REVIEWS

EXPLOITING TUMOUR HYPOXIA

IN CANCER TREATMENT
J. Martin Brown* and William R. Wilson
Solid tumours contain regions at very low oxygen concentrations (hypoxia), often surrounding areas
of necrosis. The cells in these hypoxic regions are resistant to both radiotherapy and chemotherapy.
However, the existence of hypoxia and necrosis also provides an opportunity for tumour-selective
therapy, including prodrugs activated by hypoxia, hypoxia-specific gene therapy, targeting the
hypoxia-inducible factor 1 transcription factor, and recombinant anaerobic bacteria. These
strategies could turn what is now an impediment into a significant advantage for cancer therapy.
HYPOXIA

A low oxygen level. However,

this means different levels to
different investigators depending
on the phenomenon being
investigated. For the radiation
biologist, hypoxia occurs at
levels that produce severe
radiation resistance or levels less
than 0.1% O2 in the gas phase.
Other effects of hypoxia occur at
oxygen levels above and below
this value.

*Division of Radiation and

Cancer Biology, Department
of Radiation Oncology,
Stanford, California
94305, USA.

Experimental Oncology
Group, Auckland Cancer
Society Research Centre,
Private Bag 92019,
Auckland, New Zealand.
Correspondence to J.M.B.
e-mail:
mbrown@stanford.edu
doi:10.1038/nrc1367

NATURE REVIEWS | C ANCER

The presence of HYPOXIA regions of low levels of oxygen

in human tumours was postulated by Thomlinson
and Gray some 50 years ago based on their observations
of the distribution of necrosis relative to blood vessels1. It
was known at that time that hypoxic cells were resistant to
killing by ionizing radiation2 (BOX 1), and this led to clinical trials with patients undergoing radiotherapy in
hyperbaric oxygen chambers, to try to force more oxygen into the blood and into the tumour. These trials
were not particularly successful, in part because it was
not known at that time that, in addition to the chronic
(or diffusion-limited) hypoxia postulated by
Thomlinson and Gray, acute (perfusion-limited)
hypoxia could also occur by temporary obstruction, or
variable blood flow, in tumour vessels (FIG. 1).
During the 1970s, interest in overcoming the assumed
problem of the radiation resistance of hypoxic cells in
tumours was rekindled with the discovery of small molecules nitroimidazoles that could mimic the effects
of oxygen and thereby sensitize hypoxic cells to radiation.
Clinical trials adding nitroimidazoles (metronidazole,
misonidazole and etanidazole) to radiotherapy were conducted, but in general did not result in significant
improvements over radiotherapy alone, mainly because
the toxicities of the drugs prevented them from being
given at high enough doses3. Subsequently, however, a
meta-analysis of all of the trials has shown that these
drugs did produce a small but significant improvement in
local control, compared with radiotherapy alone, particularly for head and neck cancers4. Nonetheless, the fact that
the high expectations for hypoxic radiosensitizers in

combination with radiotherapy were not realized led

researchers to question whether hypoxia was a hallmark of solid cancers and whether it affected the
outcome of radiotherapy.
The situation changed in the 1990s with the introduction of a commercially available oxygen electrode
(the Eppendorf electrode), which enabled investigators
to make accurate measurements of oxygen levels in
human tumours5. We now know that oxygen concentrations in human tumours are highly heterogeneous with
many regions at very low levels (less than 5 mmHg partial pressure of oxygen (pO2); 5 mmHg corresponds to
approximately 0.7% O2 in the gas phase or 7 M in
solution), with median values much lower than the normal tissues from which the tumours arose (TABLE 1).
Several investigators have now demonstrated unequivocally that the extent of tumour hypoxia has a negative
impact on the ability of radiotherapy to locally control
tumours, because of the resistance of hypoxic cells to
killing by radiation6,7 (BOX 1).
Hypoxic cells are also considered to be resistant to
most anticancer drugs for several reasons: first, hypoxic
cells are distant from blood vessels and, as a result, are
not adequately exposed to some types of anticancer
drugs8,9; second, cellular proliferation decreases as a
function of distance from blood vessels10, an effect that
is at least partially due to hypoxia; third, hypoxia selects
for cells that have lost sensitivity to p53-mediated apoptosis, which might lessen sensitivity to some anticancer
agents; fourth, the action of some anticancer agents (for
example, bleomycin) resembles that of radiation in that
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Box 1 | Radiation resistance of hypoxic cells

Ionizing radiation, such as that used in radiotherapy, kills cells by producing DNA damage, particularly DNA doublestrand breaks. This damage results from ionizations in or very close to the DNA that produce a radical on the DNA
(DNA ). This radical then enters into a competition for oxidation, primarily by oxygen (which fixes, or makes
permanent, the damage), or reduction, primarily by SH-containing compounds that can restore the DNA to its original
form (see figure, part a). Therefore, DNA damage is less in the absence of oxygen. This effect of oxygen in sensitizing cells
to radiation is illustrated in the cell-survival curve (see figure, part b) and is quantitated as the ratio of dose in the
absence of oxygen to dose in the presence of oxygen needed to obtain the same surviving fraction of cells. For
mammalian cells, this ratio is usually 2.53.0 (see horizontal dotted line). The oxygen partial pressure (pO2) that
produces sensitivity midway between the oxic and hypoxic responses is approximately 3 mmHg. Clinical trials,
particularly with head and neck cancers for which control of the primary tumour is the main problem, have
demonstrated that the more hypoxic tumours (typically those with a median pO2 less than 10 mmHg) are more
radioresistant than the less hypoxic tumours.

a
Ionizing
radiation

b
Restitution

DNA-H

Reduction in hypoxic conditions

DNA-H

Survivng fraction

RSH

DNA

0.1

0.01

O2 Oxidation in aerobic conditions

0.001

DNA-OO

DNA breaks

Cell
death

Damage fixation

P-GLYCOPROTEIN

A protein localized to the cell

membrane that actively pumps
many drugs out of the cell. High
levels of this protein lead to
resistance to many anticancer
drugs.
PRODRUG

A latent form of a drug that can

be activated by metabolism or
other chemical transformation
in the body.

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| JUNE 2004 | VOLUME 4

oxygen increases the cytotoxicity of the DNA lesions

they cause11,12; fifth, hypoxia upregulates genes
involved in drug resistance, including genes encoding
13,14
P-GLYCOPROTEIN
. These clear links between hypoxia
and intrinsic resistance to chemotherapy provide the
smoking gun, yet, surprisingly, clinical studies investigating the role of hypoxia in response to chemotherapy
have not been reported.
Finally, in addition to its effect on response to cytotoxic therapy, it has also been demonstrated that
hypoxia in tumours tends to select for a more malignant
phenotype15, increases mutation rates16, increases
expression of genes associated with angiogenesis17 and
tumour invasion18, and is associated with a more
metastatic phenotype of human cancers19,20. By enhancing metastasis, hypoxia can compromise curability of
tumours by surgery 21,22.
Therefore, hypoxia has a key negative role in tumour
prognosis both because it causes resistance to standard
therapies and because it promotes a more malignant
phenotype17. However, the very low levels of oxygen and
the presence of necrosis are unique features of solid
tumours under normal physiological conditions they
do not occur in normal tissues and so are potentially

10

20

30

40

Dose (Gy)
Aerobic

Hypoxic

exploitable in cancer therapy. Four general strategies are

now being developed: PRODRUGS activated by hypoxia;
hypoxia-selective gene therapy; targeting the hypoxiainducible factor 1 (HIF-1) transcription factor; and the
use of recombinant obligate anaerobic bacteria.
Hypoxia-activated prodrugs in clinical trials

A common mechanism by which a non-toxic prodrug

can be activated to a toxic drug in a hypoxia-dependent
manner is shown in FIG. 2. In essence, hypoxia-selective
cytotoxicity requires one-electron reduction of a relatively non-toxic prodrug to a radical that then becomes a
substrate for back-oxidation by oxygen to the original
compound. If the so-formed radical or downstream
products of the radical are much more toxic than the
superoxide generated by redox cycling in oxic cells,
hypoxia-dependent cytotoxicity arises. Examples of
hypoxia-activated prodrugs in clinical trials are illustrated below and those in preclinical development are
discussed in the subsequent section.
Tirapazamine. Brown and Lee discovered the
hypoxic cytotoxicity of tirapazamine (TP2; FIG.3)
almost 20 years ago, and this is the first compound to
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Summary
A characteristic feature of solid tumours is the presence of cells at very low oxygen tensions. These hypoxic cells confer
radiotherapy and chemotherapy resistance to the tumours, as well as selecting for a more malignant phenotype.
These hypoxic cells, however, provide a tumour-specific targeting strategy for therapy, and four approaches are being
investigated: prodrugs activated by hypoxia; hypoxia-selective gene therapy; targeting the hypoxia-inducible factor 1
(HIF-1) transcription factor; and the use of recombinant obligate anaerobic bacteria.
Tirapazamine is the prototype hypoxia-activated prodrug. Its toxic metabolite, a highly reactive radical that is present
at higher concentrations under hypoxia, selectively kills the resistant hypoxic cells in tumours. This makes the tumours
much more sensitive to treatment with conventional chemotherapy and radiotherapy.
Several other hypoxia-activated prodrugs, including AQ4N, NLCQ-1 and dinitrobenzamide mustards, are in
preclinical or early clinical development.
Hypoxia-activated gene therapy using hypoxia-specific promoters provides selective transcription of enzymes that can
convert prodrugs into toxic drugs. The efficacy of this approach has been shown in animal models, but clinical testing
must await better systemic delivery of vectors to hypoxic cells.
Targeting HIF-1 is a third strategy. This protein is stabilized under hypoxic conditions and promotes the survival of
tumour cells under hypoxic conditions. Several strategies to inactivate or to exploit this unique protein in tumours are
being investigated at the preclinical level.
Finally, using recombinant non-pathogenic clostridia an obligate anaerobe that colonizes tumour necrosis after
systemic administration is another strategy to exploit the unique physiology of solid tumours. This approach has
demonstrated considerable preclinical efficacy.

