EXTRACHROMOSOMAL DNA IN PATIENTS' OVARIAN CANCERS

卵巢癌是#年最常见的癌症死亡原因之一。 女人。去年，它造成了12000多名妇女的死亡 在美国的一年。大约每70名女性中就有一人会患上 疾病。这个国家1%的女性死亡是由于 卵巢癌。我们正在提出一种新的方法来识别扩增的 可能与肿瘤进展和耐药性有关的基因 在这些患者中通过靶向染色体外DNA。染色体外染色体 DNA被认为自扩增以来在肿瘤发生中起着关键作用 癌基因和耐药基因常位于染色体外 肿瘤细胞和肿瘤细胞系中的DNA。 染色体外DNA的最大形式是双微小染色体 (DM)。我们现在已经记录了DM经常出现在中期 从新鲜活检标本中获得的卵巢肿瘤的扩散。 因此，将避免延长区域性，因为DM经常在 体外培养。 我们已经开发了一种策略来分离和鉴定染色体外 位于DMS扩增的癌基因或耐药基因上。这些 存在于DM上的扩增的癌基因或耐药基因可以是 通过染色体显微切割和片段 通过聚合酶链式反应(PCR)进行体外扩增。最初，我们 将在DM上使用基于PCR的策略来识别 DM患者扩增基因起源的中期染色体 在正常细胞染色体制备上使用基因作图技术。 在确定DM的染色体图谱位置后，我们将能够 以确定我们是否发现了可能的新的未鉴定的癌基因 和/或耐药基因。那时，特定的克隆可能是 并最终确定它们的基因序列。 提出了以下具体目标： 1)利用当前最先进的染色体显微切割技术 为了确定已知的癌基因扩增在DM和DM中的发生率 开发一种基因指纹技术来表征DM。 2)利用染色体显微切割技术和聚合酶链式反应扩增技术 人类扩增的癌基因和耐药基因的特征 肿瘤。

Ovarian cancer is one of the most frequent causes of cancer deaths in women. It was responsible for the deaths of more than 12,000 women last year in the United States. About one in 70 women will develop the disease. One percent of al female deaths in this country are due to ovarian cancer. We are proposing a novel method to identify amplified genes which may be responsible for tumor progression and drug resistance in these patients by targeting extrachromosomal DNA. Extrachromosomal DNA is thought to play a pivotal role in tumorigenesis since amplified oncogenes and drug-resistant genes are often located in extrachromosomal DNA in tumor cells and tumor cell lines. The largest form of extrachromosomal DNA is the double minute chromosome (DM). We now have documented that DMs are frequently found in metaphase spreads from ovarian tumors obtained from fresh biopsy specimens. Therefore, prolonged culture will be avoided since DMs are often lost in vitro. We have developed a strategy to isolate and identify extrachromosomally located (i.e. on DMs amplified oncogenes or drug resistance genes. These amplified oncogenes or drug resistant genes present on DMs can be specifically isolated by chromosome microdissection and the fragments amplified in vitro by polymerase chain reaction (PCR). Initially, we will use PCR-based strategies on DMs to identify the precise region on metaphase chromosomes where the amplified genes of the DMs originate by using gene mapping techniques on normal cell chromosomal preparations. After determining the chromosomal map position of the DM we would be able to determine if we have identified probable new uncharacterized oncogenes and/or drug resistance genes. At that time specific clones could be isolated and their gene sequences ultimately determine. The following specific aims are proposed: 1) Utilize current state-of-the-art chromosome microdissection techniques to determine the incidence of known oncogene amplification on DMs and develop a genetic fingerprinting techniques to characterize DMs. 2) To use chromosome microdissection techniques and PCR amplification to characterize amplified oncogenes and drug-resistant genes in human tumors.