Download Radiosensitizing Nucleosides

REVIEW
Radiosensitizing Nucleosides
Cornelius J. McGinn, Donna S. Shewach, Theodore S. Lawrence*
Chemotherapeutic drugs that perturb nucleotide metabolism have the potential to produce substantial sensitization
of tumor cells to radiation treatment. The process is called
radiosensitization, and the agents that induce it are called
radiosensitizers. The clinical effectiveness of fluoropyrimidines as radiosensitizers has been proven in multiple
randomized trials. Thymidine analogues and hydroxyurea
also appear to produce clinically relevant increases in radiation sensitivity. Recent laboratory investigations have identified difluorodeoxycytidine (gemcitabine) and fludarabine
as promising agents to use in combination with radiation.
Until recently, little was known about how the biochemical
changes caused by these drugs produced radiosensitization.
However, advances in related fields, such as cell cycle checkpoint control, have permitted the development of a hypothesis that may explain the relative tumor selectivity of
fluoropyrimidine-mediated radiosensitization. In addition,
recent Findings suggest that the rational manipulation of
drug administration schedules and the use of combinations
of radiosensitizers have the potential to improve the efficacy
of the currently used agents and to establish the benefit of
new ones. [J Natl Cancer Inst 1996;88:1193-1203]
Drugs that affect nucleoside and nucleotide metabolism are
among the most effective and most widely used agents to sensitize tumor cells to radiation treatment (i.e., radiosensitizers).
These agents include fluoropyrimidines (e.g., 5-fluorouracil
[FUra], fluorodeoxyuridine [FdUrd]), thymidine analogues (e.g.,
bromodeoxyuridine [BrdUrd], iododeoxyuridine [IdUrd]), and
hydroxyurea. Radiosensitizing nucleosides that are currently
under active preclinical investigation include difluorodeoxycytidine (gemcitabine) and fludarabine. In this review, we will
focus on mechanisms of action of these agents in order to consider the hypothesis that the mechanisms by which these drugs
radiosensitize predict different clinical strategies to achieve
selective radiosensitization of tumors relative to normal tissues.
As we will discuss below, this hypothesis has reasonable support in the case of fluoropyrimidines and thymidine analogues
but has not been adequately tested for other agents. Therefore,
in addition to reviewing current data, a goal of this review will
be to suggest additional studies that might lead to improved
selectivity for this class of radiosensitizers.
For this review, radiosensitization will be defined as increased radiation sensitivity of cells (tumor or normal tissue, unless otherwise stated) in the absence of significant drug-induced
cytotoxicity. As described below, situations exist in which the
effect of radiation is enhanced by the addition of drugs administered at cytotoxic dose levels. The conclusion that this
form of interaction represents additivity does not suggest a lack
of clinical utility.
Fluoropyrimidines
FUra and FdUrd are analogues of uracil and deoxyuridine,
respectively. Randomized trials have demonstrated local control
and survival advantages with systemic FUra and radiation compared with radiation alone in patients with rectal cancer,
esophageal cancer, and pancreatic cancer [for review, see (J)].
Hepatic arterial infusions of chemotherapeutic agents (such as
fluoropyrimidines in particular) are often employed for the treatment of intrahepatic cancers, because these cancers derive most
of their blood supply from the hepatic artery as opposed to the
portal vein (2). FdUrd has been used most extensively in this
setting because it is more efficiently extracted by hepatic tissue
(95%-97% efficiency of extraction) than is FUra (60%-70% efficiency of extraction) (J), which results in a higher local concentration of drug and reduced systemic effects. A further
advantage of FdUrd in this setting relates to its high potency (it
is effective in the nanomolar range, compared with the
micromolar range for FUra), permitting it to be used in implanted infusion pumps. Indeed, hepatic arterial administration
of FdUrd produces significantly higher response rates in patients
with colorectal cancer metastases compared with systemic
administration of FdUrd or FUra (4). We have shown that the
combination of high-dose, three-dimensionally planned radiation therapy (5) and hepatic arterial infusion of FdUrd can
produce a 50% 4-year actuarial hepatic disease control rate in
patients with unresectable hepatobiliary carcinoma (6).
*Affiliations of authors: C. J. McGinn, T. S. Lawrence (Department of Radiation Oncology), D. S. Shewach (Department of Pharmacology), University of
Michigan, Ann Arbor.
Correspondence to: Theodore S. Lawrence, M.D., Ph.D., Department of
Radiation Oncology, University of Michigan, 1331 E. Ann St., Ann Arbor, MI
48109-0582.
See "Notes" section following "References."
