-
[CANCER RESEARCH 41, 73-81, January
1981]0008-5472/81/1141-OOOOS02.00
Classification of Antineoplastic Agents by their Selective
Toxicitiestoward Oxygenated and Hypoxie Tumor Cells1
Beverly A. Teicher,2 John S. Lazo, and Alan C. Salterelli
Department ot Pharmacology and Developmental Therapeutics
Program, Comprehensive Cancer Center, Yale University School of
Medicine. New Haven,Connecticut 06510
ABSTRACT
The cytotoxicities of a number of antineoplastic agents
tooxygenated and hypoxic EMT6 mouse mammary tumor cells ¡nculture
were examined. Based on the relative sensitivities ofcells under
aerobic and hypoxic conditions, drugs were placedinto three
categories. Drugs that were preferentially toxic tocells under
oxygenated conditions were classified as type 1agents; this group
includes bleomycin, procarbazine, strepto-
nigrin, actinomycin D, and vincristine. Type 2 agents werethose
preferentially toxic to cells under hypoxic conditions.These
include mitomycin C and Adriamycin. On the basis ofother published
reports, the glucose analogs, 5-thio-o-glucoseand
2-deoxy-D-glucose, and the radiosensitizers, misonidazole
and metronidazole, can also be placed in this category.
Severalantineoplastic agents showed no major preferential toxicity
tocells under the conditions of oxygénation or hypoxia used
inthese experiments and were placed in a third class. This
group(type 3) includes 1,3-bis(2-chloroethyl)-1-nitrosourea,
1-(2-chloroethyl)-3-cyclohexyl-1 -nitrosourea,
c/'s-diamminedichlo-
roplatinum(ll), 5-fluorouracil, and methotrexate. The
success
of many combination chemotherapy and combined modalitytreatments
may be due to their ability to kill both the hypoxicand aerobic
cell populations of solid tumors. Future chemo-
therapeutic regimens for the treatment of solid tumors
shouldinclude agents and modalities directed toward the hypoxic
cellpopulation of the tumor, as well as toward the proliferating
andnonproliferating tumor cell compartments; a therapeutic approach
to the selection of antineoplastic agents for use incombination
based upon physiological considerations of thearchitecture of solid
tumors is presented.
INTRODUCTION
Solid tumors are refractory to cytotoxic agents for
severalreasons including: (a) many antineoplastic agents do not
reachthe poorly vascularized regions of the tumor; and (b) the
cellularpopulations in solid tumors are physiologically more
heterogeneous with respect to oxygénationand proliferation than
arethe cellular components of hematological or particularly
well-vascularized tumors.
Most of the currently used chemotherapeutic agents aremore
active against cells in exponential growth than againstcells in the
plateau phase, in which a relatively great proportionof the cells
are not actively traversing the cell cycle (16, 68).However, little
is known about the toxicity of most antineoplas-
' This research was supported in part by USPHS Grants CA-02817
and CA-
16359 from the National Cancer Institute and a grant from the
Bristol-MyersCompany.
2 Recipient of a postdoctoral fellowship (CA-06365) from the
National Cancer
Institute To whom requests for reprints should be
addressed.Received March 17. 1980; accepted September 23, 1980.
tic agents toward cells that are hypoxic. It has been well-
established that hypoxic cells exist in solid tumors and
thatthese cells are relatively resistant to the cytotoxic effects
ofionizing radiation. Thus, the hypoxic cell population limits
thecurability of experimental animal tumors by large doses
ofradiation (52). Since hypoxic cells may be either noncycling
orslowly progressing through the cell cycle (9, 14, 54), they
arealso presumed to be relatively resistant to cell
cycle-specific
chemotherapy. To develop a therapeutic program designed
toapproach the cure of solid tumors, the use of agents
withcytotoxic actions directed toward each of the physiologic
cellular components of the tumor population would appear to
berequired.
In this study, representative compounds from several classesof
anticancer drugs were tested for cytotoxic activity towardcultured
EMT6 tumor cells under conditions of normal aerationand chronic
hypoxia to determine whether preferential cytotox-
icity toward hypoxic cells was a property of any currently
usedantineoplastic agents. Based on the results obtained from
thesestudies, agents were grouped into 3 distinct classes.
Thepossible use of these findings to fashion an approach to
theselection of agents to use in combination in the clinical
treatment of solid tumors is discussed.
