Human Artificial Chromosomes for Cancer Research and Functional Genomics

用于癌症研究和功能基因组学的人类人工染色体

• 批准号: 9556281
• 负责人: VLADIMIR LARIONOV
• 金额: $ 184.59万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556281
• 关键词: Aneuploidy; Antineoplastic Agents; Biological Assay; Biotechnology; Cell Proliferation; Cell division; Cells; Cellular Stress; Centromere; Characteristics; Chromatin; Chromosomal Instability; Chromosome Segregation; Chromosome Transfer; Chromosomes; Chromosomes, Artificial, Human; Controlled Study; DNA; DNA biosynthesis; Development; Disease; Epigenetic Process; Failure; Flow Cytometry; Fluorescence; Frequencies; Generations; Genes; Genetic Recombination; Genetic Transcription; Genetic study; Genomics; Goals; Heterochromatin; Human; Human Genome; Inherited; Integrase; Kinetochores; Knowledge; Lead; Libraries; Location; Maintenance; Malignant Neoplasms; Measurement; Measures; Mediating; Microtubule stabilizing agent; Mitosis; Mitotic; Molecular; Nucleolar Organizer Region; Paclitaxel; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Polyploidy; Process; Protein Kinase; Recombinant DNA; Repressor Proteins; Research; Role; Site; Small Interfering RNA; Structure; TOP1 gene; Tetanus Helper Peptide; Therapeutic; Topoisomerase Inhibitors; Transgenes; Work; anticancer research; base; biological adaptation to stress; cancer cell; cancer type; centromere protein A; chromosome loss; experimental study; functional genomics; gene therapy; histone modification; novel; novel therapeutic intervention; prevent; response; screening; small molecule libraries; targeted cancer therapy; transmission process; tumor growth; vector

Human Artificial Chromosomes (HACs) assembled from alphoid DNA arrays represent novel vectors that have a great potential for the study assembly and maintenance of human kinetochore as well as for gene therapy, screening of anticancer drugs and biotechnology. We previously constructed a synthetic HAC (tetO-HAC) allowing tethering of its kinetochore by different chromatin modifies fused with the tet-repressor protein. This HAC was successfully used to clarify role of different types of chromatin in functioning of the kinetochore. During past year, the same approach based on tethering of the tetO-HAC kinetochore by different fusions revealed that mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres. In another study, we demonstrated that human centromere resists silencing mediated by H3K27me3/K9me3. In separate experiments, a platform with multi-integrase recombination sites has been inserted into tetO-HAC and has been successfully used for a gene assembly in the HAC. Work is in progress to assemble synthetic nucleolar organizer region (NOR) in the HAC using TAR-isolated human rDNA units. Such a HAC module that is transferable into any type of human cells via micro-cell mediated chromosome transfer (MMCT) will be used to investigate the requirements for nucleolar location of rDNA repeats and effect of copy number of rDNA units on cell proliferation and stress response. We have also applied our tetO-HAC for measuring chromosome instability (CIN) in human cells. Whole-chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a characteristic feature of most types of cancer, thus distinguishing them from their normal counterparts. Although CIN is generally considered a driver of tumor growth, a threshold level exists whereby further increase in CIN frequency becomes a barrier against tumor growth and therefore can be exploited therapeutically. However, drugs known to increase CIN beyond this therapeutic threshold are currently few in number. In our previous work, we have developed a new quantitative assay for measuring CIN based on the use of a non-essential HAC carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows measurement of HAC loss rate in response to drug treatment by routine flow cytometry. We used this assay to rank more than 100 anticancer drugs on their effect on HAC loss. The strongest effect was observed for, taxol (microtubule-stabilizing agent), LMP744 (inhibitor of topoisomerase TOP1, developed in our branch). We also demonstrated the utility of the assay to detect increase of CIN after siRNA depletion of known genes controlling chromosome transmission. In our recent work, we modified EGFP-HAC and converted the original assay into high-throughput CIN screen of chemical libraries and siRNA libraries of human genes. Analysis of siRNAs targeting each of 720 human protein kinase genes revealed 27 CIN genes with no information on their role in chromosome transmission. Each of these new CIN genes may be considered as a new target for cancer therapy.

由阿尔法DNA阵列组装而成的人类人工染色体（Human Artificial chromosome, HACs）是一种新型载体，在研究人类着丝点的组装和维持、基因治疗、抗癌药物筛选和生物技术等方面具有很大的潜力。我们之前构建了一个合成的HAC (tetO-HAC)，允许通过与tet抑制蛋白融合的不同染色质修饰来系住其着丝点。该HAC被成功地用于阐明不同类型的染色质在着丝点功能中的作用。在过去的一年里，基于tetO-HAC着丝点的不同融合的相同方法表明，有丝分裂转录加上包括H3K9ac在内的组蛋白修饰构成了允许CENP-A组装和着丝染色质维持的“表观遗传景观”。H3K4me2是转录所必需的，H3K9ac可以在人类着丝粒上形成屏障，阻止异染色质扩散和着丝点失活。在另一项研究中，我们证明了人类着丝粒抵抗H3K27me3/K9me3介导的沉默。在单独的实验中，一个具有多整合酶重组位点的平台被插入到tetO-HAC中，并成功地用于HAC中的基因组装。利用tar分离的人类rDNA单元在HAC中组装合成核仁组织者区（NOR）的工作正在进行中。该HAC模块可通过微细胞介导的染色体转移（MMCT）转移到任何类型的人类细胞中，将用于研究rDNA重复序列对核仁位置的要求以及rDNA单位拷贝数对细胞增殖和应激反应的影响。我们还应用我们的tetO-HAC来测量人类细胞的染色体不稳定性（CIN）。全染色体不稳定性（CIN）表现为细胞分裂过程中染色体分布不均匀，是大多数类型癌症的特征，从而将其与正常肿瘤区分开来。尽管CIN通常被认为是肿瘤生长的驱动因素，但存在一个阈值水平，使得CIN频率的进一步增加成为肿瘤生长的障碍，因此可以用于治疗。然而，目前已知能使CIN升高超过这一治疗阈值的药物数量很少。在我们之前的工作中，我们开发了一种新的定量检测方法，用于测量CIN，该方法基于使用非必需HAC携带组成型表达EGFP转基因。因此，继承HAC的细胞显示绿色荧光，而缺乏HAC的细胞则没有。这允许通过常规流式细胞术测量药物治疗反应的HAC损失率。我们用这种方法对100多种抗癌药物对HAC损失的影响进行了排名。紫杉醇（微管稳定剂）和LMP744（拓扑异构酶TOP1抑制剂，我们的分支机构开发）的效果最强。我们还证明了该检测在siRNA耗尽控制染色体传递的已知基因后检测CIN增加的效用。在我们最近的工作中，我们对EGFP-HAC进行了改进，并将原来的检测方法转化为人类基因化学文库和siRNA文库的高通量CIN筛选。针对720个人类蛋白激酶基因的sirna分析显示，27个CIN基因在染色体传递中没有作用的信息。这些新的CIN基因中的每一个都可能被认为是癌症治疗的新靶点。