Study of hereditary prostate cancer and human artificial chromosomes

遗传性前列腺癌与人类人工染色体的研究

• 批准号: 8763097
• 负责人: VLADIMIR LARIONOV
• 金额: $ 160.52万
• 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/8763097
• 关键词: Affect; Aneuploidy; Annual Reports; Antineoplastic Agents; BRCA1 gene; Binding; Biological Assay; Cancer-Predisposing Gene; Cells; Centromere; Chromatin; Chromatin Structure; Chromosomal Instability; Chromosome Segregation; Chromosome Transfer; Chromosomes; Chromosomes, Artificial, Human; Cloning; DNA; Data Linkages; Development; Disease; Doxycycline; Elements; Engineering; Epigenetic Process; Evolution; Exclusion; Family; Flow Cytometry; Gene Cluster; Gene Delivery; Gene Duplication; Gene Expression; Gene Silencing; Gene Transfer; Gene Transfer Techniques; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genetic Variation; Genets; Genomic Segment; Genomics; Goals; Heterochromatin; Human; Human Genome; Immunoglobulin Variable Region; Inherited; Insertional Mutagenesis; International; Kinetochores; Knowledge; Lead; Length; Light; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mitosis; Modification; Molecular; Mutate; Mutation; NBS1 gene; Nijmegen Breakage Syndrome; Pharmaceutical Preparations; Phenotype; Plague; Polyploidy; Population; Preclinical Drug Evaluation; Predisposition; Process; Proliferating; Prostate; Publications; Regenerative Medicine; Research; Retroviral Vector; Role; Satellite DNA; Site; Structure; Susceptibility Gene; System; Techniques; Therapeutic; Transfection; Transfer RNA; Transgenes; Viral Vector; Von Hippel-Lindau Syndrome; Work; Yeasts; base; cancer cell; cancer genetics; cancer genome; chromosome loss; expression vector; functional genomics; gene correction; gene delivery system; gene function; gene therapy; human disease; in vivo; interest; novel; novel therapeutics; prevent; response; segregation; success; tool; transgene expression; tumor growth; tumor progression; vector

Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. However, the susceptibility gene in this region has not yet been identified. The strongest linkage peak of prostate cancer overlies a variable region of 750-kb at Xq27 that is enriched by large SDs carrying a cluster of SPANX genes. No mutations were detected in SPANX genes. It suggests that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. Since the last Annual Report, we have concentrated on the analysis of the 750-kb region in several X-linked families with the strongest linkage to HPCX1. Direct isolation of a set of overlapping genomic segments carrying SDs that completely covers the 750-kb region by in vivo recombination in yeast (a TAR cloning technique) were used to perform a mutational analysis of this region. The subsequent analysis of isolated fragments excluded the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. When our work was completed and submitted for publication (Genes, Chromosomes & Cancer J 2012, 51:933-948), the International Consortium for Prostate Cancer Genetics re-evaluated previous linkage data and concluded that there is no strong evidence for a major prostate cancer susceptibility gene located at Xq27-28 (Bailey-Wilson JE et al; International Consortium for Prostate Cancer Genetics. Analysis of Xq27-28 linkage in the international consortium for prostate cancer genetics (ICPCG) families BMC Med Genet 2012, 13: 46.) After exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy, the studies related to this