Initiation Patterns of DNA Replication in Cancer Cell Lines

癌细胞系中 DNA 复制的起始模式

• 批准号: 7672501
• 负责人: MANUEL SEVERO VALENZUELA
• 金额: $ 36.63万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-11 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7672501
• 关键词: Abnormal Cell; Address; Autoradiography; Bioinformatics; Biological; Biological Assay; Biological Process; Breast; Breast Cancer Cell; Cancer cell line; Cell Line; Cell Proliferation; Cells; Chromosomes; Chromosomes, Human, Pair 2; Chromosomes, Human, Pair 3; Collaborations; Coupled; Custom; DNA; DNA Microarray Chip; DNA Probes; DNA Sequence; DNA amplification; DNA biosynthesis; Data; Development; Diagnosis; Ensure; Fiber; Frequencies; Gene Expression; Genetic; Goals; Housekeeping; Human; Human Chromosomes; Human Genome; Institution; Laboratories; Length; Location; Malignant Neoplasms; Maps; Microarray Analysis; Minority; Modeling; Normal Cell; Pattern; Phase; Physiological; Positioning Attribute; Principal Investigator; Replication Initiation; Scientist; Screening for cancer; Site; Specificity; Staging; Technology; Testing; Time; Tissues; Training; United States National Institutes of Health; base; cancer cell; cell growth; cell type; chromatin modification; density; flexibility; malignant breast neoplasm; medical schools; next generation; novel; novel strategies; programs; promoter; synergism; tool; tumor

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.

描述（由申请人提供）：人类基因组的复制取决于数千个起始位点的激活，DNA 合成被编程为在这些起始位点开始。然而，尽管该领域取得了巨大进步，但尚不清楚这些位点沿每条染色体的分布情况，也不清楚这种分布是否随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图谱，以推断起始特征，从而使我们能够区分正常细胞和异常生长细胞，从而为我们提供检测异常细胞增殖早期阶段的工具。基于对 2 号染色体 99 kb 区域进行的基于 PCR 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方面与正常细胞不同。在目前的提案中，我们希望利用 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。我们假设癌细胞系中这些区域起始位点的频率、分布和时间激活与正常细胞系不同。 为了检验这一假设，我们的具体目标是：(1) 绘制并比较正常细胞系和乳腺癌细胞系中 3、17 和 20 号染色体的乳腺癌相关区域的起始位点位置。为了实现这一目标，我们计划 (a) 从异步生长的细胞中分离出短的（约 1-1.5 kb）新生 DNA 链； (b) 定量 DNA 平铺阵列上的丰度，该阵列包含 60 聚体探针，覆盖 Chr20q12-13 上的 20 Mb 区域、分别覆盖 Chr17p13 和 Chr17q23 上的两个 4Mb 区域以及 Chr3q26 上的 3 Mb 区域； (c) 比较正常细胞系和癌细胞系的概况。 (2) 评估正常和癌症乳腺细胞系中 3、17 和 20 号染色体癌症相关区域起始位点激活的时间顺序。为此，我们计划同步 G1/G0 期的细胞，并探测上述平铺路径 DNA 微阵列，在进入 S 期后的不同时间点获得短的新生 DNA 链。这些研究将为我们提供有关正常细胞和恶性细胞中 DNA 复制的起始和暂时激活的新信息，这将有助于识别肿瘤特异性起始位点。这些信息还将为诊断和控制异常细胞生长提供新方法。