MATERIALS AND METHODS
Drugs. Mitomycin C and bleomycin (Bleoxane) (1.6 units/mg) were
the gifts of Dr. Maxwell Gordon and Dr. William T.Bradner,
respectively, of the Bristol-Myers Company (New
York, N. Y.). Vincristine sulfate (Oncovin) was obtained fromEli
Lilly and Company (Indianapolis, Ind.). Streptonigrin andAdriamycin
were the gifts of Dr. John D. Douros of the Divisionof Cancer
Treatment of the National Cancer Institute (Bethesda,Md.);
c/s-diamminedichloroplatinum(ll), BCNU,3 and CCNU
were also obtained from the Division of Cancer
Treatment.Procarbazine-HCI was obtained from Hoffman-LaRoche
Inc.(Nutley, N. J.) and actinomycin D from Calbiochem-BehringCorp.
(La Jolla, Calif.). 5-Fluorouracil and methotrexate werepurchased
from Sigma Chemical Company (St. Louis, Mo.). Allother reagents
were obtained from standard chemical sources.Drugs were dissolved
in acetone, ethanol, sterile distilled water,or sterile
phosphate-buffered saline (8.0 g/l NaCI, 0.2 g/l KCI,1.15 g/l
Na2HPO4, 0.2 g/l KH2PO4) for addition to the tissueculture
system.
Tumor Cells and Cytotoxicity Studies. Experiments wereperformed
using EMT6 mouse mammary tumor cells in vitro.The techniques used
for propagating the cells and measuringtheir survival by colony
formation have been described in detailpreviously (50, 82, 83).
Cells were grown as monolayers in 25
3 The abbreviations used are: BCNU.
1,3-bis(2-chloroethylM-nitrosourea;CCNU. 1-
-
B. A. Teicher et al.
sq cm Corning plastic culture flasks in Waymouth's medium
supplemented with 15% fetal bovine serum and used for
theseexperiments when in exponential growth. To produce
hypoxia,flasks were fitted with sterile rubber sleeve serum
stoppers andexposed to a continuously flowing 95% nitrogen/5%
CO2humidified atmosphere for 4 hr at 37°prior to drug
treatment.
These conditions produce a degree of hypoxia sufficient toresult
in radiobiological resistance (oxygen concentration 10ppm or less).
Parallel flasks were maintained in humidified 95%air/5% COj. At
this time, each of the drugs or vehicle wasadded to the flasks by
injection through the rubber stopperswithout breaking the hypoxia.
After exposure to each agent for1 hr at 37°under hypoxia or normal
aeration, the cells were
washed with 3 ml of sterile phosphate-buffered saline, suspended
by treatment with 0.05% trypsin in phosphate-buffered
saline for 15 min, plated in replicate dishes at 3 dilutions
inWaymouth's medium plus 15% fetal calf serum, and the surviv
ing fraction of cells was measured by colony formation.
Nodifference existed between the survival of untreated or
vehicle-
treated cells maintained under the aerobic and hypoxic
conditions used; the plating efficiency for these control cultures
was65 to 80%. Cells exposed to hypoxic conditions appeared
tocontinue traversing the cell cycle during the course of
theseexperiments, since no decrease in the rate of
[3H]thymidine
incorporation into acid-insoluble material (data not shown)
or
in the mitotic index was observed after 4 hr of incubation in
thehypoxic atmosphere. Each drug was tested in at least 3 separate
experiments.
RESULTS AND DISCUSSION
Classification of Antineoplastic Agents. Based upon
physiological considerations, solid tumors may be envisioned
toconsist of at least 3 classes of neoplastic cells. These
include:(a) cells which are well oxygenated, are relatively rapidly
traversing the cell cycle, and may correspond in drug
sensitivitiesto logarithmically growing cells in culture; (b) non-
or slowly
proliferating oxygenated cells which may correspond in
theirsusceptibilities to anticancer agents to plateau-phase cells
in
culture; and (c) cells in various degrees of hypoxia (108,
109).This last population may be composed of neoplastic cells
witheither relatively normal or prolonged cell cycle times or
withcells blocked in their progression through the cell cycle.
Antineoplastic agents can be grouped into classes based upontheir
cytotoxicities toward the neoplastic cell populations present in
each of these compartments (Table 1). Type 1 agentsand treatment
modalities were those which were more toxic to
oxygenated cells than to chronically hypoxic cells. The
compounds grouped as type 2 were agents which were more toxicto
hypoxic cells than to cells under conditions of normal aeration.