sub-project have been completed in our lab and we have focused on other sub-projects linked with Human Artificial Chromosomes (HACs). (HACs) assembled from alphoid DNA arrays represent a novel episomal gene delivery vector for functional genomics and gene therapy. HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. We previously constructed a synthetic HAC (tetO-HAC) that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This HAC is the most advanced vector for expression of full-length genes and entire loci and for correction of genetic deficiencies in human cells. The tetO-HAC was also used as a unique system to study a role of epigenetic modifications in the human kinetochore function. The broad use of the tetO-HAC requires the knowledge of its structural organization. During the past year, we completed physical characterization of a megabase- size synthetic alphoid DNA array in the HAC that has been formed from a synthetic tetO-array. Our results provide a tool to control structural integrity of tetO-HAC during gene loading and HAC transfer into different host cells. They also shed light on a mechanism for de novo HAC formation in human cells. We previously demonstrated the utility of the synthetic HAC for delivery of full size genes and correction of genetic deficiencies in human cells. Specifically genomic copies of two cancer-associated genes, VHL mutated in von Hippel Lindau syndrome (VHL) and NBS1 mutated in Nijmegen breakage syndrome (NBS) were successfully transferred into gene deficient cells. We have also shown that phenotypes arising from stable gene expression from the HAC can be reversed when cells are cured of the HAC by inactivating its kinetochore in proliferating cell populations. During the past year, several other human genes were loaded into the HAC for gene transfer/gene expression studies, including BRCA1 and mtTOP1 gene previously discovered in LMP. In the tetO-HAC with a conditional centromere, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While this domain is permissivefor transcription, there are no studies on a long-term transgene expression within centrochromatin. In our recent study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA and tDNA, on the expression of an EGFP transgene loaded into the tetO-HAC vector. Unexpectedly, insulator function was essential for stable expression of the transgene in centrochromatin that represents open chromatin structure. A tDNA insulator (discovered in our lab) consisting of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements, such as gamma-satellite DNA and tDNA, that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. The tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, such as the tTS, to its centromeric tetO sequences. The opportunity to induce HAC loss provides a unique control for phenotypes induced by genes loaded into the alphoidtetO-HAC. However, inactivation of the HAC kinetochore requires transfection of cells by a retrovirus vector to achieve a high level of tTS expression, a step that potentially may lead to insertional mutagenesis. In our recent work, we described an approach to re-engineering the alphoidtetO-HAC vector that allows verification of phenotypic changes attributed to expression of genes from the HAC without transfecting exogenous chromatin modulators. In the new HAC vector, a tTS cassette is inserted into a gene-loading site along with a gene of interest. In the absence of doxycycline, expression