Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

• 批准号: 7049728
• 负责人: NICOLAE POPESCU
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7049728
• 关键词: DNA damage; DNA methylation; athymic mouse; cancer risk; cell growth regulation; cell line; cell senescence; chromosome aberrations; cytogenetics; fluorescent in situ hybridization; gene expression; genetic mapping; genetic markers; genetic susceptibility; genetically modified animals; laboratory mouse; molecular oncology; neoplasm /cancer genetics; neoplastic process; nucleic acid structure; oncoproteins; single nucleotide polymorphism; tissue /cell culture; transfection; tumor suppressor genes

The research program of the Molecular Cytogenetics Section of the Laboratory of Experimental Carcinogenesis is aimed at the identification and characterization of genomic modifications associated with initiation and progression of the neoplastic phenotype. Using a combined approach based on integrated use of molecular cytogenetics and molecular biology, our group identified and examined a number of recurrent chromosome alterations that led to the discovery of several new cancer-relevant genes, and to the detection of alterations in a number of known ones. Analysis of these alterations may provide markers useful for the prognosis and diagnosis of cancer and potential targets for therapy.In the past year a recurrent region of deletion on the long arm of chromosome 16q23 identified in human hepatocellular carcinoma (HCC) by comparative genomic hybridization was characterized molecularly. The 16q32 region span the second most active fragile site (FS) FRA16D and contains WWOX (WW-domain containing oxidoreductase), a candidate tumor suppressor gene. WWOX mRNA and protein expression were absent or reduced in over 60% of HCC cell lines, showing that WWOX gene is frequently altered in liver cancer and raising the possibility that this gene is implicated in hepatocarcinogenesis. The organization of DNA and its replication at FSs renders them prone to breakage and recombination as well as preferential targets for mutagenic carcinogens and integration of oncogenic viruses. For many years, attempts to link FSs and cancer generated mostly circumstantial evidence, but recently ample evidence has accumulated that DNA breakage at FSs is associated with chromosomal translocations, amplification of proto-oncogenes, deletion of tumor suppressor genes, and integration of oncogenic viruses. Alterations of WWOX gene at FRA16D provide additional support for relating damage at FSs to alterations in cancer-related genes. Several years ago, from a critical region of deletion on the short arm of chromosome 8 suspected to harbor one or more tumor suppressor genes, we isolated the DLC-1 gene, which encodes a regulator of the Rho family of small GTPases. The Rho proteins control actin cytoskeleton organization, membrane trafficking, gene expression, and are involved in cell proliferation, malignant transformation, and metastasis. In the intervening years we showed that restoration of DLC-1 expression in cells derived from