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
REVIEW 1193
FUra and FdUrd, through their metabolite fluorodeoxyuridine
monophosphate (FdUMP), can inhibit the enzyme thymidylate
synthase, which is responsible for the conversion of uridylate
monophosphate to thymidylate monophosphate (TMP) (Fig. 1).
Thymidylate synthase inhibition produces depletion of TMP
(and, ultimately, thymidylate triphosphate [TTP]), leading to
cell cycle redistribution, DNA fragmentation, and cell death (7).
Whereas clinically achievable concentrations of FdUrd produce
only DNA-mediated cytotoxic effects, FUra can also kill cells
by RNA-dependent mechanisms (8,9).
Although the cytotoxic effects of fluoropyrimidines may be
produced by DNA or RNA-directed effects, substantial evidence
suggests that it is the DNA-directed effects that are responsible
for radiosensitization [for reviews see (10,11)]. This evidence
includes the findings that 1) radiosensitization correlates with a
decrease in the rate of repair of radiation-induced DNA doublestrand breaks (12-14), 2) thymidine inhibits FdUrd-mediated
radiosensitization (12), 3) thymidine inhibits FUra-mediated radiosensitization to a greater extent than FUra-induced cytotoxicity
(75), and 4) leucovorin, which increases binding of FdUMP to
thymidylate synthase, potentiates radiosensitization (16).
Previous laboratory investigations into fluoropyrimidinemediated radiosensitization have not clearly indicated one
mechanism of action. Initial cell culture studies (17,18) involving FUra and radiation treatments suggested an interaction of effects only when cells were exposed to cytotoxic concentrations
of the drug after irradiation. The most likely explanation for this
result is that, under these conditions, FUra kills cells that are in
mid-S phase, which is a relatively radioresistant phase of the
cell cycle. This interaction is, therefore, a form of additivity
rather than of radiosensitization. In contrast, studies (12,19)
using FdUrd have found radiosensitization under noncytotoxic
conditions only when cells are exposed to the drug before radiation. It has been proposed (19,20) that exposure of cells to
fluoropyrimidines prior to radiation causes radiosensitization
because fluoropyrimidine treatment causes the cells to be redistributed into the relatively radiosensitive Gj/S and early S
TdR
EXTRACELLULAR
INTRACELLULAR
dUTP
t
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phases of the cell cycle (21,22). We (14) have recently summarized indirect evidence that this mechanism cannot fully explain fluoropyrimidine-mediated radiosensitization. To show
directly that FdUrd can radiosensitize tumor cells independently
of cell cycle redistribution, we assessed the effect of this drug
on cells in mid-S phase. We (23) found that mid-S-phase cells
sorted from a FdUrd-treated population showed significantly increased radiosensitivity compared with mid-S-phase control
cells. These data demonstrate that FdUrd radiosensitizes cells by
a mechanism other than by redistributing them into a sensitive
phase of the cell cycle.
We have constructed a model to explain fluoropyrimidinemediated radiosensitization (Fig. 2). In this model, cells treated
with fluoropyrimidine(s) generate increased levels of the cyclin
proteins that are involved in cell cycle progression and the G|/S
phase transition in particular (especially cyclin E) (24) and attempt to enter S phase. The cells then exhibit one of two types
of response: arrest at the G^S boundary or progression into S
phase (Fig. 3). Arrest at the G]/S boundary occurs in normal
cells as well as in some tumor cells. For normal cells, represented by skin fibroblasts in Fig. 2, exposure to fluoropyrimidines increases the level of p53 protein as well as its
specific binding to DNA (25,26). This leads to a negative feedback on the cyclin-dependent kinases (Cdk) that mediate the
G)/S transition. In some cases, such as represented by p53mutant SW620 human colon cancer cells in Figs. 2 and 3, inhibitors (as yet uncharacterized) prevent activation of cyclin
E-Cdk activity. Our data suggest that regardless of whether
cells arrest at the G|/S boundary by p53-independent or -dependent mechanisms, no radiosensitization results. In contrast,
in other p53 mutant cell types such as HT29 human colon cancer cells, the increase in cyclin E leads to the formation of activated cyclin E-Cdk complexes at the Gi/S boundary despite
fluoropyrimidine treatment. Cells of this type enter S phase and
are sensitized to radiation.
