy which is resistant to conventional therapy and has a poor prognosis. A greater understanding of the underlying molecular biology of oesophageal cancer and the identification of novel targets is necessary for the future treatment of this disease. This thesis focuses specifically on the ill-defined and understudied p38δ mitogen-activated protein kinase (MAPK) and its function(s) in oesophageal squamous cell carcinoma (OESCC). In contrast to the three other p38 isoforms (p38α, -β and –γ which have to-date been relatively well-studied), p38δ MAPK signalling is poorly understood. Thus, this research elucidates some of the role(s) played by p38δ MAPK in cancer progression. This work outlines how loss of p38δ MAPK expression confers greater tumourigenicity in oesophageal cancer. Restoration of p38δ MAPK expression, however, has anti-proliferative and anti-migratory effects and decreases OESCC capacity for anchorageindependent growth. Using a novel application of an enzyme-substrate fusion approach, the effect of phosphorylated p38δ (p-p38δ) MAPK expression is also considered. The work goes onto describe the effect(s) of p38δ MAPK status on the chemosensitivity of OESCC to conventional cisplatin and 5-fluorouracil (CF) versus the effectiveness of doxorubicin, cisplatin and 5-fluorouracil (ACF). ACF treatment of p38δ MAPK-negative OESCC results in decreased proliferation, migration and recovery, and increased apoptosis when compared with CF treatment. This thesis examines the potential mechanisms by which p38δ MAPK expression is lost in OESCC and identifies epigenetic regulation as the probable cause of differential p38δ MAPK expression. Also analysed is the role p38δ MAPK and p-p38δ MAPK play in the cell cycle. In summary, this research identifies p38δ MAPK as a possible molecular target and a potential predictor of response to chemotherapy in OESCC patients.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX and BAK expression as well as cleaved caspases-3, -7, -8 and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the P13K and MAPK signalling pathways

Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.

Modification of P13K- and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CBII

Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKC alpha and PKC beta II levels were increased in 25 mmol/L glucose. However, only PKC beta II inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKC beta II were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKC beta II inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKC beta II was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKC beta II-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

Modelling the MAPK signalling pathway using a two-stage identification algorithm

Signal transduction pathways describe the dynamics of cellular response to input signalling molecules at receptors on the cell membrane. The Mitogen-Activated Protein Kinase (MAPK) cascade is one of such pathways that are involved in many important cellular processes including cell growth and proliferation. This paper describes a black-box model of this pathway created using an advanced two-stage identification algorithm. Identification allows us to capture the unique features and dynamics of the pathway and also opens up the possibility of regulatory control design. In the approach described, an optimal model is obtained by performing model subset selection in two stages, where the terms are first determined by a forward selection method and then modified using a backward selection model refinement. The simulation results demonstrate that the model selected using the two-stage algorithm performs better than with the forward selection method alone.

Constitutive activation of the Raf-MAPK pathway causes negative feedback inhibition of Ras-PI3K-AKT and cellular arrest through the EphA(2) receptor

The Raf-mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)-AKT pathways are two downstream effectors of the small GTPase Ras. Although both pathways are positively regulated by Ras, the Raf-MAPK and PI3K-AKT pathways have been shown to control opposing functions within the cell, suggesting a need for cross-talk regulation. The PI3K -AKT pathway can inhibit the Raf-MAPK pathway directly during processes such as muscle differentiation. Here we describe the ability of the Raf-MAPK pathway to negatively regulate the PI3K-AKT pathway during cellular arrest. Constitutive activation of Raf or methyl ethyl ketone 1 (MEK1) leads to inhibition of AKT and cellular arrest. Furthermore, we show that activation of Raf-MEK1 signaling causes negative feedback inhibition of Ras through the ephrin receptor EphA(2). EphA(2)-mediated negative feedback inhibition is required for Raf-induced AKT inhibition and cell cycle arrest, therefore establishing the inhibition of the Ras-PI3K-AKT pathway as a necessary event for the Raf-MEK1-regulated cellular arrest.