Type 3 agents and treatment modalities were essentiallyequitoxic to
oxygenated and hypoxic cells.
Type 1 Agents. Bleomycin produces fragmentation of DMAin a
reaction which is dependent upon the presence of ferrousions and
molecular oxygen (89, 90). Reactive free radicals ofoxygen may be
responsible for the cleavage of DNA by bleo-mycin (61, 73, 99).
Sausville ef al. (90) showed that thereaction of bleomycin and
iron(ll) with adenovirus [3H]DNA in
phosphate buffer was virtually completely inhibited when
thereaction mixture was equilibrated with argon. The survival
ofEMT6 cells exposed to bleomycin for 1 hr under either oxygenated
or hypoxic conditions is shown in Chart 1. As reportedby others (7,
85), the survival-dose response curve to shorttime exposure to
bleomycin is bi- or multiphasic. At all concentrations examined,
bleomycin was more toxic to oxygenatedcells than to cells
maintained in a hypoxic atmosphere for 4 hrprior to exposure to the
drug. At 150 milliunits of the antibiotic
I 00
BLEOMYCIN
15 IO 25 50 75 100 ISO
DRUG CONCENTRATION, mil/ml
Chart 1. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of bleomycin in cell
culture.
Table 1
Classification of antineoplastic agents and treatment modalities
based on cellular oxygénation
Preferential toxic-ity to aerobic cells
(type 1)Preferential toxicity to hypoxic
cells (type 2)Minimal or no selectivity based on cel
lular oxygénation (type 3)
BleomycinProcarbazineStreptonigrinActinomycin
DVincristineIonizing radiation
Mitomycin CAdriamycinMisonidazole,
metronidazole5-Thio-o-glucose. 2-deoxy-D-glucose
5-FluorouracilMethotrexatea
c/s-Diamminedichloroplatinum (II)BCNU, CCNUHigh linear energy
transfer radiation
3 Under the test conditions used in these experiments, hypoxic
cells are still capable of DNA synthesis
and of cellular replication. These agents have cytotoxic effects
primarily on cells in the S phase of the cellcycle. Thus, in
hypoxic cells that are blocked in their progression through the
cell cycle or are cyclingslowly, agents such as these that act on
the S phase of the cell cycle would be expected to be
relativelynoncytotoxic.
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Drug Cytotoxicity and Cellular Oxygénation
per ml, a 9-fold difference in drug sensitivity was
observed.That bleomycin showed some toxicity toward chronically
hy-
poxic cells may be the result of (a) residual antibiotic
whichexerted its cytotoxic effect after the hypoxic state was
disrupted, (£>)residual oxygen in hypoxic cells, or (c) a
non-oxygen-dependent mechanism of drug cytotoxicity.
Procarbazine is rapidly oxidized in aqueous solution in
thepresence of oxygen to an azo derivative which is further
oxidized in vivo and in vitro to alkylating species (112).
Procarbazine is considerably more toxic to bacteria under
aerobicconditions than under anaerobiasis (81). As shown in Chart
2,this differential cytotoxicity is also expressed with
mammaliantumor cells, the drug being approximately 7 times more
toxicto normally aerated EMT6 cells than to these cells
maintainedunder conditions of chronic hypoxia during drug exposure.
Ata drug concentration of 1 ¡J.M,there was no kill of hypoxic
cells,yet greater than 70% of the aerobic cells were killed. It
isunclear whether the cytotoxicity of procarbazine to hypoxiccells
is due to residual drug or to a non-oxygen-dependent
mechanism.Streptonigrin appears to damage DNA through the
obligatory
intermediacy of Superoxide radical (22). As shown in Chart
3,only 20% survival of normally aerated EMT6 cells was obtainedat a
concentration of only 1 pw Streptonigrin; under theseconditions,
the survival of hypoxic EMT6 tumor cells wasunaffected. At all drug
concentrations examined, Streptonigrinwas about 10 times more toxic
to normally aerated cells thanto hypoxic cells.