An appealing feature of the proposed model is that it suggests
a mechanism for selective radiosensitization of tumor cells com-
TdR
i
TS
dUMP
TK
TMP
TTP
DNA
INHIBITION
FdUrd
FUra
1194 REVIEW
FdUMP
FdUTP
FUMP
FUTP
> RNA
Fig. 1. Metabolic pathways of fluorodeoxyuridine (FdUrd) and 5-fluorouracil
(FUra). TdR, thymidine; dUTP, deoxyuridine triphosphate; dUMP, deoxyuridine monophosphate; TS, thymidylate
synthase; TK, thymidine kinase; TMP,
thymidine monophosphate; TTP, thymidine triphosphate; FdUMP, fluorodeoxyuridine monophosphate; FdUDP,
fluorodeoxyuridine diphosphate: FdUTP,
fluorodeoxyuridine triphosphate; FUMP,
fluorouridine monophosphate; FUDP,
fluorouridine
diphosphate;
FUTP,
fluorouridine triphosphate. (With permission, from McGinn CJ. Kinsella TJ.
Curr Probl Cancer 1993:17:275-321.)
Journal of the National Cancer Institute, Vol. 88, No. 17, September4, 1996
Cdk
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Fig. 2. Model for fluoropyrimidine-mediated radiosensitization. The cell cycle and its four phases are depicted at the left. TS, thymidylate synthase; FdUrd,
fluorodeoxyuridine; mtp53, mutant p53 tumor suppressor protein: wtp53. wild-type p53 protein: HT29 and SW620, human colon cancer cell lines; Fibro, fibroblasts;
Cdk, cyclin-dependent kinase; p21, tumor suppressor protein: see text for further details.
pared with normal cells. Many colorectal cancers are p53
mutant and would be expected to progress into S phase in the
presence of fluoropyrimidines, as do HT29 cells, whereas normal cells would be anticipated to arrest at the G,/S boundary
and not be radiosensitized. Thus, the rational application of
combination fluoropyrimidines and radiation would involve
balancing this potential therapeutic index with the possibility
that normal cells could undergo apoptosis after drug and/or
radiation treatment (see below).
Although this hypothesis is consistent with our data and those
of other laboratories, it is likely an oversimplification. For instance, Lowe et a). (27) have proposed what appears to be the
opposite hypothesis, since, in their studies, p53-mutant tumors
tend to be more resistant to radiation and some chemotherapeutic agents because of a defect in the ability to carry out
apoptosis. Several other studies (28-30) have also not found an
association between p53 status and radiation sensitivity. A possible explanation for these differences concerns the relative
roles of apoptosis versus mitotic-linked clonogenic death in the
600
HT29
different model systems. The relative importance of these two
forms of radiation-induced cell death is controversial, both in
the laboratory (31) and in the clinic (32), where, for instance, it
has recently been reported that p53-mutant breast cancers
respond better to radiation than p53 wild-type cancers. Thus, it
appears that it will be difficult to make generalizations based on
the study of a limited number of model systems. However, it is
clear that the cross-fertilization between the fields of cell cycle
check point control and experimental therapeutics of radiosensitizing nucleosides will be particularly fruitful in the future.
Thymidine Analogues
The thymidine analogues BrdUrd and IdUrd have been used
as radiosensitizers in the treatment of a number of cancers including head and neck cancers (33), malignant gliomas (34-37),
brain metastases (38), soft tissue sarcomas (39,40), intrahepatic
cancers (41), and cervical cancers (42). Unfortunately, the only
completed randomized trial (55), which involved the treatment
800
SW620
600
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S
400
I
Control
% 200
200
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50
100
150
200 250
Channel Number
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50
100 150 200 250
Channel Number
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
Fig. 3. Effect of fluorodeoxyuridine
(FdUrd) on the cell cycle distribution of
HT29 and SW620 human colon cancer
cells. Cell were incubated without (—)
or with (• • ) 100 nAf FdUrd for 14 hours.
Under these conditions, FdUrd markedly
radiosensitizes HT29 cells but has little
effect on the radiation sensitivity of
SW620 cells. Cells were assessed for
DNA content using flow cytometry. Vertical arrow indicates the internal standard
(human leukocytes for HT29, chinook
salmon red blood cells for SW620). For
SW620 cells, the FdUrd and control curves are superimposed in the G| region
(represented by signal in channels numbered 70-90). This figure shows that, in
the presence of FdUrd, HT29 cells
progress into S phase, whereas SW620
cells tend to arrest at the G,/S boundary.
[From (24), with permission.]
REVIEW 1195
of patients with brain metastases, did not show an improvement
in the arm receiving BrdUrd with radiation compared with
radiation alone. However, interesting results have been obtained
at two or more institutions for anaplastic astrocytomas (using
BrdUrd) (34) and soft tissue sarcomas (using IdUrd) (40). In the
case of anaplastic astrocytomas, a phase II clinical trial involving 138 patients that assessed procarbazine, lomustine [l-(2chloroethyl)-3-cyclohexyl-l-nitrosourea
or CCNU], and
vincristine chemotherapy combined with radiation and BrdUrd
demonstrated a 4-year progression-free survival of 42% (34).
