Decrease in Abundance of Apurinic/Apyrimidinic Endonuclease Causes Failure of Base Excision Repair in Culture-Adapted Human Embryonic Stem Cells

Abstract

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in c-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling. STEM CELLS 2013;31:693-702 Disclosure of potential conflicts of interest is found at the end of this article.

Mutation Frequency Dynamics inHPRTLocus in Culture-Adapted Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Correspond to Their Differentiated Counterparts

“…of decreases in DNA maintenance associated with long-term cultivation [30] or whether other mechanisms projected to increase MF and the high MF-associated mutations are the reason for the DNA maintenance decrease in hESCs. Considering that similar effects of increases in MF up to 50% were achieved with the inhibition of APE1 in hESCs (MOX or anti-APE1 siRNA; Figs.…”

“…Interestingly, BER appears to be important not only for the amelioration of base damage but also to trigger checkpoint control mechanisms by releasing double-stranded breaks as BER-mediated clustered damage repair intermediates [30]. Thus, BER failure not only increases the base damage burden but also renders checkpoints less sensitive.…”

“…Inhibition/downregulation of apurinic/apyrimidinic endonuclease Either methoxamine (MOX; Sigma-Aldrich; 80 mM) or siRNA against apurinic/apyrimidinic endonuclease (APE1) (according to Dvorak et al [28] and Krutá et al [30]) was used to inhibit or downregulate APE1. MOX was added to the medium 1 h prior to IR treatment and was left for 24 h. siRNA treatment was performed 48 h prior to the standard HPRT assay.…”

“…Immunocytochemical analyses to determine the level of APE1 using rabbit polyclonal anti-APE/Ref1 (Trevigen) and anti-rabbit Alexa 594-conjugated secondary antibody (Invitrogen) were performed as previously described [30]. Rabbit monoclonal anti-Nanog (1:200, 4903S; Cell Signaling) and mouse monoclonal anti-Oct4 (1:250, sc-5279; Santa Cruz Biotechnology) were used.…”

Low dose effects of ionizing radiation on normal tissue stem cells

“…A mechanism involving the down-regulation of Apurinic Endonuclease 1 (APE1) and subsequent failure of BER has been described to explain this phenomenon in cultured human ESCs [105]. The authors reported that a decrease in the efficiency of the BER pathway meant that oxidative base damage was not converted by glycosylases to DNA DSBs [106;107] and therefore caused an accumulation of damage.…”