Gemcitabine (2¢,2¢-difluorodeoxycytidine) is an analog of cytarabine(Drug information on cytarabine) (cytosine arabinoside) with clinical activity against a variety of solid tumors, particularly pancreatic[1,2] and nonsmall-cell lung cancer.[3,4] Gemcitabine(Drug information on gemcitabine) is distinguished from other chemotherapeutic agents by its relatively low toxicity (typically mild fatigue and modest bone-marrow suppression), its broad spectrum of activity against a variety of cancers, and its ability to perturb deoxynucleotide metabolism at clinically achievable concentrations. Gemcitabine must be phosphorylated by deoxycytidine kinase to produce cytotoxicity. The key phosphorylated metabolites are: (1) difluorodeoxycytidine diphosphate (dFdCDP), which can inhibit ribonucleotide reductase, resulting in perturbation of deoxyribonucleoside
5¢-triphosphate (dNTP) pools, particularly d-adenosine triphosphate (dATP) pool depletion, and (2) difluorodeoxycytidine triphosphate (dFdCTP), which blocks the DNA polymerases necessary for replication by competing with dCTP.[9,10] These and other studies suggest that dFdCTP (which can become incorporated into DNA in the form of difluorodeoxycytidine monophosphate [dFdCMP]) is the metabolite responsible for cytotoxicity.
In addition to its cytotoxic effects, gemcitabine is a potent radiation sensitizer of EMT6 rodent tumor cells and a variety of human tumor cell lines[8,12,13] by a continuous exposure to noncytotoxic concentrations of gemcitabine (10 nM) for up to 16 to 24 hours. Because gemcitabine is typically administered once weekly as
an infusion, we determined whether radiosensitization could be obtained by exposing cells for 2 hours to clinically relevant concentrations of the drug. In view of the prolonged retention of the toxic metabolites,[8,14] we hypothesized that this brief treatment with gemcitabine could produce significant delayed radiosensitization. We found that radiosensitization equivalent to or greater than that resulting from a 24-hour continuous incubation with a low concentration of gemcitabine occurred 24 to 48 hours after a 2-hour exposure to 100 nM or 3 mM gemcitabine. Because plasma levels greater than 10 mM are routinely obtained in clinical infusions, these findings suggested that gemcitabine would be a clinical radiosensitizer.
Role of dATP Pool Depletion and Cell-Cycle Redistribution in Radiosensitization
Our initial efforts focused on determining which metabolite was chiefly responsible for the mechanism of sensitization: dFdCTP (the metabolite responsible for cytotoxicity) or dFdCDP (which produces ribonucleotide reductase inhibition and dNTP pool depletion). Substantial correlative evidence suggests that dFdCTP is not responsible for increased radiation sensitivity. For instance, we found that after exposure of colon cancer cells to gemcitabine, dFdCTP accumulated rapidly, reaching a plateau level within 6 hours of exposure. This rapid rise contrasted with the fact that a minimum of 4 hours was required to develop detectable sensitization, and 16 to 24 hours were needed to produce the maximum effect. This disparity of time courses suggests that dFdCTP accumulation is not responsible for sensitization.
Similarly, we found that the same radiosensitization was produced in two different pancreatic cancer cell lines despite a tenfold difference in intracellular dFdCTP concentration. In addition, direct measurement of dCMP incorporation (which results from intracellular dFdCTP) shows a poor correlation with radiosensitization.[Shewach DS, unpublished data, May 1998] These and other data show that dFdCTP levels tend to correlate well with cytotoxicity, but are not closely associated with radiosensitization.
In contrast, our data tend to support the hypothesis that the critical event in gemcitabine-mediated radiosensitization is inhibition of ribonucleotide reductase by dFdCDP, leading to perturbation of dNTP pools (in particular, dATP). We have found that dATP-pool depletion after gemcitabine treatment occurs with a time course that correlates with radiosensitization for both colon and pancreatic cancer cells. These changes were associated with a substantial change in the cell-cycle distribution, with most cells progressing into early to mid S phase.