This result was felt to reflect a substantial increase in progression-free survival, compared with prior published survival data
(43). The Radiation Therapy Oncology Group is currently testing the efficacy of BrdUrd as a radiation sensitizer for highgrade gliomas in a randomized trial.
In contrast to the fluoropyrimidines, which exert their effects
on DNA chiefly by inhibition of thymidylate synthase, BrdUrd
and IdUrd produce radiosensitization by incorporation into
DNA. Their incorporation increases the susceptibility of the
DNA to single-strand breaks from radiation-produced free radicals (44). Incorporation of these analogues into DNA may be
augmented by the ability of the triphosphate analogues to decrease competing TTP pools through feedback inhibition of
ribonucleotide reductase (45,46). Analogue incorporation produces both an increase in radiation-induced DNA damage (4749) and a decrease in the rate of DNA repair (50,57). We have
recently confirmed the latter finding using pulsed-field gel
electrophoresis (52). Incorporation of BrdUrd and IdUrd into
DNA correlates linearly with radiosensitization both in vivo and
in vitro in a wide variety of model systems, including both
tumor cells and normal cells (47-49,53,54).
This last result highlights a fundamental difference between
the thymidine analogues and the fluoropyrimidines. As described above, the fluoropyrimidines may produce a therapeutic
advantage because they exploit a difference between the control
of the G|/S phase transition in cancer cells compared with normal cells. In contrast, all S phase cells (tumor and normal) are
capable of incorporating thymidine analogues. Therefore,
therapeutic strategies using thymidine analogues have focused
on selection of tumor sites and on routes and schedules of drug
administration to achieve selective incorporation of the compounds into tumor cells compared with normal cells. Initial
strategies were designed to target tumors that were hypothesized
to proliferate more rapidly than the surrounding normal tissue.
Levels of incorporation that would be anticipated to produce a
clinical effect have subsequently been documented in the tumors
of patients with colorectal cancer metastatic to the liver (55^6)
and in unresectable extremity soft tissue sarcomas (57). With
regard to the latter group of patients, it is of interest to note that
thymidine analogues have also been shown to be chemosensitizers (58-60). Since chemotherapy appears to confer a modest
but detectable increase in survival for these patients (61), it
would be interesting to consider integration of thymidine
analogues into the treatment regimens as both radiosensitizers
and chemosensitizers for these patients.
To extend the use of thymidine analogues, we have carried
out studies to determine if these agents could be used to treat
tumors in which the radiation dose limiting-structure is rapidly
1196 REVIEW
dividing normal tissue (e.g., intestine and bone marrow). These
studies in nude mice bearing human tumor xenografts revealed
that incorporation of analogue in tumor tissue exceeded that in
the bone marrow and intestine in a "window" of time following
the completion of drug infusion (Fig. 4) (62). We used this
strategy to design clinical protocols for the treatment of
retroperitoneal sarcomas and cervical cancer using IdUrd and
BrdUrd, respectively. Patients treated on these protocols underwent tumor biopsies and biopsies of normal tissues at the completion of a 5-day drug infusion and again 3 days later to
determine the level of thymidine analogue incorporation into
DNA. These studies have revealed that, as expected, incorporation of the compound into DNA in normal tissue increases with
increasing doses of analogue. During the 3-day drug elimination
period, incorporation into the DNA of bone marrow cells fell by
approximately 50%, while incorporation into rectal mucosal
cells did not change (63). However, immunohistochemical
evaluation of the rectal biopsies demonstrated a progression of
labeled cells from the crypts to the villi (Fig. 5). Since no additional drug was infused during this 3-day interval, and the incorporation in the rectal mucosa (both crypts and villi combined)
remained unchanged, the degree of incorporation in the crypt
cells must have decreased. Since the crypt cells are required to
repopulate the intestine after irradiation, it would be anticipated
that irradiation during this drug elimination period would
produce less toxicity (42). Our preliminary data on tumor biopsies demonstrate no substantial change in incorporation during
the drug elimination period. Studies are ongoing to evaluate the
labeling index and patterns of labeling in these specimens, some
of which included a complete tumor resection. Although our ini-
24
Tumor
Intestine
1 2
3
4
5
6
7
Time (days)
Fig. 4. Incorporation of BrdUrd into intestinal ( • ) and tumor cells ( • ) after the
completion of an infusion. Athymic nude mice bearing HT29 human colon cancer xenografts were infused with BrdUrd (200 mg/kg per day) for 4 days using
an osmotic minipump. The pumps were removed, and cells prepared from the
tumors and intestine were assessed for replacement of thymidine by BrdUrd.