examples39. TPZ was a significant advance over the

previously known classes because its differential toxicity towards hypoxic cells was larger23, and combination
studies with fractionated radiation demonstrated its
ability to kill hypoxic cells in transplanted tumours24.
The mechanism for selective toxicity of TPZ to
hypoxic cells follows the general scheme outlined in FIG. 2.
However, whereas it had previously been proposed that

be developed specifically as a hypoxic cytotoxin and

for which antitumour activity has been demonstrated
in clinical trials. Before the discovery of this benzotriazine di-N-oxide, two other classes of agents were
known that produced some selective killing of hypoxic
cells: quinone-containing alkylating agents, of which
mitomycin C is the prototype; and nitroaromatic
compounds, of which misonidazole and RB 6145 are
a Normal

b Tumour

Blind
ends
Temporary
occlusion

Hypoxia

Break in
vessel walls

Red blood cells

AV shunt

Figure 1 | The vascular network of normal tissue versus tumour tissue. Tumours contain regions of hypoxia and necrosis
because their vasculature can not supply oxygen and other vital nutrients to all the cells. Whereas normal vasculature (a) is
hierarchically organized, with vessels that are sufficiently close to ensure adequate nutrient and oxygen supply to all cells, tumour
vessels (b) are chaotic, dilated, tortuous and are often far apart and have sluggish blood flow. As a consequence, areas of hypoxia
and necrosis often develop distant from blood vessels. In addition to these regions of chronic (or diffusion-limited) hypoxia, areas of
acute (or perfusion-limited) hypoxia can develop in tumours as a result of the temporary closure or reduced flow in certain vessels.
Adapted from REF. 125. AV, arteriovenous.

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Table 1 | Oxygenation of tumours and the surrounding normal tissue
Tumour type

Median tumour pO2*

(number of patients)

Median normal pO2*

(number of patients)

References

Glioblastoma

4.9 (10)
5.6 (14)

ND
ND

128
129

Head and neck

carcinoma

12.2 (30)
14.7 (23)
14.6 (65)

40.0 (14)
43.8 (30)
51.2 (65)

130
131
132

Lung cancer

7.5 (17)

38.5 (17)

Q. Le (personal
communication)

Breast cancer

10.0 (15)

ND

133

Pancreatic cancer

2.7 (7)

51.6 (7)

134

Cervical cancer

5.0 (8)
5.0 (74)
3 (86)

51 (8)
ND
ND

135
136
137

Prostate cancer

2.4 (59)

30.0 (59)

138

Soft-tissue sarcoma

6.2 (34)
18 (22)

ND
ND

139
140

*p02 measured in mmHg. Measurements were made using a commercially available oxygen
electrode (the Eppendorf electrode). The values shown are the median of the median values for
each patient. ND, not determined; pO2, oxygen partial pressure.

TOPOISOMERASE II

An enzyme that catalyses

changes in DNA topology by
transiently cleaving and
re-ligating both strands of the
double helix. This enzyme
catalyses the passage of one
DNA double-stranded molecule
through another.

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| JUNE 2004 | VOLUME 4

the damaging species was the TPZ radical itself 25, it now
seems that the toxic species is an oxidizing radical
formed by spontaneous decay of the protonated TPZ
radical; this ultimate cytotoxin has been indicated to be
either the hydroxyl radical26,27 or a benzotriazinyl (BTZ)
radical formed by loss of H2O28. The oxidizing radical
gives rise to cytotoxic DNA double-strand breaks
through a TOPOISOMERASE-II-dependent process29 (FIG. 3).
TPZ potentiates the antitumour effect of radiation
by selectively killing the hypoxic cells in the tumours.
As these are the most radiation-resistant cells in
tumours, TPZ and radiation act as complementary
cytotoxins, each one killing the cells resistant to the
other, thereby potentiating the efficacy of radiation on
the tumour. TPZ is also very effective in enhancing
the anticancer activity of the chemotherapeutic drug
cisplatin30, an interaction that again depends on
hypoxia31, but that results from an increase in cisplatin
sensitivity in non-lethally-damaged TPZ-treated cells
rather than from complementary killing of oxic and
hypoxic cells by the two agents, as is the case with
radiation. The interaction with cisplatin has been
tested in a Phase III clinical trial with advanced nonsmall-cell lung cancer and has been shown to be effective the addition of TPZ to the standard cisplatin
regimen doubled the overall response rate and significantly prolonged survival32. TPZ has also been tested
in a randomized Phase II trial with cisplatin-based
chemoradiotherapy of advanced head and neck cancer, and the preliminary results of this trial also show
improved survival in the group treated with TPZ33. A
Phase III study with cisplatin-based chemoradiotherapy is now underway. Although TPZ seems to have
clinical activity, and therefore provides important
proof of principle for this approach, the dose that can
be administered during chemoradiation is limited by
neutropaenia and other toxicities by as yet unknown
mechanisms. So, there is a clear need for improved
hypoxia-activated prodrugs.

AQ4N. The only other hypoxia-activated prodrug now

in clinical trials the anthraquinone AQ4N (FIG. 4)
was designed specifically for this purpose. It resembles
TPZ in being a di-N-oxide, but has a distinct mechanism
of activation and cytotoxicity. AQ4N is a prodrug of a
potent DNA intercalator/topoisomerase poison, AQ4,
which is formed by reduction of the two tertiary amine
N-oxide groups that mask DNA binding in the prodrug
form34. AQ4N is unusual among hypoxia-activated prodrugs in being activated by two-electron reduction,
which is effected mainly by the CYP3A members of the
cytochrome P450 family 35, which are strongly expressed
in some human tumours35. Inhibition by oxygen results
from competition between O2 and prodrug for binding
at the reduced haem group in the enzyme active site,
rather than from redox cycling36. Although AQ4 is selective for cycling cells, its long residence time in tissue
probably enables it to persist until hypoxic cells come
into cycle37. AQ4N has substantial activity against
hypoxic cells in various transplanted tumours38 and has
recently completed a Phase I clinical trial. The results of
further clinical evaluation are awaited with interest.
Hypoxia prodrugs in preclinical development

Originally the province of academic groups with an

interest in the radiation resistance of hypoxic cells3946,
several pharmaceutical companies are also now developing prodrugs for exploiting hypoxia. The strategies
that are being pursued in the development of improved
hypoxia prodrugs are outlined below.
DNA targeting. One approach is to try to increase
potency by linking DNA-targeting units to moieties
known to damage DNA in hypoxic cells. An example is

a Oxic cell
1e reductases
e

D
O2

D
O2

b Hypoxic cell
1e reductases
D

Toxic drug

Figure 2 | The usual mechanism by which prodrugs act as

hypoxia-selective cytotoxins. The non-toxic prodrug (D) must
be a substrate for intracellular one-electron (1e) reductases,
such as cytochrome P450 reductase, which add an electron to
the prodrug and therefore convert it to a free radical. a | In oxic
cells, the unpaired electron in the prodrug radical is rapidly
transferred to molecular oxygen, forming superoxide and
regenerating the initial prodrug. This futile redox cycle prevents
build-up of the prodrug radical when O2 is present. b | Hypoxiaselective cell killing is achieved if the prodrug radical that
accumulates in hypoxic cells is more cytotoxic than the
superoxide formed in oxic cells. In principle, the prodrug radical
could itself be the cytotoxin, but more commonly it undergoes
further reactions to form the ultimate toxic species.

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REVIEWS
O
N+

1e reductases

e, H +

N+

N
N

O2

TPZ

NH2

H2O

O2

NH

BTZ

NH2

Topoisomerase ll
poisoning and
DNA doublestrand breaks

OH

OH

TPZ

Figure 3 | The mechanism by which tirapazamine selectively kills hypoxic cells.

Tirapazamine (TPZ) is a substrate for one-electron (1e) reductases. The resulting free radical
(TPZ) undergoes spontaneous decay to an oxidizing hydroxyl radical (OH) or an oxidizing
benzotriazinyl radical (BTZ). When oxygen is present, the TPZ radical is back-oxidized to the
parent compound, producing a superoxide radical (O2 ) which might be responsible for the
muscle cramps seen in patients given the drug126. The available evidence is that the doublestrand breaks are not caused directly by the oxidizing radical (OH or BTZ), but, at least in part,
through poisoning of topoisomerase II29. This could be the result of radical damage directly to
the topoisomerase II enzyme, therefore poisoning it midway through its catalytic cycle and
producing a double-strand break in much the same way as etoposide; or the radical damage
to DNA could act as a substrate for topoisomerase II, so producing double-strand breaks

the linkage of a chloroquinoline DNA-targeting unit to

2-nitroimidazole, as in the prodrug NLCQ-1 (FIG. 4),
which shows hypoxia-selective cytotoxicity in cell culture and a favourable interaction with radiotherapy
and chemotherapy in transplanted tumours47. The relatively low DNA-binding affinity of the chloroquinoline
unit is probably important in allowing adequate penetration through tumour tissue, which can be severely
constrained by avid DNA binding48. It is not yet clear

ELECTROPHILE

A chemical group that reacts

with electron-rich centres in
molecules.
FREE RADICAL

A compound with an unpaired

electron and that is usually very
reactive because of this feature.

