Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34+ cells treated with etoposide

OBJECTIVE: Understanding the molecular events that contribute to survival of and drug-induced apoptosis in hematopoietic stem and progenitor cells (HSC/P) can have impact on more rational approaches to blood cancer therapeutic design, as well as on strategies to minimize toxic side effects of chemotherapeutic drugs. Here we sought to systematically evaluate the basic molecular components and main pathways that govern and mediate cellular response initiated within human CD34(+) cells to etoposide-induced apoptosis. MATERIALS AND METHODS: Human CD34(+) cells were isolated from umbilical cord blood (CB) and expanded in vitro. Expression of apoptosis-related genes in the control and etoposide treated cells was determined using cDNA array and quantitative real-time RT-PCR. RESULTS: We identified a set of apoptosis-related genes expressed in highly purified normal human CB CD34(+) cells and determined how the expression of these genes changed in response to etoposide treatment. In addition, TRAIL does not induce apoptosis of normal human CD34(+) cells, and it has no cytotoxic effect on human CD34(+) cells that are undergoing apoptosis in response to growth factor withdrawal. This may be due to upregulation of cytotoxic receptors as well as the decoy receptor for TRAIL, and c-FLIP. CONCLUSION: p53, c-Myc, and BAFF pathways are main pathways utilized by CD34(+) cells to arrest cell-cycle progression at multiple checkpoints, to halt proliferation, and to induce apoptosis as part of their cellular response to etoposide. Multiple known pro-survival and pro-apoptotic pathways are simultaneously activated in etoposide-treated CD34(+) cells. Also, TRAIL, used alone or in concert with chemotherapeutic drugs, may be of use as a safe blood cancer therapeutic with no or low toxicity for HSC/P.     

Profiling of differentially expressed genes in human gastric carcinoma by cDNA expression array

AIM: To investigate the expression of cancer related genes in gastric carcinoma (GC) through the use of Atlas Human Cancer Array membranes with 588 well-characterized human genes related to cancer and tumor biology.METHODS: Hybridization of cDNA blotting membrane was performed with (32)P-labeled cDNA probes synthesized from RNA isolated from gastric carcinoma and adjacent noncancerous gastric epithelial tissue. AtlasImage, which is a software specific to array, was used to analyze the result.RESULTS: The differentially expression cell cycle/growth regulator in GC showed a stronger tendency toward cell proliferation with 2.7-fold up-regulation of CK1. The promoter genes of apoptosis were down-regulated, including caspase-8 precursor, caspase-9 and caspase-10. Among the oncogene/tumor suppressor genes, ABL2 was down-regulated. In addition, some genes were up-regulated, including matrix metalloproteinse 2(MMP-2), MMP-16(MT3-MMP), SKY, CD9 and semaphorin V. A number of genes were down-regulated, including neuroendocrine-dlg (NE-dlg), retinoic acid receptor gamma and tumor suppressor DCC colorectal. In general, The expression of the cancer progression genes were up-regulated, while the expression of anti-cancer progression genes were down-regulated.CONCLUSION: Investigation of these genes should help to disclose the molecular mechanism of the onset, progression and prognosis of GC. Several genes are reported herein to be altered in GC for the first time. The quick and high-throughout method of profiling gene expression by cDNA array provides us with an overview of key factors that may involved in GC, and may aid the study of GC carcinogenesis and provide molecular targets for diagnosis and therapy. The precise relationship between the altered genes and gastric carcinogenesis is a matter for further investigation.     

Profiling of differentially expressed genes in human Gastric carcinoma by cDNA expression array

