title: 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.
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. alfrodriguezg@yahoo.com.|Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, D.F., México. alfrodriguezg@yahoo.com.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Rodríguez
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. ledactorres@gmail.com.
authors.forename: Leda
authors.initials: L
authors.lastname: Torres
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. ehatlujf@gmail.com.
authors.forename: Ulises
authors.initials: U
authors.lastname: Juárez
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. biol.deivid@gmail.com.
authors.forename: David
authors.initials: D
authors.lastname: Sosa
authors.affiliation: Instituto de Ecología, Universidad Nacional Autónoma de México, D.F., México. emazpeitia@gmail.com.|C3, Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México, D.F., México. emazpeitia@gmail.com.|Current address: INRIA, Virtual Plants Project Team, UMR AGAP, Montpellier, France. emazpeitia@gmail.com.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Azpeitia
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. benildirin@yahoo.com.mx.
authors.forename: Benilde
authors.initials: B
authors.lastname: García-de Teresa
authors.affiliation: Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, D.F., México. cobe@xanum.uam.mx.
authors.forename: Edith
authors.initials: E
authors.lastname: Cortés
authors.affiliation: Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, D.F., México. arom@xanum.uam.mx.
authors.forename: Rocío
authors.initials: R
authors.lastname: Ortíz
authors.affiliation: Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., México. anamsm@biomedicas.unam.mx.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Salazar
authors.affiliation: Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., México. ostrosky@unam.mx.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ostrosky-Wegman
authors.affiliation: C3, Centro de Ciencias de la Complejidad, Universidad Nacional Autónoma de México, D.F., México.|Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., México.
authors.forename: Luis
authors.initials: L
authors.lastname: Mendoza
authors.affiliation: Laboratorio de Citogenética, Departamento de Investigación en Genética Humana, Instituto Nacional de Pediatría, D.F., México. sarafrias@biomedicas.unam.mx.|Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, D.F., México. sarafrias@biomedicas.unam.mx.
authors.forename: Sara
authors.initials: S
authors.lastname: Frías
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D003720:Densitometry; D005199:Fanconi Anemia / Q000473:pathology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D053263:Gene Regulatory Networks / Q000187:drug effects; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D009154:Mutation / Q000235:genetics
chemical_list: D016685:Mitomycin