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Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells.

ABSTRACT: Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.

SUBMITTER: Ji W

PROVIDER: S-EPMC4233979 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells.

Project description:Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells.

| S-EPMC4280677 | biostudies-literature

Molecular structure of double-minute chromosomes bearing amplified copies of the epidermal growth factor receptor gene in gliomas.

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Gemcitabine eliminates double minute chromosomes from human ovarian cancer cells.

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| S-EPMC3750019 | biostudies-literature

Double minute chromosomes in glioblastoma multiforme are revealed by precise reconstruction of oncogenic amplicons.

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Project description:BackgroundDouble minute chromosomes are circular fragments of DNA whose presence is associated with the onset of certain cancers. Double minutes are lethal, as they are highly amplified and typically contain oncogenes. Locating double minutes can supplement the process of cancer diagnosis, and it can help to identify therapeutic targets. However, there is currently a dearth of computational methods available to identify double minutes. We propose a computational framework for the idenfication of double minute chromosomes using next-generation sequencing data. Our framework integrates predictions from algorithms that detect DNA copy number variants, and it also integrates predictions from algorithms that locate genomic structural variants. This information is used by a graph-based algorithm to predict the presence of double minute chromosomes.ResultsUsing a previously published copy number variant algorithm and two structural variation prediction algorithms, we implemented our framework and tested it on a dataset consisting of simulated double minute chromosomes. Our approach uncovered double minutes with high accuracy, demonstrating its plausibility.ConclusionsAlthough we only tested the framework with three programs (RDXplorer, BreakDancer, Delly), it can be extended to incorporate results from programs that 1) detect amplified copy number and from programs that 2) detect genomic structural variants like deletions, translocations, inversions, and tandem repeats. The software that implements the framework can be accessed here: https://github.com/mhayes20/DMFinder

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Chromosomes trapped in micronuclei are liable to segregation errors.

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Met promotes the formation of double minute chromosomes induced by Sei-1 in NIH-3T3 murine fibroblasts.

Project description:BackgroundSei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated.ResultsDMs formation significantly increased during serial passage in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3.Materials and methodsWe employed NIH-3T3 cell line with Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure.ConclusionsMet is an important promotor of DMs formation.

| S-EPMC5302943 | biostudies-literature

Novel functional MAR elements of double minute chromosomes in human ovarian cells capable of enhancing gene expression.

Project description:Double minute chromosomes or double minutes (DMs) are cytogenetic hallmarks of extrachromosomal genomic amplification and play a critical role in tumorigenesis. Amplified copies of oncogenes in DMs have been associated with increased growth and survival of cancer cells but DNA sequences in DMs which are mostly non-coding remain to be characterized. Following sequencing and bioinformatics analyses, we have found 5 novel matrix attachment regions (MARs) in a 682 kb DM in the human ovarian cancer cell line, UACC-1598. By electrophoretic mobility shift assay (EMSA), we determined that all 5 MARs interact with the nuclear matrix in vitro. Furthermore, qPCR analysis revealed that these MARs associate with the nuclear matrix in vivo, indicating that they are functional. Transfection of MARs constructs into human embryonic kidney 293T cells showed significant enhancement of gene expression as measured by luciferase assay, suggesting that the identified MARS, particularly MARs 1 to 4, regulate their target genes in vivo and are potentially involved in DM-mediated oncogene activation.

| S-EPMC3272018 | biostudies-literature

Sei-1 promotes double minute chromosomes formation through activation of the PI3K/Akt/BRCA1-Abraxas pathway and induces double-strand breaks in NIH-3T3 fibroblasts.

Project description:Sei-1 is a potential oncogene that plays an important role in promoting genomic instability. Double minute chromosomes (DMs) are hallmarks of gene amplification and contribute to tumorigenesis. Defects in the DNA double-strand break (DSB) repairing pathways can lead to gene amplification. To date, the mechanisms governing the formation of DMs induced by Sei-1 are not fully understood. We established DMs induced by Sei-1 in the NIH-3T3 cell line. RNA-sequencing was used to identify key characteristics of differentially expressed genes. Metaphase spreads were used to calculate DM numbers. Immunofluorescence was employed to detect ?H2AX foci. Western blot and Akt pathway inhibition experiments were performed to reveal the role of the PI3K/Akt/BRCA1-Abraxas pathway in Sei-1-induced DMs. Luciferase reporter assay was employed to explore the regulatory mechanisms between Sei-1 and BRCA1. DM formation was associated with a deficiency in DSB repair. Based on this finding, activation of the PI3K/Akt/BRCA1-Abraxas pathway was found to increase the DM population with passage in vivo, and inhibition resulted in a reduction of DMs. Apart from this, it was shown for the first time that Sei-1 could directly regulate the expression of BRCA1. Our results suggest that the PI3K/Akt/BRCA1-Abraxas pathway is responsible for the formation of DMs induced by Sei-1.

| S-EPMC5832785 | biostudies-literature

Chromosomes missegregated into micronuclei contribute to chromosomal instability by missegregating at the next division.

Project description:Micronuclei (MNi) are extranuclear DNA-containing structures that form upon mitotic exit from unsegregated chromosome fragments or anaphase lagging (whole) chromosomes (LCs). MNi formed from whole chromosomes are of particular interest because LCs are observed in both cancer and non-cancer cells, and are recognized as a major source of chromosomal instability (CIN) in cancer cells. Here, we generated a PtK1 cell line expressing a photoactivatable H2B histone to study the behavior of whole chromosome-containing MNi at the mitosis following their formation. Importantly, MNi of PtK1 cells did not display the membrane rupture or transport defects reported for other cell types. Despite this, we found that most micronucleated cells displayed some kind of chromosome segregation defect and that the missegregating chromosome was the one derived from the MN. Moreover, condensation of the chromosome within the MN was frequently delayed and associated with failure to align at the metaphase plate. Finally, the defective condensation of the MN-derived chromosomes could also explain the frequent occurrence of cytokinesis failure in micronucleated cells. In summary, we find that chromosomes from MNi may trigger a CIN phenotype by missegregating at the mitosis following MN formation.

| S-EPMC6505630 | biostudies-literature

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