Actinomycin D binds to double-stranded DNA, permitting
initiation of RNA synthesis but blocking the elongation
process.The cytotoxicity of actinomycin D appears to be primarily
aconsequence of this interaction with DNA (36, 110). Chart 4shows
that, at concentrations less than 0.01 /¿M,no majordifference was
observed in the cytotoxicity of this antibiotictoward normally
aerated and hypoxic cells; however, at drugconcentrations
approaching 1 /ÃŒM,oxygenated cells are almost
i.20 r PROCARBAZINE
IO 100 1000
DRUG CONCENTRATION, pM
lO.OOO
100-fold more sensitive to the lethal actions of actinomycin
D
than are hypoxic cells. Thus, there may be a secondary
oxygen-dependent cytotoxic mechanism of action for actinomycin
D at relatively high drug concentrations.
STREPTONIGRIN
COjH
OOOOOOI OOOOOI OOOOI OOOI 001 01
DRUG CONCENTRATION, ^M
Chart 3. Survival of aerobic {•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of Streptonigrin in
cell culture.
OOOOOI OOOOI OOOI 001
DRUG CONCENTRATION,
O.I 1.0
Chart 2. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of procarbazine in cell
culture.
Chart 4. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of actinomycin D in
cell culture.
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S. A. Teicher et al.
Although the cytotoxicity of vincristine is attributed to
itsability to interrupt cell division in metaphase (113), other
effectsmay also contribute to cell death (23). In the range of
vincristineconcentrations tested (i.e., 0.01 to 50 /IM), the drug
showedminimal cytotoxicity to hypoxic cells, while toxicity to
oxygenated cells was very clearly a dose-related process (Chart
5).
This finding cannot be attributed to a decrease in mitoses inthe
hypoxic cells, since the mitotic index was 5.79 ±0.89%for aerobic
cells and 6.59 ± 1.69% for cells after 4 hr ofhypoxia.
Agents classified as type 1 are primarily drugs that
requiremolecular oxygen or oxidative biotransformation to exert
acytotoxic effect and includes the treatment modality
ionizingradiation (52). Additionally, some drugs may be
accumulatedor retained intracellularly by energy-requiring
processes thatdepend upon oxidative metabolism; therefore,
intracellular concentrations of these drugs may be different in
oxygenated andhypoxic cells. In this test system, streptonigrin
showed thelargest differential kill of aerobic cells, being greater
than 1000times more toxic to oxygenated cells at 50% survival
andapproximately 10,000 times more toxic to aerobic cells at
20%survival. Procarbazine required greater than 5000 times moredrug
to achieve a 50% kill of hypoxic cells than to kill 50% ofaerobic
cells. The degree of differential kill achieved by bleo-
mycin, actinomycin D, and vincristine was similar,
rangingbetween 50 to 100 times more toxic to oxygenated EMT6
cellsthan to their hypoxic counterparts at 50% survival levels.
Type 2 Agents. Early studies established that DNA is
theprincipal target for the expression of the antineoplastic
activityof mitomycin C (46, 47). The term bioreductive alkylating
agenthas been used by this laboratory to describe the class of
drugswhich require reductive transformation to exhibit
alkylatingactivity (57, 58), and mitomycin C can be considered to
be anaturally occurring bioreductive alkylating agent.
Enzymaticreduction of this agent to the hydroquinone results in the
lossof methanol to give an aziridinomitosene derivative (24).
Spon-
IOO -
O 080
U<K
060 -
040 -
020 -
OOI O.I I.O IO
DRUG CONCENTRATION, ¿iM
taneous rearrangement of the reduced molecule can
thentheoretically lead to the production of a highly reactive
quinonemethide intermediate capable of alkylating cellular
molecules(46, 47, 60, 92). Schwartz (91) demonstrated that liver
contains an enzymatic system capable of metabolizing mitomycinC
under anaerobic conditions; this enzyme system is found inboth the
microsomal and nuclear fractions (51). The bioacti-
vation of mitomycin C to an alkylating species can occur
inneoplastic cells in a reaction requiring anaerobiasis and
anNADPH-generating system (50, 52). In agreement with these
findings, mitomycin C was considerably more toxic to
hypoxiccells than to oxygenated cells over a wide range of
concentrations (Chart 6). This difference in sensitivity reached a
maximum of about 10-fold at relatively high concentrations of
drug;
in addition, however, at a concentration range of 0.001 to
0.1/¿Mmitomycin C, little or no measurable cytotoxic activity
occurred in oxygenated cells, while 50 to 90% of the hypoxiccells
were unable to replicate.
The action of Adriamycin may involve activation via
metabolicreduction, either by one- or 2-electron transfer (29, 30,
62).
