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 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: gj.peters@vumc.nl