AIM: To investigate the expression of cancer relatedgenes in gastric carcinoma (GC) through the use of AtlasHuman Cancer Array membranes with 588 well-characterized human genes related to cancer and tumorbiology.METHODS: Hybridization of cDNA blotting membranewas performed with 32P-labeled cDNA probessynthesized from RNA isolated from gastric carcinomaand adjacent noncancerous gastric epithelial tissue.AtlasImage, which is a software specific to array, wasused to analyze the result.RESULTS: The differentially expression cell cycle/growth regulator in GC showed a stronger tendencytoward cell proliferation with 2.7-fold up-regulation ofCK1. The promoter genes of apoptosis were down-regulated, including caspase-8 precursor, caspase-9and caspase-10. Among the oncogene/tumorsuppressor genes, ABL2 was down-regulated. Inaddition, some genes were up-regulated, includingmatrix metalloproteinse 2(MMP-2), MMP-16(MT3-MMP), SKY, CD9 and semaphorin V. A number of geneswere down-regulated, including neuroendocrine-dlg(NE-dig), retinoic acid receptor gamma and tumorsuppressor DCC colorectal. In general, The expressionof the cancer progression genes were up-regulated,while the expression of anti-cancer progression geneswero down-regulated.CONCLUSION: Investigation of these genes should helpto disclose the molecular mechanism of the onset,progression and prognosis of GC. Several genes arereported herein to be altered in GC for the first time.The quick and high-throughout method of profiling geneexpression by cDNA array provides us with an overviewof key factors that may involved in GC, and may aid thestudy of GC carcinogenesis and provide moleculartargets for diagnosis and therapy. The preciserelationship between the altered genes and gastriccarcinogenesis is a matter for further investigation.     

MCP-1基因多态性与结核性脓胸发病风险的关系

目的探讨中国河北地区MCP-1(单核细胞趋化因子蛋白-1)基因-2518A/G和-362G/C单核苷酸多态性(SNP)与结核性脓胸的关系。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法对结核性脓胸患者500例和健康对照组500例进行MCP-1基因-2518A/G和-362G/C SNPs检测。结果与MCP-1-2518A/A基因型相比,携带A/G和G/G基因型均可增加结核性脓胸发病风险(OR=2.254和OR=8.728)。进行分层分析发现,无卡介苗(BCG)接种史、体质量指数(BMI)18.5均增加结核性脓胸发病风险(OR=3.309和OR=2.767)。MCP-1-362G/C SNP无统计学意义。结论 (1)MCP-1基因-2518A/G SNP可能与结核性脓胸的发病风险有关。(2)-362G/C SNP可能与结核性脓胸无关。     

Isolation of differentially expressed genes in NPM-ALK-positive anaplastic large cell lymphoma

In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis-1 (HIAP-1), Bax inhibitor-1 and MCL-1, or DNA repair, such as poly (ADP-ribose) polymerase 1 (PARP-1), X-associated protein-1 (XAP-1), SUMO-1 (sentrin-1) and RanGTPase-activating protein 1 (RanGAP-1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM-ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM-ALK-driven transformation process, and that moesin and cytohesin-1 may be more specifically implicated in a signalling pathway involving PLCgamma and PI3K.     

雌二醇诱导人成骨样细胞差异表达基因筛查

目的 快速筛查17β雌二醇(E2)诱导人成骨肉瘤MG—63细胞株差异表达cDNA片段,寻找雌激素相关基因。方法 改良cDNA代表性差异分析法(cDNA RDA)分离E2干预MG—63细胞株表达上调cDNA片段,Southern杂交证实后,制备阵列膜行点杂交,然后挑取阳性差异克隆测序,经同源比较分析,最后选取部分克隆标记探针行Northern印迹杂交。结果经过4轮消减和动力性富集后得到5个E2诱导人成骨肉瘤MG—63细胞表达上调的差异cDNA条带,Southern杂交证明这些表达上调的cDNA片段来自E2干预的MG—63细胞;共得到600余个含有阳性插入片段的cDNA克隆,点杂交筛查得到120个差异表达克隆。选20个克隆测序得到15个序列,其中1个经Northern杂交证实E2干预后表达上调。结论 cDNA RDA能有效筛查差异表达cDNA片段,联合cDNA阵列点杂交可快速筛查差异表达基因,E2诱导人成骨肉瘤MG—63细胞某些基因表达上调。     