metastatic breast adenocarcinomas lacking endogenous gene expression caused significant growth inhibition and prevented the development of tumors in athymic nude mice. In the past year, evidence that DLC-1 also acts as a tumor suppressor gene in liver and lung cancer was provided. Transfection experiments demonstrated that the restoration of DLC-1 expression in HCC cells resulted in caspase 3 mediated apoptosis, inhibition of cell growth and invasiveness in vitro as well as a reduction of the ability of the cells to form tumors in athymic nude mice. In HCC exonic missense mutations and intronic insertions/deletions of DLC-1 were found only in subset of the tumors. Several single nucleotide polymorphisms (SNP) were identified that could be used for determining whether DLC-1 is linked to liver cancer susceptibility loci. Other investigators, using high-throughput SNP genotyping and gene expression profiling, identified DLC-1 as a strong candidate for a breast cancer susceptibility gene. In non-small cell lung carcinoma (NSCLC), we showed that DLC-1 is frequently inactivated by aberrant DNA methylation and restoration of its expression results in suppression of in vitro and in vivo tumor cell growth. Stable transfer of DLC-1 abolished the tumorigenicity of cultured cells derived from NSCLC in nude mice. Hypermethylation of the DLC-1 promoter region has been detected in a number of cases in various types of cancer, and the DLC-1 methylation profile may serve as a useful marker for early detection, prediction of cancer risk and prognosis of the disease. Also, it may represent a potential target for pharmacological re-expression as a novel method for lung cancer treatment. The mouse DLC-1 gene was also isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and loss of heterozygosity analysis. To provide an animal model system for investigating the biological functions of DLC-1 in vivo, we successfully used homologous recombination in embryonic stem cells to generate mice with a disrupted DLC-1 gene. The generation of the DLC-1-knock out mice, and initial characterization of this model revealed that the DLC-1 protein is also essential for normal embryonic development. The mice heterozygous for the mutated DLC-1 allele will be useful for analysis of the role of DLC-1 in tumorigenesis.In addition to DLC-1, alterations of other cancer-related genes were detected by examining the gene expression profile in human NSCLC cells exposed to the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Down-regulation of tyrosine protein kinase ABL2 (ABL2) gene and up-regulation of hint/protein kinase C inhibitor 1 (Hint/PKCI-1), DVL1, TIMP-1 and TRP-1 genes were recurrent in NSCLC cell lines. Transfer of Hint/PKCI-1 gene in cell lines exhibiting 5-aza-dC-induced up-regulation of the gene resulted in a significant in vitro growth inhibition and significantly reduced in vivo tumorigenicity of NSCLC cells. Cancer is considered a disorder of aging organisms. Various types of damage and mutations may accumulate during the lifetime rendering the cells more susceptible to developing