Abstract Lung cancer is one of the most common cancer death in Taiwan. The dead population of lung cancer are over five thousands per year. High mortality and bad prognosis displayed the severity of the lung cancer. RAPD (Random Amplified Polymorphic DNA), a simple technique for the detection of genomic instability, has been used in this research. We inquire into genetic variation in carcinogenesis, and find out genes association with NSCLC. Three or more than three DNA fragment patterns of normal and lung cancer samples exhibited by RAPD from the seven arbitrary were classified as genomic instability. Four out of seven arbitrary primers have been used for lung cancer RAPD analysis and three of them were newly designed in this investigation. Analysis of genomic instability with these seven random primers in twenty-seven NSCLC patients revealed that 81.48% of NSCLC exhibited genomic instability. The RAPD reproducibilities of primer 6 and primer 7 were the best among the seven primers used in this study. Therefore, the variable DNA fragments of primer 6 and primer 7 in RAPD analysis were subcloned and sequenced for the study of the possible mutated genes in NSCLC. Results showed that DNA gains or losses were found in chromosomes 2, 4, 6, 14 and 22. After bioinformatic searching and alignments with human Genebank, some oncogenes (such as RABL2B, c-fos, n-myc and mas1) and tumor suppressor gene (AR) were found located nearby the locus of these subclones. Genomic instability was investigated in relation with the clinical-pathological features such as age, stage, tumor size, metastasis, differential status, survival days and cancer types. Results, evaluated by the X2 test, were not significant except tumor stage.

Replication fork stalling is a source of potentially tumourigenic genomic instability. The RecQ family helicase, Rqh1, is critical for the prevention of replication fork collapse and the formation of potentially deleterious recombination intermediates following fork stalling. Previous work in our lab with Schizosaccharomyces pombe (fission yeast) has shown that rqh10/rqh10 diploids are inherently unstable and show rapid reversion to the haploid state. The current work exploits this characteristic of fission yeast rqh10 mutants in a screen for genes that normally promote replication associated genomic instability. The rqh10rad30 mutant strains employed in this work incorporate the checkpoint deficiency caused by a lack of Rad3, so as to exacerbate the genomically unstable nature of this model. The current work describes the lithium acetate transformation based random mutagenesis by non-homologous integration of the ura4+ selectable marker into the rqh10rad30 fission yeast strains. This random mutagenesis generated extensive (24,500 – 50,000) mutant libraries. The quality of the libraries was assessed by can1 mutant assay, confirming an adequately extensive mutagenesis for the proposed screen. The process to be employed in the screen would involve the crossing of the mutant libraries, with the hope of generating diploids that will have two mutant copies of the same gene. Some of these diploids would appear unusually stable, showing a normal sporulation phenotype. This would indicate the mutation of a gene that normally promotes genomic instability following replication fork stalling. The practicality of the proposed screen of a vast number of diploids was assessed and described in detail in the current work. A technique involving inverse PCR (IPCR) adopted from previous work to identify mutants of interest, was also investigated. The investigation of this technique, and the work of others, suggests that transformation using such selectable marker fragments results in most apparent transformants containing extrachromosomal ura4+ fragments. These fragments are thought to provide the predominant template for IPCR, rendering the process unsuccessful at identifying the mutation in the current screen. However, with the mutant libraries generated, and the screen procedure detailed, the stage is set to conduct the screen once a more appropriate mutation location technique is identified. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-05-31 22:25:14.009

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Genomic instability and aneuploidy, which are ubiquitous hallmarks of cancer cells, encompass both structural and numerical chromosome aberrations. Strikingly, cancer cells often display recurrent patterns of aneuploidy which are thought to be contingent on selection pressures within the tumour microenvironment maintaining advantageous karyotypes. However, it is currently unknown if individual chromosomes are intrinsically vulnerable to missegregation, and therefore whether chromosome bias may also contribute to pathological aneuploidy patterns. Moreover, the earliest responses to chromosome missegregation in non-transformed cells, and how these are overcome in cancer, has remained elusive due to the difficult nature of isolating nascent aneuploid cells. Results. Individual chromosomes displayed recurrent patterns of biased missegregation in response to a variety of cellular stresses across cell lines. Likewise, a small subset of chromosomes accounted for a large fraction of segregation errors following one specific mechanism driving aneuploidy. This was supported by the discovery that chromosomes 1 and 2 are strikingly susceptible to the premature loss of sister chromatid cohesion during prolonged prometaphase arrest. Additionally, I have elucidated the arrangement of individual metaphase human chromosomes, highlighting missegregation vulnerabilities occurring at the metaphase plate periphery following nocodazole wash-out. Finally, I have developed a novel system for isolating nascent aneuploid cells, suggesting the earliest transcriptome responses to chromosome missegregation in non-transformed human cells involve ATM and BCL2-mediated apoptosis.

