Induction of Circles of Heterogeneous Sizes in Carcinogen-Treated Cells: Two-Dimensional Gel Analysis of Circular DNA Molecules

Abstract

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.

Small polydispersed circular DNA contains strains of mobile genetic elements and occurs more frequently in permanent cell lines of malignant tumors than in normal lymphocytes

“…Those analyses were performed with DNA of patients with genetic diseases, for example Fanconi's anemia, in comparison with DNA of normal skin fibroblasts (1,3). Other reports have shown that the elevated amount of spcDNA was apparently connected with increased aging (17,18) and with transformation of cells (1,4,15). Until now there is only one report that directly compares the spcDNA of normal skin fibroblasts with those of a colon carcinoma tissue and of the tumor cell line HeLa.…”

“…By this technique, DNA molecules are separated both according to their size and their structure. Therefore, a population of molecules that are of the same structure, but of heterogeneous molecular mass, generates a continuous arc (1,15). The structural identity of the DNA in each arc can be determined by electron microscopy (15) and verified by molecular techniques (6,7,15,16).…”

Cell cycle checkpoint defects contribute to genomic instability in PTEN deficient cells independent of DNA DSB repair

“…This was achieved by analyzing telomeric DNA using neutral-neutral 2D gel electrophoresis, which separates telomeric restriction fragments first by size and then by shape. 20,21 The knockdown of BRCA1 by BRCA1 siRNA in MCF-7 cells has been described previously. 5 The results presented are the mean and standard error from three independent experiments.…”

FRA7G extends over a broad region: coincidence of human endogenous retroviral sequences (HERV-H) and small polydispersed circular DNAs (spcDNA) and fragile sites

“…spcDNAs can also be induced or increased by DNA damaging agents (Cohen and Lavi 1996;Cohen et al, 1997) and inhibitors of DNA synthesis, including the common fragile site inducer aphidicolin (Sunnerhagen et al, 1989). The presence of sequences with considerable homology to spcDNA right in the middle of two of the common fragile sites may therefore not be coincidental.…”