O
OH

OH

N+

HN

HN

N+

AQ4N

CH3
CH3

CH3
CH3

NO2
HN

OH

HN

NO2

1e reductases

HN

CH3
CH3

Topoisomerase ll
poisoning
OH

O2

O2

CH3
CH3

NO
N

DNA-targeted
reactive
electrophile

O2

NLCQ-1

Cl

2e reductases
(CYP3A)

NO2
2

NO2
CONH2

NH 2

CONH2

CONH2

1e reductases

O2N

O2N
N

Cl

Cl

O2

O2

Cl

DNA
crosslinks

O2N
N

N
Cl

Cl

Cl

SN 23862

Figure 4 | Mechanisms of activation under hypoxia of prodrugs. a | AQ4N is reduced by a

2-electron (2e) process to form the potent DNA intercalator and topoisomerase inhibitor AQ4.
b | With NLCQ-1, DNA binding of the chloroquinoline unit is considered to target reactive
species, arising from reduction of the 2-nitroimidazole moiety to DNA. c | SN 23862 is a latent
nitrogen mustard in which reduction (predominantly at the 2-nitro group) to the corresponding
amine under hypoxic conditions greatly increases the reactivity of the mustard moiety. CYP3A,
cytochrome P450 3A.

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whether the tumour activity of NLCQ-1 is primarily

because of radiosensitization or hypoxic cytotoxicity,
but its toxicology is now being evaluated in anticipation
of a Phase I clinical trial. The linkage of TPZ-like benzotriazines to DNA intercalators has also recently been
shown to greatly increase hypoxic cytotoxicity 49, and
analogues with a range of DNA-binding affinities are
under investigation50.
Prodrugs of relatively stable cytotoxins that provide
bystander effects. Most of the first-generation hypoxic
cytotoxins (AQ4N is an exception) were quinones, nitro
compounds or aromatic N-oxides, which are metabolized to reactive ELECTROPHILES or FREE RADICALS that are not
able to escape from the hypoxic cells in which they are
generated. Much of the recent research is focused on the
development of prodrugs that release more stable cytotoxins on reduction. These prodrugs can be considered
to comprise three modular domains: a trigger unit that
is reduced selectively under hypoxia; an effector, which
is the drug moiety responsible for cell killing (or other
desired effect); and a linker, which transmits the triggering event to the effector (for example, by fragmentation
or through an electronic change)43,51.
The release of an active drug that can diffuse from
the cell of origin to generate a BYSTANDER EFFECT offers a
way of killing more than just the hypoxic subpopulation
in tumours42, and by partially decoupling activation
from killing an important problem in exploiting
hypoxia as a tumour-selective target is solved. Although
it is certainly a valid generalization that hypoxia is more
severe in tumours than in normal tissues, there is a heterogeneous distribution of oxygen in many tissues. The
normal tissues that are known or suspected to include
regions of mild (physiological) hypoxia include liver,
bone marrow, skin, testis, retina and cartilage. The oxygen concentration required to inhibit the activation of
TPZ by 50% (KO2) is approximately 13 M52,53, which
is considerably higher than for most quinones54, nitro
compounds52,55 and transition metals56, which typically
have KO2 values 0.1 M. The high KO2 value of TPZ is
both a strength and a weakness. On the one hand, it
ensures activation in mildly hypoxic tumour cells at
intermediate O2 concentrations (125 M) that are
considered to be the most important in limiting the
response to radiation therapy57. But on the other hand,
this relative insensitivity to O2 allows some activation in
normal tissues, as illustrated by the irreversible toxicity
of TPZ to physiologically hypoxic photoreceptor cells in
the retinal tissue of mice58. Retinal damage has not been
a problem in humans, but it is possible that hypoxia in
the stem-cell compartment of bone marrow59,60 contributes to the myelotoxicity of TPZ. The newer generation of prodrugs offers a way out of this dilemma. Using
trigger units that are activated only at very low O2 concentrations should make it possible to confine activation
to regions of severe hypoxia, which are essentially
unique to tumours, whereas release of an effector that
can cause bystander killing makes it possible to eliminate adjacent radioresistant cells at higher pO2. This
more sophisticated approach effectively redefines the
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Box 2 | CB 1954: prodrug extraordinaire

CB 1954 first came to attention because of its marked curative activity against the Walker rat tumour118. It was subsequently
shown to be a bioreductive prodrug, activated within the tumours by rat DT-diaphorase (DTD), which reduces its 4-nitro
group to the corresponding hydroxylamine119,120; following reaction of the hydroxylamine with acetyl CoA, the latter
becomes a very potent DNA-crosslinking agent119. CB 1954 is even more efficiently activated by an Escherichia coli
nitroreductase (NTR, the product of the nfsB gene)121, and has recently entered a clinical trial122 as gene-dependent enzyme
prodrug therapy (GDEPT) using a non-replicating adenoviral vector that expresses NTR. NTR reduces either (but not
both) nitro groups of CB 1954, and recent data indicate that the 2-amino reduction product might be the key metabolite
responsible for bystander effects in NTR-GDEPT116. CB 1954 is also activated by one-electron (1e) reductases, such as
cytochrome P450 reductase (P540R), selectively under hypoxia123, but the related dinitrobenzamide mustards (see main
text) seem to have greater selectivity for hypoxia because of their lack of sensitivity to activation by DTD124.
NHOH

NTR

CONH2

O2N

DNA
mono-adducts

O2N

NO2
2

NH2

CONH2

2e reductases:

CONH2

O2N

NO2

2e reductases:
NTR, DTD
1e reductases:
P450R

BYSTANDER EFFECT

Influence of a drug on
untargeted cells, in the present
context by diffusion of an
activated cytotoxin from
hypoxic cells to surrounding
cells at higher oxygen
concentrations.
NITROGEN MUSTARD

DNA-crosslinking alkylating
agents containing a bis(Xethyl)amine group, where X is an
electrophile that can react with
nucleophiles such as the N7
position of guanine.

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| JUNE 2004 | VOLUME 4

NO2

CONH2

CONH2
O

Acetyl CoA

HOHN
N

therapeutic target as not simply hypoxia in any cell, but

as cells adjacent to regions of severe (pathological)
hypoxia, therefore sparing physiologically hypoxic
normal tissues.
A class of hypoxia-activated prodrugs that fits this
profile is the dinitrobenzamide mustards, illustrated by
SN 23862 (FIG. 4). This is a NITROGEN MUSTARD analogue of
an aziridine prodrug, CB 1954, which has long fascinated
experimental oncologists (BOX 2). Reduction of either
nitro group of SN 23862 acts as an electronic switch,
redistributing electron density in the aromatic ring
(linker) to activate the nitrogen mustard61. The 2-amine
reduction product is a key metabolite under hypoxia, and
shows a 2000-fold increase in alkylating reactivity and
cytotoxic potency relative to the parent prodrug 62. This
metabolite is known to provide an efficient bystander
effect in three-dimensional cultures of WiDr cells when
SN 23862 is metabolized by Escherichia coli nitroreductase63 and recent studies confirm its bystander effect when
activated by endogenous reductases under hypoxia (S. M.
Pullen, A.V. Patterson and W.R.W., unpublished observations). Importantly, activation of SN 23862 by endogenous one-electron reductases is readily inhibited by very
low concentrations of oxygen. An analogue development
programme based on SN 23862 is well advanced; a watersoluble derivative (SN 28343) with excellent activity
against hypoxic cells in transplanted murine and human
tumours is in preclinical development.
Other prodrugs that can release well-defined cytotoxins on reduction in hypoxic cells include nitrobenzyl phosphoramidate mustards64, nitroheterocyclic
methylquaternary salts 65, cobalt(III) complexes66
and indoloquinones67.

N
H

DNA
crosslinks

Radiation-activated prodrugs. One of the limitations

in restricting prodrug activation to severely hypoxic
tissue is that a large proportion of such tissue is
necrotic, lacking the enzymes and cofactors needed to
reduce prodrugs. It would therefore be very attractive
to activate prodrugs under hypoxia by reducing them
with ionizing radiation (which is widely used to treat
tumours despite the presence of significant hypoxia)
rather than enzymes. Radiolysis of water generates
the aquated electron (e aq), which is a much more
powerful reductant than enzymes and is readily scavenged by O2 in oxic cells (to form superoxide). Such
radiation-activated prodrugs offer several theoretical
advantages in addition to expanding the extent of the
hypoxic zone that can be used for prodrug activation68. In particular, the fact that radiotherapy focuses
the radiation field on the tumour and a small volume
of surrounding tissue provides tumour specificity
additional to hypoxia alone. In addition, the lack of
requirement of enzymatic activation makes the
approach independent of the reductase expression
profile in the target tumour. However the low yield of
eaq during radiotherapy (approximately 20 mol/kg
over a typical course of 70 Gy), coupled with competition with endogenous electron acceptors, will
require release of very potent cytotoxins if this
theoretically attractive approach is to be realized.
Three prodrug systems have been described that are
efficiently activated by ionizing radiation under
hypoxia nitrobenzyl quaternary ammonium
salts69, cobalt(III) complexes70, and oxypropyl-substituted 5-fluorouracil derivatives71 but none have
yet provided convincing activity in transplanted
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a Oxygenated tissue
Promoter

Prodrug-metabolizing enzyme gene

Hypoxiaresponsive
elements
Prodrug

Toxic drug

b Hypoxic tissue
HIF-1
dimer

mRNA

Enzyme

Prodrug

Toxic drug

Figure 5 | Rationale for hypoxia-dependent gene

therapy. Shows how the hypoxic environment of tumours,
which produces high levels of the hypoxia-inducible factor 1
(HIF-1) transcription factor, could be used in gene therapy to
produce tumour-specific expression of an enzyme that can
metabolize a non-toxic prodrug into a toxic drug selectively in
the tumour. a | In oxygenated tissue, there is little or no HIF-1
transcription factor. Also, oxygen inhibits HIF-1
transactivation. Consequently, no prodrug-metabolizing
enzyme is produced, so little or none of the prodrug is
converted to the toxic drug. b | In hypoxic tumour tissue, HIF1 is produced and downstream genes are transcribed
following binding of HIF-1 to the hypoxia-responsive
elements (HREs) in the promoter region of the genes.
Therefore, the prodrug-activating enzyme with HREs in its
promoter will be transcribed and, after translation, activate
the prodrug to the toxic drug selectively in the tumour.

tumours. Use of effectors with much greater cytotoxic potency than those that have been investigated
so far will probably be needed.
Hypoxia-selective gene therapy

GDEPT

(Gene-directed enzyme prodrug

therapy). A cancer treatment
strategy that aims to deliver a
prodrug-activating enzyme
specifically to tumour cells using
gene therapy. The anticancer
effect would be achieved by
subsequent systemic
administration of the non-toxic
prodrug, which would be
converted to a toxic drug
preferentially in the tumour cells.