Although these findings demonstrate that dATP pools and ribonucleotide reductase activity are important factors in radiosensitization, we have also found that sensitization appears to be affected by cell-cycle phase. For instance, the maximum sensitization we could achieve using a 4-hour exposure in HT29 human colon cancer cells was an enhancement ratio of 1.4, whereas during a 24-hour exposure to gemcitabine 10 nM, an enhancement ratio of 1.8 was obtained. This longer exposure was accompanied by redistribution of cells into S phase.
Additional evidence supporting the role of cell-cycle redistribution comes from our study of radiosensitization after gemcitabine removal. We found that maximum radiosensitization was obtained 24 hours after drug exposure, when there was concurrent dATP-pool depletion and redistribution of cells into S phase. There was detectable dATP depletion 72 hours after drug exposure, but cell cycle had normalized and no significant radiosensitization was detected. In vivo studies of mouse intestine demonstrate similar kinetics. These findings are also in agreement with a recent study that directly assessed the role of cell-cycle phase in gemcitabine-mediated radiosensitization using synchronized V79 cells. Although gemcitabine increased the radiation sensitivity of all cell-cycle phases, the effect was greatest in S-phase cells.
Role of Apoptosis in Radiosensitization
The critical lesion produced by ionizing radiation appears to be the DNA double-strand break. Hence, we used pulsed-field gel electrophoresis to assess the effect of gemcitabine on the induction and repair of radiation-induced DNA damage in HT29 human colon cancer cells under two conditions that produced substantial radiosensitization (immediately after a 24-hour exposure to gemcitabine 10 nM or 24 hours after a 2-hour exposure to 100 nM). Both of these conditions produced a radiation enhancement ratio of 1.8. In contrast to our findings with other antimetabolites, such as bromodeoxyuridine and fluorodeoxyuridine, gemcitabine treatment did not increase radiation-induced damage nor did it decrease damage repair during the first 4 hours after radiation. This result has recently been confirmed. These findings suggest that gemcitabine does not affect the primary DNA lesion, but the results of this lesion.
Given the lack of effect of gemcitabine on the induction and immediate repair of radiation damage, we hypothesized that gemcitabine could act as a radiation sensitizer by lowering the threshold for radiation-induced apoptosis. There is mounting evidence that several chemotherapeutic drugs, including gemcitabine, activate the cellular apoptotic machinery, and that alterations in the expression of p53, bcl-2, and bcl-x directly affect the sensitivity of cancer cells to chemotherapy.[23-25]
To begin to assess the role of apoptosis in gemcitabine-mediated radiosensitization, we investigated the effect of gemcitabine on radiation-induced apoptosis in HT29 and SW620 human colon cancer cells, UMSCC-6 human head and neck squamous cancer cells (which are sensitized by gemcitabine), and A549 human lung cancer cells (which are not sensitized by gemcitabine). We have found that gemcitabine significantly enhances apoptosis in cell lines that are radiosensitized (Figure 1). We also found that although apoptosis played a relatively minor role in the clonogenic death produced by radiation alone, it accounted for a substantial fraction of the loss of clonogenicity produced by the combination of gemcitabine and radiation (data not shown). These findings suggest that gemcitabine shifts the pattern of radiation-induced cell death from a nonapoptotic to an apoptotic mechanism.
We have performed similar experiments with A549 lung cancer cells (Figure 2). Although HT29 and A549 cells show similar sensitivity to the cytotoxic effects of gemcitabine, A549 cells are not radiosensitized by noncytotoxic concentrations of gemcitabine. Importantly, exposure of A549 cells to gemcitabine prior to radiation does not result in an important increase in radiation-induced apoptosis (Figure 3). We believe this represents strong correlative evidence that apoptosis plays a key role in gemcitabine-mediated radiosensitization.