Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects and cancer, particularly leukemia and squamous cell carcinomas involving the cervix or head and neck. Although the FANC proteins have been shown to be involved in DNA repair, the pathogenesis of bone marrow failure and other developmental abnormalities is still unclear, but presumably, reflects abnormalities in senescence and apoptosis. The canonical ATM-p53 pathway is a pivotal mediator of the DNA damage response, which may act as a barrier to cancer through cell cycle and apoptosis regulation but has not been fully studied in FA. To address this, we first analyzed the integrity of the ATM-p53 axis in primary fibroblasts derived from FA patients. By immunoblot assays, we found that expression and phosphorylation of ATM and p53 were all up-regulated in two different FANCA-mutant fibroblasts following irradiation (IR) or mitomycin C (MMC) treatment, when compared to identically treated isogenic mutant cells transduced with wild-type FANCA cDNA. We reasoned that these results might be explained by up-regulation of the basal levels of ATM protein in the mutant fibroblasts. By real-time PCR, we confirmed that ATM gene expression was increased in the mutant line in comparison to the gene-corrected control. We also confirmed the up-regulation of both ATM protein and transcription in a FANCA-mutant EBV-immortalized lymphoblastoid cell line in comparison to its gene-corrected control. In order to determine if these changes in ATM gene expression were directly caused by FANCA depletion, we transfected HCT116 cells (known to be p53 wild-type) with two different siRNAs against FANCA. For these experiments, we used a plasmid expression vector (OriGene Technologies, Rockville MD) that drives 29-mer short hairpin RNAs targeting FANCA. In both transient and stable transfection experiments with either siRNA, we found that knockdown of FANCA expression was associated with down-regulation of ATM expression, as assessed by real-time PCR, when compared to cells transfected with an empty vector control. These results have led us to hypothesize that knockdown of FANCA leads to down-regulation of ATM, whereas cells with mutations in FANCA up-regulate ATM, perhaps as a result of chronic genotoxic stress not seen in the knockdown cells. Up-regulation of the ATM-p53 axis, in turn, may contribute to the hypo-proliferative characteristics seen in both primary fibroblasts and hematopoietic stem cells from FA patients.
Disclosure: No relevant conflicts of interest to declare.