NATURE REVIEWS | C ANCER

A key limitation of present day gene therapy of cancer is the lack of specificity of the gene-delivery
system. Accordingly, essentially all of the protocols
now being investigated in cancer gene therapy involve
local administration of the delivery vectors directly
into the tumour, usually by needle injection.
Although this might be useful in some cases, it has
limited applicability to cancer in general because
metastases from the primary tumour are usually too
numerous, inaccessible or undetected to allow for
direct injection. An alternative to direct targeting of
tumours is to have the therapeutic gene transcribed
or translated by a tumour-specific property so that

expression of a particular protein would be tumour

specific. One way to do this is to use the fact that the
transcription factor HIF-1 is expressed at high levels
in most tumours, but not generally in normal tissues 72,73. HIF-1 comprises a dimer of HIF-1 and
HIF-1, and it is the former that is increased in
tumour cells both by increased transcription by
transformed cells and by stabilization of the protein
under hypoxic conditions 17. HIF-1 expression is
also associated with poor prognosis and resistance to
therapy in head and neck cancer, ovarian cancer and
oesophageal cancer72,73.
HIF-1 stimulates the transcription of a large
number of genes involved in such processes as oxygen
transport, angiogenesis, glycolysis and stress
response 17. Transcription of all of these genes is
effected by the binding of the HIF-1 dimer to
sequences known as hypoxia-responsive elements
(HREs) in the promoter regions of the target genes.
Therefore, the strategy indicated to obtain hypoxiaspecific transcription of a therapeutic gene would be
to develop a promoter that is highly responsive to
HIF-1 that would therefore drive the expression of
the therapeutic gene specifically in tumours (FIG. 5).
Expression of an enzyme that is not normally found
in the human body (for example, cytosine deaminase
derived from E. coli) could, under the control of a
hypoxia-responsive promoter, convert a non-toxic
prodrug into a toxic drug in the tumour. Promoters
using HREs from hypoxia-responsive genes have
been developed 74,75, and in vivo activity has been
obtained in experimental tumour systems either by
direct injection of adenoviral vectors containing the
HRE promoters76 or using tumour cells stably transfected with HRE-regulated prodrug-activating
enzymes 77,78. Unfortunately the latter systems, in
which 100% of the tumour cells carry the hypoxiaresponsive gene, are not realistic to achieve in a clinical situation. However, this might not be a crucial
limitation: various investigators have shown that,
provided the active drug can diffuse from the cell in
which it is generated to kill surrounding cells (the socalled bystander effect discussed above), efficient
antitumour activity can be obtained with much lower
percentages of transformed cells63,79. A further potential strategy for exploiting hypoxia in gene therapy is
to deliver the gene encoding a one-electron reductase
such as cytochrome P450 reductase (P450R) as the
prodrug-activating therapeutic gene, so confining
prodrug activation to hypoxic regions; this can be
combined with HIF-1 regulation of P450R expression to further enforce tumour selectivity78. A similar
approach relies on hypoxia-selective metabolism of
AQ4N by CYP3A4 as an enzymeprodrug system for
gene-directed enzyme prodrug therapy (GDEPT)80.
A challenge with these approaches will be achieving
efficient systemic delivery of vectors to cells expressing
HIF-1 and/or hypoxic cells, which are generally found
in regions distant from blood vessels. One possibility
for delivery of the HRE-driven therapeutic protein to
tumours would be to take advantage of the fact that
VOLUME 4 | JUNE 2004 | 4 4 3

REVIEWS

HIF-1 staining

Blood vessels
Active drug
Clostridia-filling
necrosis

Figure 6 | Clostridial-dependent enzyme prodrug

therapysimulation of how it might work.
A photomicrograph of a section of a human head and neck
cancer biopsy immunostained for the hypoxia-inducible
factor-1 (HIF-1). The likely distribution of viable anaerobic
bacteria (clostridia) and the concentration of active drug
formed from the reaction of the prodrug with the enzyme
expressed by the recombinant clostridia in the tumour have
been simulated by drawing over the necrotic regions. The
concentrations of the active drug will probably be highest
next to areas of necrosis and far from blood vessels.
Photomicrograph modified with permission from REF. 127
(2001) American Association for Cancer Research.

macrophages are often recruited to tumours and that

such macrophages show increased levels of HIF-1 in
various human tumours81,82.
Targeting HIF-1

The characteristics, functions and possibilities for

targeting HIF-1 in cancer therapy have been recently
reviewed83,84. Its role in angiogenesis, glucose utilization and tumour-cell survival85, its association with
poor prognosis17, and the fact that growth of mouse
xenografts is inhibited by loss of HIF-1 activity85 all
make it a potentially attractive tumour-specific
target. It should be noted that the expression of
HIF-1 is not restricted to hypoxic cells alone in many
tumours, but is also upregulated by oncogenic mutations in RAS, SRC or ERBB2 (also known as
HER2/NEU). Therefore, targeting HIF-1 could
potentially target the better oxygenated cells in the
tumours 84. Three general approaches could be used
to exploit the high levels of HIF-1 in cancers.
First, inhibition of transactivation of HIF-1 target
genes (such as the angiogenesis inducer vascular
endothelial growth factor) would be expected to have
an antitumour effect. Proof of principle of this
approach comes from studies by Kung and colleagues, who showed that tumour cells infected with
a polypeptide that disrupted the binding of HIF-1
to its transcriptional coactivators p300/CREB,
thereby inhibiting hypoxia-induced transcription,
markedly reduced the growth of these cells when
transplanted into nude mice86. These data have led
investigators to screen for small molecules that

444

| JUNE 2004 | VOLUME 4

inhibit HIF-1 transcription, and early reports have

indicated that such compounds exist 87. However,
anticancer effects directly attributable to inhibition of
HIF-1 transactivation have yet to be reported.
A second approach is to suppress HIF-1 protein
levels, either by destabilizing the protein or inhibiting
its production. The heat-shock protein 90 inhibitor
geldanamycin has been shown to reduce HIF-1
protein levels by promoting its oxygen and VHLindependent degradation through the proteasome88,89.
However, it has yet to be demonstrated that this
occurs in vivo or that the antitumour activity of this
compound is a direct result of reduced levels of HIF-1,
as many other proteins are also affected. Targeting of
HIF-1 by direct injection of an antisense construct to
HIF-1 has been shown to eradicate a small transplanted thymic lymphoma and to increase the efficacy
of immunotherapy against larger tumours 88.
However, small-molecule inhibitors of HIF-1 would
be preferable, and two groups have reported success.
Mabjeesh and colleagues reported that microtubule
inhibitors such as 2-methoxyestradiol, vincristine and
paclitaxel reduce HIF-1 levels in vitro apparently by
inhibiting translation of HIF-1 mRNA90. These
compounds can also reduce tumour growth and vascularity, but whether this is an effect of reduced levels
of HIF-1 or a direct effect on microtubules is not
known. The second small molecule that has been
reported to reduce HIF-1 levels and inhibit tumour
growth is the soluble guanylyl cyclase stimulator YC-1
(REF. 91). Soluble guanylyl cyclase is the receptor for
nitric oxide (NO) a molecule involved in many signalling pathways, including those regulating vascular
tone and platelet function. However, the authors
attribute the antitumour and anti-angiogenic effects
of YC-1 to a reduction in HIF-1 protein levels (by an
unknown post-translational effect) rather than to an
effect on NO signalling.
A third approach would be to screen for compounds that are preferentially toxic to cells expressing
HIF-1. At present this is a theoretical possibility
with no published data demonstrating its efficacy.
Recombinant anaerobic bacteria

Brown and colleagues first indicated that the necrotic

regions in human solid tumours could be used to target cancer therapy to tumours using a genetically
engineered non-pathogenic strain of the bacterial
genus Clostridium9294. This genus comprises a large
and heterogeneous group of Gram-positive, sporeforming bacteria that become vegetative and grow
only in the absence (or at very low levels) of oxygen.
Malmgren and Flanagan were the first to demonstrate this phenomenon by observing that tumourbearing mice died of tetanus within 48 hours of
intravenous injection of C. tetani spores, whereas
non-tumour-bearing animals were unaffected 95.
Mse and Mse96,97 later reported that a nonpathogenic clostridial strain, C. butyricum M-55,
localized and germinated in solid Ehrlich tumours in
mice, causing extensive lysis without any concomitant
www.nature.com/reviews/cancer

