Initiation Patterns of DNA Replication in Cancer Cell Lines

描述(申请人提供)：人类基因组的复制依赖于数千个DNA合成程序开始的起始点的激活。然而，尽管这一领域取得了很大的进步，但目前尚不清楚这些位点在每条染色体上的分布情况，也不清楚这种分布是否会随着细胞的生理状态而变化。我们的长期目标是获得人类染色体上这些位置的详细图谱，以推断出可能允许我们区分正常和异常生长的细胞的起始信号，从而为我们提供一个工具来检测异常细胞增殖的早期阶段。基于对2号染色体99kb区域的基于聚合酶链式反应的新生链丰度分析，我们已经获得了初步数据，表明癌细胞在起始点的分布上与正常细胞不同。在目前的提案中，我们希望使用DNA微阵列技术将这些研究扩展到与乳腺癌相关的更长的染色体区域。我们假设这些区域的起始点的频率、分布和时间激活在癌细胞系中与正常细胞系相比是不同的。 为了验证这一假设，我们的具体目标是：(1)定位并比较正常细胞和乳腺癌细胞系中与乳腺癌相关的3、17和20号染色体区域的起始点的位置。为了实现这一目标，我们计划：(A)从异步生长的细胞中分离短的(约1-1.5kb)新生DNA链；(B)在DNA拼接阵列上定量它们的丰度；(B)在DNA拼接阵列上定量它们的丰度，所述DNA拼接阵列包含60聚体探针，分别覆盖Chr20q12-13上的20Mb区域、Chr17p13和Chr17q23上的两个4Mb区域以及Chr3q26上的3Mb区域；以及(C)比较正常细胞和癌细胞株的图谱。(2)评估正常乳腺细胞系和癌乳腺细胞系中3、17和20号染色体癌相关区域起始点激活的时间顺序。为此，我们计划同步G1/G0中的细胞，并探测上述平铺路径基因芯片，在进入S阶段后的不同时间点获得新生的短DNA链。这些研究将为我们提供关于正常和恶性细胞中DNA复制的启动和瞬时激活的新信息，这将有助于确定肿瘤特异性的起始点。这些信息也将为细胞异常生长的诊断和控制提供新的途径。

DESCRIPTION (provided by applicant): Duplication of the human genome depends on the activation of thousands of initiation sites where DNA synthesis is programmed to start. However, in spite of great advances in the field it is not clear what the distribution is of these sites along each one of the chromosomes, nor whether this distribution changes with the physiological state of the cell. Our long-term goal is to obtain a detailed map of these sites along human chromosomes to deduce initiation signatures that may allow us to distinguish normal from abnormally growing cells, thus providing us with a tool to detect early stages of abnormal cell proliferation. Based on a PCR-based nascent strand abundance assay on a 99 kb region of chromosome 2, we have obtained preliminary data suggesting that cancer cells differ from their normal counterparts in the distribution of initiation sites. In the present proposal, we wish to use DNA microarray technology to expand these studies to longer chromosome regions that are relevant in breast cancer. We hypothesize that the frequency, distribution, and temporal activation of initiation sites in these regions is different in cancer cell lines compared to their normal counterparts. To test this hypothesis, our specific aims are: (1) to map and compare the location of initiation sites on breast cancer-relevant regions of chromosomes 3, 17 and 20, in both normal and breast cancer cell lines. To accomplish this objective we plan to (a) isolate short (about 1-1.5 kb) nascent DNA strands from asynchronously growing cells; (b) quantitate their abundance on DNA tiling arrays containing 60-mer probes covering a 20 Mb region on Chr20q12-13, two 4Mb regions on Chr17p13 and Chr17q23, respectively, and a 3 Mb region on Chr3q26; and (c) compare the profiles of normal and cancer cell lines. (2) To assess the temporal order of activation of initiation sites along cancer-relevant regions of chromosomes 3, 17 and 20 in both normal and cancer breast cell lines. To this end we plan to synchronize cells in G1/G0 and probe the tiling path DNA microarray described above, with short nascent DNA strands obtained at different time points after entrance into the S phase. These studies will provide us with novel information about the initiation and temporal activation of DNA replication in both normal and malignant cells which will help identify tumor-specific initiation sites. This information will also offer new approaches for the diagnosis and control of abnormal cell growth.