Data are expressed as the mean values ± standard error determined from at least
three animals. Error bars not visualized are within the symbol; * indicates that
tumor cell incorporation significantly (two-sided P<.05) exceeds intestinal cell
incorporation.
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
Fig. 5. Human rectal biopsy specimens prepared with immunohistochemical stains for incorporated BrdUrd at the completion
of a 5-day infusion of BrdUrd at
a dose of 1.0-1.6 g/m" per day
(A) and following a 3-day drug
elimination period during which
no additional drug was infused
(B).
tial clinical trials (40,42) suggest that these incorporation results
may translate into improved outcome, additional clinical evaluation will be required to determine efficacy.
Hydroxyurea
Although hydroxyurea is not a nucleoside analogue, its
primary mechanism of cytotoxicity is related to its inhibition of
ribonucleotide reductase, a key enzyme for the transformation of
ribonucleotides to deoxyribonucleotides (64). Its role in the
treatment of hematologic malignancies and myeloproliferative
disorders is well established (65). The use of hydroxyurea as a
radiosensitizer has been investigated in the clinic since the
1960s in patients with head and neck cancer (66), malignant
glioma (43), and cervical cancer (67). In the former two sites,
the contribution of hydroxyurea as a radiosensitizer is difficult
to discern, since it has often been part of a multidrug regimen
(43,68). Perhaps the most convincing trials suggesting clinically
relevant radiosensitization by hydroxyurea involve patients with
cervical cancer. Since hydroxyurea has little or no activity as a
chemotherapeutic agent in patients with advanced squamous
cell carcinoma of the cervix, it has been assumed that any positive result would represent radiosensitization rather than additive effects. A series of randomized trials from Piver et al.
(69,70) at the Roswell Park Cancer Center have reported improvements in survival for patients who received hydroxyurea in
addition to standard radiotherapy. A randomized trial (71) from
the Gynecological Oncology Group (GOG) has also shown an
advantage to the use of hydroxyurea over misonidazole with respect to progression-free survival and overall survival. Although
there was no local control advantage and the overall survival ad-
vantage was only marginally significant in that trial (P - .066),
hydroxyurea remains in the control arm of current GOG trials.
The mechanism of hydroxyurea radiosensitization has been
attributed to the inhibition of DNA synthesis resulting from the
inhibition of ribonucleotide reductase (72). Exposure prior to irradiation blocks cells at the G^S phase border, although it is uncertain if this redistribution alone causes increased radiation
sensitivity (see the discussion of fluoropyrimidines above). Exposure during or after irradiation also results in radiosensitization, presumably by inhibiting the repair of DNA damage. Yet,
as with the halogenated pyrimidines, there is no differential effect
on tumor cells compared with normal cells. However, tumors that
proliferate more rapidly than surrounding normal tissue might be
preferentially sensitized to radiation. This preferential radiosensitization may be particularly important to reconsider now that
techniques of cell kinetics analysis can be applied in the clinic to
investigate more successful application of this cell cycle-specific
agent. In addition, it remains possible that, as is the case for
fluoropyrimidines, hydroxyurea-mediated radiosensitization occurs
in cells that progress through the Gj/S boundary.
Recently initiated studies on the mechanism of hydroxyurea
radiosensitization may also guide a more rational use of this
agent as a radiosensitizer. These investigations (73,74) are
focusing more closely on the role of ribonucleotide reductase
rather than disruption of cell cycle kinetics, based on work (75)
that has further defined the interaction of hydroxyurea with
ribonucleotide reductase. Further understanding of the cell cycle
regulation of ribonucleotide reductase, its response to DNA
damaging agents (including radiation) (76), and its interrelationship to other enzymes related to DNA synthesis (77,78) may
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
REVIEW 1197
also provide additional information on the interaction of hydroxyurea and ionizing radiation.
Hydroxyurea can also modulate fluoropyrimidine-mediated
radiosensitization of tumor cells. Hydroxyurea depletes intracellular deoxyuridine monophosphate pools, thus increasing the
ability of FdUMP to bind with thymidylate synthase. Synergism
between the two drugs has been demonstrated in experimental
systems {79). On the basis of these data, Vokes et al. (68) have
initiated trials of FUra/hydroxyurea chemotherapy and radiation
in patients with head and neck cancer. A similar study (71) has
also been initiated by the GOG for patients with cervical cancer.
