MOLECULAR CLONING OF A T(15;19) IN LUNG CANCER CELL LINE

这是为Thao Dang医学博士申请K08奖项的申请，该奖项旨在支持为期五年的实验室培训，以进一步发展她在分子遗传学方面的技能，并探索癌症发生的新机制。恶性转化是基因异常累积的结果。特定的染色体易位是血液系统恶性肿瘤癌基因激活的主要机制，但在更常见的上皮性肿瘤中尚未被描述。我们已经建立了一个细胞系，HCC2429，来自一种侵袭性的转移性肺癌，除了15q和19p染色体之间的单一易位外，它具有正常的核型。通过定位克隆，我们证明了19号染色体上的断裂点位于Notch3起始点的上游约40kb，Notch3是Notch原癌基因家族的成员。这种易位与Notch3的大量过度表达有关，支持了t(15；19)易位导致这一假定的细胞原癌基因失控的假设。此外，我们还展示了Notch3在一组肺癌细胞系中的过度表达，并表明它与涉及19P的易位高度相关。因此，我们确定了肺癌中癌基因激活的一种新的复发机制，以及一种以前未知的与人类癌症相关的假定癌基因。在David Carbone博士的指导下，Dang博士将完成已确定的t(15；19)易位的分子鉴定，测定Notch3受体的光谱和在肺癌和正常组织中的配体表达，并进行研究，以表征Notch3的转化性质及其对肺癌下游信号通路的影响。范德比尔特英格拉姆癌症中心的研究环境具有卓越的水平，将为Dang博士提供与经验丰富的分子生物学家和遗传学家互动的机会。K08奖项给予的支持将使Dang博士能够在现有知识的基础上，在竞争激烈的环境中促进她向独立研究人员的转变。

This is an application for a K08 award for Thao Dang, M.D. designed to support five-years of laboratory training to further develop her skills in molecular genetics and to explore a novel mechanism of carcinogenesis. Malignant transformation is the result of an accumulation of genetic abnormalities. Specific chromosomal translocations are a major mechanism for oncogene activation in hematopoietic malignancies, but have not been described in the much more common epithelial tumors. We have established a cell line, HCC2429, from an aggressive, metastatic lung cancer that has a normal karyotype except for a single translocation between chromosomes 15q and 19p. Using positional cloning we demonstrated that the breakpoint on chromosome 19 lies approximately 40 kb upstream from the start site of Notch3, a member of the Notch proto- oncogene family. This translocation is associated with massive overexpression of Notch3, supporting the hypothesis that the t(15;19) translocation results in the deregulation of this putative cellular proto-oncogene. Furthermore, we have also demonstrated Notch3 over-expression in a panel of lung cancer cell lines and shown that it is highly correlated with translocations involving 19p. We have therefore identified a novel recurring mechanism for oncogene activation in lung cancer as well as a putative oncogene not previously known to be involved in human cancer. Under the mentorship of Dr. David Carbone, Dr. Dang will complete the molecular characterization of the identified t(15;19) translocation, determine the spectrum of the Notch3 receptor and ligand expression in lung cancer and normal tissues, and perform studies to characterize the transforming nature of Notch3 and its effects on downstream signaling pathways in lung cancer. The research environment at the Vanderbilt Ingram Cancer Center is of exceptional caliber and will provide Dr. Dang with the opportunity to interact with experienced molecular biologists as well as geneticists. The support given by this K08 award will allow Dr. Dang to build on her existing knowledge and promote her transition to an independent investigator in a highly competitive environment.