REVIEWS

CDEPT

(Clostridial-dependent enzyme
prodrug therapy). A cancer
therapy using the nonpathogenic species of the
obligate anaerobe genus
clostridia that have been
genetically engineered to express
a prodrug-activating enzyme.
This is used to activate a prodrug
within the hypoxic/necrotic
regions that are colononized by
the bacterium.
ADEPT

(Antibody-directed enzyme
prodrug therapy). A cancer
treatment strategy that involves
conjugation of a prodrugactivating enzyme (such as
cytosine deaminase, which
converts the non-toxic prodrug
5-fluorocytosine to the
anticancer drug 5-fluorouracil)
to a tumour-targeting antibody.
VASCULAR-TARGETING AGENT

Drugs that damage existing

blood vessels and therefore
interfere with blood flow in
tumours.

effect on normal tissues. Such observations were soon

confirmed and extended by several investigators using
tumours in mice, rats, hamsters and rabbits98,99, and
were followed by clinical studies with patients with
cancer100102. Although the anaerobic bacteria did not
significantly alter tumour control or eradication, these
clinical reports demonstrated that spores of nonpathogenic strains of clostridia could be given safely,
that the spores germinate in the necrotic regions of
tumours, and that lysis in these tumour regions can
occur. This is an important distinction over the similar approach using genetically modified, live attenuated Salmonella, which, although producing excellent
colonization of transplanted tumours in mice103, produced only marginal colonization of human tumours
in a Phase I clinical trial104. The reasons for the difference between the rodent and human tumours in
colonization by Salmonella are unknown. However,
colonization by clostridia is different from that of
Salmonella in being dependent on hypoxic necrotic
regions, which are equally common in human and
rodent tumours. In addition, as noted above, excellent
colonization of human tumours has been reported
following intravenous injection of clostridial spores.
The Clostridium used in the clinical studies was a
strain of C. sporogenes, renamed C. oncolyticum to
reflect the lysis that was produced in human tumours.
This strain has been genetically modified to express
the E. coli enzyme cytosine deaminase, which can convert the non-toxic 5-fluorocytosine to the toxic anticancer drug 5-fluorouracil. Animal experiments have
demonstrated the efficacy of this approach105 and clinical studies are planned. In addition, other
enzymeprodrug systems for arming clostridia are in
development, including CB 1954 (BOX 2), which, when
activated by E. coli nitroreductase, kills non-cycling
cells efficiently106 and is therefore expected to have
greater activity against cells in hypoxic regions. (See
a recent review for further details of possible
enzymeprodrug combinations that can be used with
clostridial targeting of tumours 107.) Although
clostridial-dependent enzyme prodrug therapy (CDEPT)
is similar to the strategy of antibody-dependent
enzyme prodrug therapy (ADEPT), which is now under
clinical evaluation, it has several significant advantages, not the least of which is its favorable intratumour distribution. Because the prodrug-activating
enzyme from clostridia will be at its highest concentration in areas adjacent to necrosis and far from
blood vessels (FIG. 6), this guarantees the highest activedrug concentrations in the distant cells and also minimizes the problem of leakage of activated drug back
into the blood vessels, which has been reported to be a
problem for ADEPT108.
Improving CDEPT with vascular targeting drugs. A
way in which targeting of clostridia to solid tumours
might be improved still further, and perhaps
extended to very small tumours that have not
yet developed necrotic areas, is by the addition of
VASCULAR-TARGETING AGENTS. Such agents include DMXAA

NATURE REVIEWS | C ANCER

(5,6-dimethylxanthenone-4-acetic acid), which acts

primarily by inducing expression of tumour-necrosis
factor in tumours 109,110, and the tubulin-binding
agent combretastatin 4A and its analogue ZD 6126.
These agents produce a rapid and selective occlusion
of tumour blood vessels, leading to necrosis within
1624 hours of administration. Phase I clinical trials
of DMXAA, combretastatin 4A and ZD 6126 have
recently been completed and have demonstrated
reduced blood flow in human tumours 111,112. The
ability of vascular-targeting agents to produce
tumour necrosis has been shown to increase the colonization113 and antitumour activity114 of clostridial
spores injected intravenously. This is a significant
development for CDEPT because even if only a modest increase in tumour necrosis occurs with the clinical use of vascular-targeting agents, this could be
exploited to great advantage with clostridial spores
that grow exclusively in necrotic areas.
Future directions

The benefits of exploiting tumour hypoxia have yet to

be fully realized. Despite this, the positive clinical
results with the combination of the hypoxic cytotoxin
tirapazamine with cisplatin to treat advanced nonsmall-cell lung cancer and with chemoradiotherapy to
treat advanced head and neck cancer demonstrate the
potential of this approach. There is good reason to
expect that future drugs or strategies will do better: in
particular, we know that the efficacy of tirapazamine
and other hypoxic cytotoxins is reduced by their limited diffusion through tumour tissue to reach all of
the hypoxic cells115. We now have tools for quantifying
the ability of prodrugs, and their activated metabolites, to diffuse in tumour tissue48,63,115,116, so designing
second-generation prodrugs with properly optimized
micropharmacokinetic properties is a clear possibility
for the future.
The other strategies discussed above hypoxia
activated gene therapy, targeting HIF-1 and the use of
recombinant clostridia are certainly promising
and have demonstrated antitumour efficacy in preclinical studies. What is now needed is the development of the optimum drug or vector combination for
each strategy and their clinical testing. Relevant to the
latter is the appropriate selection of patients with
whom to use hypoxia-directed treatments. As with
any targeted anticancer strategy, hypoxia-directed
therapy can only be effective on those tumours
expressing the target or, in this case, with sufficient
levels of hypoxia. Performing clinical trials on unselected patients who have a mixture of hypoxic and
better-oxygenated tumours runs the clear risk of
rejecting a treatment that could be of significant benefit to a subset of patients. The most appropriate
means of assessing tumour hypoxia to perform such
a selection is under active investigation 117. Clearly,
there is much to be done to exploit the unique features of hypoxia, HIF-1, other molecular targets
upregulated under hypoxia and necrosis in human
solid tumours, but the future is bright.
VOLUME 4 | JUNE 2004 | 4 4 5

REVIEWS
1.

2.

3.
4.

5.

6.

7.

8.

9.
10.

11.

12.

13.

14.

15.

16.
17.
18.

19.
20.

21.

22.

23.

24.

25.

446

Thomlinson, R. H. & Gray, L. H. The histological structure

of some human lung cancers and the possible
implications for radiotherapy. Br. J. Cancer 9, 539549
(1955).
Gray, L. H., Conger, A. D., Ebert, M., Hornsey, S. & Scott, O. C.
Concentration of oxygen dissolved in tissues at the time of
irradiation as a factor in radiotherapy. Br. J. Radiol. 26,
638648 (1953).
References 1 and 2 are classic papers describing both
the general nature of the oxygen effect in reducing
radiation sensitivity (reference 2) and the fact that the
hypoxic cells almost certainly are present in human
tumours (reference 1).
Brown, J. M. Clinical trials of radiosensitizers: what should we
expect? Int. J. Radiat. Oncol. Biol. Phys. 10, 425429 (1984).
Overgaard, J. Clinical evaluation of nitroimidazoles as
modifiers of hypoxia in solid tumors. Oncol. Res. 6, 509518
(1994).
Vaupel, P., Schlenger, K., Knoop, C. & Hockel, M.
Oxygenation of human tumors: evaluation of tissue
oxygen distribution in breast cancers by computerized O2
tension measurements. Cancer Res. 51, 33163322
(1991).
Nordsmark, M., Overgaard, M. & Overgaard, J.
Pretreatment oxygenation predicts radiation response in
advanced squamous cell carcinoma of the head and neck.
Radiother. Oncol. 41, 3140 (1996).
Brizel, D. M., Dodge, R. K., Clough, R. W. & Dewhirst, M. W.
Oxygenation of head and neck cancer: changes during
radiotherapy and impact on treatment outcome. Radiother.
Oncol. 53, 113117 (1999).
Tannock, I. F. Conventional cancer therapy: promise
broken or promise delayed? Lancet 351 (Suppl. 2), 916
(1998).
Durand, R. E. The influence of microenvironmental
factors during cancer therapy. In vivo 8, 691702 (1994).
Tannock, I. F. The relation between cell proliferation and the
vascular system in a transplanted mouse mammary tumour.
Br. J. Cancer 22, 258273 (1968).
Teicher, B. A., Lazo, J. S. & Sartorelli, A. C. Classification of
antineoplastic agents by their selective toxicities toward
oxygenated and hypoxic tumor cells. Cancer Res. 41,
7381 (1981).
Batchelder, R. M., Wilson, W. R., Hay, M. P. & Denny, W. A.
Oxygen dependence of the cytotoxicity of the enediyne antitumour antibiotic esperamicin A1. Br. J. Cancer Suppl. 27,
S52S56 (1996).
Comerford, K. M. et al. Hypoxia-inducible factor-1dependent regulation of the multidrug resistance (MDR1)
gene. Cancer Res. 62, 33873394 (2002).
Wartenberg, M. et al. Regulation of the multidrug resistance
transporter P-glycoprotein in multicellular tumor spheroids
by hypoxia-inducible factor (HIF-1) and reactive oxygen
species. FASEB J. 17, 503505 (2003).
Graeber, T. G. et al. Hypoxia-mediated selection of cells with
diminished apoptotic potential in solid tumours. Nature 379,
8891 (1996).
Describes how hypoxia in tumours selects against
wild-type p53 by causing apoptosis in these cells.
Yuan, J. & Glazer, P. M. Mutagenesis induced by the tumor
microenvironment. Mutat. Res. 400, 439446 (1998).
Harris, A. L. Hypoxia a key regulatory factor in tumour
growth. Nature Rev. Cancer 2, 3847 (2002).
Pennacchietti, S. et al. Hypoxia promotes invasive growth by
transcriptional activation of the met protooncogene. Cancer
Cell 3, 347361 (2003).
Rofstad, E. K. Microenvironment-induced cancer
metastasis. Int. J. Radiat. Biol. 76, 589605 (2000).
Subarsky, P. & Hill, R. P. The hypoxic tumour
microenvironment and metastatic progression. Clin. Exp.
Metastasis 20, 237250 (2003).
Hockel, M. et al. Association between tumor hypoxia and
malignant progression in advanced cancer of the uterine
cervix. Cancer Res. 56, 45094515 (1996).
Brizel, D. M. et al. Tumor oxygenation predicts for the
likelihood of distant metastases in human soft tissue
sarcoma. Cancer Res. 56, 941943 (1996).
Zeman, E. M., Brown, J. M., Lemmon, M. J., Hirst, V. K. &
Lee, W. W. SR-4233: a new bioreductive agent with high
selective toxicity for hypoxic mammalian cells. Int. J. Radiat.
Oncol. Biol. Phys. 12, 12391242 (1986).
The initial paper describing the hypoxic selectivity of
tirapazamine for cell killing.
Brown, J. M. & Lemmon, M. J. Potentiation by the hypoxic
cytotoxin SR 4233 of cell killing produced by fractionated
irradiation of mouse tumors. Cancer Res. 50, 77457749
(1990).
Brown, J. M. SR 4233 (tirapazamine): a new anticancer drug
exploiting hypoxia in solid tumours. Br. J. Cancer 67,
11631170 (1993).

