Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process

Abstract

BackgroundThe FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage.MethodsWe performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot.ResultsComputational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA.ConclusionsOur results show that FA cells, despite extensive DNA damage, do not loss the capacity to express the transcriptional and protein components of checkpoint recovery that might eventually allow their division with unrepaired DNA damage. This might allow cell survival but increases the genomic instability inherent to FA individuals and promotes cancer.

Primary Ovarian Insufficiency Induced by Fanconi Anemia E Mutation in a Mouse Model

“…Efficient and accurate DNA repair is likely to be essential to maintain genomic integrity in germ cells, thereby protecting the coding sequences of the germ line. At least 18 FA proteins (FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q,-S, and UBE2T) and 4 Fanconi-associated proteins (FAAPs; FAAP16, FAAP20, FAAP24, and FAAP100) are involved in the FA pathway [ 8 , 9 , 27 ]. FANCE is a component of the FA core complex (composed of 8 FA proteins and 2 FA-associated proteins), and activation of this complex controls the FA pathway.…”

The genomics of inherited bone marrow failure: from mechanism to the clinic

“…The FA/BRCA DNA repair pathway functions in repair of DNA interstrand crosslinks (ICLs), covalent links between strands of DNA that create an obstacle to DNA replication and transcription. ICLs are produced by endogenous chemicals, such as reactive aldehydes, and environmental exposures, including natural psoralens and DNA crosslinking drugs, such as cisplatin (Deans & West, ; Rodriguez et al , ). FANCA, ‐B, ‐C, ‐E, ‐F, ‐G, ‐L and ‐M comprise the FA “core complex” which functions as a multi‐subunit E3 ubiquitin ligase.…”

Analysis of polymorphisms in genes associated with the FA/BRCA pathway in three patients with multiple primary malignant neoplasms

“…Whether patients with this molecular subtype are associated with the FA/BRCA pathway requires further examination. The FA/BRCA pathway can regulate DNA damage repair in the body, and defects in expression of genes in this pathway increase genome instability in FA patients and promote cancer occurrence [30]. In contrast, various subtypes in the FA family are all sensitive to DNA cross-linking agents such as mitomycin [31], suggesting that all FA proteins elicit their effects through a common pathway.…”