is an analog of cytarabine (cytosine arabinoside) with clinical
activity against a variety of solid tumors, particularly
pancreatic[1,2] and nonsmall-cell lung cancer.[3,4] 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
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.
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