Cell-type-specific consequences of nucleotide excision repair deficiencies: Embryonic stem cells versus fibroblasts
Introduction
Preserving the genome is of prime importance for all living organisms since endogenous and exogenous agents (e.g. UV-light, X-rays, oxidative stress and many chemicals) continuously induce a wide variety of DNA lesions. Replication of the damaged template may lead to mutations, potentially resulting in cancer. Alternatively, persistent DNA damage can hinder cellular key processes like transcription and replication, which may cause cell malfunctioning, ultimately leading to cell death. Evidence is accumulating that this overall functional decline of the genome may contribute to aging (reviewed in [1], [2], [3]). To counteract genotoxic stress, cells are equipped with an elaborate genome caretaking network, including different DNA repair mechanisms with partly overlapping substrate specificity: (i) non-homologous end joining and homologous recombination, for repair of double strand breaks, (ii) a variety of base excision repair (BER) enzymes, coping with small base modifications, like methylation and oxidation, and (iii) nucleotide excision repair (NER), for removal of UV-induced and other helix-distorting lesions [4], [5].
The NER machinery consists of two sub-pathways, namely global genome NER (GG-NER) and transcription-coupled NER (TC-NER). GG-NER removes DNA damage from the entire genome. TC-NER specifically and efficiently removes DNA damage from the transcribed strand of active genes, thereby releasing transcription arrest, caused by RNA polymerase II stalled at lesions. Defects in TC-NER are associated with the rare inherited disorder Cockayne syndrome (CS), while GG-NER defects or total NER deficiencies are interrelated with xeroderma pigmentosum (XP) (for review see [6]). In addition to repair, transient cell cycle arrest provides the cell with a time window to fix damaged DNA before lesions are converted into permanent genetic changes [7]. When repair fails or takes too long, cells carrying too much genetic damage can be eliminated via apoptosis or become senescent [8], [9].
It has been suggested that, depending on cell type, differentiation-stage and age, cells may have a different need to withstand genotoxic stress and therefore might have different priorities in the use of the various genome-caretaking processes. For example, exposure of pregnant mice to very low doses of ionizing radiation causes severe apoptosis in the embryo, while apoptosis is not observed in extra-embryonic tissue [10]. A similar observation has been made for the small intestine, where ionizing radiation-induced apoptosis is restricted to stem cells in the crypt and is absent in the more differentiated cells in the villi [11]. An example of cell-type-specific usage of various repair pathways is the preference for error-free repair of double strand breaks by homologous recombination in ES cells, while double strand break repair in more differentiated cells largely depend on the error prone non-homologous end joining [12]. It is even possible that certain repair mechanisms are only present in, and adapted to specific cell types. For example, neurons are post-mitotic cells that do not divide and as a consequence will not use the gross of their genome anymore. Therefore, it has been suggested that these cells do not need to keep their overall genome error-free, but instead only have to properly maintain their transcribed genes. Indeed, it has been shown that neurons have low levels of GG-NER, while TC-NER is normal [13]. Since in the absence of GG-NER, damage could accumulate on the non-transcribed strand of active genes (serving as a template for the TC-NER reaction) also the non-transcribed strand of active genes should be kept lesion-free. A specific mechanism, differentiation associated repair (DAR), has been described to complement TC-NER by repair of damage in the non-transcribed strand of active genes in neurons [13], [14].
Embryonic stem cells (ES cells) are the ultimate stem cells, and therefore can be considered at the far end of the spectrum ranging from undifferentiated to differentiated cells. Tolerance of DNA damage in pluripotent ES cells could have detrimental consequences. For example, clonal expansion of mutated cells to considerable parts of the organism can lead to huge malformations. As part of the genome-caretaking network, a hyperapoptotic response of ES cells to various genotoxic agents has been shown [15], [16], [17], [18]. NER seems to contribute to some extent in protection against DNA damage [18]. To gain more insight in the contribution of TC-NER and GG-NER in genome caretaking in pluripotent stem cells and in (partially) differentiated somatic cells, we established mouse ES cells and mouse embryonic fibroblasts (MEFs) with defects in TC-NER (Csb−/−), GG-NER (Xpc−/−) or both pathways (Xpa−/−), as well as wild type cells. We show that, in comparison to MEFs, ES cells are hypersensitive to a wide variety of genotoxic agents. Interestingly, inactivation of specific repair genes has different effects on the damage response in ES cells and MEFs. Our data suggest that the contribution of damage in the global genome is the major determinant for the ES cell response to helix-distorting lesions. Therefore, in contrast to the situation in MEFs, the main contributor to apoptosis induction in Xpa−/− ES cells is the deficiency for GG-NER, rather than the TC-NER-defect. Finally, our study provides evidence for a double safeguard mechanism against mutations in ES cells.
