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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

基本信息

批准号：
7965305
负责人：
VLADIMIR LARIONOV
金额：
$ 149.97万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7965305
关键词：
Adopted Affect Affinity Aneuploidy Antigens Back Binding Brothers CD4 Positive T Lymphocytes CD8B1 gene Cancer Patient Cell Nucleus Cells Centromere Chickens Chimeric Proteins Chinese Hamster Ovary Cell Chromatin Chromatin Structure Chromosome Segregation Chromosome Structures Chromosome Transfer Chromosomes Chromosomes, Artificial, Human Code Colcemid Color Complement Complex DNA Dendritic Cells Disease Dissection Elements Epigenetic Process Epitopes Equilibrium Evolution Family Frequencies Future Gene Deletion Gene Duplication Gene Expression Genes Genetic Genetic Recombination Genetic Variation Genome Genomic Segment Genomics Germ Lines Goals HLA-A2 Antigen Hamsters Hematopoietic Heterochromatin Histocompatibility Antigens Class I Human Human Chromosomes Human Development Human Genome Immunization In Vitro Individual Inherited Interphase Kinetochores Knowledge Laboratories Lead Left Length Link Malignant Neoplasms Malignant neoplasm of prostate Malignant neoplasm of testis Mammalian Chromosomes Maps Mediating Melanoma Cell Methods Mitosis Modification Molecular Mouse Cell Line Mutation Nucleic Acid Sequence Homology PRC1 Protein Patients Pattern Play Pluripotent Stem Cells Polycomb Polyploidy Predisposition Process Proteins Research Role Saccharomycetales Satellite DNA Sequence Analysis Specificity Stretching Structure Susceptibility Gene System T-Lymphocyte Techniques Tetanus Helper Peptide Tetracyclines Therapeutic Therapeutic Studies Transcription Repressor/Corepressor Transgenes Universities Work anti-cancer therapeutic base cancer cell cell type gene delivery system gene therapy genetic linkage homologous recombination human disease in vivo killings novel novel strategies prevent response sperm cell success tumor progression

