The degradation profile of extrachromosomal circular DNA during cisplatin-induced apoptosis is consistent with preferential cleavage at matrix attachment regions

Abstract

Extrachromosomal circular DNA molecules are prevalent in cancer cells and harbor amplified genes, such as oncogenes and drug resistance genes, that can provide a selective growth advantage to cancer cells. These circular DNA structures include double minute chromosomes (dmin), which can be detected with light microscopy following Giemsa staining, and submicroscopic circular DNA structures referred to as episomes. In this study, we investigated the fate of dmin and episomes in multidrug-resistant human epidermoid KB-VI cells undergoing cisplatin-induced apoptosis-a mode of cell death initially characterized by the fragmentation of chromosomal DNA, while the nuclear membrane remains intact. The circular DNA structures carry amplified copies of the multidrug resistance gene (MDR1). During cisplatin-induced apoptotic cell death, episomes and dmin, as well as native chromosomes, were degraded into high molecular weight DNA fragments of approximately 50 kb in length. DNA fragments in this size range appear to result from the preferential cleavage of matrix-associated regions in chromatin with the subsequent release of 20-30 nm loop domains of chromatin from the nuclear scaffold. Scanning electron microscopy studies were performed and confirmed the presence of 30 nm filaments in a higher-order DNA packing of MDR1-containing dmin and episomes. These combined data provide strong evidence that the higher-order DNA packing of episomes, as well as dmin, is similar to that of native chromosomes and underscore the potential for extrachromosomal DNA amplicons to study the structural and functional organization of chromatin. We discuss the implications of extra-chromosomal DNA matrix associated regions competing with native chromosomal DNA for binding to the nuclear matrix in tumor cells.

Loop domain organization of the p53 locus in normal and breast cancer cells correlates with the transcriptional status of the TP53 and the neighboring genes

“…For example, sites of chromosomal breakpoints preferentially localize to MARs [Schoenlein et al, 1999] and aberrant binding of DNA to the nuclear matrix may destabilize the expression of a host of genes that maintain cells in a noncancerous state [Linnemann and Krawetz, 2009a]. In the present study, we investigated for the first time the role of MARs in the regulation of transcription of the TP53 gene domain in breast cells.…”

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR

“…34 There have been a number of reports in the literature suggesting that initiation of apoptotic DNA cleavage occurs at matrix attachment regions. 6,7,[35][36][37] The first supporting evidence came from studies that establish that high molecular weight DNA fragments of 50 and 300 kb, produced in early stages of apoptosis, represent structural domains of chromatin, loops and rosettes, respectively. 1,7 It was then suggested that topoisomerase II, located at matrix attachment sites, is responsible for this cleavage since inhibition of its activity generated the same pattern of DNA fragments.…”

“…The cleavage sites have not been precisely mapped, due to a significant heterogeneity in MAR sequence and size as well as errors in sizing the apoptotic DNA fragments which, under the PFGE resolution, could be several kilobases. 7,36,37 Our approach was to use LM-PCR to map precisely the cleavage sites in the genomic sequences selected based on their high similarity to the SATB1-binding site sequence within the IgH gene enhancer region, their base content (i.e., A+T-rich, clustering of ATC exclusively on one strand) and location in noncoding regions of the genome. These features are considered to be characteristic for the core-unwinding element of BURs.…”

Downregulation of retinoblastoma protein is involved in the enhanced cytotoxicity of 4-hydroxytamoxifen plus mifepristone combination therapy versus antiestrogen monotherapy of human breast cancer

“…Briefly, total oligonucleosomal DNA obtained from 2x10 7 cells was loaded into one lane of a 2% agarose gel and subjected to conventional gel electrophoresis. To detect high molecular weight (HMW) DNA degradation, pulsed field gel electrophoresis analysis was performed as previously described (34)(35)(36). In brief, chromosomal DNA was embedded into agarose plugs and deproteinized in situ in EST lysis buffer [500 mM EDTA (pH 9.0) 1.0 % sodium lauroylsarcosine, 2 mg/ml proteinase K] (Boehringer Mannheim, Indianopolis, IN).…”