ag-2034 and purine

4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non-tumorigenic prostatic epithelial cells, RWPE-1.. Cells were cultured in medium containing 10 nM 5-methyl-tetrahydrofolate supplemented with/without 1.7 microM hypoxanthine/1.5 microM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. Total ATP was quantified by reverse-phase HPLC and [(14)C]-glycine incorporation and [(3)H]-hypoxanthine conversion into ATP by liquid scintillation counting. Protein expression levels were determined by Western blotting, cell cycle analysis by propidium iodide staining and cell-senescence by beta-galactosidase staining. AG2034 inhibited LNCaP cell proliferation causing death in the absence of hypoxanthine and cytostasis in its presence. However, RWPE-1 cells were resistant to AG2034 when hypoxanthine was present. AG2034 elevates AMP/ATP ratios but is unable to activate AMPK in RWPE-1 when hypoxanthine is present. Drug exposure increased expression levels of p53, p21, p27, and p16 in both cell lines and increased senescence-associated-beta-gal staining in LNCaP with/without hypoxanthine, but primarily in its absence in RWPE-1.. LNCaP cells primarily depend upon de novo while RWPE-1 cells largely favor salvage synthesis for maintenance of their ATP pools. With AG2034 treatment, ATP synthesis via hypoxanthine salvage is insufficient to support growth of LNCaP but enough to restore ATP levels and support RWPE-1 growth. The anti-proliferative effect of AG2034 involves increasing phosphorylation of AMPK. These results indicate that AG2034 activates p53 and AMPK mediating the induction of signaling pathways leading to senescence.

Topics: Adenocarcinoma; Adenosine Triphosphate; AMP-Activated Protein Kinase Kinases; Cell Line; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Epithelial Cells; Glutamates; Humans; Hypoxanthine; Male; Prostate; Prostatic Neoplasms; Protein Kinases; Purines; Pyrimidines; Signal Transduction; Tumor Suppressor Protein p53

We studied the effects of purine depletion on the cell cycle using a specific inhibitor of de novo purine biosynthesis, AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT).. Cytotoxicity was determined by clonogenic assays, and cell cycle perturbations by flow cytometry. Ribonucleotide pools were measured by anion exchange high-pressure liquid chromatography, and DNA strand-breaks were determined by alkaline elution and by the TUNEL assay.. When cells were maintained in standard tissue culture medium, which contained 2.2 microM folic acid, AG2034 was cytostatic in all the cell lines tested. Under low-folate conditions (50 nM folic acid), AG2034 caused up to 50% cell death in cell lines that possessed a functional G1 checkpoint (A549, MCF-7), but was only cytostatic to the remaining cells, even at very high concentrations (100 microM). In contrast, AG2034 at 10 nM or 100 nM killed all the cells in cultures of HeLa/S3 or SW480 cells, which lack a functional G1 checkpoint. Flow cytometry studies indicated that in G1 checkpoint-competent cells, AG2034 caused a G1 arrest. Those cells (up to 50%) that were already in S phase died, but the cells that were in G1 arrest maintained viability, based upon clonogenic assays, for many days. In G1 checkpoint-deficient cells, no G1 arrest was seen after AG2034 treatment, all cells progressed into S phase, and all cells died. Measurement of DNA strand-breaks, either by alkaline elution or by the dUTP end-labelling technique, indicated no DNA strand-breaks 24 h after AG2034 treatment, indicating that purine nucleotide depletion can trigger the G1 checkpoint in the absence of DNA damage.. Purine depletion causes slow cell death in cells that have passed the G1 checkpoint, but cytostasis in cells that are arrested at the G1 checkpoint. The GARFT inhibitor, at physiological folate concentrations, thus causes selective cytotoxicity to cells lacking a functional G1 checkpoint.

Topics: Animals; DNA, Neoplasm; Enzyme Inhibitors; Folic Acid; G1 Phase; Glutamates; HeLa Cells; Humans; Hydroxymethyl and Formyl Transferases; Mice; Phosphoribosylglycinamide Formyltransferase; Purines; Pyrimidines