| JUNE 2004 | VOLUME 4

26. Daniels, J. S. & Gates, K. S. DNA cleavage by the antitumor

agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (SR4233):
Evidence for involvement of hydroxyl radical. J. Am. Chem.
Soc. 118, 33803385 (1996).
27. Zagorevskii, D. et al. A mass spectrometry study of
tirapazamine and its metabolites. insights into the
mechanism of metabolic transformations and the
characterization of reaction intermediates. J. Am. Soc. Mass
Spectrom. 14, 881892 (2003).
28. Anderson, R. F., Shinde, S. S., Hay, M. P., Gamage, S. A. &
Denny, W. A. Activation of 3-amino-1,2,4-benzotriazine 1,4dioxide antitumor agents to oxidizing species following their
one-electron reduction. J. Am. Chem. Soc. 125, 748756
(2003).
29. Peters, K. B. & Brown, J. M. Tirapazamine: a hypoxiaactivated topoisomerase II poison. Cancer Res. 62,
52485253 (2002).
30. Dorie, M. J. & Brown, J. M. Tumor-specific, scheduledependent interaction between tirapazamine (SR 4233) and
cisplatin. Cancer Res. 53, 46334636 (1993).
In vivo data showing that tirapazamine and cisplatin
have a marked hypoxia and schedule-dependent
synergism.
31. Kovacs, M. S. et al. Cisplatin anti-tumour potentiation by
tirapazamine results from a hypoxia-dependent cellular
sensitization to cisplatin. Br. J Cancer 80, 12451251
(1999).
32. von Pawel, J. et al. Tirapazamine plus cisplatin versus
cisplatin in advanced non-small-cell lung cancer: A report of
the international CATAPULT I study group. J. Clin. Oncol. 18,
13511359 (2000).
Clinical data showing the efficacy of tirapazamine in
combination with cisplatin in a randomized
multicentre Phase III trial.
33. Rischin, D. et al. Preliminary results of TROG 98.02a
randomized phase II study of 5-fluorouracil, cisplatin and
radiation versus tirapazamine, cisplatin and radiation for
advanced squamous cell carcinoma of the head and neck.
Proc. Am. Soc. Clin. Oncol. 22, A1992 (2003).
34. Patterson, L. H. Rationale for the use of aliphatic N-oxides of
cytotoxic anthraquinones as prodrug DNA binding agents: a
new class of bioreductive agent. Cancer Metastasis Rev. 12,
119134 (1993).
35. Patterson, L. H. Bioreductively activated antitumor
N-oxides: the case of AQ4N, a unique approach to hypoxiaactivated cancer chemotherapy. Drug Metab. Rev. 34,
581592 (2002).
36. Patterson, L. H. & Murray, G. I. Tumour cytochrome P450
and drug activation. Curr. Pharm. Des. 8, 13351347
(2002).
37. Patterson, L. H. & McKeown, S. R. AQ4N: a new approach
to hypoxia-activated cancer chemotherapy. Br. J. Cancer
83, 15891593 (2000).
38. Patterson, L. H. et al. Enhancement of chemotherapy and
radiotherapy of murine tumours by AQ4N, a bioreductively
activated anti-tumour agent. Br. J. Cancer 82, 19841990
(2000).
Demonstration of potentiation of radiation and
chemotherapy by the bioreductive agent AQ4N.
39. Adams, G. E. & Stratford, I. J. Bioreductive drugs for cancer
therapy: the search for tumor specificity. Int. J. Radiat.
Oncol. Biol. Phys. 29, 231238 (1994).
40. Brown, J. M. & Siim, B. G. Hypoxia-specific cytotoxins in
cancer therapy. Semin. Rad. Onc. 6, 2236 (1996).
41. de Groot, F. M., Damen, E. W. & Scheeren, H. W. Anticancer
prodrugs for application in monotherapy: targeting hypoxia,
tumor-associated enzymes, and receptors. Curr. Med.
Chem. 8, 10931122 (2001).
42. Denny, W. A. & Wilson, W. R. Bioreducible mustards: a
paradigm for hypoxia-selective prodrugs of diffusible cytotoxins
(HPDCs). Cancer Metastasis Rev. 12, 135151 (1993).
43. Naylor, M. A. & Thomson, P. Recent advances in
bioreductive drug targeting. Mini Rev. Med. Chem. 1, 1729
(2001).
44. Rauth, A. M., Melo, T. & Misra, V. Bioreductive therapies: an
overview of drugs and their mechanisms of action. Int. J.
Radiat. Oncol. Biol. Phys. 42, 755762 (1998).
45. Rockwell, S. Use of hypoxia-directed drugs in the therapy of
solid tumors. Semin. Oncol. 19, 2940 (1992).
46. Workman, P. & Stratford, I. J. The experimental
development of bioreductive drugs and their role in cancer
therapy. Cancer Metastasis Rev. 12, 7382 (1993).
47. Papadopoulou, M. V. & Bloomer, W. D. NLCQ-1 (NSC
709257): exploiting hypoxia with a weak DNAintercalating bioreductive drug. Clin. Cancer Res. 9,
57145720 (2003).
48. Hicks, K. O., Pruijn, F. B., Baguley, B. C. & Wilson, W. R.
Extravascular transport of the DNA intercalator and
topoisomerase poison N-[2-(Dimethylamino)ethyl]acridine4-carboxamide (DACA): diffusion and metabolism in

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

multicellular layers of tumor cells. J. Pharmacol. Exp. Ther.