Profiling of differentially expressed genes in haemophilia A with inhibitor

Inhibitor development is the most significant complication in the therapy of haemophilia A (HA) patients. In spite of many studies, not much is known regarding the mechanism underlying inhibitor development. To understand the mechanism, we analysed profiles of differentially expressed genes (DEGs) between inhibitor and non-inhibitor HA via a microarray technique. Twenty unrelated Korean HAs were studied: 11 were non-inhibitor and nine were HA with inhibitor (≥5 BU mL(-1)). Microarray analysis was conducted using a Human Ref-8 expression Beadchip system (Illumina) and the data were analysed using Beadstudio software. We identified 545 DEGs in inhibitor HA as compared with the non-inhibitor patients; 384 genes were up-regulated and 161 genes were down-regulated. Among them, 75 genes whose expressions were altered by at least two-fold (>+2 or <-2) were selected and classified via the PANTHER classification method. The expressions of signal transduction and immunity-related genes differed significantly in the two groups. For validation of the DEGs, semi-quantitative RT-PCR (semi-qRT-PCR) was conducted with the six selected DEGs. The results corresponded to the microarray data, with the exception of one gene. We also examined the expression of the genes associated with the antigen presentation process via real-time PCR. The average levels of IL10, CTLA4 and TNFα slightly reduced, whereas that of IFNγ increased in the inhibitor HA group. We are currently unable to explain whether this phenomenon is a function of the inhibitor-inducing factor or is an epiphenomenon of antibody production. Nevertheless, our results provide a possible explanation for inhibitor development.     

筛选转化生长因子β1刺激肝星状细胞差异表达基因的研究

目的 筛选转化生长因子β 1(TGF β 1)刺激大鼠肝星状细胞(Hsc)的差异表达基因,以揭示TGF β1介导肝纤维化的分子发病机制. 方法分别用Trizol法抽提TGF β1刺激的HSC及磷酸盐缓冲液刺激为对照的HSC总RNA,逆转录合成双链cDNA,制备掺入生物素标记的cDNA探针,与人基因表达谱芯片杂交,用Agilent扫描仪对芯片结果进行扫描,利用软件对差异表达基因进行生物信息学分析. 结果 从13824条目的 基因中筛选出177条差异表达基因,其中123条基因表达上调,其中包括:结缔组织生长因子,微管蛋白ε 1,V型胶原α2,连环蛋白6 2,钙粘蛋白6,2型,Smad3,丝裂源活化蛋白激酶4,生长因子受体结合蛋白7,丝裂原活化蛋白激酶相互作用/丝氨酸/苏氨酸激酶1等;54条基因表达下调,包括:肿瘤坏死因子受体相关因子4,干扰素调节因子7,干扰素诱生蛋白p78,骨形态发生蛋白7,基质gLa蛋白,人类丝氨酸蛋白酶抑制剂进化支B成员8,干扰素刺激基因2.0×104,死亡相关蛋白6,金属硫蛋白1H,超氧化物歧化酶2等;同时筛选到8个未知功能蛋白. 结论 应用基因表达谱芯片技术成功筛选了TGF β 1刺激HSC的差异表达基因,初步揭示了TGF β1致肝纤维化的分子机制是诸多因素共同作用的结果,为进一步寻找新的基因治疗靶点奠定了基础.     

Detection of differentially expressed genes in lymphomas using cDNA arrays: identification of clusterin as a new diagnostic marker for anaplastic large-cell lymphomas