cancer. Localization of double-strand breaks strand (DBS) by immunostaining with g-H2AX antibody and fluorescence in situ hybridization (FISH) with a telomeric probe on human and mouse cells undergoing senescence generated evidence on the genomic distribution of the DBS repair factors, the role of accumulation and repair of DNA damage during the process of aging and provided a useful marker for discerning human and animal aging cells. We previously localized a human mitochondrial topoisomerase1 gene and recent FISH localization of the murine homologue showed that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. GC-binding factor 2 (GCF), a transcriptional repressor overexpressed in cancer cells that plays a role in regulating the expression of EGFR, was mapped at a region a frequent genomic alterations in several disorders and might be important in pathogenesis of malignant lymphomas.

实验性致癌实验室的分子细胞遗传学部分的研究计划旨在鉴定和表征与肿瘤表型的起始和进展相关的基因组修饰。使用基于分子细胞遗传学和分子生物学的综合使用方法的组合方法，我们的小组鉴定并检查了许多复发性染色体的改变，从而发现了几种新的癌症含量的基因，并发现了许多已知的染色体基因。对这些变化的分析可能会为癌症的预后和诊断和治疗潜在靶标提供有用的标记。在过去的一年中，在人类肝细胞癌（HCC）中鉴定出的16q23染色体16q23的复发区域通过比较基因组杂交在人类肝细胞癌（HCC）中鉴定出来。 16q32区域跨越了第二高活跃的脆弱位点（FS）FRA16D，并包含WWOX（含有WW-domain的氧化还原酶），一种候选肿瘤抑制基因。在超过60％的HCC细胞系中，WWOX mRNA和蛋白质表达不存在或降低，表明WWOX基因在肝癌中经常改变，并提高了该基因与肝癌发生有关的可能性。 DNA的组织及其在FSS中的复制使它们容易破裂和重组，以及诱变致癌物的优先靶标和致癌病毒的整合。多年来，尝试将FSS和癌症联系起来的尝试主要产生了间接证据，但是最近有足够的证据积累，FSS的DNA断裂与染色体易位，原始癌基因的扩增，肿瘤抑制基因的缺失以及Oncenitic病毒的整合有关。 WWOX基因在FRA16D上的改变为将FSS的损害与癌症相关基因的改变相关的额外支持。几年前，我们从怀疑具有一个或多个抑制肿瘤基因的染色体的短臂上的一个关键缺失区域，我们分离了DLC-1基因，该基因编码了小型GTPase的Rho家族的调节剂。 Rho蛋白控制肌动蛋白细胞骨架组织，膜运输，基因表达，并参与细胞增殖，恶性转化和转移。在随后的几年中，我们表明在缺乏内源基因表达的转移性乳腺腺癌衍生的细胞中DLC-1表达的恢复引起了显着的生长抑制，并阻止了无胸腺裸鼠肿瘤的发展。在过去的一年中，提供了DLC-1在肝癌和肺癌中充当肿瘤抑制基因的证据。转染实验表明，HCC细胞中DLC-1表达的恢复导致caspase 3介导的细胞凋亡，体外抑制细胞生长和侵入性，以及降低细胞在裸裸小鼠中形成肿瘤的能力。在HCC外显子义突变和内含子插入/DLC-1的缺失仅在肿瘤的子集中发现。确定了几种单核苷酸多态性（SNP），可用于确定DLC-1是否与肝癌易感性基因座有关。使用高通量SNP基因分型和基因表达分析的其他研究人员将DLC-1确定为乳腺癌易感基因的强大候选者。在非小细胞肺癌（NSCLC）中，我们表明DLC-1经常被异常的DNA甲基化和恢复其表达的恢复导致体外和体内肿瘤细胞生长的抑制。 DLC-1的稳定转移消除了裸鼠NSCLC衍生的培养细胞的致瘤性。在多种类型的癌症中，已经在许多病例中检测到了DLC-1启动子区域的高甲基化，并且DLC-1甲基化谱可能是早期发现，预测癌症风险和疾病预后的有用标记。同样，它可能代表了药理学重新表达的潜在靶标，作为一种新型的肺癌治疗方法。还分离了小鼠DLC-1基因，并表征了外显子/内含子组织。鉴定出基因内多态微卫星标记物，可用于连锁映射和杂合性分析的丢失。为了提供一个用于研究DLC-1在体内生物学功能的动物模型系统，我们成功地使用了胚胎干细胞中的同源重组来生成具有破坏的DLC-1基因的小鼠。 DLC-1敲除小鼠的产生，该模型的初始表征表明，DLC-1蛋白对于正常的胚胎发育也是必不可少的。突变的DLC-1等位基因的杂合小鼠将有助于分析DLC-1在肿瘤发生中的作用。在DLC-1的外，通过检查针对人NSCLC中的NSCLC中的基因表达谱检测到其他与癌症相关的基因的变化，该基因表达谱图暴露于5-aza-2'-脱氧剂5-aza-2'-二氧化物的人NSCLC细胞（5c）。酪氨酸蛋白激酶ABL2（ABL2）基因的下调以及提示/蛋白激酶C抑制剂1（HINT/PKCI-1），DVL1，TIMP-1和TRP-1基因的上调在NSCLC细胞系中复发。在表现出5-Aza-DC诱导的基因上调的细胞系中提示/PKCI-1基因的转移会导致显着的体外生长抑制作用，并显着降低了NSCLC细胞的体内肿瘤性。癌症被认为是衰老生物的疾病。在一生中，可能会积累各种类型的损害和突变，从而使细胞更容易受到癌症的影响。通过用G-H2AX抗体和荧光原位杂交（FIS）进行免疫染色，与对人和小鼠细胞的端粒探针进行免疫染色，从而在人和小鼠细胞上进行触发性探针来定位双链断裂链（DB），这产生了衰老的证据，这证明了DBS修复因子的基因组分布，在衰减过程中的积累和dick ail agranning and and Markerning and Arimernning a Agranning and sage and and and and and and and and and and and and and and and and and and and and and Marker and Marker and Markerning。我们以前将人的线粒体拓扑异构酶1基因定位，而最近的鱼类同源物的鱼类定位表明，线粒体DNA拓扑酶（IB型）的基因仅在脊椎动物中存在。 GC结合因子2（GCF）是一种在癌细胞中过表达的转录阻遏物，在调节EGFR的表达中起作用，在某个区域绘制了几种疾病的频繁基因组改变，在恶性淋巴瘤的发病机理中可能很重要。