Anaerobic incubation of Adriamycin with liver microsomes inthe
presence of NADPH results in the appearance of an electron spin
resonance signal attributed to the semiquinone freeradical (1-3,
40, 88). Although the direct reaction of the
anthracycline radical with tissue constituents has not
beenreported, Bachur et al. (3) have observed widespread apparently
covalent binding of Adriamycin to tissue proteins. Anexplanation
for this alkylating ability which involves 2-electron
reduction of the quinone nucleus of Adriamycin followed by
theelimination of the daunosamine sugar moiety with
subsequentformation of a quinone methide alkylating species has
beenproposed by Moore (71 ). As shown in Chart 7, Adriamycin
wasmore toxic to hypoxic cells at all of the concentrations of
the
OOOI O.OI O.l I.O
DRUG CONCENTRATION,/
IOO
Chart 5. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of vincristine in cell
culture.
Chart 6. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of mitomycin C in cell
culture.
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Drug Cytotoxicity and Cellular Oxygénation
5-FLUOROURACIL
0.0001 0.001 0.01 O.I 1.0
DRUG CONCENTRATION, ¿iM
Chart 7. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of Adriamycin in cell
culture.
antibiotic tested. At high drug levels, the differential kill
ofhypoxic cells was 10 times greater than the kill of
oxygenatedcells. It appears that, under hypoxic conditions,
Adriamycinmay act as a bioreductive alkylating agent, while under
conditions of normal aeration, an oxygen-dependent mechanism of
cytotoxicity may be operative. In contrast to these
findings,Smith et al. (94) reported that hypoxic Chinese hamster
ovarycells were more resistant to Adriamycin than their
oxygenatedcounterparts, and Harris and Schrieve (41), using an
EMT6cell line, found no difference in the toxicity of Adriamycin
tooxygenated and hypoxic cells. The latter authors indicate thatthe
sensitivity of their line of EMT6 to antineoplastic agentsdiffered
from that of others. The results of these studies,however, indicate
that all tumor cells may not be capable ofcarrying out the
reductive reactions necessary to activateAdriamycin under hypoxic
conditions.
Except for glucose analogs, all agents classified as type 2seem
to use mechanisms that involve enzymatic reduction ofa functional
group on the drug molecule to express theircytotoxic activity. The
nitroheterocyclic radiosensitizers, exemplified by metronidazole
and misonidazole, are selectivelytoxic to hypoxic cells in the
absence of irradiation (27, 33-35,67, 102, 107). This selective
toxicity corresponds to the preferential formation of relatively
large amounts of N-hydroxy andamine metabolites formed by nitro
group reduction by hypoxiccells (8, 74, 103, 107). To attain
cytotoxicity by the nitroimi-
dazoles requires prolonged contact times of several hr
andrelatively high drug concentrations (e.g., 1 to 5 rriM
misonidazole) (32, 70, 115, 11 6); consequently, the selective
action ofthese agents on hypoxic cells may not be exploitable in
thetreatment of human cancer (1 5, 26).
A prominent metabolic difference between aerobic and hypoxic
cells is their degree of dependence upon the metabolism
1.20 -
1.00 -
O
rru_
0.60 -
0.40 -
020 -
I.O 10 100
DRUG CONCENTRATION,
1000 10,000
Chart 8. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of 5-fluorouracil in
cell culture.
of glucose for survival; aerobic cells are more resistant
todepletion of the glucose supply or to inhibition of
glycolysis(96). Since hypoxic cells derive their energy primarily
by anaerobic glycolysis, they are particularly vulnerable to either
adiminished availability of glucose or an inhibition of
glycolysis.5-Thio-D-glucose and 2-deoxy-o-glucose, potent
inhibitors ofglycolysis, are preferentially cytotoxic to hypoxic
cells (95-
98). Glucose analogs may be of limited clinical utility,
however,because of the relatively large quantities required to
producelethality by creating a shortage of glucose in hypoxic
tumorcells.
Type 3 Agents. The mechanism by which 5-fluorouracil
killsneoplastic cells is believed to require conversion to
5-fluoro-2'-deoxyuridylic acid, which forms a stable ternary
complex
with the cofactor, 5,10-methylenetetrahydrofolate and the en
zyme thymidylate synthetase (42, 56, 64), leading to a state
of"thymineless death" (21, 44, 87). As shown in Chart 8, no
major difference was observed in the cytotoxicity of 5-fluo
rouracil to oxygenated and hypoxic cells. The aerobic andhypoxic
cells used in this study incorporate [3H]thymidine into
acid-insoluble material at equal rates, suggesting that
cells
under both of these conditions are actively traversing the
cellcycle during the course of the experiment. This
presumablyexplains the sensitivity of oxygenated and hypoxic cells
to thisantimetabolite.