Finally, hydroxyurea may have a role as a biochemical modulator of IdUrd radiosensitization of tumor cells (74). Exposure to
minimally cytotoxic doses of hydroxyurea (10-100 \iM) with
IdUrd (2 \\M) for one cell cycle prior to irradiation has been
shown to enhance IdUrd-DNA incorporation and radiosensitization in the human bladder cancer cell line 647V. This effect is
presumably the result of inhibition of ribonucleotide reductase
with subsequent direct or indirect interactions with thymidylate
synthase. These data suggest a clinical strategy to further differentiate the radiation response of rapidly proliferating tumor
cells from more slowly proliferating cells in normal tissue.
Gemcitabine
Gemcitabine (2',2'-difluoro-2'-deoxycytidine or dFdCyd) is
an analogue of deoxycytidine which, unlike cytarabine (Ara-C),
has demonstrated effectiveness as a single agent against solid
tumors (80), including pancreatic cancer (81), non-small-cell
lung cancer (82,83), head and neck cancer (84), and breast cancer (85). The cytotoxic metabolite of gemcitabine is the triphosphate form for which the rate-limiting step of its production is
the initial phosphorylation by deoxycytidine kinase (86,87). The
cytotoxic activity of gemcitabine is related to the incorporation
of difluorodeoxycytidine monophosphate (dFdCMP) into DNA
(88,89), an effect that is enhanced by the reduction of deoxyribonucleotide pools following difluorodeoxycytidine diphosphate (dFdCDP) inhibition of ribonucleotide reductase (90).
Analogue incorporation into DNA results in termination of
DNA elongation after the addition of one more deoxynucleotide, and dFdCMP in the template strand increases the
frequency of base mismatching in the de novo synthesized
strand (88,9J). Several other self-potentiating actions of gemcitabine have been demonstrated, including the inhibition of
deoxycytidine monophosphate (dCMP) deaminase activity,
which, in turn, results in decreased difluorodeoxycytidine
triphosphate (dFdCTP) catabolism (92). Recent studies demonstrate that the deamination product of gemcitabine, dFdUrd, is a
moderate radiosensitizer at clinically achievable concentrations
(Salinas F, Lawrence TS, Hertel LW, Shewach DS: manuscript
submitted for publication).
We investigated the radiosensitizing potential of gemcitabine
in human solid tumor cell lines after it was reported (93) that
this compound could radiosensitize a rodent tumor cell line.
These studies demonstrated that prolonged exposure to gemcitabine results in potent radiosensitization of colon (94),
pancreas (95), breast (regardless of multidrug resistance status)
(Shewach D, Lawrence T: unpublished results), and head and
1198 REVIEW
neck cancer cell lines (Fig. 6). When it became clear that the
majority of the clinical studies involved chemotherapy with
bolus drug administration, we decided to investigate if an in
vitro 2-hour exposure of cells to clinically relevant concentrations of gemcitabine could produce radiosensitization. We have
found that radiosensitization is detectable 4 hours after a short
exposure, that it peaks 24 hours later, and that it is detectable for
up to 48 hours after drug treatment (94). These data have been
used to design a phase I clinical trial, into which patients are
now being accrued, using escalating doses of gemcitabine in
combination with standard-dose radiation (70 Gy in 2-Gy fractions) for the treatment of patients with locally advanced head
and neck cancer. On the basis of the clinical results using gemcitabine alone and the preclinical data regarding radiosensitization of cells by gemcitabine, it would be logical to design
similar combination gemcitabine-radiation trials for patients
with pancreatic or breast cancer.
The mechanism by which gemcitabine radiosensitizes tumor
cells is not yet clear. Our preliminary studies indicate that the
observed radiosensitization is not associated with either an increase in the radiation-induced DNA double-strand breaks or
with a slowing of DNA double-strand break repair. This suggests that radiosensitization by gemcitabine is unlike that produced by the fluoropyrimidines and the thymidine analogues.
However, radiosensitization is associated with the depletion of
deoxyadenosine triphosphate pools (but not with the intracellular concentration of dFdCTP) (94,95), suggesting that inhibition of ribonucleotide reductase may be relevant, as is the case
with hydroxyurea. It is interesting to note that all of the radiosensitizers discussed here are capable of depleting one or more
of the endogenous deoxynucleoside triphosphate pools, which
may be more important in radiosensitization than previously
recognized. The relationships between gemcitabine radiosensitization and DNA incorporation, alterations in DNA synthesis,
or alteration in cell cycle kinetics remain to be investigated. In
addition, it would be logical to investigate the role of apoptosis
in gemcitabine-mediated radiosensitization, since this mechanism of cell death has been shown to be the pathway by which
the drug exerts its cytotoxic action, at least in lymphoid cell
lines (89).
