The mechanism of action of TNP-470, which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21WAF1/CIP1, respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21WAF1/CIP1 by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase -- the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid -- showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced following TNP-470 treatment. Addition of guanosine at the start of G1 phase causes an doubling in GTP levels that persists to the G1/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather a restoration of a drug-induced GTP deficit. The mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.
- Received April 8, 2010.
- Revision received June 19, 2010.
- Accepted June 21, 2010.
- The American Society for Pharmacology and Experimental Therapeutics