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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010413-12
Application #
8552689
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2012
Total Cost
$1,732,292
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Lee, Hee-Sheung; Lee, Nicholas C O; Kouprina, Natalay et al. (2016) Effects of Anticancer Drugs on Chromosome Instability and New Clinical Implications for Tumor-Suppressing Therapies. Cancer Res 76:902-11
Kim, Jung-Hyun; Lee, Hee-Sheung; Lee, Nicholas C O et al. (2016) Development of a novel HAC-based ""gain of signal"" quantitative assay for measuring chromosome instability (CIN) in cancer cells. Oncotarget 7:14841-56
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Kouprina, Natalay; Earnshaw, William C; Masumoto, Hiroshi et al. (2013) A new generation of human artificial chromosomes for functional genomics and gene therapy. Cell Mol Life Sci 70:1135-48
Kouprina, Natalay; Lee, Nicholas C O; Pavlicek, Adam et al. (2012) Exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1-linked families. Genes Chromosomes Cancer 51:933-48
Bergmann, Jan H; Jakubsche, Julia N; Martins, Nuno M et al. (2012) Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function. J Cell Sci 125:411-21

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