Molecular Cytogenetics of Solid Tumors

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。在过去的一年里，癌细胞的分子细胞遗传学和分子生物学研究取得了重大进展。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的作用的证据。是identifid . .分离了小鼠DLC-1基因，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LOH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。尽管DLC-1 mRNA水平降低，但干扰等位基因杂合的小鼠存活且表型正常。杂合动物交配未获得纯合突变体后代，说明DLC-1缺失导致胚胎致死性。定时妊娠分析显示，DLC-1-/-胚胎不能存活超过妊娠10.5天，组织学检查显示神经管、脑、心脏和胎盘存在缺陷。野生型小鼠胚胎中DLC-1 mRNA的原位杂交定位显示，在突变型胚胎中发现异常时，DLC-1 mRNA在胚胎和胚胎外组织中广泛表达。这种分布与DLC-1在正常发育中的作用是一致的。纯合子突变胚培养成纤维细胞显示肌动蛋白丝组织和局灶粘连的改变。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的缺失可能会通过对细胞粘附和迁移产生不利影响而干扰细胞的发育，鉴定参与癌症转移扩散的生物学机制的分子特征是开发能够消除这一过程的治疗干预措施所必需的。在此之前，我们在胸腺裸鼠中证明，在缺乏内源性基因表达的转移性乳腺腺癌细胞系中，恢复DLC-1的表达会导致显著的生长抑制，并阻止肿瘤的发展。与佛罗里达大学的Steve Goodison博士和他的同事合作，研究了DLC-1在转移中的作用。是检查。单克隆细胞系M4A4和NM2C5是MDA-MB235的自发亚系，它们在生长、侵袭、传播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在稳定和等基因模型中对转移过程中的细胞和分子事件进行比较研究成为可能。与转移表型相关的171个基因表达特征突出了几种GTPase信号传导成分。其中一个组成部分，DLC 1基因，在转移细胞中相对于非转移细胞被发现下调。

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.