Cc. Zhang et al., AN INHIBITOR OF GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE IS SELECTIVELY CYTOTOXIC TO CELLS THAT LACK A FUNCTIONAL G1 CHECKPOINT, Cancer chemotherapy and pharmacology, 41(3), 1998, pp. 223-228
Purpose: 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). Met
hods: Cytotoxicity was determined by clonogenic assays, and cell cycle
perturbations by flow cytometry. Ribonucleotide pools were measured b
y anion exchange high-pressure liquid chromatography, and DNA strand-b
reaks were determined by alkaline elution and by the TUNEL assay. Resu
lts: When cells were maintained in standard tissue culture medium, whi
ch contained 2.2 mu M folic acid, AG2034 was cytostatic in all the cel
l 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 mu M). In contrast, AG20
34 at 10 nM or 100 nM killed all the cells in cultures of HeLa/S3 or S
W480 cells, which lack a functional G1 checkpoint. Flow cytometry stud
ies indicated that in G1 checkpoint-competent cells, AG2034 caused a G
1 arrest. Those cells (up to 50%) that were already in S phase died, b
ut the cells that were in G1 arrest maintained viability, based upon c
lonogenic 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 b
y alkaline elution or by the dUTP end-labelling technique, indicated n
o DNA strand-breaks 24 h after AG2034 treatment, indicating that purin
e nucleotide depletion can trigger the G1 checkpoint in the absence of
DNA damage. Conclusion: Purine depletion causes slow cell death in ce
lls that have passed the G1 checkpoint, but cytostasis in cells that a
re arrested at the G1 checkpoint. The GARFT inhibitor, at physiologica
l folate concentrations, thus causes selective cytotoxicity to cells l
acking a functional G1 checkpoint.