EXTRACHROMOSOMAL DNA IN PATIENTS' OVARIAN CANCERS

卵巢癌是癌症死亡的最常见原因之一， 妇女 去年，它造成了12,000多名妇女的死亡。 年在美国。 大约每70名女性中就有一人会患上 疾病 这个国家百分之一的女性死亡是由于 卵巢癌 我们提出了一种新的方法来识别扩增的 可能导致肿瘤进展和耐药性的基因 在这些患者中通过靶向染色体外DNA。 染色体外 DNA被认为在肿瘤发生中起着关键作用，因为扩增的 癌基因和耐药基因通常位于染色体外， 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）。 我们现在已经证明，DM经常出现在中期 从新鲜活检标本中获得的卵巢肿瘤扩散。 因此，将避免长时间培养，因为DM通常在培养过程中丢失。 体外 我们已经开发出一种策略， 位于（即在DM上）扩增的癌基因或耐药基因。 这些 DM上存在的扩增的癌基因或耐药基因可以是 通过染色体显微切割特异性分离， 通过聚合酶链反应（PCR）体外扩增。 最初我们 将在DM上使用基于PCR的策略，以确定DM上的精确区域。 中期染色体，其中DM的扩增基因起源于 在正常细胞染色体制备物上使用基因定位技术。 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知癌基因 和/或耐药基因。 在那个时候，特定的克隆可以 基因序列最终决定了。 建议的具体目标如下： 1)利用当前最先进的染色体显微切割技术 确定DM中已知癌基因扩增的发生率， 开发基因指纹技术来表征DM。 2)利用染色体显微切割技术和PCR扩增技术， 表征人类中扩增的癌基因和耐药基因 肿瘤的

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.