Methotrexate is a potent inhibitor of dihydrofolate reducÃ-ase(1
2); interference with this enzyme activity leads to a decreasein
the rate of synthesis of cellular DMA, RNA, and protein (10,11). As
shown in Chart 9, methotrexate was similar to 5-
fluorouracil, in that little difference was observed in the
degreeof cytotoxicity exhibited toward EMT6 cells which was
dependent upon their state of oxygénation.
The neutral platinum complexes interact with DNA to impairits
function as a template for further DNA replication (80).
cis-Diamminedichloroplatinum(ll) has been shown to cross-link
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ß.A. Teicher et al.
METHOTREXATEi.oo -
001 0.1 1.0 IO 100
DRUG CONCENTRATION, MM
1000
Chart 9. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of methotrexate in cell
culture.
cis-DIAMMINEDICHLOROPLATINUM (H)
IOO -
oooooi ooooi IOO0.001 0.01 O.I 10
DRUG CONCENTRATION,/iM
Chart 10. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of
c/s-diamminedichloroplatinum(ll) in cell culture.
DMA in mammalian cells (79). As might be expected for anagent
which can covalently bind to critical molecules in cellsregardless
of their physiological state, no difference in thecytotoxicity of
c/s-diamminedichloroplatinum(ll) toward oxy
genated and hypoxic cells was observed (Chart 10).The
2-haloethylnitrosoureas, BCNU and CCNU, are chemi
cally reactive compounds that decompose nonenzymatically
atrelatively rapid rates under physiological conditions (28, 43,69,
78). Furthermore, BCNU and related 2-haloethylnitrosoureas
covalently cross-link DMA (48, 49, 93). As shown in Chart
11, BCNU has little selective cytotoxicity that is based uponthe
cellular state of oxygénation. A similar result was obtainedwith
CCNU.
100
BCNU
O.I IO IO
DRUG CONCENTRATION, MM
IOO
Chart 11. Survival of aerobic (•)and hypoxic (O) EMT6 cells
treated for 1 hrwith various concentrations of BCNU in cell
culture.
Antineoplastic agents considered to be type 3 were those inwhich
the ratio of drug concentrations required to achieveeither a 50% or
20% survival of oxygenated and hypoxic cellsranged between 0.5 and
5. Drugs in this category includealkylating agents, such as the
2-haloethylnitrosoureas and the
neutral platinum compounds, and since in the methodologyused in
this study both oxygenated and hypoxic cells appearto be actively
traversing the cell cycle, the S-phase inhibitors5-fluorouracil and
methotrexate fall into this group (45). It isessential to
recognize, however, that long-term chronically
hypoxic cells which are either noncycling or are slowly
movingthrough the cycle would not be expected to be sensitive
tothese agents. High linear energy transfer radiation also has
nooxygen requirement for cytotoxic activity and may be placed
inthis class.
Penetration. A property of major importance for hypoxic
celldirected chemotherapeutic agents is the ability of the drugs
topenetrate to poorly vascularized regions of the tumor to
reachtarget hypoxic cells in therapeutically useful
concentrations.Furthermore, the intracellular concentrations of the
cytotoxicagents which can be achieved may differ for oxygenated
andhypoxic cells. Although molecular oxygen penetrates only ashort
distance through tumor tissue, largely because of itsrapid
metabolic utilization, some dyes and some drugs candiffuse into the
tumor mass over much greater distances (108,109). Although the
nitroimidazole radiosensitizers and the glucose analogs are both
type 2 agents, since they are notexceedingly potent cytotoxic
agents for hypoxic cells, largedoses would appear to be required to
achieve effective drugconcentrations in the tumor. Adriamycin is
exceedingly effective against hypoxic cells, but this agent has
been reported tohave poor tumor penetrating properties (101). In
contrast,mitomycin C, also a potent type 2 agent, has been shown
tohave the ability to penetrate to hypoxic regions of a rodentsolid
tumor (84). Therefore, we believe that at present mitomycin C is
the most promising agent with which to attack thehypoxic cell
compartment of solid tumors currently availablefor clinical
use.