Section snippets
Cell lines
Isolation of primary Csb−/− (FVB/129Ola), Xpa−/− (hybrid C57BL/6J and 129ola) MEFs and corresponding wild type cell lines has been described [19], [20]. Xpc−/− (hybrid C57BL/6J and 129ola) MEFs were isolated in a similar manner. Cells were cultured in F10/DMEM (1:1) (Gibco) medium, supplemented with 10% fetal calf serum and 50 μg/ml penicillin and streptomycin (Gibco). Spontaneously immortalized cell lines were obtained by continuous subculturing of primary MEFs. ES cells were isolated from wild
ES cells are hypersensitive to a variety of genotoxic treatments
To investigate whether ES cells are more sensitive to genotoxic stress than other cell types, we compared sensitivities of ES cells and spontaneously transformed MEFs to various genotoxic agents. Although, we never observed effects of the genetic background in MEFs (hybrid C57BL/6J-129ola, hybrid FVB-129ola and C57BL/6J) on UV-sensitivity, we isolated for this study ES cells in a pure C57BL/6J background to completely rule out the influence of differences in genetic background. As shown in Fig.
A hypersensitive response of ES cells to a wide variety of genotoxic treatments
In this study, we show that pluripotent undifferentiated ES cells respond differently to genotoxic stress when compared to more differentiated mouse embryonic fibroblasts. ES cells are more sensitive to UV (causing helix-distorting photoproducts), gamma rays (inducing double strand breaks and oxidative lesions) and mitomycin C (evoking mono-adducts, oxidative lesions, intra- and inter-strand cross-links), than MEFs. Since the UV-sensitivity of spontaneously immortalized MEFs compares well to
Conflict of interest statement
None.
Acknowledgements
This research was supported by the Netherlands Organization for Scientific Research (NWO) through the foundation of the Research Institute Diseases of the Elderly as well as grants from SenterNovem IOP-Genomics (IGE03009), the NIH (AG17242-02 and RFA-ES-00-005) the Dutch Cancer Society (UU 97-1531) and the EC (QRTL-1999-02002 and QRLT-CT-1999-00181).
References (53)
- et al.
Divide and conquer: nucleotide excision repair battles cancer and ageing
Curr. Opin. Cell. Biol.
(2003) - et al.
DNA repair, genome stability, and aging
Cell
(2005) DNA structure dependent checkpoints as regulators of DNA repair
DNA Repair (Amst)
(2002)- et al.
DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis
Mutat. Res.
(2002) DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling
Biochem. Pharmacol.
(2003)- et al.
DNA repair in terminally differentiated cells
DNA Repair (Amst)
(2002) - et al.
ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage
Curr. Biol.
(1998) - et al.
Defective transcription-coupled repair in Cockayne syndrome B mice is associated with skin cancer predisposition
Cell
(1997) - et al.
Cell type-specific hypersensitivity to oxidative damage in CSB and XPA mice
DNA Repair (Amst)
(2003) - et al.
Xeroderma pigmentosum group F caused by a defect in a structure-specific DNA repair endonuclease
Cell
(1996)
Excision repair in Cockayne syndrome
Mutat. Res.
Anti-tumour compounds illudin S and Irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways
DNA Repair (Amst)
Protecting genomic integrity in somatic cells and embryonic stem cells
Mutat. Res.
Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair
Mol. Cell
Sequential assembly of the nucleotide excision repair factors in vivo
Mol. Cell
DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation
Mutat. Res.
Cancer predisposition in mutant mice defective in multiple genetic pathways: uncovering important genetic interactions
Mutat. Res.
p53-independent DNA repair and cell cycle arrest in embryonic stem cells
FEBS Lett.
Strand-specific mutation spectra in repair-proficient and repair-deficient hamster cells
Mutat. Res.
UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand
Mutat. Res.
Testicular germ cell tumors: molecular understanding and clinical implications
Mol. Med. Today
Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformation and death
Mutat. Res.
Aging and genome maintenance: lessons from the mouse?
Science
Genome maintenance mechanisms for preventing cancer
Nature
DNA damage and repair
Nature
Nucleotide Excision Repair Syndromes: Xeroderma Pigmentosum, Cockayne Syndrome and Trichothiodystrophy
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Current address: Laboratory of Clinical Chemistry and Hematology, Jeroen Bosch Hospital, POB 90153, 5200 ME ‘s-Hertogenbosch, The Netherlands.