Fludarabine
Fludarabine is an analogue of adenine arabinoside (Ara-A), a
well-studied DNA damage repair inhibitor (96). Many similarities exist between gemcitabine and fludarabine; both compounds require phosphorylation for activation, with the initial
step mediated exclusively by deoxycytidine kinase, and the incorporation of the corresponding nucleotides into DNA results
in potent inhibition of DNA synthesis. Fludarabine has clinical
activity against hematologic cancers such as chronic lymphocytic leukemia and follicular non-Hodgkin's lymphoma (97).
The mechanism of cytotoxicity has been attributed to inhibition
of enzymes critical for DNA synthesis and repair, including
DNA polymerases, ribonucleotide reductase, DNA primase, and
DNA ligase (95). Incorporation of fludarabine at the DNA chain
terminus results in gene deletions and increased mutational frequency (99). Fludarabine also inhibits RNA synthesis and induces apoptosis (98).
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
10"
Control
4-1
u
30nM dFdCyd
Control
c
o
10
1
10
-2.
60
lOnM dFdCyd
3
30nM dFdCyd
CD
10"
10"
2
4
6
8
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4
6
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Dose (Gy)
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2
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Fig. 6. Gemcitabine radiosensitizes breast cancer and head and
neck cancer cells. Head and neck squamous cancer cells (UMSCC1, A) and breast cancer cells MCF7-WT (wild-type parental cells)
and MCF7-PDR (pleiotropic drug resistant cells), (B and C, respectively) were exposed to control conditions, i.e., no drug (fj) or 10
nM gemcitabine (A) and 30 nM gemcitabine ( • ) . Cells were
treated with gemcitabine for 24 hours prior to irradiation, then irradiated and assessed for clonogenic survival. The results of single
experiments are shown.
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60
C
With regard to radiosensitization, Kim et al. (100) initially
reported that treatment with fludarabine and radiation substantially increased the control rate of Meth-A fibrosarcomas in
BALB/c mice compared with radiation treatment alone. In a
recent series of studies (101-103), investigators from The
University of Texas M. D. Anderson Cancer Center have investigated the mechanism by which fludarabine can potentiate the
effects of radiation in three solid tumor model systems. They
(70/) found that the administration of fludarabine at a dose of
800 mg/kg 1 hour prior to radiation treatment lengthened
regrowth delay over a range of conditions, but particularly if irradiation was performed 24 hours after drug treatment. Subsequent studies suggested that this delay in radiosensitization
reflected a fludarabine-induced loss of radioresistant S-phase
cells (through apoptosis) and partial synchronization of cells
into Gj/M 24 hours later (102). In further experiments using this
murine model system, a beneficial therapeutic ratio was reported
following a single fraction of radiation (103).
Summary and Clinical Perspective
Although one must use great care in generalizing results from
laboratory studies to the clinic, we feel the data presented above
support the following concepts, which are summarized in Table
1, and elaborated on below.
Fluoropyrimidines
Since the duration of action is short, it would be anticipated
that maximal radiosensitization of tumor cells will be obtained
by using continuous infusion rather than bolus therapy. In this
manner, tumor cells may be sensitized to each radiation fraction
rather than just the few fractions delivered on the days of bolus
treatment. An infusion would need to begin at least 8-12 hours
before radiation and be continued during the course of treatment. This general approach is supported by the results of a
North Central Cancer Treatment Group trial (104) that revealed
improved survival with protracted venous infusions of FUra
compared with bolus FUra during postoperative radiotherapy for
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
REVIEW 1199
Table 1. Summary of the main features regarding use of radiosensitizing nucleosides in cancer therapy
Drug*
Disease
site
Limiting
tissue
Drug
administration
Biology underlying
therapeutic index
FUra/FdUrd
Gastrointestinal
tract
Intestine
Continuous with
radiotherapy
Normal cell arrest at Gi/S
Tumor cells progress into S
BrdUrd/IdUrd
Liver
Brain
Cervix
Sarcoma
Liver
Brain
Intestine
Continuous before/during
radiotherapy
Continuous, alternating with
radiotherapy
Incorporation in tumor tissue greater
than in normal tissue
Retention in tumor tissue greater than
in normal tissue
Hydroxyurea
Cervix
Before radiotherapy
Related to relative rates of
proliferation
Gemcitabine
Pancreas
Head/neck
Bolus
Radiotherapy <48 h later
Unknown
Fludarabine
Not tested
Intestine
Oral mucosa
Unknown
*FUra = 5-fluorouracil; FdUrd = fluorodeoxyuridine; BrdUrd = bromodeoxyuridine; IdUrd = iododeoxyuridine.
patients with stage II or III rectal cancer. Our proposed model
(Fig. 2) suggests that the therapeutic benefit occurs because
tumor cells do not respect the G]/S phase boundary in the
presence of fluoropyrimidines, progress into S phase, and find
themselves unable to effectively repair radiation-induced DNA
damage.