项目摘要

Genetic linkage studies implicate a gene or genes at Xq27 in hereditary prostate cancer susceptibility (HPCX). The corresponding region spans 750 kb and includes five SPANX genes (SPANX-A1, -A2, -B, -C, and D), 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 gene region. Due to their recent amplification, both SPANX coding and flanking sequences in the SDs are nearly identical throughout the SPANX-A/D cluster, which complicates sequence analysis of these genes by PCR-based methods in the search for mutations. However, we recently succeeded in performing such an analysis of the Xq27-linked SPANX genes from prostate cancer patients, using the transformation-associated recombination (TAR) technique, which makes it possible to directly isolate large genomic segments from complex genomes. This analysis revealed frequent gene deletion/duplication and homology-based sequence transfers involving SPANX genes at Xq27, suggesting that SD-mediated homologous recombination involving the SPANX genes might lead to increased genetic instability and possibly to a higher level of genetic diversity in SPANX genes in germ lines. In our recent work, a search for inversions in the SPANX region was undertaken using three-color interphase FISH. This analysis was performed with prostate cancer patients and unaffected controls from several extensively studied Finish and JHU families with X-linked hereditary prostate cancer. An inversion in the region including SPANX-B, SPANX-C, SPANX-A1, SPANX-A2, and the LDOC1 gene was detected in affected but not in unaffected brothers. Our results are consistent with the hypothesis that this inversion is causally-related to susceptibility to prostate cancer in the affected patients. However, the molecular basis of such a causal relationship is not yet known. Therefore, future work will focus on mapping of breakpoint(s) of the inversion and on the analysis how the inversion alters expression of genes at/near breakpoints which could lead to malignancy. We hypothesize that X-linked predisposition to prostate cancer is caused by activation of expression of SPANX proteins that may play a role in cancer progression in multiple human cell types. Because of their expression pattern, SPANX genes are among the few CT antigens that are considered to be potential targets for anticancer therapeutics. Thus, we initiated a project to assess the potential of SPANX antigens as anticancer therapeutics. Our studies demonstrated that SPANX-B-specific T cell precursors are present in cancer-free individuals. Furthermore, after in vitro immunization with SPANX-B -treated dendritic cells (DCs), the precursor T cells give rise to helper CD4+ T cells and cytolytic CD8+ T cells (i.e. CTLs). Importantly, human melanoma cells express SPANX-B at a very high level and process and present at least two immunodominant HLA-A2 restricted epitopes of SPANX-B on MHC class I molecules, which activate CTLmediated killing of melanoma cells. Taken together, these studies indicated that SPANX proteins could be novel targets of anticancer therapeutics, which may be particularly relevant to prostate cancer. The role of chromatin structure in kinetochore function has been studied intensively but remains poorly understood. However we have generated a HAC in human HT1080 cells with a conditional centromere, which we expect to be instrumental in resolving this question. This HAC is the first chromosome outside of budding yeast with a regulated centromere. The HAC includes approximately 6,000 copies of the tetracycline operator (tet-O) sequence. Because tet-O is bound with very high affinity and specificity by the tet repressor (tet-R), the array of tet-O sequences in this HAC can be targeted efficiently with tet-R fusion proteins. The power of this system is that it allows the specific manipulation of the protein complement of a single kinetochore in vivo, while leaving all other kinetochores unperturbed. This system has been used to target chromatin modifying proteins into the HAC and to demonstrate that a balance between open and condensed chromatin is critical for kinetochore function. The strongest effect on the synthetic kinetochore was observed after targeting of a transcriptional repressor (the tTS) inducing HP1alpha-repressive chromatin. Our recent collaborative studies with William Earnshaws laboratory at Edinburgh University showed that not all types of repressive chromatin are inconsistent with kinetochore function. Thus, establishment of PRC1 (Polycomb complex) repressive chromatin within the centromere has no effect on kinetochore structure or function. We also have shown that the disruption of kinetochore structure by a transcriptional repressor reflects a hierarchical disassembly of kinetochore components. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore. To extend studies of epigenetic modification of this HAC and to adopt it for expression of full length human genes, a Lox-P - 5 HPRT - Hyg - TK cassette was inserted into the HAC by homologous recombination in chicken DT40 cells. Clones with the lox-P cassette were transferred back into hamster CHO cells deficient in HPRT via microcell-mediated chromosome transfer (MMCT). We demonstrated that 20 kb transgenes can be efficiently and accurately inserted into the retrofitted lox-P HAC in CHO cells and the transgenes are stably expressed. Because CHO cells form microcells at high frequency in response to colcemid, the HAC can be easily moved from donor CHO cells into other recipient human or mouse cell lines via MMCT. The development of this HAC system is important, because of its potential future use in gene therapy research. This system may also be useful to generate pluripotent stem cells and for studies of chromatin dynamics in a functional human centromere. Alpha-satellite DNA, which can promote kinetochore formation in human cells, is the only human centromeric DNA with a known function. Non-alphoid DNA repeats have also been identified in human centromeres. However, the precise sequence and order of non-alphoid pericentromeric repetitive DNA in human chromosomes remains mostly undetermined. Because the spreading of heterochromatin is an active process, the multi-domain organization of the human centromere suggests the presence of specific elements demarcating the different chromatin states. So far, very few elements with such activity, known as chromatin barriers, have been described. In our recent work, we have shown that pericentromeric regions of all human chromosomes contain gamma-satellite DNA. Using a new experimental system, we demonstrated that human pericentromeric gamma-satellite DNA functions as a heterochromatin arresting element. In hematopoietic cells, the anti-silencing and heterochromatin-arresting activities of gamma-satellite DNA require the binding of Ikaros, a protein that regulates hematopoeiesis. The ability of gamma-satellite DNA arrays to promote open chromatin structures in non-hematopoietic cells (i.e. where Ikaros is not expressed) suggests that another protein(s) may bind to these repeats in other cell types. We propose that the primary role of human gamma-satellite DNA may be to prevent the spreading of pericentric heterochromatin into chromosoma [summary truncated at 7800 characters]