The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

The evolution of prostate cancer from normal epithelium via the preneoplastic lesion of high-grade prostatic intraepithelial neoplasia to invasive carcinoma is characterised by a number of particular genomic abnormalities that are predominantly generated in the preneoplastic phase. Whilst there are numerous candidates for the cause of these alterations, telomere dysfunction is thought to be a major contributor. Telomeres are the terminal ends of human chromosomes, and when dysfunctional can lead to break-fusion-bridge cycles and multi-polar mitoses that generate numerical and structural chromosomal instability. The results presented reinforce the association of telomere dysfunction with the generation of certain markers of genomic instability such as abnormalities of the arms of chromosome 8. Furthermore, this work clarifies that the TMPRSSS2-ERG aberrations are not telomere related phenomena and are associated with a genomic deletion in a proportion of cases. Similarly, the PTEN microdeletions did not appear to have an association with telomere attrition. A previously unrecognised association between the telomere length in various types of prostatic epithelia and adjacent stroma is defined, suggesting evidence of a micro-environmental field effect in the generation of prostatic neoplasia. Finally, when examined retrospectively, it appears that telomere attrition, both in the HPIN epithelium and the stroma has independent prognostic value in the diagnosis of prostate cancer after a previous diagnosis of HPIN. Taken together, the research presented suggests important avenues for further research to determine the nature of barriers to the evolution of prostatic carcinogenesis such as oncogene- and telomere-induced senescence that may be exploited for therapeutic gain. These understandings may also help tailor management for prostate cancer such as risk stratification for men with HPIN and the use of targeted agents such as AKT inhibitors and telomerase inhibitors. In more advanced disease, translational application of this work has enabled a clinical trial of cytarabine in the treatment of metastatic hormone refractory prostate cancer.

Cancer is a disease that involves genomic instability, to which c-Myc contributes during its initiation and progression. Over 70% of all human cancers show deregulated levels of c-Myc protein. The term genomic instability refers to genetic and/or epigenetic changes that alter the normal organization and function of genes and chromosomes. Genomic instability is a hallmark of cancer and often is associated with cancer. Deregulated c-Myc expression generates genomic instability by initiating intra- and extrachromosomally locus-specific gene amplification, gene rearrangements and karyotypic instability that includes translocations, fusions, insertions and deletions. Out of the several outlined pathways by which deregulated levels of c-Myc can lead to genomic instability, the work described in this thesis focuses on three with direct relevance to tumorigenesis; gene amplification (increase in gene copy number), remodeling of the chromosomal and telomeric structures in the interphase nucleus and comparing the effect of Myc to that of Epstein Bar virus (EBV) infection in remodeling the nuclear structure that may lead to genomic instability.

Cancer is a disease that involves genomic instability, to which c-Myc contributes during its initiation and progression. Over 70% of all human cancers show deregulated levels of c-Myc protein. The term genomic instability refers to genetic and/or epigenetic changes that alter the normal organization and function of genes and chromosomes. Genomic instability is a hallmark of cancer and often is associated with cancer. Deregulated c-Myc expression generates genomic instability by initiating intra- and extrachromosomally locus-specific gene amplification, gene rearrangements and karyotypic instability that includes translocations, fusions, insertions and deletions. Out of the several outlined pathways by which deregulated levels of c-Myc can lead to genomic instability, the work described in this thesis focuses on three with direct relevance to tumorigenesis; gene amplification (increase in gene copy number), remodeling of the chromosomal and telomeric structures in the interphase nucleus and comparing the effect of Myc to that of Epstein Bar virus (EBV) infection in remodeling the nuclear structure that may lead to genomic instability.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive functions of FA signaling is important for the study of Fanconi anemia, inherited cancers, and sporadic cancers. The FA network functions as a genome guardian throughout the cell cycle. In addition to the well-established roles of FA proteins in interphase DNA replication/repair, the FA pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure proper chromosome segregation. The SAC consists of several tumor suppressors, including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However, the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the SAC are unclear. To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC dysfunction. Importantly, these mice were prone to developing hematologic malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of Fanconi anemia. Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC function of FANCC during mitosis. Our study highlights the essential role of compromised chromosome segregation in the development of leukemia due to impaired FA signaling. This work furthers our knowledge of FANCC signaling at the SAC, and has implications for future use of mitotic-centered therapies for FA-associated tumors. / 2 years