Project description:The immunodeficiency, centromere instability and facial anomalies (ICF) syndrome is associated with mutation of the DNA methyl-transferase DNMT3B, resulting in a reduction of enzyme activity. Aberrant expression of immune system genes and hypomethylation of pericentromeric regions accompanied by chromosomal instability were determined as alterations driving the disease phenotype. However, so far only technologies capable of analyzing single loci were applied to determine epigenetic alterations in ICF patients. In the current study, we performed whole-genome bisulphite sequencing to assess alteration in DNA methylation at base-pair resolution. Whole-genome bisulphite sequencing was performed to assess alteration in DNA methylation of one ICF patient and one healthy control sample at base-pair resolution.
Project description:Preservation of a balanced chromosomal content is regarded to be a key point for the success of multicellular organisms. Chromosomal segregation takes place under the strict control of well-orchestrated cell-cycle checkpoints, consequently leading to accurate transmission of intact chromosomes. Estimates of the whole chromosomal error rates per cell division based on cytogenetic analyses of newborns and products of conception, range between 4.57x10-5 and 3.42x10-4. Recent sporadic studies of single cell genome wide CNV analysis suggested that the error rate might be higher than currently estimated. To obtain accurate measures of chromosomal error rates, we plated single fibroblast and analyzed the two daughter sister cells following a single cell division. In total 14 single fibroblasts derived from 7 mitoses carried segmental aneuploidies in a total of 178 cells from 5 different cell lines that were analyzed after a single cell division, indicating a mean frequency of 7.9% in vitro. In conclusion, the chromosomal stability is hundreds times lower than the current dogma, showing that chromosomal instability is a common place and putting the efficacy of the DNA-repair mechanisms and control checkpoints in question.
Project description:Cancer development is associated with multiple genetic alterations and genomic instability either at the chromosomal or base pair level is generally thought to underlie these changes. However, it is still unknown whether genetic instability is absolutely required for tumorigenesis. Here we investigated the genomic instability status of four cytogenetically stable diploid cell lines CAL51, SK-UT-1B, A204 and CH1. We applied high resolution 500K single nucleotide polymorphism (SNP) array analysis and found that all the four cell lines have some sub-microscopic genomic copy number changes. Interestingly, there were many more sub-microscopic chromosome alterations in A204 and CH1 than in CAL51 and SK-UT-1B. Twenty-four-color fluorescence in situ hybridization (FISH) was used to analyse a large number of metaphases from CAL51, SK-UT-1B and CH1 cells. The rate of de novo chromosome rearrangements was significantly higher in CH1 than CAL51 and SK-UT-1B. Although this increased rate did not lead to many clonal cytogenetically apparent chromosome alterations in CH1 cells, it is consistent with a first step towards chromosomal instability. It is more striking that both cell lines CAL51 and SK-UT-1B which have a similar de novo chromosomal change rate to that of normal lymphocytes were microsatellite instability positive by BAT-26 microsatellite analysis. This study further strengths the current concept that genomic instability is associated with tumor development. Keywords: DNA copy number changes
Project description:Cancer development is associated with multiple genetic alterations and genomic instability either at the chromosomal or base pair level is generally thought to underlie these changes. However, it is still unknown whether genetic instability is absolutely required for tumorigenesis. Here we investigated the genomic instability status of four cytogenetically stable diploid cell lines CAL51, SK-UT-1B, A204 and CH1. We applied high resolution 500K single nucleotide polymorphism (SNP) array analysis and found that all the four cell lines have some sub-microscopic genomic copy number changes. Interestingly, there were many more sub-microscopic chromosome alterations in A204 and CH1 than in CAL51 and SK-UT-1B. Twenty-four-color fluorescence in situ hybridization (FISH) was used to analyse a large number of metaphases from CAL51, SK-UT-1B and CH1 cells. The rate of de novo chromosome rearrangements was significantly higher in CH1 than CAL51 and SK-UT-1B. Although this increased rate did not lead to many clonal cytogenetically apparent chromosome alterations in CH1 cells, it is consistent with a first step towards chromosomal instability. It is more striking that both cell lines CAL51 and SK-UT-1B which have a similar de novo chromosomal change rate to that of normal lymphocytes were microsatellite instability positive by BAT-26 microsatellite analysis. This study further strengths the current concept that genomic instability is associated with tumor development. Keywords: DNA copy number changes DNA from A204, CH1, CAL51 and SK-UT-1B were analyzed by Affymetrix Genechip Mapping 500K Set array. Data were compared with normal samples from HAPMAP database.
Project description:Together with TEAD4 the Hippo pathway effector YAP stimulates chromosomal instability. Verteporfin is known to disrupt the physical YAP/TEAD interaction and tehrefore might prevent from chromosomal instability in liver cancer. Immortalized HCC cell line hepG2 is treated with Verteporfin for 24 hours.
Project description:Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH. The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines displayed a higher degree of instability. We subsequently assessed the clonal evolution of a single cell in two aneuploid cell lines, SW480 and HT-29, which both have near-triploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter cell lines, as assessed by multiplex FISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, and had little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter cell lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter cell lines.. When analyzing the evolutionary development over time, most single cell-derived daughter cell lines maintained their major clonal pattern, with the exception of one daughter of SW480 that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis.
2018-05-01 | GSE102647 | GEO
Project description:Whole genome sequencing of bulk and single RPE-1 cells with induced chromosomal instability