2000 — 2008 |
Wang, Yuh-Hwa |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Studies of Cancer Specific Fragile Sites @ Univ of Med/Dent Nj-R W Johnson Med Sch
Instability of chromosomal fragile sites is related directly to many cancers. Three types of fragile sites generated under three different culture conditions: FRA3B, an aphidicolin-inducible fragile site, FRA11B, a folate-sensitive site, and FRA 16B, a distamycin-A-inducible site, are involved in the formation of cancers. The CCG repeating sequence found in the FRA11B site along with four other folate-sensitive sites, has been shown to resist assembly into nucleosomes and the degree of CpG methylation further excludes nucleosome assembly proportionally. These results provide an intriguing model for the nature of fragile sites in which nucleosome exclusion at these sites would create unusual chromatin structure to allow chromosomal rearrangement and viral integration, resulting in fragile site- specific tumorigenesis. Three goals are proposed : (1) The chromatin structure of DNA sequences derived from fragile sites will be investigated in vitro. We showed that the CCG repeating DNA of the FRA11B site excludes nucleosome. We will employ the same techniques for two other different groups of fragile sites, the FRA3B and FRA16B. Further, nucleosome arrays formed over large DNAs of three fragile sites will be characterized by nuclease probing and electron microscopy to determine the stability and physical structure of nucleosomes formed over these sites. (2) The in vivo chromatin structure of these three fragile sites will be examined. Nucleosome position will be mapped over fragile site DNAs in cell lines from fragile site-expressing individuals or an EBV episomal system. The effect of the fragile site-inducing chemicals on the nucleosome pattern will also be examined. (3) The location of fragile sites in higher-order chromatin domains will be explored. Various cell lines will be used to map the location of each fragile site and its adjacent region relative to MARs/SARs. This association will be further investigated for its cell-cycle dependency, the nature of the interaction, and the involvement of topoisomerase II cleavage sites. These experiments will contribute information about the nature of these fragile sites and their role in the formation of cancer, and also address some fundamental biological questions, such as determinants of chromatin structure.
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2006 — 2018 |
Wang, Yuh-Hwa |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of Fragile Sites in Ret/Ptc Rearrangement
? DESCRIPTION (provided by applicant): Chromosomal rearrangements are a common genomic abnormality found in tumor cells, which can alter the normal function of the genes involved, thus contributing to cancer development. Despite their prevalence, much remains unknown about the mechanisms that form translocations. Several commonly occurring chromosomal rearrangements, RET/PTC, AKAP9/BRAF, STRN/ALK, ETV6/NTRK3 and several NTRK1 gene fusions, contribute to the development of papillary thyroid carcinomas (PTC), a major type of thyroid cancer. In the US general population, thyroid cancer is the fastest increasing type of cancer. Its incidence has increased 4 to 7-fold in US Air Force active-duty personnel compared to the general population, even though overall cancers are less frequent in military personnel. We have demonstrated for the first time that DNA breaks at chromosomal fragile sites participate directly in the generation of oncogenic RET/PTC1 rearrangements in human thyroid cells. Fragile sites are sensitive to a range of chemicals, and have been identified in the regions of deletions and chromosomal rearrangements. All partner genes participating in PTC rearrangements are located in known fragile sites. Several environmental exposures, which are daily encountered by the general population and/or intensified in military personnel, such as benzene (in explosives, jet and automobile exhausts, and napalm) and diethylnitrosamine (in cigarette smoke and pesticides), also induce fragile site breakage. Many of them, including benzene, show a positive association with the risk of thyroid cancer. We hypothesize that these agents alone or in combination contribute to the increased incidence of thyroid cancer. In this proposal, we will investigate whether environmental exposures generate RET fragility, leading to RET/PTC rearrangements in human thyroid cells by examining a variety of environmental and therapeutic agents known to induce fragile sites, and will expand this study to other PTC-specific rearrangements as well. Next, we will identify the mechanism of fragile site-mediated RET/PTC rearrangements in response to these environmental exposures, by determining whether the formation of secondary structures during DNA replication contribute to RET gene instability in the formation of RET/PTC rearrangements. Then, to work towards clinical application of DNA fragility to a DNA diagnostic test, we will test whether breakage of RET and other rearrangement-participating genes in normal cells of PTC patients with rearrangements is higher than that in normal individuals, as a means to evaluate individual susceptibility to PTC. This proposal will have significant impact on our understanding of the direct role of environmental factors in the RET/PTC and other rearrangements of thyroid cancers. These mechanistic studies will provide new knowledge to better understand fragile site breakage and its role in sporadic cancer initiation. Further, using data on preferential breakage properties of rearrangement-participating genes to create a DNA test can potentially be extended to other cancers caused by fragile site-mediated rearrangements.
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2013 — 2021 |
Wang, Yuh-Hwa |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genome-Wide Dna Secondary Structure Analysis to Investigate Dna Fragility
Project Summary/Abstract: Both physiological DNA breaks occurring during DNA metabolic processes and pathological DNA breaks responding to a wide range of stresses, contribute to the outcome of human genome instability. DNA fragility generated by alternative DNA secondary structures is a known cause of many human diseases, and also occurs in normal DNA processes. Formation of these structures can arise from single-stranded DNA when the DNA duplex is unwound during DNA processes such as replication and transcription, and thus can be affected by cellular activities, nucleotide sequences, and chemical exposures. Here we will examine if DNA regions having potential to form stable secondary structures when unwound during cell processes, can serve as signals for topoisomerase II (TOP2) to recognize and cleave, and lead to the removal of the excessive supercoiling. We have carried out a computational evaluation of the entire available human genome sequence for optimal ability to fold single-stranded sequences into multiple-hairpin structures, and identified sites of highly stable DNA secondary structures throughout the genome. We will measure TOP2-mediated DNA breaks at these sites upon changes in DNA supercoiling from cell activities, then analyze TOP2-cleaved sites to identify structural features, and examine if DNA secondary structures influence the removal of TOP2 cleavage complexes. TOP2-mediated breaks are also often associated with pathological damage due to the use of TOP2 inhibitors as anticancer drugs. Many DNA secondary structure-rich and fragile regions are located within cancer- specific translocation-participating gene regions, including acute myeloid leukemia (AML)-rearranged regions. We will determine whether DNA fragility at these regions can serve as a biomarker for assessing the potential development of cancer-causing rearrangements. We will first test if DNA fragility at gene regions of AML rearrangements is sensitive to various chemotherapeutic agents, and if this sensitivity leads to the formation of AML rearrangements in human cells. Then, to test if this sensitivity can predict the rearrangement formation in patients, we will examine DNA breakage at these regions in normal cells of AML patients with the AML rearrangements, compared to that of normal individuals, as a means to evaluate individual susceptibility to AML. These experiments will facilitate the clinical application of using DNA fragility as a biomarker. With personalized medicine in mind, we will evaluate the effect of naturally occurring sequence variants on the fragility of the break-prone and AML translocation-participating gene regions, to further identify structure characteristics contributing to DNA fragility, and to reveal an unexploited consequence of non-coding variants. Our preliminary results suggest that sequence variants can influence DNA break frequency of the region by changing the extent or the type of secondary structure forming ability. This proposal will elucidate the mechanistic and functional features of DNA structure-driven fragility and provide a foundation for future clinical use of fragile site breakage in disease diagnostics.
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