Cell Cycle Modulation Enhances the Cytotoxicity of Thymidylate Synthase Inhibitors

Thymidylate synthase (TS) is a cell cycle regulated enzyme. It increases during proliferation and has a higher activity during the S-phase in order to provide the cell with sufficient dTTP to facilitate DNA synthesis. Hence inhibition of cyclin dependent kinases (CdK) may lead to a decrease of TS and enhance the inhibition of TS. A number of CdKs control progress of the cell cycle together with checkpoint kinases (ChK1 and ChK2) which are activated by phosphorylation mediated by protein kinases such as protein kinase C (PKC). Both staurosporine (STS) and UCN-01 are inhibitors of PKC, but STS also inhibits CdK2, while UCN-01 inhibits CdK2, 4 and 6 as well as ChK1, cyclin D and pRb. We investigated the interaction between 5-fluorouracil (5FU) and STS or UCN-01 in syngeneic colon cancer cells, either wild type for p53 (LovoB2) or mutated (Lovo175x2). Cell growth inhibition was evaluated using the sulforhodamine B (SRB) test, synergism was evaluated using the multiple drug effect analysis yielding combination indices (CI: synergism < 0.9; antagonism: > 1.1), cell cycle distribution and cell death by FACS analysis, cell cycle proteins by western blots, while TS expression was measured by radioactive assays. 5FU was combined with STS or UCN-01 using simultaneous and sequential schedules. Cytotoxicity of 5FU was enhanced by UCN-01 (LovoB2, CI=0.4; Lovo175x2, CI=0.2) but less for STS (CI=0.8-0.9). At IC80 values, 5FU (5 μM) induced an S-phase arrest (2-3 fold) in both cell lines, 0.5 μM UCN-01 a slight decrease in G2-M arrest but 0.05 μM STS not. 5FU and UCN-01 combinations all decreased G2-M phase. STS and 5FU combinations led to a similar S-phase accumulation as with 5FU. Induction of cell kill after 48 hr by UCN-01 was independent of p53, as it was 2-3 fold higher (25%) in Lovo175x2 cells compared to LovoB2, for STS this was similar for both cell lines (5-10%), as well as for 5FU (2-5%). Combinations of 5FU and STS or UCN-01 resulted in additive cell kill in both cell lines. At a molecular level 5FU caused an increase in TS levels (predominantly as the ternary complex), STS downregulated TS partially, but UCN-01 completely, which was associated with a similar decrease in E2F. STS, UCN-01 and 5FU treatment also decreased TS catalytic activity in both cell lines. 5FU caused a transient appearance of pChK expression at 24 hr which was enhanced by UCN-01 and STS. In conclusion: the effect of 5FU can be enhanced by the cell cycle modulators UCN-01 and STS, by abrogation of either the S or G2M checkpoints. Correspondence : Dr. GJ Peters, Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, the Netherlands; T: +31-20-4442633; F: +31-20-4443844; Email: [email protected]