Heightened Activation of the p53-ATM Axis in Fanconi Anemia Mutant-Derived Primary Cells: Implications for Carcinogenesis.

Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects and cancer, particularly leukemia and squamous cell carcinomas involving the cervix or head and neck. Cancer predisposition suggests that at least one function of the FA gene products is tumor suppression, however, the molecular basis for this is unclear. FA gene products promote chromosome integrity, and p53-mediated apoptosis is known to provide a crucial tumor suppressive mechanism to eliminate cells susceptible to genomic instability. Consequently, we have sought to analyze the integrity of the p53-ATM axis in primary (untransformed) fibroblasts derived from FA-A and FA-C patients or Fanca-null mouse embryonic fibroblasts (MEFs). We found that in FANCA (two different) or FANCC-mutant primary fibroblasts, p53 expression, Ser15 phosphorylation of p53, ATM expression and phosphorylation were all upregulated following irradiation or mitomycin C treatment, when compared to identically treated isogenic mutant cells transduced with wild-type FANCA or FANCC. ATM is activated in response to DNA double-strand breaks, but the sequence and mechanism of its activation are only partially understood. Checkpoint kinase-2 (CHK2) expression and phosphorylation were not significantly different, nor were there consistent differences in putative upstream regulators of ATM activation such as MRE11/RAD50/NBS1, 53BP1, MDC1, or TRF2. To extend our analysis of the p53 axis to primary murine cells, we first transduced human p53 into Fanca-null MEFs, with the intent of creating cells with defined basal levels of p53 (since endogenous murine p53 was nearly absent in the MEFs). As in the human patient fibroblasts, p53 expression and Ser15 phosphorylation of p53 were markedly upregulated in Fanca −/− MEFs transduced with human p53 when compared to Fanca +/+ MEFs transduced with human p53. These latter experiments also imply that upregulation of p53 levels may be due to enhanced protein stability rather than transcription. In previous work, we had reported that FANCC-mutant primary fibroblasts transduced with wild-type FANCC were at least 10-fold less prone (than parental cells) to form SV40 virus-induced transformation foci. By contrast, our present data, which indicate p53-ATM upregulation in mutant cells, suggest intact tumor suppressor proapoptotic pathways in mutant cells responding to DNA damage. Acquired functional (qualitative) p53 defects or defects in p53-independent mechanisms of tumor suppression may therefore underlie carcinogenesis in FA.