Functional analysis and methylation regulation of Dermo1 and Twist transcription factors in human ovary, breast and gastric cancers

第一部分Dermo1和Twist在人类卵巢癌、乳腺癌、胃癌中的表达及功能分析 高度保守的转录因子Dermo1和Twist都属于碱性Helix-loop-helix(b-HLH)蛋白家族，它们具有结构的高度同源性，功能的部分相似性。b-HLH家族蛋白在细胞分化、胚胎发育的调控中具有重要作用，同时广泛参与多种组织的形成，如：骨、肌肉、神经、心脏、造血组织等。 最新研究成果表明Twist能促进细胞存活，抑制非恶性细胞分化，诱导非恶性细胞肿瘤发生，促进恶性细胞肿瘤形成、发展和转移，也参与多种难治性肿瘤抗药性（破坏微管药物如taxol和vincristine）的形成。近来发现，乳腺癌、恶性黑色素瘤以...Part I Expression and functional analysis of Dermo1 and Twist in human cancers Dermo1 and Twist are highly conserved transcription factors that belong to the basic Helix-loop-helix (b-HLH) protein family. These two proteins share structural homology and functional similarity. The b-HLH proteins acting as positive or negative regulators play key roles during developmental process such as neuro...学位：理学博士院系专业：生命科学学院生物学系_细胞生物学学号：B20032601

酪氨酸激酶抑制药GW2974诱导乳腺癌细胞BT474耐药的代谢机制研究

目的通过比较酪氨酸激酶抑制药GW2974给药后母本细胞株BT474与耐药株r BT474之间代谢相关因子mRNA表达的差异,探讨r BT474可能的耐药机制。方法收集对数生长期r BT474和BT474细胞,接种于96孔板,加入含有GW2974的培养基,分别于孵育0,12,24和48 h后,通过噻唑蓝法检测BT474与r BT474细胞的增殖情况。用逆转录实时定量-聚合酶链反应检测12种相关代谢因子葡萄糖转运体4(GLUT4)、2,6-二磷酸果糖激酶(PFK-2)、丙酮酸激酶2(PKM2)、乳酸脱氢酶(LDHA)、丙酮酸羧化酶(PC)、6-磷酸果糖激酶(G-6-PD)、脂肪酸合成酶(FASN)、脂酰肉碱转移酶1 (CPT1A)、葡萄糖调节蛋白75(GRP75)、电压依赖性阴离子通道蛋白1 (VDAC1)、葡萄糖调节蛋白78(GRP78)和钙网蛋白(CALR)的mRNA在BT474细胞和r BT474细胞中给药前后的表达水平。结果给予GW2974后,母本细胞BT474中糖脂代谢相关因子GLUT4、PFK-2、PKM2、LDHA、PC、G-6-PD、FASN和CPT1A的mRNA表达水平均明显下调,线粒体应激因子GRP75、VDAC1与内质网应激因子CALR表达水平均明显下调,内质网应激因子GRP78表达明显上调。而在耐药株r BT474中给药后这12种代谢相关因子的mRNA表达水平均明显上调。结论耐药株r BT474可能通过调控糖脂代谢以及细胞应激等过程使细胞耐受性增强。福建省自然科学基金面上项目(2012J01417

