Interactive antiproliferative mechanisms when 5-fluorouracil is combined with cisplatin or ionizing radiation : an in vivo and in vitro experimental study

Abstract: Results from clinical studies and experimental investigations have given evidence of a synergistic effect when 5- fluorouracil (5-FU) has been combined with radiation or cisplatin (CDDP). From earlier experiments performed by the research team we know, that addition of S-FU to CDDP can synergistically increase the antiproliferative effect. Inhibition of DNAsynthesis and passage through G2 are eliminated when 5-FU is combined with CDDP. The frequency of DNA-damage is not increased by the combination and the repair unchanged. From earlier we also know, that addition of 5-FU to radiation abolishes the radiation-induced G2-arrest. The mechanisms underlying the potentiated or synergistic increase of cytotoxicity, when 5-FU is added to CDDP or irradiation, remain unclear. The aim in this project have been to further elucidate these mechanisms. In this study we found, that the combination of CDDP and 5-FU did not cause misincorporation of nucleotides in DNA and that misincorporation was not the reason for synergistic effect of the combination. When 5-FU was added after CDDP in mouse ascites sarcoma cells in vivo the CDDP-induced G2-arrest was eliminated and the inhibition of p34cdc2 was abolished. Change of phosphorylation in Wee1 and Cdc25C could account for change of p34cdc2-activity When 5-FU was added to radiation in pregnant mice there was evidence of synergy in fetal toxicity measured as inhibition of proliferation, excess of malformations and death. 5-FU did abolish radiation-induced G2-arrest. When 5-FU was added to radiation in mouse ascites sarcoma cells in vivo the mitosis regulating enzymes were influenced. The activity of p34cdc2 in total cells decreased unexpectedly. When the nuclear and cytoplasmic fractions were analysed separately, it was evident, that 5-FU mediated normalization of the distribution of cyclin B1 in nucleus and cytoplasm and influenced activity in regulating enzymes, which could provide conditions for formation of functional cyclin B1-p34cdc2-complexes in nucleus and explain why mitosis was initiated. We conclude, that the synergy when 5-FU is added to CDDP is not due to misincorporation of nucleotides in newly synthesized DNA. Furthermore, addition of 5-FU to CDDP or radiation can shorten or eliminate cell cycle arrest in G2 and increase cytotoxicity. 5-FU influences the G2-M checkpoint regulation. Mitosis can be unphysiologically promoted by facilitated transport of cyclin B1 from cytoplasm to nucleus after irradiation in a situation where the cell requires G2-arrest to repair DNA damage. We have also found evidence, that 5-FU can influence the phosphorylation state in checkpoint enzymes regulating activity in the p34cdc2-kinase The results indicate that the influence by 5-FU on the mechanism for initiation of mitosis is central and can explain the synergy when 5-FU is combined with cisplatin or irradiation.