Current Status of Radiation Sensitization by Fluoropyrimidines

Introduction

The fluoropyrimidines fluorouracil(Drug information on fluorouracil) (5-FU) and floxuridine (5-fluoro-2'-deoxyuridine, FdUrd [FUDR]) are the most widely used radiation sensitizers in clinical practice. A large number of retrospective and prospective studies have suggested that the combination of fluorouracil and radiation is a more efficacious treatment for many malignancies (particularly gastrointestinal cancers) than either modality alone, and it has been hypothesized that the benefit from this combination is due to radiosensitization. In addition, the related nucleoside floxuridine has been used in combination with low-dose whole liver irradiation and with high-dose partial liver irradiation in the treatment of patients with intrahepatic malignancies.

In this article, we will briefly summarize laboratory studies aimed at determining the mechanism of fluoro- pyrimidine-mediated radiosensitization. (for more detailed reviews, readers are directed to references 1-3). We will then focus on clinical studies demonstrating the efficacy of the combination of radiation and fluoropyrimidines.

Fluoropyrimidine Cytotoxicity

DNA-Mediated Mechanisms

Fluorouracil and floxuridine are analogs of uracil and deoxyuridine, respectively (Figure 1). These compounds are metabolized to form fluorodeoxyuridine monophosphate (FdUMP) (Figure 2), which is ultimately responsible for the DNA-directed effects of both drugs. Fluorodeoxyuridine monophosphate, along with a folate cofactor, binds to and irreversibly inhibits thymidylate synthase (TS), the enzyme responsible for the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). Thymidylate synthase inhibition leads to depletion of the product dTMP (and, ultimately, thymidine triphosphate) and to the accumulation of the substrate dUMP and other deoxyuridine nucleotides, particularly deoxyuridine triphosphate (dUTP). The resultant increase in the ratio of dUTP to deoxythymidine triphosphate (dTTP) results in misincorporation of dUTP into DNA. The importance of dUTP levels in cytotoxicity is suggested by the finding that human colon cancer cells transfected with the dUTPase gene, which express increased enzyme activity, are relatively protected from floxuridine cytotoxicity [4].

Prolonged TS inhibition leads ultimately to DNA fragmentation and cell death. Two patterns of DNA fragmentation have been noted. The first involves the production of a wide spectrum of fragments with molecular weights ranging from 50 Kb to 5 Mb, and is associated with a morphologic pattern consistent with necrosis. The second pattern results in discrete fragments weighing between 50 and 200 Kb. It resembles programmed cell death (ie, apoptosis), and suggests that the formation of oligosomal ladders is not an essential part of this phenomenon [5].

RNA-Mediated Mechanisms

Whereas clinically achievable concentrations of floxuridine produce only DNA-mediated cytotoxicity, fluorouracil can also kill cells by RNA-dependent mechanisms. Flurouracil can be metabolized to fluorouracil monophosphate (FUMP) and ultimately to fluorouracil triphosphate (FUTP), which is incorporated into RNA in place of UTP. This misincorporation of FUTP can affect several critical aspects of messenger RNA (mRNA) function (including transcription, translation, and splicing).

The relative importance of DNA- vs RNA-mediated cytotoxicity varies considerably from one cell line to another. Leucovorin can act as a folate cofactor and increase the binding of fluorouracil to TS (see above), which tends to increase fluorouracil's DNA-directed actions.

Fluoropyrimidine-Radiation Interactions

Initial cell culture studies with fluorouracil suggested that sensitization occurred when cells were exposed to cytotoxic drug concentrations after irradiation [1]. Other investigators have studied floxuridine, with its single mode of action rather than the more complex fluorouracil with its RNA- and DNA-directed actions. Floxuridine is a potent radiosensitizer of human colon cancer cells [6,7] (Figure 3). Floxuridine exposure prior to radiation produces significantly greater radiosensitization than treatment after irradiation.

Radiosensitization by floxuridine is related to TS inhibition, and is accompanied by both a decrease in DNA double-strand break [6] and sublethal damage repair [8]. These findings probably also describe fluorouracil-mediated radiosensitization, since it appears to derive from the drug's DNA-directed effects [9]. Also, leucovorin potentiates fluorouracil radiosensitization [2], presumably by increasing TS inhibition.

Various mechanisms have been proposed for fluoropyrimidine-mediated radiosensitization. These include:

1. Alterations in the nucleotide pools;
2. Redistribution of cells to a relatively radiosensitive phase of the cell cycle (ie, cells accumulate at the relatively radiosensitive G1/S boundary following exposure to floxuridine); and
3. Fluorodeoxyuridine triphosphate incorporation into DNA.

Although each of these proposed mechanisms may play a role, none alone explains fluoropyrimidine-radiation interactions.10 Furthermore, recent evidence11 is inconsistent with the hypothesis that cytotoxicity and radio- sensitization are closely correlated [2,12]. We recently proposed that sensitization depends on the inappropriate progression of cells through the G1/S boundary and into S phase during exposure to fluoropyrimidines [10,11]. Research into the mechanisms of fluoropyrimidine-induced cytotoxicity and sensitization is driven by the need to develop methods of overcoming clinical drug resistance.