Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells

Abstract

Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin-C (MMC) on FA patient-derived lymphoblastoid cell lines from groups FA-A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC-induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well-known role in replication fork stalling during S-phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen. 56:457-467, 2015. © 2015 Wiley Periodicals, Inc.

Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia

“…The mutant FANCA cDNAs (p.His913Pro and p.Arg951Gln/Trp) were generated by site-directed mutagenesis using specific primers (available upon request) and transfected, using calcium phosphate, in 293T cells treated with 2 mM hydroxyurea (HU) for 24 hours. 17 Protein whole and fractionated cell extracts were prepared using M-PER™ Mammalian Protein Extraction Reagent and NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), respectively. Primary antibodies were used as follows: anti-FANCA (Savino et al ., 18 1:500), anti-FANCD2 (Santa Cruz, sc-20022, 1:500), add anti-HSP90 (Santa Cruz, sc-7947, 1:4000), anti-ORC2 (Abcam, ab68348; 1:500), anti-FLAG (OctA Probe H5, Santa Cruz, 1:500), and anti-α Tubulin (Santa Cruz, sc-5286, 1:2000).…”

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

“…The laboratory diagnosis of FA is made by evaluating peripheral blood lymphocytes for chromosomal breakage in the presence of a clastogen, such as mitomycin C or diepoxybutane (Esmer et al , ; Auerbach, ; Molina et al , ). Patients with FA have significantly increased numbers of chromosome breaks and radial forms when compared with controls.…”

Germ‐Line TP53 Mutation in an Adolescent With CMML/Atypical CML and Familiar Cancer Predisposition

“…Consistently, cell lines that harbor Fanconi Anemia mutations (ie, DNA repair mutations) are often resistant to HU treatment. 16 In conclusion, we described here a peculiar case of unclassifiable MDS/MPN which is however suggestive of CMML/atypical CML. We hypothesize that the occurrence of his leukemia was related to a germline TP53 mutation, which was also found in his first grade cousin, who developed AML, in his uncle, who developed follicular lymphoma and intra-pancreatic mucinous neoplasm, and his father, who is at present clinically silent, thus indicating an incomplete penetrance.…”

“…Effective DNA repair is crucial in order to kill tumor cells with HU. Consistently, cell lines that harbor Fanconi Anemia mutations (ie, DNA repair mutations) are often resistant to HU treatment 16 …”