Molecular Cytogenetics of Solid Tumors

实验致癌实验室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启动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的组合方法，我们的小组鉴定并检查了一些复发性染色体改变，这些改变导致发现了几个新的癌症相关基因，并检测了一些已知基因的改变。对这些改变的分析可以提供对癌症的预后和诊断有用的标记物和潜在的治疗靶点。在过去的一年中，癌细胞的分子细胞遗传学和分子生物学取得了重大进展。新的复发性染色体改变和DLC-1基因在胚胎发育和人乳腺癌转移中作用的证据。被识别..分离小鼠DLC-1基因，并表征外显子-内含子组织。一个基因内的多态性微卫星标记被确定，是有用的连锁作图和洛分析。为了提供一个动物模型系统，研究DLC-1的生物学功能在vivo，我们成功地使用同源重组在胚胎干细胞中产生的小鼠与DLC-1基因的破坏。破坏的等位基因的杂合子小鼠是可行的，表型正常，但DLC-1 mRNA水平降低。从杂合动物的交配中没有获得纯合突变后代，表明DLC-1缺陷导致胚胎致死。定时妊娠的分析表明，DLC-1-/-胚胎不能存活超过10.5天妊娠，组织学检查显示神经管，大脑，心脏和胎盘缺陷。DLC-1 mRNA在野生型小鼠胚胎中的原位杂交定位显示，在突变胚胎中发现异常时，在胚胎和胚外组织中广泛表达。这种分布与DLC-1在正常发育中的作用一致。从纯合子突变胚胎培养的成纤维细胞显示肌动蛋白丝和局灶性粘连的组织的改变。这些结果表明，DLC-1蛋白在细胞骨架和细胞粘附复合物的组装中起着重要作用，DLC-1的缺失可能通过不利地影响细胞粘附和迁移而干扰发育。鉴定癌症转移扩散中所涉及的生物机制的分子特征对于开发能够消除该过程的治疗干预是必需的。以前，我们证明，恢复DLC-1表达的细胞系来源于转移性乳腺癌缺乏内源性基因表达引起显着的生长抑制，并防止肿瘤的发展在无胸腺nude mice.In collaboaration与博士史蒂夫古迪逊从佛罗里达大学和他的同事的DLC-1在转移中的作用。进行了检查。单克隆细胞系M4 A4和NM 2C 5是MDA-MB 235的自发发生的亚系，其在生长、侵袭、播散和原位自发转移效率方面表现出许多表型差异。这些细胞系的共同起源使得能够在稳定和同源模型中比较研究转移过程中的细胞和分子事件。与转移表型相关的171个基因表达特征突出了几个GT3信号传导组分。其中一个成分DLC 1基因在转移癌细胞中表达下调。

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 significant progress has been made molecular cytogenetics and molecular biology of cancer cells. New recurrent chromosome alterations and evidence regarding the role of DLC-1 gene in embryonic development and human breast cancer metastasis. were identifid.. The mouse DLC-1 gene was isolated, and the exon-intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and LOH 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. Mice heterozygous for the disrupted allele were viable and phenotypically normal, although with reduced levels of DLC-1 mRNA. No homozygous mutant progeny was obtained from mating of heterozygous animals, indicating that DLC-1 deficiency resulted in embryonic lethality. Analysis of timed pregnancies showed that DLC-1-/- embryos did not survive beyond 10.5 days gestation , and histological examination revealed defects in the neural tube, brain, heart, and placenta. In situ hybridization localization of DLC-1 mRNA in the wild type mouse embryo showed a widespread expression in embryonic and extraembryonic tissues as at the time when abnormalities were found in mutant embryos. This distribution is consistent with a role for DLC-1 in normal development. Cultured fibroblasts from homozygous mutant embryos displayed alterations in the organization of actin filaments and focal adhesions. These results suggest that the DLC-1 protein plays an important role in the assembly of the cytoskeleton and cell adhesion complexes and loss of DLC-1 may interfere with development by adversely affecting cell adhesion and migration Identification of molecular signatures characteristic of the biological mechanisms involved in the metastasis spread of cancer is required for the development of therapeutic interventions able to abrogate the process. Previously , we demonstrated that restoration of DLC-1 expression in cell lines derived from metastasic breast adenocarcinomas lacking endogenous gene expression caused significant growth inhibition and prevented the development of tumors in athymic nude mice.In collaboaration with Dr. Steve Goodison from University of Florida and his colleagues the role of DLC-1 in metastasis. was examined. Monoclonal cell lines M4A4 and NM2C5 are spontaneously occurring sublines of the MDA-MB235 which exhibit many phenotypic differences in growth, invasion, dissemination and spontaneous metastatic efficiency from an orthotopic site. The common origin of these cell lines enables the comparative investigation of cellular and molecular events in the metastatic process in a stable and isogeneic model. A 171-gene expression signature that correlated with metastatic phenotype highlighted several GTPase signaling components. One of these components, DLC 1 gene, was found down-regulated in the metastatic relative to the non-metastatic cells.