TOPOISOMERASE I INHIBITOR, CAMPTOTHECIN, INDUCES APOPTOSIS OF HUMAN EMBRYONIC STEM CELLS

CAROLINA GARCIA; GUILLERMO VIDELA RICHARDSON; NICOLAS DIMOPOULOS; DIEGO RIVA; DARÍO FERNÁNDEZ ESPINOSA; LEONARDO ROMORINI; SANTIAGO G. MIRIUKA; GUSTAVO SEVLEVER; MARÍA ELIDA SCASSA

Boston

Congreso; International Society of Stem Cell Research; 2013

ISSCR

Human embryonic stem cells (hESC) possess two unique properties that distinguish them from many other cell types: the ability to self-renew indefinitely in culture under permissive conditions and the capability of giving rise to all cell types of embryonic lineage under the guidance of appropriate developmental cues (pluripotency). Maintaining genomic integrity is vital for stem cells and their function as primary progenitors, since mutations will severely compromise all derived cell lineages. Currently, the mechanisms that protect the genome in rapidly proliferating hESCs are minimally understood. The pathways controlled by the ataxia telangiectasia-mutated (ATM) and ATM-related (ATR) proteins represent one of the main pathways by which cells react to DNA damage in somatic cells. In a previous study, we determined that the ATM checkpoint signaling cascade is intact in WA09 hESCs. Here, we investigated how WA09 hESC line reacts to replication mediated double-strand DNA breaks triggered by the topoisomerase I inhibitor, camptothecin (CPT), and whether this genomic insult evokes DNA repair pathways and/or cell death. Using immunofluorescence microscopy we found that hESCs respond to DNA damage by rapidly inducing caspase-3 activation and PARP-1 cleavage concomitantly with phospho-H2AX foci formation. Western blot analysis revealed that p53 phosphorylation on serine 15 occurs within 3 h after CPT addition. Although a marked and sustained increase in p21WAF1 transcripts was observed by qPCR (5 fold) upon damage, p21WAF1 protein levels were undetectable at all tested time points. We also found that the majority of hESCs were undergoing cell death via caspase-related apoptosis following induction of DNA damage. During the cellular DNA damage response, p53 can either promote cell survival by activating cell cycle arrest and DNA repair to maintain genomic integrity or direct cells to undergo apoptosis to eliminate extensively damaged cells. Our data suggest that following CPT treatment, hESCs initiate a p53-dependent programmed cell death to ensure that its genomic integrity will not be compromised. The absence of p21WAF1 protein to sustain cell cycle arrest supports this premise.