A defect in thep53 response pathway induced by GART inhibition

The class of folate antimetabolites typified by 6R-dideazatetrahydrofolate (DDATHF) are specific inhibitors of de novo purine synthesis due to potent inhibition of glycinamide ribonucleotide formyltransferase (GART). GART inhibitors are rare compounds in that they produce cytotoxicity in tumor cells without incurring detectable DNA damage. As such, they are a test case of the concept that the tumor suppressor p53 senses cellular ribonucleotide depletion in the absence of DNA strand breaks and invokes a p21-dependent G₁ cell cycle arrest in response. This concept was investigated. Cell cycle progression was slowed by DDATHF, but tumor cells with wild type, mutant, or null p53 function entered and completed one S phase in the presence of drug. This inability of p53 to initiate a G₁ arrest after GART inhibition was reflected in the independence of DDATHF-induced cytotoxicity on p53 function, for p53 +/+ and p53 −/− HCT116 cells were equi-sensitive to DDATHF. Interestingly, in response to DDATHF or second and third generation GART inhibitors, p53 accumulated in HCT116, MCF7, and A549 carcinoma cells, but transcription of several p53 targets, including p21, was impaired. The p53 accumulating in these cells was nuclear, but was not phosphorylated at serines 6, 15, or 20, nor acetylated at lysines 373 or 382, residues that are post-translationally modified by DNA-damaging agents. DDATHF-stabilized p53 bound to the p21 promoter in vitro and in vivo and recruited RNA polymerase II, but failed to activate histone acetylation over the p53 binding sites on this promoter, an integral part of the transcriptional response mediated by the DNA damage pathway. Thus, it appears the robust initial response of the p53 pathway to GART inhibition is not propagated due to a defect in p53 post-translational modifications and a failure to open chromatin structure at the p21 promoter; this renders tumor cells functionally p53 null with regard to the G₁ checkpoint. Further clinical utility of the GART inhibitors may be realized in combination with other chemotherapeutic compounds that exploit the generation of a p53 null phenotype or protein kinase inhibitors whose cytotoxicity may be potentiated when administered with a specific ATP-depleting compound.