This study reports the first use of gene array technology for the identification of a tumor-specific marker in lymphoid neoplasms. The differential gene expression of 31 hematopoietic cell lines, representing most major lymphoma subgroups of B- and T-cell origin, was assessed by hybridizing labeled complementary DNA to Atlas human expression arrays containing 588 genes. Genes known to be specific for B, T, or myelomonocytic lineages were appropriately identified in the arrays, validating the general utility of this approach. One gene, clusterin, not previously known to be expressed in lymphoid neoplasms, was specifically found in all 4 anaplastic large-cell lymphoma (ALCL) cell lines, but not in any of the 27 remaining tumor lines. Using a monoclonal antibody against clusterin, its differential expression was confirmed by Western blotting and immunohistochemistry. A total of 198 primary lymphomas (representing most major lymphoma subtypes), including 36 cases of systemic ALCL, were surveyed for clusterin expression by immunohistochemistry and Western blotting. All of the 36 ALCL cases marked for clusterin, with most cases showing moderate to strong staining in the majority of neoplastic cells. Clusterin expression was not related to expression of anaplastic lymphoma kinase-1. With 2 exceptions, none of the remaining 162 non-ALCL cases marked with the clusterin antibody, including Hodgkin disease and primary cutaneous ALCL. In reactive lymphoid tissues, only follicular dendritic cells and fibroblastic reticular cells exhibited staining. Clusterin is a highly conserved glycoprotein implicated in intercellular and cell matrix interactions, regulation of the complement system, lipid transport, stress responses, and apoptosis. Although its function in ALCL is unknown, the unique expression of clusterin within this category of lymphoma provides an additional marker for the diagnosis of ALCL. This study illustrates the enormous potential of gene array technologies for diagnostic marker discovery. (Blood. 2000;96:398-404)     

Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.     

MCP-1基因多态性与老年2型糖尿病患者并发肾功能衰竭的相关性研究

目的 探讨单核细胞趋化蛋白-1(MCP-1)基因-2518A/G多态性与老年2型糖尿病患者并发肾功能衰竭之间的相关性.方法 采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测56例2型糖尿病伴肾功能衰竭患者及56例2型糖尿病(无肾损伤)患者、50名健康对照者的MCP-1基因-2518位点的基因型分布及基...     

野生型K-ras2诱导结肠癌细胞基因表达谱改变的特征分析

目的：应用基因芯片技术,检测野生型K-ras2基因在结肠癌Caco2细胞中表达诱发的基因谱改变,进一步阐明野生型K-ras2基因可能的分子生物学功能.方法：抽取正常人的静脉血,提取RNA,反转录成cDNA,以此为模板,采用PCR方法克隆K-ras2野生型全编码cDNA序列.以常规的分子生物学技术将获得的K-ras2 cDNA克隆到T Easy载体中进行核苷酸序列的测定,构建真核表达载体pCI-neo-K-ras2,以脂质体转染结肠癌细胞系Caco2,提取mRNA逆转录为cDNA与转染空白表达载体pCI-neo的Caco2细胞进行cDNA芯片分析.结果：构建的表达载体经过限制性内切酶分析和DNA序列测定证实准确无误,提取高质量的RNA逆转录为cDNA进行DNA芯片技术分析.在135个差异表达基因中发现有24个基因表达水平显著上调,121个基因表达水平显著下调.差异表达基因与细胞增殖、分化、凋亡和信号传导等有关.结论：应用基因表达谱芯片技术成功筛选了野生型K-ras2转染结肠癌细胞后差异表达基因,反映出野生型K-ras2对细胞增殖、代谢、转录调控等过程的负性调控状态,为进一步阐明野生型K-ras2可能的生物学功能提供了新的线索.     

氯沙坦和替米沙坦保护靶器官与抑制单核细胞趋化蛋白1及受体2表达有关

目的 研究血管紧张素受体拮抗剂(ARBs)氯沙坦与替米沙坦对原发性高血压靶器官的保护作用可能的降压外作用与抑制单个核细胞趋化蛋白1(MCP-1)和趋化细胞因子受体2(CCR2)有关.方法 自发性高血压大鼠(SHR)24只,随机服用低剂量氯沙坦[LL,5 mg/(kg·d),n=6]、高剂量氯沙坦[HL,30 mg/(kg·d),n=6]、替米沙坦[T,30 mg/(kg·d),n=6],未治疗SHR及WKY大鼠6只为对照(NC)组.左室称质量,测量大鼠胸主动脉中膜及内径,胸主动脉周围纤维化面积,RT-PCR检测心、肾、胸主动脉MCP-1、CCR2的表达,对外周血单个核细胞(PBMCs)进行MCP-1的趋化功能实验,ELISA观察大鼠血清MCP-1蛋白含量.结果 低剂量氯沙坦不能降血压(176.7±15.3 vs 174.2±17.1 mm Hg,P＞0.05).高剂量氯沙坦及替米沙坦可明显降压并显著抑制SHR左室增重、血管中膜增厚、血管周围纤维化,且动脉、心、肾和循环中MCP-1及CCR2的表达及PBMCs对MCP-1的趋化性下降(P＜0.05).低剂量氯沙坦尽管不能抑制左室肥厚及血管中膜增厚,但与高剂量氯沙坦同样可降低靶器官MCP-1,CCR2的表达及PBMCs对MCP-1的趋化性(P＜0.01).结论 SHR与WKY组相比,各靶器官组织MCP-1/CCR2的表达显著升高,ARBs能够发挥对靶器官的保护作用,同时抑制MCP-1/CCR2在靶器官及外周血的表达,并且ARBs能在不影响血压的条件下即能发挥这一作用,说明ARBs对MCP-1,CCR2的抑制作用是独立于其降压作用的.     

