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

Abstract

Background: The 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.

Methods: We 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.

Results: Computational 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.

Conclusions: Our 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.

Fig. 1

The latest FA/BRCA network. In response to an ICL, the FA/BRCA network responds by blocking the cell cycle through the ATR and ATM checkpoint kinases and their downstream target p53. Similarly, the FA core complex (FAcore) becomes activated and ubiquitinates FANCD2I complex, which in turn recruits DNA endonucleases (NUC1 and NUC2). These endonucleases unhook the ICL generating a DNA adduct (ADD) and a double strand break (DSB). Translesion synythesis (TLS) takes over the ADD while the DSB can be rejoined either by FA/BRCA-dependent Homologous Recombination (FAHRR), FA/BRCA-independent Homologous Recombination (HRR2), or by the error prone Non-Homologous End-Joining (NHEJ) pathways. Finally, we predict that the CHKREC node, composed by the G2/M transcriptional program and checkpoint recovery proteins, turns off the checkpoint and DNA repair proteins. Rectangles represent proteins or protein complexes, pointed arrows are positive regulatory interactions, and dashed lines with blunt arrows are negative regulatory interactions. Readers may refer to [38] for a more detailed description of the FA/BRCA pathway

Fig. 2

FA network simulations. a The current information regarding the FA/BRCA pathway have not uncovered the mechanism that allows the resolution of the G2/M checkpoint after DNA damage and further cell division. b Trajectories and attractor of the wild type FA/BRCA network under an ICL pulse. In this simulation wild type cells repair DNA damage through the FA/BRCA pathway and arrive to CCP attractor after activating the CHKREC node once the damage has been fixed. The inclusion of the CHKREC node, as a checkpoint negative regulator, allows to explore the mechanisms behind cell division after checkpoint resolution. c In response to a continuous ICL DNA damage, wild type cells arrive to a CCA attractor with activation of the checkpoint and DNA damage repair nodes,the CHKREC node becomes eventually activated in this attractor. d Under and ICL pulse FAcore mutant cells activate the NHEJ pathway to repair DNA damage and arrive to a CCP attractor. e In response to a continuous ICL DNA damage, FAcore mutant cells concomitantly activate the checkpoint and the CHKREC nodes. Node names are indicated at the topmost row. The leftmost column indicates simulation time steps in arbitrary units. Time steps corresponding to trajectories are indicated and time steps corresponding to attractors are indicated by shaded gray and “ATT”. For illustrative purpose cyclic attractors are represented twice

Fig. 3

FA cells arrest at G2 phase in response to MMC. a Flow cytometry analysis showing accumulation of FA cells in G2 in response to MMC (left panel) and diminished number of FA mitotic cells compared to normal cells (right panel). b FA cells activate CHK1 kinase in response to MMC treatment. c FA cells increase the expression of p21 mRNA as showed by qRT-PCR analysis (n = 3 independent experiments, p < 0.05)

Fig. 4

FA cells divide despite MMC treatment and cell cycle arrest. a The number of mononucleated cells (upper panel), binucleated cells (middle panel) and tetranucleated cells (bottom panel) was quantified after exposure to MMC (24 h) and Citochalasin B (48 h). The number of basal binucleated and tetranucleated cells was counted without Citochalasin B treatment and rested from the total number (data not shown). b Despite G2 arrest FA cells arrive to mitosis and divide with unrepaired DNA damage as demonstrated by two independent DNA damage analyses, increased chromosome aberrations (upper panel) and increased micronuclei in cells that have reached one division (bottom panel) (n = 3 independent experiments, p < 0.05)

Fig. 5

FA cells have a gene expression pattern compatible with checkpoint resolution despite DNA damage. Gene expression analysis of the genes belonging to the G2 transcriptional program did not show differences in the expression of these genes despite MMC treatment. Cyclin A2 (a), Cyclin B1 (b), WIP1 (c), FOXM1 (d) and PLK1 (e) (n = 3 independent experiments). No statistically significant differences were found in the gene expression patterns among groups

Fig. 6

FA and normal cells co-express checkpoint and CHKREC proteins. a Western Blot analysis of checkpoint proteins. b Western blot analysis of CHKREC proteins. FA cells increase the amount of some G2 blockage proteins, but have a reduction in others. Although CHK1 (Fig. 4 b) and MYT1 show increased signal, WEE1, γH2AX and p21 protein appear as diminished in FA cells, this weakens the checkpoint blockage, which is eventually overwhelmed by CHKREC signaling (n = 3 independent experiments, see also Fig.7)

Fig. 7

Western Blot densitometry analysis. Checkpoint proteins (a–e) and CHKREC proteins (f–i). (n = 3 independent experiments). No statistically significant differences were found in the protein expression patterns among groups

Fig. 8

CHKREC components activation in normal and FA cells. After DNA damage induction, the cell activates the DNA damage integrity checkpoints, the G2/M checkpoint specifically avoids the transition of the cell from G2 to M phase with unrepaired DNA damage. Once the DNA has been repaired, the G2/M checkpoint is satisfied and the cell activates the CHKREC, a process that inactivates checkpoint proteins and promotes cell division. Upper panel. Normal cells activate CHKREC with repaired chromosomes. Bottom panel. FA cells activate CHKREC despite unrepaired chromosomes. The specific mechanism triggering this inappropriate CHKREC activation in FA cells remains unknown