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.

实验癌变实验室分子细胞遗传学部门的研究项目旨在鉴定和表征与肿瘤表型的开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综合使用的综合方法，我们的小组确定并检查了一些复发性染色体改变，这些改变导致了几个新的癌症相关基因的发现，并检测了一些已知基因的改变。分析这些改变可能为癌症的预后和诊断提供有用的标记物和潜在的治疗靶点。在过去的一年中，通过比较基因组杂交技术在人肝细胞癌（HCC）中发现了16q23染色体长臂上的一个复发性缺失区域。16q32区域横跨第二活跃的脆弱位点（FS） FRA16D，含有WWOX （ww结构域含氧化还原酶），这是一种候选的肿瘤抑制基因。在超过60%的HCC细胞系中，WWOX mRNA和蛋白表达缺失或降低，表明WWOX基因在肝癌中经常发生改变，并提高了该基因参与肝癌发生的可能性。DNA的组织及其在FSs中的复制使其易于断裂和重组，并且是致突变致癌物和致癌病毒整合的优先目标。多年来，将FSs与癌症联系起来的尝试大多是间接证据，但最近积累的大量证据表明，FSs的DNA断裂与染色体易位、原癌基因扩增、肿瘤抑制基因缺失和致癌病毒整合有关。fr16d处WWOX基因的改变为FSs损伤与癌症相关基因的改变提供了额外的支持。几年前，我们从8号染色体短臂上一个怀疑含有一个或多个肿瘤抑制基因的关键缺失区域分离出DLC-1基因，该基因编码Rho家族小gtpase的调节因子。Rho蛋白控制肌动蛋白骨架组织、膜运输、基因表达，并参与细胞增殖、恶性转化和转移。在这期间的几年中，我们发现在缺乏内源性基因表达的转移性乳腺腺癌细胞中恢复DLC-1表达会导致胸腺裸鼠显著的生长抑制并阻止肿瘤的发展。在过去的一年中，有证据表明DLC-1在肝癌和肺癌中也可以作为肿瘤抑制基因。转染实验表明，恢复DLC-1在HCC细胞中的表达可导致caspase 3介导的细胞凋亡，抑制细胞生长和体外侵袭性，并降低胸腺裸小鼠细胞形成肿瘤的能力。在HCC中，DLC-1的外显子错义突变和内含子插入/缺失仅在部分肿瘤中发现。发现了几个单核苷酸多态性（SNP），可用于确定DLC-1是否与肝癌易感位点相关。其他研究人员使用高通量SNP基因分型和基因表达谱，确定DLC-1是乳腺癌易感基因的强候选基因。在非小细胞肺癌（NSCLC）中，我们发现DLC-1经常因异常DNA甲基化而失活，其表达的恢复导致体外和体内肿瘤细胞生长的抑制。DLC-1的稳定转移可消除裸鼠非小细胞肺癌培养细胞的致瘤性。DLC-1启动子区域的高甲基化已经在各种类型的癌症的许多病例中被检测到，DLC-1甲基化谱可以作为早期发现、预测癌症风险和疾病预后的有用标记。此外，作为一种治疗肺癌的新方法，它可能代表着药物再表达的潜在靶点。同时对小鼠DLC-1基因进行了分离，并对其外显子/内含子结构进行了表征。发现了一个基因内多态性微卫星标记，可用于连锁定位和杂合性损失分析。为了为研究DLC-1在体内的生物学功能提供一个动物模型系统，我们成功地在胚胎干细胞中使用同源重组技术产生了DLC-1基因断裂的小鼠。DLC-1敲除小鼠的产生和该模型的初步表征表明，DLC-1蛋白对正常胚胎发育也是必不可少的。突变的DLC-1等位基因的小鼠杂合将有助于分析DLC-1在肿瘤发生中的作用。除了DLC-1外，通过检测暴露于去甲基化剂5-aza-2'-脱氧胞苷（5-aza-dC）的人非小细胞肺癌细胞的基因表达谱，还检测了其他癌症相关基因的改变。酪氨酸蛋白激酶ABL2 （ABL2）基因下调和提示/蛋白激酶C抑制剂1 （hint/ PKCI-1）、DVL1、TIMP-1和TRP-1基因上调在NSCLC细胞系中反复出现。在5-aza- dc诱导的基因上调细胞系中转移Hint/PKCI-1基因，可显著抑制NSCLC细胞的体外生长，并显著降低其体内致瘤性。癌症被认为是机体老化的一种疾病。各种类型的损伤和突变可能在一生中积累，使细胞更容易患癌症。利用g-H2AX抗体免疫染色和端粒探针荧光原位杂交（FISH）在人和小鼠衰老细胞上定位双链断裂链（DBS），为DBS修复因子的基因组分布、衰老过程中DNA损伤的积累和修复作用提供了证据，为识别人和动物衰老细胞提供了有用的标记。我们之前定位了人类线粒体拓扑异构酶1基因，最近对小鼠同源物的FISH定位表明，线粒体DNA拓扑异构酶基因（IB型）仅存在于脊椎动物中。gc结合因子2 （GCF）是一种在癌细胞中过度表达的转录抑制因子，在调节EGFR的表达中起作用，在几种疾病中经常发生基因组改变的区域被定位，可能在恶性淋巴瘤的发病机制中起重要作用。