Thymidine Analogues
Two distinct strategies for selective sensitization of tumors
compared with normal tissues have evolved on the basis of the
estimated relative rates of proliferation of tumor and normal tissues. With a proliferative tumor surrounded by a slowly dividing normal tissue (e.g., colorectal cancer metastases in the liver,
sarcomas of the extremities, brain tumors), a continuous infusion beginning 4-7 days prior to radiation treatment should
produce incorporation and subsequent sensitization of tumor to
a greater extent than the normal tissue. The appropriate duration
of this infusion during the course of radiation treatment remains
a subject of debate {105,106). Conversely, the use of a drug
elimination period should increase the therapeutic index of
thymidine analogues when the tumor is surrounded by more rapidly proliferating normal tissue such as intestine or bone marrow, as
is the case with retroperitoneal sarcomas and cervical cancer.
Hydroxyurea
Like the thymidine analogues, current evidence does not
demonstrate that hydroxyurea provides selective radiosensitization of tumor cells compared with normal tissue. Therefore, as a
single agent radiosensitizer, its use may be limited. However,
recent data {74,107) suggest that it may be used in combination
with other nucleoside analogues to potentiate radiosensitization.
In this manner, it may be clinically valuable, particularly since it
can be administered orally throughout a course of radiotherapy.
Clinical trials to test the value of hydroxyurea combined with
FUra are under way.
Gemcitabine
Although laboratory data suggest that radiosensitization can
be achieved through long-term exposure to noncytotoxic concentrations of drug, the clinical experience has focused chiefly
1200 REVIEW
on weekly (cytotoxic) bolus administration of the drug on a
schedule of 3 weeks out of 4 weeks. If this schedule of administration is used for combined modality studies, it would be
logical to administer drug early in the week to permit as many
radiation fractions as possible to be delivered when radiosensitization might occur. Furthermore, it would be interesting to
consider twice-weekly schedules. Since radiosensitization can
occur 1-2 days after exposure, this approach might sensitize
tumor cells to all radiation fractions. However, the biological
basis for a therapeutic index is not yet certain.
Fludarabine
We are unaware of any clinical trials that have assessed or are
assessing the combination of fludarabine and radiation for the
treatment of solid tumors, but such trials would seem warranted
based on the interesting preclinical results described above.
Conclusion
We would like to note again, however, that the design of
clinical trials needs to balance the results of cell culture and
animal studies with clinical estimates of normal tissue toxicity.
Cell culture studies serve to define mechanisms of drug-radiation interaction, whereas animal studies allow investigators to
address issues of therapeutic index and to investigate alternative
drug-radiation schedules for initial human trials. These phase
I/II trials are typically designed and conducted using conventional doses of radiation combined with drug that is usually administered at a fraction (perhaps 25%) of the maximum
tolerated dose when the drug is given alone. This strategy will
then define the relevant clinical toxic effects and is particularly
important for drugs that are potent radiosensitizers in preclinical
studies under noncytotoxic conditions. We strongly recommend
that tumor and normal tissue biopsies be obtained during these
trials in order to generate data that may validate or refute the initial hypotheses formed from preclinical work. The results of
these early phase I/II trials, including relevant biopsy data, may
then allow refinement of these radiosensitizing strategies.
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
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Notes
Supported by Public Health Service grants CA53440, CA42671, and
CA46592 from the National Cancer Institute, RR00042 from the National Center for Research Resources, National Institutes of Health, Department of Health
and Human Services, and by American Cancer Society grant CH-520.
We thank Mary Davis, Jonathan Maybaum, and Vernon Sondak for their helpful comments and Marlene Langley for her secretarial assistance.
Manuscript received February 8, 1996: revised May 31, 1996; accepted June
10, 1996.
This match is out.
Orisifl
g through these hills, see,
and the air was as d e a n and as
mountain stream, and the sun
nd it kept thundering through
Only you can prevent forest fires.
I A Public Service of the USDA Forest Service and your Slate Forester.
Journal of the National Cancer Institute, Vol. 88, No. 17, September 4, 1996
REVIEW 1203