Abstract
As the source of all cells in the developing embryo proper, embryonic stem cells (ESC) bear the unique responsibility to prevent mutations from being propagated throughout the entire organism and the germ line. It is likely for this reason that ESC and induced pluripotent stem cells (iPSC) maintain a dramatically lower mutation frequency than cultured somatic cells. Multiple mechanisms for this enhanced genomic surveillance have been proposed, including hypersensitivity of DNA damage response signaling pathways and increased activity of error-free DNA repair pathways, such as homologous recombination. However, the effect of loss of function of DNA repair pathways in these cells remains poorly understood. The Fanconi Anemia (FA) pathway is a DNA repair pathway that is required for the repair of DNA interstrand crosslink damage and also promotes repair of DNA double-strand breaks by homologous recombination . Genetic defects in this pathway cause a disease characterized by bone marrow failure and extreme cancer incidence. Several recent studies have revealed that the FA pathway is required for efficient somatic cell reprogramming to iPSC and suggest that FA cells undergo cell death during this process. Another recent study found that the growth of FA patient-specific iPSC was attenuated with a G2/M arrest when compared to control iPSC, suggesting that these cells arrest upon failed DNA repair. In this study, we sought to determine the effects of acute loss of function of the FA pathway in iPSC through the generation of FA patient-derived iPSC with inducible complementation of the defective FA gene.
Fibroblasts were cultured from skin biopsies of multiple FA patients and transduced with a lentiviral vector expressing the complementing FA gene product under DOX-inducible control. Cells were then reprogrammed to iPSC using episomal transfection. These cells formed iPSC colonies only when reprogramming was carried out in the presence of DOX, confirming that the FA pathway is required for efficient reprogramming. Once cell lines were obtained, DOX-dependent FA functionality was verified based on FANCD2 monoubiquitination and nuclear focus formation after treatment with DNA damaging agents. We then cultured the iPSC for extended periods of time in the presence and absence of DOX. Interestingly, the cultures underwent profound cell death and cell cycle arrest within 7 days of DOX-withdrawal and completely failed to expand after one passage. EdU cell cycle analysis confirmed cell cycle arrest in the G2/M phase. Furthermore, cleaved caspase 3 staining confirmed that the number of apoptotic cells increased by 3-fold in the -DOX culture. Despite these effects, cells cultured in both the presence and absence of DOX formed teratomas in nude mice, thus indicating the maintenance of full differentiation capacity in the absence of the FA pathway. In order to determine the mechanisms underlying G2/M arrest and cell death, expression of p53 and its target genes was detected by both western blot analysis and qRT-PCR. Only a slight increase in p53 activation was observed by 7 days post DOX-withdrawal. Furthermore, knockdown of p53 resulted in rescue from apoptosis to normal levels but not rescue from cell cycle arrest. Increased ATM and ATR DNA damage sensor kinase activities were also detected in –DOX cells, concominant with increased phosphorylation of the ATM-target Chk2 and reduced abundance of the G2/M checkpoint protein CDC25A. These results reveal hyperactive DNA damage responses upon FA loss which may underlie the attenuated cell cycle progression of FA-iPSC independent of p53. Remarkably, effects in this FA model system appear equivalent to those responsible for the depletion of HSC in the bone marrow of FA patients. Thus, iPSC models may be useful for future studies of the mechanisms underlying FA stem cell arrest and for the development of therapeutics that alleviate these phenotypes.
Disclosures
No relevant conflicts of interest to declare.
Design, Synthesis and Pharmacological Evaluation of Three Novel Dehydroabietyl Piperazine Dithiocarbamate Ruthenium (II) Polypyridyl Complexes as Potential Antitumor Agents: DNA Damage, Cell Cycle Arrest and Apoptosis Induction