Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells

Abstract

Embryonic stem cells (ESCs) need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT). We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs) phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21(Waf1), a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21(Waf1). The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development.

Enabling large-scale genome editing by reducing DNA nicking

“…We transfected HEK 293T cells with plasmids expressing pCas9_GFP and LINE-1 30 targeting gRNAs to disrupt the two key enzymatic domains of ORF-2: endonuclease (EN) and reverse transcriptase (RT) ( Fig. 2A and table S4).…”

“…Clone K was the highest edited stable clonal population and its targeted CàT mutation frequency from day 11 to 30 was confirmed. 30 By subjecting the top edited single cell isolate Clone K to another round of nCBE4-gam editing ( fig. S7A) we detected cells with up to 36.26 % Cà T nucleotide conversion on day 2, and four living clones were isolated with mutation frequencies ranging from 2.43% to 5.04%corresponding to about 643 to 1315 edits ( fig.…”

“…S10). 30 The nucleotide composition of all bases in the gRNA and PAM are displayed for the highest edited clone and parental 293T control for each BE condition used, showing very low nonspecific nucleotide conversions for both nBEs and dBEs at LINE-1 ( fig. S11).…”

“…These DSBs contribute to its high genome-editing efficiency by potently triggering endogenous processes that repair them with random or user-specified variations, but high numbers of concurrent DSBs overwhelm these processes and cause cell death. Recently, however, two types 30 of CRISPR/Cas9 "base editors" (BEs) were developed (table S1) by fusing variants of Cas9 that are either "dead" (dCas9; both nuclease domains inactivated) or "nicking" (nCas9; one nuclease domain inactivated), in which the DSB-generating nuclease domains are disabled, to a nucleotide deaminase. Cytidine base editors (CBEs: either dCBEs or nCBEs (18)) employ cytidine deaminases and convert C:G base pairs to T:A, while adenine base editors (ABEs: either dABEs 35 or nABEs(19)) use adenine deaminases and convert A:T base pairs to G:C. Using properly designed gRNAs, C->T conversions may be used to generate stop codons to knock-out protein coding genes of interest (14).…”

Full biological characterization of human pluripotent stem cells will open the door to translational research

“…Treatment with camptothecin, a topoisomerase 1 inhibitor, induces DSB during S-phase, thereby promoting apoptosis via caspases 3 and 9. Furthermore, ATM and DNA-PKcs, which act complementary and are necessary to avoid errors during DNA repair (Martin et al 2012), are translocated to sites of DSB and activate downstream signaling cascades (γH2AX foci, p53 phosphorylation and stabilization)-indicating functional PI3KK pathways (Garcia et al 2014).…”