Study of hereditary prostate cancer and human artificial chromosomes

遗传连锁研究表明Xq27-q28上的一个或多个基因与遗传性前列腺癌易感性(HPCX1)有关。相应的区域大小为750kb，包括5个SPANX基因，这些基因编码在精子核和各种癌细胞中表达的蛋白质。每个SPANX基因都嵌入到最近形成的长达100kb的片段重复(SD)中，导致该区域长段重复DNA的广泛丰富。我们先前的分析揭示了频繁的基因缺失、重复和基于同源的序列转移，涉及到Xq27上的span x基因，这表明SD介导的该区域的同源重组可能是遗传性前列腺癌的易感性来源。在过去的一年里，我们继续对前列腺癌易感家系的候选区域进行突变分析。十二烷基硫酸酯的大小和它们的序列相似性使使用标准方法检查该区域的可能重排变得困难。为了解决这一问题，通过在酵母中体内重组直接分离一组基因组片段(一种焦油克隆技术)，对X连锁家庭的750kb区域进行了突变分析。通过分析一组重叠的TAR克隆，我们没有检测到该区域内的疾病特异性重排。CGH和Southern-Blot杂交分析的结果与TAR克隆数据一致。因此，我们的数据不支持Xq27遗传性前列腺癌是一种基因组疾病？的假设。由这一地区的不稳定造成的。此外，还进行了转录组和计算分析，以寻找Xq27-q28区域内的新的非注释基因，这可能与前列腺癌的遗传易感性有关。我们鉴定了两个候选基因，其中一个是新基因SPANX-L，它代表了SPANX基因家族中一个高度分化的成员，以及先前描述的在正常和恶性前列腺细胞中高水平表达的CDR1基因，并定位了SPANX基因簇上游210kb的区域。在这些基因中没有发现疾病特异性的改变。综上所述，我们的结果排除了Xq27上750 kb的遗传不稳定区域作为前列腺癌的候选基因。相邻区域似乎是最有可能识别难以捉摸的HPCX1基因座的候选基因。由Alphid DNA阵列形成的人类人工染色体(HAC)代表着一种新的用于功能基因组学和基因治疗的附体基因传递载体。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困扰病毒载体的宿主染色体而导致的插入突变。我们先前构建了一种人工合成的HAC(AlPhoidteO-HAC)，它可以通过灭活其条件动粒而很容易地从细胞群中消除。这种HAC是表达全长基因和整个基因座以及纠正人类细胞遗传缺陷的最先进的载体。该系统也被用来研究表观遗传修饰在人类动粒功能中的作用。α-HAc的广泛使用需要了解其结构组织。在过去的一年里，我们在HAC中完成了一个百万碱基大小的合成α-DNA阵列的物理表征，该阵列是由合成的α-O-阵列形成的。我们的分析表明，HAC的形成是由多个50kb的输入DNA拷贝组装而成的，其中很大一部分在组装前进行了重排。大小从25kb到150kb的合成α-DNA阵列的串联和反向重复序列被组织为1.1Mb的连续巨型阵列序列。我们的结果提供了一个工具来控制基因装载和HAC转移到不同宿主细胞过程中的结构完整性，并揭示了人类细胞从头开始形成HAC的机制。我们之前证明了人工合成的HAC用于传递完整大小的基因和纠正人类细胞中的遗传缺陷。将von Hippel Lindau综合征(VHL)突变的VHL和Nijmegen断裂综合征(NBS)突变的NBS1两个肿瘤相关基因的基因组拷贝成功地转移到基因缺陷细胞中。我们还表明，当细胞通过灭活增殖细胞群体中的动粒而治愈HAC时，由HAC稳定基因表达产生的表型可以逆转。在过去的一年里，其他几个人类基因被加载到HAC中进行基因转移/基因表达研究，包括以前在LMP中发现的mtTOP1基因。我们还开始将我们的HAC系统应用于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，但最近的发现指出，CIN存在一个阈值水平，超过这个阈值，CIN就会成为肿瘤生长的障碍。我们的目标是开发一种新的检测方法，用于鉴定提高癌细胞中CIN水平的药物。为此，利用Cre-loxP重组技术将绿色荧光蛋白转基因载体装载到α-HAc中。绿色荧光蛋白的存在使得用流式细胞术测量HAc损失成为可能。我们已经成功地使用这个系统测量了在HDAC抑制剂SAHA和有丝分裂抑制剂紫杉醇的反应下，EGFP-HAC的错误分离增加。该系统也适用于发现控制人类细胞染色体分离的新基因。一种高通量的基于荧光的分析将被开发为一种筛选工具，用于未来鉴定新的治疗药物，这些药物可以促进癌细胞中CIN的增加，从而促进致命的非整倍体。

Genetic linkage studies implicate a gene or genes at Xq27-q28 in hereditary prostate cancer susceptibility (HPCX1). The corresponding region spans 750 kb and includes five SPANX genes, which encode proteins that are expressed in sperm nuclei and a variety of cancer cells. Each SPANX gene is embedded in a recently formed segmental duplication (SD) up to 100 kb in size, resulting in extensive enrichment in long stretches of repeated DNA in this region. Our previous analysis revealed frequent gene deletion, duplication and homology-based sequence transfers involving SPANX genes at Xq27, suggesting that SD-mediated homologous recombination in this region might be a source for predisposition to hereditary prostate cancer. During the past year, we have continued our work on mutational analysis of the candidate region in families with the predisposition to prostate cancer. The large size of the SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region by analyzing a set of overlapping TAR clones. The results of CGH and Southern-blot hybridization analyses were in agreement with the TAR cloning data. Thus, our data do not support the hypothesis that hereditary prostate cancer at Xq27 is a ?genomic disorder? caused by instability of this region. In addition, transcriptome and computational analyses were performed to search for new non-annotated genes within the Xq27-q28 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANX-L that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. To summarize, our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.Human artificial chromosomes (HACs) formed 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 (alphoidtetO-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 alphoidtetO-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 alphoidtetO-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 alphoidtetO-array. Our analysis showed that the HAC formation resulted from the assembly of multiple 50 kb input DNA copies, a significant part of which was rearranged before assembling. Both tandem and inverted repeats of synthetic alphoid DNA arrays with the size from 25 to 150 kb are organized as a 1.1 Mb continuous mega-array sequence. Our results provide a tool to control structural integrity of alphoidtetO-HAC during gene loading and HAC transfer into different host cells and 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 mtTOP1 gene previously discovered in LMP. We have also initiated work to apply 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 is to develop a new assay for identification of drugs that elevate CIN in cancer cells. For this purpose, the EGFP transgene was loaded into the alphoidtetO-HAC using Cre-loxP recombination. The presence of EGFP allows measuring of the HAC loss by flow cytometry. We have successfully used this system to measure increased mis-segregation of a EGFP-HAC in response to an HDAC inhibitor, SAHA and to a mitotic inhibitor, taxol. The system is also applicable to identify new genes controlling chromosome segregation in human cells. A high throughput fluorescence based assay will be developed as a screening tool for future identification of novel therapeutic agents that drive up CIN in cancer cells to promote lethal aneuploidy.