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.

实验致癌实验室分子细胞遗传学部分的研究项目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。我们的小组采用基于分子细胞遗传学和分子生物学综合应用的组合方法，识别并检查了许多反复出现的染色体改变，从而发现了一些新的癌症相关基因，并检测了许多已知基因的改变。对这些改变的分析可能为癌症的预后和诊断以及潜在的治疗靶标提供有用的标记。 去年，通过比较基因组杂交在人类肝细胞癌（HCC）中鉴定出染色体 16q23 长臂上的重复缺失区域，并对其进行了分子表征。 16q32 区域跨越第二个最活跃的脆弱位点 (FS) FRA16D，并包含 WWOX（含氧化还原酶的 WW 结构域），这是一种候选肿瘤抑制基因。超过 60% 的 HCC 细胞系中 WWOX mRNA 和蛋白表达缺失或减少，表明 WWOX 基因在肝癌中频繁发生改变，并增加了该基因与肝癌发生有关的可能性。 DNA 的组织及其在 FS 上的复制使它们易于断裂和重组，也是诱变致癌物和致癌病毒整合的优先目标。多年来，将 FS 与癌症联系起来的尝试大多产生了间接证据，但最近积累的大量证据表明 FS 处的 DNA 断裂与染色体易位、原癌基因扩增、抑癌基因缺失和致癌病毒整合有关。 FRA16D WWOX 基因的改变为 FS 损伤与癌症相关基因改变的关联提供了额外的支持。几年前，我们从怀疑含有一个或多个肿瘤抑制基因的 8 号染色体短臂上的一个关键缺失区域中分离出了 DLC-1 基因，该基因编码小 GTP 酶 Rho 家族的调节因子。 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，我们发现，缺乏内源基因表达的转移性乳腺腺癌来源的细胞中 DLC-1 表达的恢复导致了显着的生长抑制，并阻止了无胸腺裸鼠肿瘤的发展。去年，有证据表明 DLC-1 还在肝癌和肺癌中发挥抑癌基因的作用。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，抑制细胞生长和体外侵袭性，并降低细胞在无胸腺裸鼠中形成肿瘤的能力。在 HCC 中，仅在部分肿瘤中发现了 DLC-1 的外显子错义突变和内含子插入/缺失。鉴定出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分析，确定 DLC-1 是乳腺癌易感基因的有力候选者。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，其表达恢复可抑制体外和体内肿瘤细胞生长。 DLC-1的稳定转移消除了裸鼠中非小细胞肺癌培养细胞的致瘤性。 DLC-1启动子区的高甲基化已在多种类型的癌症中检测到，DLC-1甲基化谱可作为早期检测、预测癌症风险和疾病预后的有用标记。此外，它可能代表药理学重新表达的潜在靶标，作为肺癌治疗的新方法。还分离了小鼠 DLC-1 基因，并表征了外显子/内含子组织。鉴定出可用于连锁作图和杂合性丢失分析的基因内多态性微卫星标记。为了提供研究 DLC-1 体内生物学功能的动物模型系统，我们成功地在胚胎干细胞中使用同源重组来产生 DLC-1 基因被破坏的小鼠。 DLC-1 敲除小鼠的产生以及该模型的初步表征表明 DLC-1 蛋白对于正常胚胎发育也是必需的。突变 DLC-1 等位基因杂合的小鼠将有助于分析 DLC-1 在肿瘤发生中的作用。除了 DLC-1 之外，通过检查暴露于去甲基化剂 5-aza-2'-deoxycytidine (5-aza-dC) 的人 NSCLC 细胞中的基因表达谱，还检测到其他癌症相关基因的改变。在 NSCLC 细胞系中，酪氨酸蛋白激酶 ABL2 (ABL2) 基因的下调和 Hint/蛋白激酶 C 抑制剂 1 (Hint/PKCI-1)、DVL1、TIMP-1 和 TRP-1 基因的上调反复出现。将 Hint/PKCI-1 基因转移到表现出 5-aza-dC 诱导的基因上调的细胞系中，导致 NSCLC 细胞的体外生长显着抑制，并显着降低体内致瘤性。癌症被认为是一种衰老有机体的疾病。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。通过 g-H2AX 抗体免疫染色和端粒荧光原位杂交 (FISH) 对人类和小鼠衰老细胞进行双链断裂链 (DBS) 定位，为 DBS 修复因子的基因组分布、衰老过程中 DNA 损伤积累和修复的作用提供证据，并为辨别人类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，最近对鼠同源物的 FISH 定位表明，线粒体 DNA 拓扑异构酶（IB 型）基因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表达中发挥作用，被定位在多种疾病中基因组频繁改变的区域，并且可能在恶性淋巴瘤的发病机制中发挥重要作用。