Abstract
Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue.Our results show that DNA DSBs repair activity is up regulated during the early commitment into adipogenesis due to an up-regulation of DNA-PK expression and activity. In opposition to the general view that DNA DSBs repair is decreased during differentiation, our results demonstrate that an up-regulation of this process might be observed in post-mitotic long-lived cells.
Highlights
The integrity of DNA is constantly being challenged by exogenous DNA-damaging agents and by endogenously produced radicals, stalled replication forks and by spontaneous formation of abasic sites in DNA
It has been initially proposed that the number of double strand breaks (DSBs) correlates to that of cH2AX foci within a 1:1 ratio [29,30,31] and that the foci number could be used as an indirect marker of DSBs occurrence
These results suggest that the efficiency in DNA DSBs repair is increased in adipocytes as compared to pre-adipocytes
Summary
The integrity of DNA is constantly being challenged by exogenous DNA-damaging agents and by endogenously produced radicals, stalled replication forks and by spontaneous formation of abasic sites in DNA. Mammalian cells have evolved a number of repair systems to deal with these various types of DNA damage. Defects in one of these DNA repair mechanisms can result in increased cell death and genomic instability leading to disease such as cancer [3]. Differentiated cells are still transcriptionnally active and there is a need to maintain genome integrity of the transcribed genome through the life span. DNA repair activities are needed to protect cells from death and to ensure tissue homeostasis. It has been previously described that differentiated cells use specific strategies such as increase efficiency of the repair of active genes to ensure the maintenance of the transcriptionnally active domains in the presence of bulky adducts and photoproducts [4,5]. Not all DNA repair systems are coupled to transcription, and to date NER (the pathway involved in the reparation of these lesions) is the only pathway for which transcription coupled repair has been formally demonstrated
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