297, 10881098 (2001).
Delahoussaye, Y. M., Hay, M. P., Pruijn, F. B., Denny, W. A. &
Brown, J. M. Improved potency of the hypoxic cytotoxin
tirapazamine by DNA-targeting. Biochem. Pharmacol. 65,
18071815 (2003).
Hay, M. P. et al. DNA-targeted 1,2,4-benzotriazine 1,4dioxides as hypoxiaselective analogues of tirapazamine.
J. Med. Chem. 47, 475488 (2004).
Denny, W. A., Wilson, W. R. & Hay, M. P. Recent
developments in the design of bioreductive drugs. Br. J.
Cancer Suppl. 27, S32S38 (1996).
Koch, C. J. Unusual oxygen concentration dependence of
toxicity of SR-4233, a hypoxic cell toxin. Cancer Res. 53,
39923997 (1993).
Hicks, K. O., Siim, B. G., Pruijn, F. B. & Wilson, W. R.
Oxygen dependence of the metabolic activation and
cytotoxicity of tirapazamine: Implications for exptravascular
transport and activity in tumors. Radiat. Res. (in the press).
Marshall, R. S. & Rauth, A. M. Oxygen and exposure
kinetics as factors influencing the cytotoxicity of
porfiromycin, a mitomycin C analogue, in Chinese hamster
ovary cells. Cancer Res. 48, 56555659 (1988).
Siim, B. G., Atwell, G. J. & Wilson, W. R. Oxygen
dependence of the cytotoxicity and metabolic activation of
4-alkylamino-5-nitroquinoline bioreductive drugs. Br. J.
Cancer 70, 596603 (1994).
Wilson, W. R., Moselen, J. W., Cliffe, S., Denny, W. A. &
Ware, D. C. Exploiting tumor hypoxia through bioreductive
release of diffusible cytotoxins: the cobalt(III)-nitrogen
mustard complex SN 24771. Int. J. Radiat. Oncol. Biol.
Phys. 29, 323327 (1994).
Wouters, B. G. & Brown, J. M. Cells at intermediate oxygen
levels can be more important than the hypoxic fraction in
determining tumor response to fractionated radiotherapy.
Radiat. Res. 147, 541550 (1997).
Lee, A. E. & Wilson, W. R. Hypoxia-dependent retinal toxicity
of bioreductive anticancer prodrugs in mice. Toxicol. Appl.
Pharmacol. 163, 5059 (2000).
Allalunis, M. J., Chapman, J. D. & Turner, A. R. Identification
of a hypoxic population of bone marrow cells. Int. J. Radiat.
Oncol. Biol. Phys. 9, 227232 (1983).
Cipolleschi, M. G., Dello Sbarba, P. & Olivotto, M. The role of
hypoxia in the maintenance of hematopoietic stem cells.
Blood 82, 20312037 (1993).
Siim, B. G., Denny, W. A. & Wilson, W. R. Nitro reduction as
an electronic switch for bioreductive drug activation. Oncol.
Res. 9, 357369 (1997).
Helsby, N. A. et al. Effect of nitroreduction on the alkylating
reactivity and cytotoxicity of the 2,4-dinitrobenzamide-5aziridine CB 1954 and the corresponding nitrogen mustard
SN 23862: distinct mechanisms of bioreductive activation.
Chem. Res. Toxicol. 16, 469478 (2003).
Wilson, W. R. et al. Quantitation of bystander effects in
nitroreductase suicide gene therapy using three-dimensional
cell cultures. Cancer Res. 62, 14251432 (2002).
Demonstrates the use of three-dimensional cell
cultures to show bystander effects from GDEPT.
Borch, R. F. et al. Synthesis and evaluation of
nitroheterocyclic phosphoramidates as hypoxia-selective
alkylating agents. J. Med. Chem. 43, 22582265 (2000).
Tercel, M. et al. Hypoxia-selective antitumor agents. 16.
Nitroarylmethyl quaternary salts as bioreductive prodrugs of
the alkylating agent mechlorethamine. J. Med. Chem. 44,
35113522 (2001).
Wilson, W. R., Moselen, J. W., Cliffe, S., Denny, W. A. &
Ware, D. C. Exploiting tumor hypoxia through bioreductive
release of diffusible cytotoxins: the cobalt(III)-nitrogen
mustard complex SN 24771. Int. J. Radiat. Oncol. Biol.
Phys. 29, 323327 (1994).
Everett, S. A. et al. Modifying rates of reductive elimination of
leaving groups from indolequinone prodrugs: a key factor in
controlling hypoxia-selective drug release. Biochem.
Pharmacol. 63, 16291639 (2002).
Wilson, W. R., Tercel, M., Anderson, R. F. & Denny, W. A.
Radiation-activated prodrugs as hypoxia-selective
cytotoxins: model studies with nitroarylmethyl quaternary
salts. Anticancer Drug Des. 13, 663685 (1998).
Kriste, A. G., Tercel, M., Anderson, R. F., Ferry, D. M. &
Wilson, W. R. Pathways of reductive fragmentation of
heterocyclic nitroarylmethyl quaternary ammonium prodrugs
of mechlorethamine. Radiat. Res. 158, 753762 (2002).
Ahn, G., Ware, D. C., Denny, W. A. & Wilson, W. R.
Optimization of the auxiliary ligand shell of cobalt(III)(8hydroxyquinoline) complexes as model hypoxia-selective
radiationactivated prodrugs. Radiat. Res. (in the press).
Shibamoto, Y., Zhou, L., Hatta, H., Mori, M. & Nishimoto, S.
A novel class of antitumor prodrug, 1-(2-oxopropyl)-5fluorouracil (OFU001), that releases 5-fluorouracil upon
hypoxic irradiation. Jpn J. Cancer Res. 91, 433438 (2000).

www.nature.com/reviews/cancer

REVIEWS
72. Zhong, H. et al. Overexpression of hypoxia-inducible factor 1
in common human cancers and their metastases. Cancer
Res. 59, 58305835 (1999).
73. Talks, K. L. et al. The expression and distribution of the
hypoxia-inducible factors HIF-1 and HIF-2 in normal
human tissues, cancers, and tumor-associated
macrophages. Am. J. Pathol. 157, 411421 (2000).
74. Shibata, T., Akiyama, N., Noda, M., Sasai, K. & Hiraoka, M.
Enhancement of gene expression under hypoxic conditions
using fragments of the human vascular endothelial growth
factor and the erythropoietin genes. Int. J. Radiat. Oncol.
Biol. Phys. 42, 913916 (1998).
75. Greco, O. & Dachs, G. U. Gene directed enzyme/prodrug
therapy of cancer: historical appraisal and future
prospectives. J. Cell Physiol. 187, 2236 (2001).
76. Binley, K. et al. Hypoxia-mediated tumour targeting. Gene
Ther. 10, 540549 (2003).
77. Shibata, T., Giaccia, A. J. & Brown, J. M. Hypoxia-inducible
regulation of a prodrug-activating enzyme for tumor-specific
gene therapy. Neoplasia 4, 4048 (2002).
78. Patterson, A. V. et al. Oxygen-sensitive enzyme-prodrug
gene therapy for the eradication of radiation-resistant solid
tumours. Gene Ther. 9, 946954 (2002).
79. Trinh, Q. T., Austin, E. A., Murray, D. M., Knick, V. C. &
Huber, B. E. Enzyme/prodrug gene therapy: comparison of
cytosine deaminase/5- fluorocytosine versus thymidine
kinase/ganciclovir enzyme/prodrug systems in a human
colorectal carcinoma cell line. Cancer Res. 55, 48084812
(1995).
80. McCarthy, H. O. et al. Bioreductive GDEPT using
cytochrome P450 3A4 in combination with AQ4N. Cancer
Gene Ther. 10, 4048 (2003).
81. Griffiths, L. et al. The macrophage: a novel system to deliver
gene therapy to pathological hypoxia. Gene Ther. 7,
255262 (2000).
82. Burke, B. et al. Expression of HIF-1 by human
macrophages: implications for the use of macrophages in
hypoxia-regulated cancer gene therapy. J. Pathol. 196,
204212 (2002).
83. Semenza, G. L. Targeting HIF-1 for cancer therapy. Nature
Rev. Cancer 3, 721732 (2003).
84. Giaccia, A., Siim, B. G. & Johnson, R. S. HIF-1 as a target
for drug development. Nature Rev. Drug Discov. 2, 803811
(2003).
85. Semenza, G. L. Involvement of hypoxia-inducible factor 1 in
human cancer. Intern. Med. 41, 7983 (2002).
86. Kung, A. L., Wang, S., Klco, J. M., Kaelin, W. G. &
Livingston, D. M. Suppression of tumor growth through
disruption of hypoxia-inducible transcription. Nature Med. 6,
13351340 (2000).
87. Rapisarda, A. et al. Identification of small molecule inhibitors
of hypoxia-inducible factor 1 transcriptional activation
pathway. Cancer Res. 62, 43164324 (2002).
88. Sun, X. et al. Gene transfer of antisense hypoxia
inducible factor-1 enhances the therapeutic efficacy
of cancer immunotherapy. Gene Ther. 8, 638645
(2001).
89. Mabjeesh, N. J. et al. Geldanamycin induces degradation of
hypoxia-inducible factor 1 protein via the proteosome
pathway in prostate cancer cells. Cancer Res. 62,
24782482 (2002).
90. Mabjeesh, N. J. et al. 2ME2 inhibits tumor growth and
angiogenesis by disrupting microtubules and dysregulating
HIF. Cancer Cell 3, 363375 (2003).
91. Yeo, E. J. et al. YC-1: a potential anticancer drug targeting
hypoxia-inducible factor 1. J. Natl Cancer Inst. 95, 516525
(2003).
92. Lemmon, M. J. et al. Anaerobic bacteria as a gene delivery
system to tumors. Proc. Am. Assoc. Cancer Res. 35, 374
(1994).
93. Fox, M. E. et al. Anaerobic bacteria as a delivery system for
cancer gene therapy: activation of 5-fluorocytosine by
genetically engineered clostridia. Gene Ther. 3, 173178
(1996).
94. Lemmon, M. L. et al. Anaerobic bacteria as a gene delivery
system that is controlled by the tumor microenvironment.
Gene Ther. 4, 791796 (1997).
95. Malmgren, R. A. & Flanigan, C. C. Localization of the
vegetative form of Clostridium tetani in mouse tumors
following intravenous spore administration. Cancer Res. 15,
473478 (1955).
96. Mse, J. R. & Mse, G. Onkolyseversuche mit apathogenen
anaeroben Sporenbildern am Ehrlich Tumor des Maus.
Z. Krebsforsch 63, 6374 (1959).
97. Mse, J. R. & Mse, G. Oncolysis by clostridia. I. Activity of
Clostridium butyricum (M-55) and other nonpathogenic
clostridia against the Ehrlich carcinoma. Cancer Res. 24,
212216 (1964).
98. Thiele, E. H., Arison, R. N. & Boxer, G. E. Oncolysis by
clostridia. III. Effects of clostridia and chemotherapeutic
agents on rodent tumors. Cancer Res. 24 (1964).