氯沙坦和替米沙坦保护靶器官与抑制单核细胞趋化蛋白1及受体2表达有关

目的研究血管紧张素受体拮抗剂(ARBs)氯沙坦与替米沙坦对原发性高血压靶器官的保护作用可能的降压外作用与抑制单个核细胞趋化蛋白1(MCP-1)和趋化细胞因子受体2(CCR2)有关。方法自发性高血压大鼠(SHR)24只,随机服用低剂量氯沙坦[LL,5mg/(kg.d),n=6]、高剂量氯沙坦[HL,30mg/(kg.d),n=6]、替米沙坦[T,30mg/(kg.d),n=6],未治疗SHR及WKY大鼠6只为对照(NC)组。左室称质量,测量大鼠胸主动脉中膜及内径,胸主动脉周围纤维化面积,RT-PCR检测心、肾、胸主动脉MCP-1、CCR2的表达,对外周血单个核细胞(PBMCs)进行MCP-1的趋化功能实验,ELISA观察大鼠血清MCP-1蛋白含量。结果低剂量氯沙坦不能降血压(176.7±15.3vs174.2±17.1mmHg,P>0.05)。高剂量氯沙坦及替米沙坦可明显降压并显著抑制SHR左室增重、血管中膜增厚、血管周围纤维化,且动脉、心、肾和循环中MCP-1及CCR2的表达及PBMCs对MCP-1的趋化性下降(P<0.05)。低剂量氯沙坦尽管不能抑制左室肥厚及血管中膜增厚,但与高剂量氯沙坦同样可降低靶器官MCP-1,CCR2的表达及PBMCs对MCP-1的趋化性(P<0.01)。结论SHR与WKY组相比,各靶器官组织MCP-1/CCR2的表达显著升高,ARBs能够发挥对靶器官的保护作用,同时抑制MCP-1/CCR2在靶器官及外周血的表达,并且ARBs能在不影响血压的条件下即能发挥这一作用,说明ARBs对MCP-1,CCR2的抑制作用是独立于其降压作用的。     

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis

INTRODUCTION Tumor metastasis is an incident involving multiple genes. However, the number of metastasis related genes available nowadays is very limited to elucidate the puzzling process of metastasis. Therefore, more attentions have been paid to screen …     

Polymorphisms of the MCP-1 and HSP70-2 Genes in Korean Patients with Alcoholic Chronic Pancreatitis

Alcoholic chronic pancreatitis (ACP) develops in only a small number of alcoholics. Monocyte chemotactic protein-1 (MCP-1) and heat-shock protein 70-2 (HSP70-2) polymorphisms have been reported to be associated with the severity of acute pancreatitis. However, their role in pathogenesis of ACP has not been investigated. A genetic association study for susceptibility and severity was performed on 79 male Korean ACP patients and 82 male controls. MCP-1 and HSP70-2 genotypes were determined using a fluorescence polarization detection method. The genotypes and G allele frequencies were no different in patients and controls. However, MCP-1 G allele had an effect on the development of severe ACP, when its frequency was compared in mild to moderate and severe ACP (29.6 vs. 56.0%, P = 0.02). The MCP-1 and HSP70-2 polymorphisms do not play a major role in the development of ACP in Koreans. However, MCP-1 polymorphism may be associated with the severity of ACP.