Publications (6)19.62 Total impact
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Article: A new assay for measuring chromosome instability (CIN) and identification of drugs that elevate CIN in cancer cells.
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ABSTRACT: BACKGROUND: Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed 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 and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory. METHODS: We have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry. RESULTS: Using the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A. CONCLUSION: Thus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells.BMC Cancer 05/2013; 13(1):252. · 3.01 Impact Factor -
Article: Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere.
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ABSTRACT: Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoid(tetO)-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoid(tetO)-HAC that has been formed from a ~50 kb synthetic alphoid(tetO)-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoid(tetO)-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells.ACS synthetic biology. 12/2012; 1(12):590-601. -
Article: Exclusion of the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy in HPCX1-linked families.
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ABSTRACT: Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of 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. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL 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. 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.Genes Chromosomes and Cancer 06/2012; 51(10):933-48. · 3.31 Impact Factor -
Article: Condensin dysfunction in human cells induces nonrandom chromosomal breaks in anaphase, with distinct patterns for both unique and repeated genomic regions.
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ABSTRACT: Condensin complexes are essential for chromosome condensation and segregation in mitosis, while condensin dysfunction, among other pathways leading to chromosomal bridging in mitosis, may play a role in tumor genomic instability, including recently discovered chromotripsis. To characterize potential double-strand breaks specifically occurring in late anaphase, human chromosomes depleted of condensin were analyzed by γ-H2AX ChIP followed by high-throughput sequencing (ChIP-seq). In condensin-depleted cells, the nonrepeated parts of the genome were shown to contain distinct γ-H2AX enrichment zones 75% of which overlapped with known hemizygous deletions in cancers. Furthermore, some tandemly repeated DNA sequences, analyzed separately from the rest of the genome, showed significant γ-H2AX enrichment in condensin-depleted anaphases. The most commonly occurring targets of such enrichment included simple repeats, centromeric satellites, and rDNA. The two latter categories indicate that acrocentric human chromosomes are especially susceptible to breaks upon condensin deficiency. The genomic regions that are specifically destabilized upon condensin dysfunction may constitute a condensin-specific chromosome destabilization pattern.Chromosoma 12/2011; 121(2):191-9. · 3.85 Impact Factor -
Article: tRNA genes protect a reporter gene from epigenetic silencing in mouse cells.
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ABSTRACT: It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.Cell cycle (Georgetown, Tex.) 08/2011; 10(16):2779-91. · 5.36 Impact Factor -
Article: Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation.
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ABSTRACT: Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.PLoS ONE 02/2009; 4(8):e6831. · 4.09 Impact Factor
Top co-authors
Top Journals
- Genes Chromosomes and Cancer (1)
- Chromosoma (1)
- BMC Cancer (1)
- Cell cycle (Georgetown, Tex.) (1)
- PLoS ONE (1)
Institutions
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2012
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NCI-Frederick
Frederick, MD, USA
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2011–2012
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National Cancer Institute (USA)
- Laboratory of Molecular Pharmacology
Bethesda, MD, USA
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2009
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Eunice Kennedy Shriver National Institute of Child Health and Human Development
Rockville, MD, USA
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