Commentary (Blackstock/Tepper): Current Status of Radiation Sensitization by Fluoropyrimidines

The authors present a thorough review of the mechanisms and clinical effects of fluoropyrimidine radiation sensitization. As they discuss, abundant clinical data from multiple tumor sites show improved outcome when radiation is delivered in combination with fluoropyrimidines. Although strong evidence exists in favor of a sensitizing effect, it is not certain from in vivo data whether this improvement indicates an additive effect of two independent therapies or an interaction between the two. However, we agree with the authors that the data support an interaction between radiation and fluoropyrimidines.

Several in vitro studies [1,2] have shown that when radiation and fluorouracil are given concurrently, the observed cytotoxicity is much greater than would be expected if the agents were acting independently. These studies have also suggested that the timing of the combination is critical, further supporting the possibility of an interaction between the agents. Our laboratory (unpublished data) and other researchers have observed through in vitro studies that the increase in cell kill seen with combined-modality therapy is lost if there is more than a 2-hour delay after administering the first agent. Although timing may be critical in obtaining a combined-modality effect, most clinical studies have paid little, if any, attention to the timing of fluoropyrimidine administration relative to radiation delivery.

Mechanism of Interaction Explored

One major area of research has been directed toward understanding the mechanism of interaction between radiation and the fluoropyrimidines. Efforts in this area have been complicated by the fact that although both fluorouracil and floxuridine (FUDR) have been investigated, these two fluoropyrimidines can have different mechanisms of action. As the authors point out, floxuridine produces only DNA-mediated cytotoxicity, while fluorouracil can also kill cells by RNA-dependent mechanisms. However, since fluorouracil is the agent used clinically for all infusion studies except hepatic therapy, an understanding of the mechanism of fluorouracil is the most germane to clinical practice.

Several researchers have observed that enhanced cell kill occurs only if radiation precedes the administration of fluorouracil [3,4]. This suggests a sensitization to fluorouracil killing by radiation, rather than a sensitization to radiation killing by fluorouracil. Recent in vivo fluorine-19 nuclear magnetic resonance (NMR) experiments have provided a method to obtain additional data on the interaction between these two agents. Using fluorine-19 NMR spectroscopy, we have found that tumor clearance of fluorouracil was delayed in tumors immediately pretreated with a single dose of ionizing radiation [3], compared with those treated with fluorouracil alone. This finding suggests that radiation, by prolonging tumor exposure to active fluorouracil, may be acting as a chemotherapy enhancer. Although Byfield has previously proposed that radiation is sensitizing cells to fluorouracil cytotoxicity, his mechanism of interaction differs from the one we propose.

Importance of Apoptosis Recognized

As the authors note, it is well known that fluoropyrimidines bind to and inhibit thymidylate synthase (TS). This enzyme catalyzes the central reaction in the de novo synthesis of thymine nucleosides and nucleotides, and its inhibition triggers their depletion. The shortage of thymine DNA precursors, the cessation of DNA synthesis, and the simultaneously ongoing RNA and protein synthesis have been viewed as unbalanced growth and the basis for cell kill, or "thymineless death" [5].

Inhibition of TS may not be the only DNA-associated event, however. Recently, researchers have linked thy-mineless death to programmed cell death (apoptosis). Only recently has the importance of apoptosis as a cellular response to cancer therapeutic modalities, such as radiation and chemo- therapy, been recognized. This interest has been heightened by the observation that apoptosis is highly regulated at the biochemical and molecular level, raising the possibility that the apoptotic response may be subject to external modulation for therapeutic gain.

Apoptosis has been documented in a variety of tumor types in response to treatment with certain chemotherapeutic drugs and/or radiation. For example, it is possible to induce apoptosis in rat prostate adenocarcinoma cells (AT-3) after exposing the cells to a 100-mm concentration of floxuridine [6]. Other researchers have induced programmed cell death in experiments that involve in vitro and in vivo exposures to fluoropyrimidines [7]. Thus, under certain clinical situations, apoptosis could be a relevant mechanism of cell death with fluorouracil and radiation.

Continuous-Infusion vs Bolus Fluorouracil

This review also points out that a recent adjuvant rectal cancer trial has shown an advantage of continuous-infusion over bolus fluorouracil. A number of reasons for this improvement have been postulated, such as simply more total drug exposure. However, it is likely that the manner of cell kill is different with long-term continuous-infusion therapy than with bolus fluorouracil. Clinically, the side effects seen following the administration of bolus fluorouracil are different from the toxicity seen with continuous-infusion therapy. In addition, in vitro data in certain systems have demonstrated that cells that are resistant to killing by bolus fluorouracil can still retain sensitivity to prolonged fluorouracil exposure [8].

RNA-associated cytotoxicity may be an important cause of cell death with continuous-infusion fluorouracil, in addition to the expected TS inhibition. This observation is strengthened by data showing that fluorouracil incorporation into RNA is a time- and concentration-dependent process [9]. These data suggest that continuous-infusion fluorouracil is not simply "more TS inhibition."

The data reviewed by the authors clearly demonstrate the beneficial clinical effects seen when fluorouracil and radiation are used together. Although the data we present above suggest a mechanism different from those usually proposed, both the authors and we have tried to show that the mechanism of this interaction is unknown. A better understanding of the mechanism of the interaction will further enhance our use of these two agents in combination during routine clinical practice.

References:

1. Byfield JE, Calabro-Jones P, Klisak I, et al: Pharmacologic requirements for obtaining sensitization of human tumor cells in vitro to combined 5-fluorouracil or ftorafur and x-rays. Int J Radiat Oncol Biol Phys 8:1923-1933, 1982.

2. Weinberg M, Lapointe T, Rauth A: Growth delay in a murine squamous cell tumor after local radiation and concurrent infusional 5-fluOrouracil treatment. Int J Radiat Oncol Biol Phys 12(8):1449-1452, 1986.

3. Blackstock AW, Kwock L, Tepper JE, et al: Using nuclear magnetic resonance spectroscopy to observe the tumor retention of 5-FU in vivo. Int J Radiat Oncol Biol Phys (submitted for publication).

4. Hughes LL, Luengas J, Rich TA, et al: Radiosensitization of cultured human colon adenocarcinoma cells by 5-fluorouracil: Effects on cell survival, DNA repair, and cell recovery. Int J Radiat Onc Biol Phys 23:983-991, 1992.

5. Rueckert RR, Mueller GC: Studies on unbalanced growth in tissue culture: I. Induction and consequences of thymidine deficiency. Cancer Res 20:1584-1591, 1960.

6. Kyprianou N, Issacs JT: 'Thymineless' death in androgen-independent prostate cancer cells. Biochem Biophys Res Commun 165:73-81, 1989.

7. Sakaguchi Y, Stephens LC, Makino M, et al: Apoptosis in normal tissue induced by 5-FU: Comparison between bolus injection and continuous infusion. Anticancer Res 14(4a):1489-1492, 1994.

8. Sobreoro AF, Aschele C, Allessandra P, et al: Synergism and lack of cross-resistance between short-term and continuous exposure to fluorouracil in human colon adenocarcinoma cells. J Natl Cancer Inst 85(23):1937-1944, 1993.

9. Spears CP, Shani J, Shahinian AH, et al: Assay and time course of 5-fluorouracil incorporation into RNA of L1210 ascites cells in vivo. Mol Pharmacol 27:302-307, 1985.