Expression of pin1 gene in lung cancer and its significance

背景与目的:Pin是人类高度保守的特异性磷酸化肽基脯氨酰顺及异构酶,它作用于脯氨酸所形成的肽键,并且仅使磷酸化pSer/Thr-Pro发生异构化,这一磷酸化后调控机制能诱导磷酸蛋白的构像变化,使其发挥功能。近来的研究显示这种新的调控机制在许多生理过程中起重要的调节作用,一旦失调便导致一系列人类疾病。如癌症患者体内Pin1表达异常增高,并调控多种癌基因的信号通路。本研究探讨pin1基因在非小细胞肺癌(NSCLC)中的表达及其意义。方法:利用免疫组织化学方法、定量PCR方法分别检测肺癌组织和正常肺组织中Pin1基因在翻译水平和mRNA水平上的表达差异。结果:在蛋白质水平上,Pin1的表达在正常/癌两种不同的肺组织中,表达量差异有显著性;而在mRNA水平上,Pin1的表达量在正常/癌两种不同的肺组织中,差异无显著性。结论:Pin1在NSCLC组织中的表达,可能是在翻译水平受到了调控;我们的研究进一步证实:Pin1在NSCLC组织中存在蛋白水平的过量表达,这对利用Pin1作为NSCLC组织检测标志物提供了理论依据。Background and purpose:Pin1 is a highly conservative enzyme that isomerizes only the phosphorylated Ser/Thr-Pro bonds in certain proteins, thereby inducing conformational changes. Recent results indicate that such conformational changes following phosphorylation are a novel signaling mechanism pivotal in regulating many cellular functions. Overexpression of Pin1 is prevalently found in human cancers.We studied expression of pin1 in non-small cell lung cancer and its implication in clinical. Methods:We detected the differential expression level of Pin1 protein in lung cancer specimen and normal lung tissues by immunohistochemical staining. Real-time quantitative PCR was also applied to detect the mRNA differential expression level in lung cancer tissues and normal lung tissues. Results:Pin1 protein is overexpressed in lung cancer at the protein level. On the other hand, the expression level of Pin1 mRNA in lung cancer tissue has no significant change compared with normal tissue. Conclusion:It demonstrated that the Pin1 expression in lung cancer might be regulated by translation mechanism.Our data implicate that Pin1 may serve as a valuable molecular marker for human cancer

Expression and Phosphorylation of Rad17 Checkpoint Protein during Spermiogenesis in Mouse Testis

Rad17是细胞周期检控点信号转导过程中的一个关键检控蛋白,在DNA损伤检控和DNA复制检控中具有重要功能。但Rad17在细胞减数分裂中的检控作用还不是很清楚。因细胞减数分裂在睾丸组织中非常活跃,应用Western印迹检测Rad17在不同发育时期的小鼠睾丸组织中的表达及其磷酸化水平,并应用免疫组化的方法检测小鼠睾丸组织不同时期生殖细胞内Rad17的表达变化。结果显示Rad17在小鼠睾丸组织内高表达,而在肝、肾等组织中表达水平较低;Rad17在不同周龄的小鼠睾丸组织中均高水平表达,但在4周龄以后的小鼠睾丸组织中其磷酸化水平明显升高;免疫组化结果显示Rad17在精原细胞、精母细胞的细胞核中高表达,但在成熟精子细胞中消失。这些结果提示Rad17在小鼠睾丸生殖细胞减数分裂过程中也起重要检控作用。Rad17 is one of critical checkpint proteins in cell cycle checkpoint singling pathways and playsvital roles in DNA damage checkpoint and DNA replication checkpoint. However, the role of Rad17 in meioticcheckpoint is poorly understood. Since meiosis is very active in adult testis, we investigated the expression andphosphorylation change of Rad17 in mouse testis at different stages by Western blot analysis, and examined theexpression of Rad17 in the germ cells at different meiotic stages in testis by Immunohistochemistry. Our resultsrevealed that Rad17 protein was overexpressed in mouse testis tissues, while other tissues such as liver and kidneyexpressed much lower Rad17. Although Rad17 expression remained high level in mouse testis at different develop-ment stages, the phosphorylation level of Rad17 was significantly increased after four weeks of birth. Immunohis-tochemical analysis indicated that Rad17 protein was present primarily in the nuclei of spermatogonia and spermatocyte,and disappeared in the matured spermatids. These results showed that Rad17 also played an important role in meioticcheckpoint during germline development in testis.国家自然科学基金(No.30170463、No.30370307、No.30400239);; 福建省青年科技人才创新专项重点项目(No.2003J017);; 厦门大学科学研究基金(No.2003XDYY31)资助~

ITRAQ based quantitative proteomics approach validated the role of calcyclin binding protein (CacyBP) in promoting colorectal cancer metastasis

