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起始位点的激活，这些起始位点是DNA合成程序开始的地方。然而，尽管在这一领域取得了很大的进展，但人们还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点的详细地图，以推断起始特征，这可能使我们能够区分正常和异常生长的细胞，从而为我们提供一种检测异常细胞增殖早期阶段的工具。基于基于pcr的2号染色体99 kb区域的新生链丰度测定，我们获得了初步数据，表明癌细胞在起始位点的分布上与正常细胞不同。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更长的染色体区域。我们假设这些区域起始位点的频率、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。

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.