Research Article: Error-prone nonhomologous end joining repair operates in human pluripotent stem cells during late G2

Date Published: June 12, 2011

Publisher: Impact Journals LLC

Author(s): Alexandra N. Bogomazova, Maria A. Lagarkova, Leyla V. Tskhovrebova, Maria V. Shutova, Sergey L. Kiselev.



Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay, we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives, but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response, revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK, a key NHEJ component, by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast, NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus, DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells.

Partial Text

Pluripotent human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of spare blastocysts and are able to differentiate into various cell types. Therefore, these cells are often used as an in vitro model of the ICM. Recent studies suggest that a chromosomally aberrant cell population is present in nearly all human spare embryos at the cleavage stage [1-3]. However, newborns are characterized by a reduced frequency of chromosomal abnormalities when compared to preimplantation embryos [4]. In vivo, the pluripotent cell state is maintained for a very limited time; however, hESCs can be grown indefinitely in culture and their capacity to self renew and to differentiate into any cell type can be preserved for prolonged periods of time. These unique properties make hESCs very attractive as a potential source of cells for therapeutic usage. Clearly, the genome stability of hESCs is an important issue to be considered prior to use in clinical applications because even small genomic changes can significantly impair cell functionality and safety. Several reports have provided evidence of remarkable karyotype stability maintained by some hESC lines over the course of more than 140 -180 passages in vitro [5-6]. However, high-resolution karyotyping methods have established that hESCs acquire chromosomal abnormalities during long-term passaging in vitro, namely new sites of heterozygosity loss (LOH) and changes in copy-number variations (CNVs) [7, 8]. It is possible that the chromosomal aberrations observed in hESCs might reflect events similar to those that occur in a developing embryo at the blastocyst stage. Later in development, cells with normal karyotypes are selected by an unknown mechanism, but hESCs accumulate chromosomal alterations during culturing in vitro. Repair of DNA double strand breaks (DSBs) by homologous recombination (HR) could be the source of the LOH arising in hESCs during cultivation while CNVs could potentially result from DSB repair by non-allelic homologous recombination (NAHR), non-homologous end joining (NHEJ) or microhomology-mediated end joining [9, 10]. A recent study aimed at characterizing DNA repair in hESCs indicates that HR is the major, if not the sole, mechanism of DSB repair in pluripotent human cells compared to differentiated somatic cells, which typically use NHEJ [11]. However, more recently Adams et al. [12] provided evidence demonstrating NHEJ functionality in hESCs and showed that two closely-spaced DSBs induced by I-Sce endonuclease can be repaired with high fidelity by NHEJ in hESCs. NHEJ activity can result in chromosomal rearrangements when multiple DSBs coincide in space and time [13]. The aim of this study is to determine the repair accuracy of multiple radiation-induced DSBs in human pluripotent cells. To investigate the level of DSB misrejoining in pluripotent and somatic cells, we used a G2-chromosomal radiosensitivity assay [14]. We analyzed radiation-induced chromosomal aberrations in solid-stained metaphases 2 hours following irradiation, i.e., the cytogenetic analysis involved only cells irradiated during the late G2 stage of the cell cycle after transition through the G2/M checkpoint [15]. The design of this G2-assay allowed us to overcome the prominent differences in sensitivity to irradiation of pluripotent and somatic cells observed by Filion et al. [16] and their differences in cell cycle structure and regulation demonstrated by Momčilović et al. [17]. In addition, cytogenetic analysis provides a unique opportunity to estimate the frequency of misrejoining during DSB repair. We used the G2-assay to compare the accuracy of repair in pluripotent cells, isogenic somatic cells and HS27 primary fibroblasts. We show that DNA-PK-dependent NHEJ suppresses the formation of chromatid breaks after irradiation during late G2, and most of the radiation-induced chromatid exchanges observed in hESCs result from DNA-PK activity. These data elucidate the mechanisms involved in the formation of radiation-induced chromatid aberrations and propose that these mechanisms contribute to chromosome instability in pluripotent cells in vivo.





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