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Inhibition of gene amplification in telomerase deficient immortalized mouse embryonic fibroblasts
No full textMutations in genes important for the preservation of genome stability can increase the frequency of gene amplification, a process relevant to tumor development. To investigate whether telomerase, the enzyme deputed to telomere maintenance, also plays a role in gene amplification, we studied the amplification of the carbamyl-P-synthetase, aspartate transcarbamilase, dihydro-orotase (CAD) gene in immortalized embryonic fibroblasts derived from telomerase knockout mice (mTERC(-/-)) of the first and of the sixth generation. As expected, in 9 out of 10 N-(phosphonacetyl)-L-aspartate (PALA) resistant clones derived from wild-type cells, CAD was amplified; in contrast, in none of the 30 PALA resistant clones isolated from the three mTERC(-/-) cell lines we could detect CAD amplification, indicating that, in the absence of telomerase activity, gene amplification is inhibited. The causal relationship between mTERC deficiency and lack of gene amplification was demonstrated by the restoration of CAD gene amplification in two of the three deficient cell lines transfected with mTERC. The lack of amplification in mTERC deficient cells could be related to a defect in the stabilization of the ends of the amplified chromosomes in the absence of telomerase, to a more general effect of telomerase in the regulation of gene expression, including genes involved in amplification, or to a possible interaction of the telomerase RNA with proteins involved in gene amplification
Chromosomal end-to-end fusions in immortalized mouse embryonic fibroblasts deficient in the DNA-dependent protein kinase catalytic subunit
No full textTelomeres constitute the ends of the linear eukaryotic chromosomes and are essential for the maintenance of chromosome stability and genome integrity. One of the consequences of an altered telomere structure is the formation of telomeric fusions (TFs), that is aberrant chromosomes in which two elements are fused at their telomeres. Proteins involved in the non-homologous end joining pathway for the repair of the DNA double strand breaks, as the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), contribute to the formation of a functional telomere. To investigate the role of DNA-PKcs in telomere functionality, we studied the frequency of TFs in mouse embryonic fibroblasts obtained from animals in which the DNA-PKcs gene had been inactivated; the analysis was performed prior and after spontaneous immortalization in culture. Our results suggest that DNA-PKcs deficiency has a limited effect, if any, on TF formation in primary cells, while it further increases chromosomal instability in immortalized cells. In fact, the frequency of TFs was significantly higher in immortalized DNA-PKcs mutant cells compared to wild type cells. Together with TFs, we also found metacentric or submetacentric chromosomes in which no telomeric sequences were detected at the joining site. The frequency of this anomaly, that resembles the Robertsonian translocations observed in wild mice, was independent of the DNA-PKcs genotype. This suggests that the formation of these rearranged chromosomes does not rely on a functional DNA-PKcs
Chromosome instability in stem cells
No full textStem cells (SCs) are able to differentiate into many different cell types of the body during early life and growth. They are unspecialized cells capable of self-renewal and, when chal-lenged, they can be induced to differentiate into specific and mature cell types. SCs can be classified into two different types: somatic stem cells (SSCs) and embryonic stem cells (ESCs). One major problem concerning in vitro SC culture is the difficulty to maintain their genetic and karyotype integrity during long-term culture. This chapter describes the different types of abnormalities accumulated by ESCs, derived from different species, and SSCs, of different somatic origin, during long-term culture in vitro. It outlines the main processes that are implicated in the onset of chromosome abnormalities in cells during culture. Among these processes, only one has been so far described in SCs
Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture
No full textIncreased gene amplification in immortal rodent cells deficient for the DNA-dependent protein kinase catalityc subunit.
No full textGene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N-(phosphonacetyl)-L-aspartate (PALA) resistance is considered a measure of gene amplification because it is mainly attributable to amplification of the carbamyl-P-synthetase aspartate transcarbamylase dihydro-orotase (CAD) gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs gene, displays also an increased frequency of gene amplification. In these cells, we found that the amplification of the CAD gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs(-/-) MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that gene amplification occurs in a genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify
Achilles' heel of pluripotent stem cells: genetic, genomic and epigenetic variations during prolonged culture
No full textPluripotent stem cells differentiate into almost any specialized adult cell type of an organism. PSCs can be derived either from the inner cell mass of a blastocyst-giving rise to embryonic stem cells-or after reprogramming of somatic terminally differentiated cells to obtain ES-like cells, named induced pluripotent stem cells. The potential use of these cells in the clinic, for investigating in vitro early embryonic development or for screening the effects of new drugs or xenobiotics, depends on capability to maintain their genome integrity during prolonged culture and differentiation. Both human and mouse PSCs are prone to genomic and (epi)genetic instability during in vitro culture, a feature that seriously limits their real potential use. Culture-induced variations of specific chromosomes or genes, are almost all unpredictable and, as a whole, differ among independent cell lines. They may arise at different culture passages, suggesting the absence of a safe passage number maintaining genome integrity and rendering the control of genomic stability mandatory since the very early culture passages. The present review highlights the urgency for further studies on the mechanisms involved in determining (epi)genetic and chromosome instability, exploiting the knowledge acquired earlier on other cell types
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