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5-FU is itself inactive and requires intracellular conversion to form active metabolites. Its three major active metabolites are : 1. 5-fluorodeoxyuridylate (5-FdUMP) , which inhibits thymidylate synthase (TS), the ratelimiting step in DNA synthesis. 2. 5-fluorouridine triphosphate (5-FUTP), which incorporates into RNA and causes alterations in its processing. 3. 5-fluorodeoxyuridine triphosphate (5-FdUTP), which incorporates into DNA instead of deoxythymidine triphosphate(dTTP), the usual aubstrate for DNA polymerase.
5-FU is a cell-cycle-specific agent whose major effects occur during the S phase.
However, it is known that at any one time only 3% of colorectal cancer cells are in the S phase. 5-FU has a short plasma half-life of 10-12 minutes and TS inhibition after a bolus dose is short, with a dissociation half-life of 6 hours. For these reasons, the infusional delivery 5-FU has been developed , with the aim of increasing the binding TS. It is also reported that 5-FU induces G1-S phase arrest, and the cytotoxic effects of 5-FU are attributed to apoptosis via a P53-dependent pathway43. A recent study showed that p53 mutants are recessive to wild-type for growth arrest, but were selectively negatively dominant for induction of apoptosis after DNA damage. However, the relationship between the status of the p53 gene and cell cycle regulation under exposure to 5-FU remains unclear.
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We examined the effects of various concentrations of 5-FU on cell cycle regulation using three human colorectal carcinoma cell lines with or without mutation of the P53 gene in 2001 22. The in vitro study showed that the PMC regimen is based on targeting at least two different phase of the cell cycle. These two different pathways depend on the integrity of the schedule-oriented cell cycle check points ; G2-M-phase arrest and mitotic catastrophe at a lower dose of 5-FU , and G1-S-phase arrest and apoptosis at a higher dose of 5-FU. In our study, G2-M-phase arrest and mitotic catastrophe at a lower dose of 5-FU were induced via a P53-independent pathway. We also found that the14-3-3σgene influenced G2-M-phase arrest and mitotic catastrophe via a P53-independent pathway34 (Fig.1). Actually, marked nuclear cyclin B1 accumulation was observed with low dose exposure in this study , regardless of the status of the p53 gene, possibly reflecting mitotic catastrophe, and apoptotic cells after PMC showed no expression of cyclin B1 , which suggests the coexistence of mitotic catastrophe and apoptosis in the surgical specimens. This result was consistent with the in vitro data. Therefore, enhancement of 5-FU concentration once a week in combination with a lower, sustained level of 5-FU seems to contribute to the drastic cytotoxic effect, and not only the cytoplastic effect, independent of p53 mutation gene. Development of a modified PMC regimen in combination with chemotherapeutic agents that target different checkpoints may heighten the efficacy and broaden the selectivity of 5-FU.
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In our clinical trial , we showed efficacy independent of p53 status, ascertained by cell kinetic analysis in vitro, which may lead to a novel concept of schedule-oriented biochemical modulation of this drug. It will be possible to modify the regimen in accordance with the malignant potential of each cancer case, as a Phase Ⅱ study of PMC plus leucovorin for patients with unresectable and metastatic colorectal carcinoma is ongoing .
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