10.1074/mcp.M112.023085Molecular and Cellular Proteomics1271865-1880MCPO

Twist2 promotes ovarian cancer cell survival through activation of Akt

National Natural Science Foundation of China [30872515, 31071187, 81272721]; Fundamental Research Funds for the Central Universities of China [2011121062]; Natural Science Foundation of Fujian Province of China [2012J01417]Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is an important prognostic factor in ovarian carcinoma. Hypoxia contributes to tumor progression and is involved in the epithelial-mesenchymal transition (EMT). Twist2 is an EMT regulator, however, it remains poorly understood in ovarian carcinoma. The present study evaluated the expression of HIF-1 alpha and Twist2 and further investigated whether Twist2 is involved in hypoxia-induced apoptosis in ovarian cancer. A series of matched paraffin-embedded tissue sections from human primary ovarian cancer and normal ovarian tissues were examined through immunohistochemical analysis, a Twist2-overexpressing stable ovarian cancer cell line was established and deferoxamine (DFO) was introduced to simulate hypoxic conditions. DFO-induced apoptosis was examined by fluorescence microscopy, MTT assays and flow cytometry. In addition, a western blot analysis was performed to examine the molecular mechanism(s) of action. Twist2 increased in epithelial ovarian cancers associated with HIF-1 alpha expression. The acquired expression of Twist2 was able to promote the survival of ovarian cancer cells through Akt phosphorylation under DFO-induced hypoxic stress. The results suggest that Twist2 activates the PI-3K-Akt pathway to protect cells from apoptosis in a hypoxic environment. Moreover, Twist2 may be involved in the HIF-1 alpha signaling pathway in ovarian cancer

Significance of Heterogeneous Twist2 Expression in Human Breast Cancers

Background: Twist2 (Dermo1) has been shown to mediate the epithelial-mesenchymal transition (EMT) to promote tumor invasion and even metastasis. However, the involvement of EMT in breast cancer progression is highly debated, partially due to clinical observations showing that the majority of human breast carcinoma metastases express E-cadherin and maintain their epithelial morphology. The molecular mechanism by which Twist2 participates in EMT of breast cancer in vivo remains poorly understood. Methods: We examined Twist2 expression pattern in human breast carcinomas by western blot and tissue microarray, and analyzed Twist2 cellular localization by confocal microscopy, cell fractionation and other approaches. Results: Twist2 expression was significantly increased in breast cancer. Cytoplasmic Twist2 positive cancer cells expressing E-cadherin on the cellular membrane were mainly located at tumor center of primary carcinomas and lymph metastases, while cancer cells with nuclear Twist2 clearly showed loss of E-cadherin and were detected at the invasive front in ductal breast carcinomas. In addition, ectopically stable-expressed Twist2 was found to localize in the cytoplasm of cancer cells. Collectively, these data indicate that upregulation of cytoplasmic Twist2 is correlated with tumor histological type and tumor metastasis in human breast cancers. Conclusion: The differential cellular distribution of Twist2 may be associated with tumor progression. The cytoplasmic Twist2 in cancer cells at tumor center of primary carcinomas and lymph metastases contributes to the maintenance of epithelial cancer characteristics expressing E-cadherin in a noninvasive state, while the nuclear Twist2 at the cancer invasion front activates EMT to deprive epithelial property of neoplastic cells, thus facilitating invasion and metastasis. These findings suggest that heterogeneous expression of Twist2 in tumors may have a functional link to tumor progression.National Natural Science Foundation of China [30872515]; Fundamental Research Funds for the Central Universities of China [2011121062]; Natural Science Foundation of Fujian Province of China [2012J01417

Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression

Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-X-L, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 mu M) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 mu M curcumin, while the expression of the anti-apoptotic factor Bcl-X-L Was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients

重离子束流横向剂量分布测量探测器及其二维成像方法

本发明涉及重离子束(包括质子束)治疗肿瘤技术的领域，尤其涉及到重离子束流横向剂量分布测量探测器及其二维成像方法，其主要特点是包括气体密封腔(1)，其内设有电离室内芯(2)，与电离室内芯(2)电连接的多路信号转接板(3)；所述的气体密封腔(1)由主体框架(1-1)和入射窗(1-2)、出射窗(1-3)组成；所述的电离室内芯(2)由两组电离室单元组成，每个单元电离室均由信号极(2-1)、绝缘垫板(2-2)和高压极(2-3)组成；所述的多路信号转接板(3)的一端设有接触端(3-3)插入气体密封腔(1)的密封口(1-5)与电离室内芯(2)的信号极(2-1)相连，另一端设有多芯连接器(3-2)为束流剖面监测探测器的信号输出端口

Truncated ErbB2 Expressed in Tumor Cell Nuclei Contributes to Acquired Therapeutic Resistance to ErbB2 Kinase Inhibitors

Department of Defense [34 W81WXH-09-0065]; Sisko Foundation; Balderacchi GiftErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185 ErbB2). In addition to p185 ErbB2, truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2(+) breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2(+) breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2(+) breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs. Mol Cancer Ther; 10(8); 1367-74. (C) 2011 AACR