NATURE REVIEWS | C ANCER

99. Engelbart, K. & Gericke, D. Oncolysis by clostridia V.

Transplanted tumors of the hamster. Cancer Res. 24,
239243 (1964).
100. Carey, R. W., Holland, J. F., Whang, H. Y., Neter, E. & Bryant, B.
Clostridial oncolysis in man. Europ. J. Cancer 3, 3746 (1967).
101. Heppner, F. & Mose, J. R. The liquefaction (oncolysis) of
malignant gliomas by a non pathogenic clostridium. Acta
Neuro. 12, 123125 (1978).
102. Heppner, F., Mose, J., Ascher, P. W. & Walter, G. Oncolysis
of malignant gliomas of the brain. 13th Int. Cong.
Chemother. 226, 3845 (1983).
103. Pawelek, J. M., Low, K. B. & Bermudes, D. Tumor-targeted
Salmonella as a novel anticancer vector. Cancer Res. 57,
45374544 (1997).
104. Toso, J. F. et al. Phase I study of the intravenous
administration of attenuated Salmonella typhimurium to
patients with metastatic melanoma. J. Clin. Oncol. 20,
142152 (2002).
105. Liu, S. C., Minton, N. P., Giaccia, A. J. & Brown, J. M.
Anticancer efficacy of systemically delivered anaerobic
bacteria as gene therapy vectors targeting tumor
hypoxia/necrosis. Gene Ther. 9, 291296 (2002).
First data demonstrating in vivo efficacy of CDEPT.
106. Bridgewater, J. A. et al. Expression of the bacterial
nitroreductase enzyme in mammalian cells renders them
selectively sensitive to killing by the prodrug CB1954. Eur. J.
Cancer 31A, 23622370 (1995).
107. Minton, N. P. Clostridia in cancer therapy. Nature Rev.
Microbiol. 1 (2003).
108. Martin, J. et al. Antibody-directed enzyme prodrug therapy:
pharmacokinetics and plasma levels of prodrug and drug in
a phase I clinical trial. Cancer Chemother. Pharmacol. 40,
189201 (1997).
109. Joseph, W. R. et al. Stimulation of tumors to synthesize
tumor necrosis factor- in situ using 5,6dimethylxanthenone-4-acetic acid: a novel approach to
cancer therapy. Cancer Res. 59, 633638 (1999).
110. Zhao, L., Ching, L. M., Kestell, P. & Baguley, B. C. The
antitumour activity of 5,6-dimethylxanthenone-4-acetic acid
(DMXAA) in TNF receptor-1 knockout mice. Br. J. Cancer
87, 465470 (2002).
111. Ching, L. M. et al. Induction of endothelial cell apoptosis by
the antivascular agent 5,6-Dimethylxanthenone-4-acetic
acid. Br. J. Cancer 86, 19371942 (2002).
112. Galbraith, S. M. et al. Effects of 5,6-dimethylxanthenone-4acetic acid on human tumor microcirculation assessed by
dynamic contrast-enhanced magnetic resonance imaging.
J. Clin. Oncol. 20, 38263840 (2002).
113. Theys, J. et al. Improvement of Clostridium tumour targeting
vectors evaluated in rat rhabdomyosarcomas. FEMS
Immunol. Med. Microbiol. 30, 3741 (2001).
114. Dang, L. H., Bettegowda, C., Huso, D. L., Kinzler, K. W. &
Vogelstein, B. Combination bacteriolytic therapy for the
treatment of experimental tumors. Proc. Natl Acad. Sci.
USA 98, 1515515160 (2001).
115. Hicks, K. O., Pruijn, F. B., Sturman, J. R., Denny, W. A. &
Wilson, W. R. Multicellular resistance to tirapazamine is due
to restricted extravascular transport in HT29 multicellular
layer cultures: A pharmacokinetic/pharmacodynamic study.
Cancer Res. 63, 59705977 (2003).
116. Helsby, N. A., Ferry, D. M., Patterson, A. V., Pullen, S. M. &
Wilson, W. R. 2amino metabolites are key mediatiors of CB
1954 and SN 23862 bystander effects in nitroreductase
GDEPT. Br. J.Cancer 90, 10841092 (2004).
117. Bussink, J., Kaanders, J. H. A. M. & van der Kogel, A. J.
Tumor hypoxia at the micro-regional level: clinical
relevance and predictive value of exdogenous and
endogenous hypoxic cell markers. Radiother. Oncol. 67,
315 (2003).
118. Cobb, L. M. et al. 2,4-Dinitro-5-ethyleneiminobenzamide
(CB 1954): A potent and selective inhibitor of the growth of
the Walker carcinoma 256. Biochem. Pharmacol. 18,
15191527 (1969).
119. Knox, R. J., Friedlos, F., Jarman, M. & Roberts, J. J. A new
cytotoxic, DNA interstrand crosslinking agent, 5-(Aziridin-1YL)-4-hydroxylamino-2-nitrobenzamide, is formed from 5(Aziridin-1-YL)-2,4-dinitrobenzamide (CB 1954) by a
nitroreductase enzyme in Walker carcinoma cells. Biochem.
Pharm. 37, 46614669 (1988).
120. Knox, R. J. et al. The nitroreductase enzyme in Walker cells
that activates 5-(Aziridin-1-YL)-2,4-dinitrobenzamide
(CB 1954) to 5-(Aziridin-1-YL)-4-hydroxylamino-2nitrobenzamide is a form of NAD(P)H dehydrogenase
(quinone) (EC 1. 6. 99. 2). Biochem. Pharmacol. 37,
46714677 (1988).
121. Anlezark, G. M. et al. The bioactivation of 5-(Aziridin-1-YL)2,4-dinitrobenzamide (CB 1954)-I Purification and properties
of a nitroreductase enzyme from Escherichia Coli: A
potential enzyme for antibody-directed enzyme prodrug
therapy (ADEPT). Biochem. Pharmacol. 44, 22892295
(1992).

122. Chung-Faye, G. et al. Virus-directed, enzyme prodrug

therapy with nitroimidazole reductase: a phase I and
pharmacokinetic study of its prodrug, CB1954. Clin. Cancer
Res. 7, 26622668 (2001).
123. Stratford, I. J., Williamson, C., Hoe, S. & Adams, G. E.
Radiosensitizing and cytotoxicity studies with CB 1954
(2,4-dinitro-5-aziridinylbenzamide). Radiat. Res. 88,
502509 (1981).
124. Palmer, B. D., Wilson, W. R., Cliffe, S. & Denny, W. A. Hypoxiaselective antitumor agents. 5. Synthesis of water-soluble
nitroaniline mustards with selective cytotoxicity for hypoxic
mammalian cells. J. Med. Chem. 35, 32143222 (1992).
125. Brown, J. M. & Giaccia, A. J. The unique physiology of solid
tumors: Opportunities (and problems) for cancer therapy.
Cancer Res. 58, 14081416 (1998).
126. Wouters, B. G. et al. Mitochondrial dysfunction after aerobic
exposure to the hypoxic cytotoxin tirapazamine. Cancer
Res. 61, 145152 (2001).
127. Aebersold, D. M. et al. Expression of hypoxia-inducible
factor-1: a novel predictive and prognostic parameter in
the radiotherapy of oropharyngeal cancer. Cancer Res. 61,
29112916 (2001).
Shows that HIF-1 can be used as an endogenous
marker of tumour hypoxia to predict response to
radiotherapy.
128. Rampling, R., Cruickshank, G., Lewis, A. D., Fitzsimmons, S. A.
& Workman, P. Direct measurement of pO2 distribution and
bioreductive enzymes in human malignant brain tumors. Int.
J. Radiat. Oncol. Biol. Phys. 29, 427431 (1994).
129. Collingridge, D. R., Piepmeier, J. M., Rockwell, S. & Knisely, J. P.
Polarographic measurements of oxygen tension in human
glioma and surrounding peritumoural brain tissue. Radiother.
Oncol. 53, 127131 (1999).
130. Nordsmark, M., Bentzen, S. M. & Overgaard, J. Measurement
of human tumour oxygenation status by a polarographic
needle electrode. Acta Oncol. 33, 383389 (1994).
131. Becker, A. et al. Oxygenation of squamous cell carcinoma of
the head and neck: comparison of primary tumors, neck
node metastases, and normal tissue. Int. J. Radiat. Oncol.
Biol. Phys. 42, 3541 (1998).
132. Le, Q. T. et al. Comparison of the comet assay and the
oxygen microelectrode for measuring tumor oxygenation in
head-and-neck cancer patients. Int. J. Radiat. Oncol. Biol.
Phys. 56, 375383 (2003).
133. Vaupel, P., Briest, S. & Hockel, M. Hypoxia in breast cancer:
pathogenesis, characterization and biological/therapeutic
implications. Wien. Med. Wochenschr. 152, 334342 (2002).
134. Koong, A. C. et al. Pancreatic tumors show high levels of
hypoxia. Int. J. Radiat. Oncol. Biol. Phys. 48, 919922 (2000).
135. Lyng, H., Sundfor, K. & Rofstad, E. K. Oxygen tension in
human tumours measured with polarographic needle
electrodes and its relationship to vascular density, necrosis
and hypoxia. Radiother. Oncol. 44, 163169 (1997).
136. Fyles, A. W. et al. Oxygenation predicts radiation response
and survival in patients with cervix cancer. Radiother. Oncol.
48, 149156 (1998).
137. Nordsmark, M. et al. Measurements of hypoxia using
pimonidazole and polarographic oxygen-sensitive
electrodes in human cervix carcinomas. Radiother. Oncol.
67, 3544 (2003).
138. Movsas, B. et al. Hypoxia in human prostate carcinoma: an
Eppendorf PO2 study. Am. J. Clin. Oncol. 24, 458461 (2001).
139. Brizel, D. M. et al. Radiation therapy and hyperthermia
improve the oxygenation of human soft tissue
sarcomas. Cancer Res. 56, 53475350 (1996).
140. Nordsmark, M. et al. The relationship between tumor
oxygenation and cell proliferation in human soft tissue
sarcomas. Int. J. Radiat. Oncol. Biol. Phys. 35, 701708
(1996).

Acknowledgements

The authors work is funded by grants from the United States

National Institutes of Health (J.M.B. and W.R.W.) and the Health
Research Council of New Zealand (W.R.W.).

Competing interests statement

The authors declare competing financial interests: see web version

for details.

Online links
DATABASES
The following terms in this article are linked online to:
Cancer.gov: http://cancer.gov/
head and neck cancer | non-small-cell lung cancer | oesophageal
cancer | ovarian cancer
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
CYP3A4 | ERBB2 | HIF-1 | HIF-1 | P450R | p53 | SRC
Access to this interactive links box is free online.

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