of the tTS generates a self-regulating fluctuating heterochromatin on the alphoidtetO-HAC that induces a fast and strong silencing of the genes on the HAC without significant effect on HAC segregation. This silencing is reversible, and therefore expression of the HAC-encoded genes can be readily recovered by adding doxycycline. The newly modified alphoidtetO-HAC-based system has the potential for multiple applications in gene function studies. We have also applied our HAC system for screening of drugs affecting chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth. Our goal was to develop a new quantitative assay for identification of drugs that elevate CIN in cancer cells. For this purpose, the EGFP transgene was loaded into the tetO-HAC using Cre-loxP recombination. The presence of EGFP allows measuring of the HAC loss by flow cytometry. We have successfully used this assay to measure increased mis-segregation of chromosomes in response to different anticancer drugs. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.

几项连锁研究提供了 Xq27-q28 处遗传性前列腺癌基因座 HPCX1 存在的证据。然而，该区域的易感基因尚未确定。前列腺癌的最强连锁峰覆盖 Xq27 处 750 kb 的可变区，该可变区由携带 SPANX 基因簇的大 SD 富集。 SPANX 基因中未检测到突变。这表明前列腺癌的易感性可能是由SD之间的重组相互作用引起的基因组疾病。自上一份年度报告以来，我们重点分析了与 HPCX1 连锁最强的几个 X 连锁家族中的 750 kb 区域。通过酵母体内重组（TAR 克隆技术）直接分离一组携带 SD 的重叠基因组片段，完全覆盖 750 kb 区域，用于对该区域进行突变分析。随后对分离片段的分析排除了 Xq27 的 750 kb 遗传不稳定区域作为前列腺恶性肿瘤的候选位点。当我们的工作完成并提交发表时（Genes, Chromosomes & Cancer J 2012, 51:933-948），国际前列腺癌遗传学联盟重新评估了之前的连锁数据，并得出结论认为，没有强有力的证据表明主要前列腺癌位于 Xq27-28 的癌症易感基因（Bailey-Wilson JE 等人；国际前列腺癌遗传学联盟。分析国际前列腺癌遗传学联盟 (ICPCG) 家族中的 Xq27-28 连锁 BMC Med Genet 2012, 13: 46。）在排除 Xq27 上的 750-kb 遗传不稳定区域作为前列腺恶性肿瘤的候选位点后，相关研究该子项目已在我们的实验室完成，我们重点关注与人类人工染色体（HAC）相关的其他子项目。由 alphoid DNA 阵列组装而成的 HAC（HAC）代表了一种用于功能基因组学和基因治疗的新型附加型基因递送载体。 HAC 避免了有限的克隆能力、缺乏拷贝数控制以及由于整合到宿主染色体中而困扰病毒载体的插入突变。我们之前构建了一种合成 HAC (tetO-HAC)，可以通过使其条件动粒失活来轻松地将其从细胞群中消除。该 HAC 是最先进的载体，用于表达全长基因和整个基因座以及纠正人类细胞中的遗传缺陷。 tetO-HAC 还被用作研究表观遗传修饰在人类动粒功能中的作用的独特系统。 tetO-HAC 的广泛使用需要了解其结构组织。在过去的一年里，我们完成了 HAC 中由合成 tetO 阵列形成的兆碱基大小的合成 alphoid DNA 阵列的物理表征。我们的结果提供了一种在基因加载和HAC转移到不同宿主细胞期间控制tetO-HAC结构完整性的工具。他们还揭示了人类细胞中 HAC 从头形成的机制。我们之前展示了合成 HAC 在传递全尺寸基因和纠正人类细胞遗传缺陷方面的实用性。具体而言，两种癌症相关基因的基因组拷贝，即冯希佩尔林道综合征（VHL）中的 VHL 突变和奈梅亨断裂综合征（NBS）中的 NBS1 突变，被成功转移到基因缺陷细胞中。我们还表明，当通过灭活增殖细胞群中的着丝粒来治愈细胞中的 HAC 时，HAC 稳定基因表达所产生的表型可以逆转。在过去的一年中，其他几个人类基因被加载到HAC中用于基因转移/基因表达研究，包括先前在LMP中发现的BRCA1和mtTOP1基因。在具有条件着丝粒的 tetO-HAC 中，基因加载位点被插入对着丝粒组装和维护至关重要的中心染色质结构域中。虽然该结构域允许转录，但尚无关于中心染色质内长期转基因表达的研究。在我们最近的研究中，我们比较了三种染色质绝缘体（cHS4、γ-卫星 DNA 和 tDNA）对加载到 tetO-HAC 载体中的 EGFP 转基因表达的影响。出乎意料的是，绝缘子功能对于代表开放染色质结构的中心染色质中转基因的稳定表达至关重要。由两个功能性 tRNA 基因拷贝组成的 tDNA 绝缘子（我们实验室发现的）表现出最高的屏障活性。我们推断，接近中心染色质并不能保护缺乏染色质绝缘体的基因免受表观遗传沉默。因此，防止中心染色质基因沉默的屏障元件，例如 γ-卫星 DNA 和 tDNA，将有助于优化使用 HAC 载体的转基因。 tetO-HAC 比其他 HAC 载体具有优势，因为它可以通过染色质修饰剂（例如 tTS）与其着丝粒 tetO 序列的结合灭活 HAC 动粒，从而轻松地从细胞中消除。诱导 HAC 损失的机会为加载到 alphoidtetO-HAC 中的基因诱导的表型提供了独特的控制。然而，HAC 着丝粒的失活需要通过逆转录病毒载体转染细胞以实现高水平的 tTS 表达，这一步骤可能会导致插入突变。在我们最近的工作中，我们描述了一种重新设计 alphoidtetO-HAC 载体的方法，该方法允许验证 HAC 基因表达引起的表型变化，而无需转染外源染色质调节剂。在新的 HAC 载体中，tTS 盒与感兴趣的基因一起插入基因加载位点。在没有强力霉素的情况下，tTS 的表达会在 alphoidtetO-HAC 上产生自我调节的波动异染色质，从而诱导 HAC 上基因的快速而强烈的沉默，而不会对 HAC 分离产生显着影响。这种沉默是可逆的，因此通过添加强力霉素可以轻松恢复 HAC 编码基因的表达。新改良的基于 alphoidtetO-HAC 的系统具有在基因功能研究中多种应用的潜力。我们还应用 HAC 系统来筛选影响染色体不稳定性 (CIN) 的药物。虽然 CIN 可以作为癌症基因组进化和肿瘤进展的驱动因素，但最近的研究结果表明存在一个阈值水平，超过该阈值水平，CIN 就会成为肿瘤生长的障碍。我们的目标是开发一种新的定量检测方法来鉴定提高癌细胞中 CIN 的药物。为此，使用 Cre-loxP 重组将 EGFP 转基因加载到 tetO-HAC 中。 EGFP 的存在允许通过流式细胞术测量 HAC 损失。我们已经成功地使用这种测定法来测量因不同抗癌药物而增加的染色体错误分离。增加染色体错误分离率的新化合物的鉴定应该会加快针对癌细胞中 CIN 表型的新治疗策略的开发。