Initiation Patterns of DNA Replication in Cancer Cell Lines

描述（由申请人提供）：人类基因组的复制依赖于数千个DNA合成启动的起始位点的激活。然而，尽管这一领域取得了很大的进展，但这些位点沿每条染色体沿着的分布是什么，以及这种分布是否随细胞的生理状态而变化，仍不清楚。我们的长期目标是获得这些位点沿沿着人类染色体的详细图谱，以推断起始特征，从而使我们能够区分正常和异常生长的细胞，从而为我们提供检测异常细胞增殖的早期阶段的工具。基于PCR的新生链丰度测定的99 kb区域的2号染色体上，我们已经获得了初步的数据表明，癌细胞不同于他们的正常同行的起始位点的分布。在目前的建议中，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较长染色体区域。我们假设，频率，分布和时间激活的起始位点在这些地区是不同的癌细胞系相比，他们的正常同行。 为了验证这一假设，我们的具体目标是：（1）在正常和乳腺癌细胞系中，定位并比较3号、17号和20号染色体上乳腺癌相关区域的起始位点。为了实现这一目标，我们计划（a）隔离短（B）在含有60-mer探针的DNA平铺阵列上定量它们的丰度，所述60-mer探针分别覆盖Chr 20 q12 -13上的20 M B区、Chr 17 p13和Chr 17 q23上的两个4 M B区以及Chr 3 q26上的3 M B区;和（c）比较正常和癌细胞系的谱。(2)评估正常乳腺细胞系和乳腺癌细胞系中3号、17号和20号染色体上沿着癌症相关区域的起始位点激活的时间顺序。为此，我们计划使G1/G 0期的细胞同步化，并使用在进入S期后的不同时间点获得的短新生DNA链探测上述平铺路径DNA微阵列。这些研究将为我们提供有关正常和恶性细胞中DNA复制的起始和时间激活的新信息，这将有助于确定肿瘤特异性起始位点。这些信息也将为诊断和控制异常细胞生长提供新的方法。

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.