Evidence for the Clinical Utility of the Concept. A variety
of
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Drug Cytotoxicity and Cellular Oxygénation
clinical trials have used mitomycin C or Adriamycin, the 2agents
which appear to be the most efficacious against thehypoxic cell
component of solid tumors, in admixture withdrugs capable of
attacking cells in the oxygenated compartments of solid tumors.
These studies encourage the utilizationof combinations of drugs
specifically designed to attack cellularcompartments based upon the
physiological status of the neo-
plastic cells. They include combination chemotherapy
withmitomycin C, particularly mitomycin C in admixture with
5-fluorouracil, an agent capable of attacking well-oxygenated
tumor cells in cycle, in the treatment of advanced carcinoma
ofthe stomach, pancreas, and colon. This combination
(oftenincluding additional agents) has produced significant
increasesin both the percentage and duration of responses as
comparedto either drug alone (17-19, 25, 39, 55, 63, 77). Mitomycin
C
in combination with bleomycin, an agent whose cytotoxicity
isdirected primarily against oxygenated cells, or in admixturewith
bleomycin and vincristine, also a type 1 agent, appears torepresent
a very significant advance in the chemotherapy ofadvanced squamous
cell cervical cancer (4, 5, 66). Radiationtherapy, a type 1
modality, plus chemotherapy consisting of 5-
fluorouracil and mitomycin C has been reported to result in
noevidence of disease in 3 patients presenting with
surgicallyinoperable epidermoid carcinoma of the anus (72).
MitomycinC in combination chemotherapy or alone has also been
reported to produce objective response in the treatment of
liver(53, 65), breast (114), and lung cancer (86).
Adriamycin, a type 2 agent, is synergistic with several
alkyl-ating agents; thus, in clinical trials in advanced breast
cancer,Adriamycin in combination with alkylating agents has
resultedin a high percentage of responses to treatment (59, 76,
104,111). Adriamycin in combination chemotherapy has also
beeneffective in the treatment of advanced lung (20),
bronchogenic(105), and testicular carcinoma (31). The treatment of
advanced epidermoid carcinoma of the cervix with this agent
incombination with bleomycin gave poor results at the
primarylesion; however, a satisfactory response rate was observed
inmetastatic growths (37). Response rates achieved with Adriamycin
therapy appear to be of relatively short duration, perhapsdue to
inadequate penetration of the drug to the hypoxic tumorcells most
distal from the tumor blood supply.
Attack of Nonproliferating Cells. The nonproliferating cellular
compartment of solid tumors includes, in addition to aportion of
the hypoxic cell subpopulation, a cellular componentconsisting of
oxygenated cells comparable to the plateau phasein culture. Since
plateau-phase cells differ markedly from ex
ponentially growing cells in their sensitivities to
antineoplasticagents, it would be expected that such a cellular
component ofa solid tumor would limit response and would require
specialconsideration in the fashioning of a combination
chemothera-peutic regimen. Agents which might be useful in
attacking thiscellular compartment include bleomycin and CCNU and
BCNU(6, 7, 13, 38, 100, 106). Among other treatment
modalities,hyperthermia has been reported to be more toxic to
plateau-phase cells (75).
An Approach to the Design of Chemotherapeutic Regimens.
Chemotherapeutic regimens for the treatment of solidtumors can be
designed based upon a consideration of thephysiological status of
the cellular components of the tumormass. Such a therapeutic
approach would appear to requirethe combination of agents and/or
modalities directed toward
each of the cell types present in the tumor, including
cyclingand nonclycing populations of oxygenated and hypoxic
compartments. Selection of combinations of drugs (or other
treatment modalities) based upon these concepts should include:(a)
a bioreductive alkylating agent designed to attack thehypoxic cell
compartment by exploitation of the capacity ofthese cells to
accomplish reductive reactions; mitomycin Cwould appear to be the
most efficacious agent of this classpresently available for
clinical use. To maximize the differentialtoxicity of this agent to
hypoxic cells, it should be given inrelatively low doses over a
relatively long period. In addition, itshould be administered prior
to the component(s) of the drugcombination used to kill oxygenated
cells, to minimize the lossof effectiveness of this agent through
reoxygenation of thehypoxic cell compartment after kill of aerobic
cells, a processthat is capable of occurring relatively rapidly
(52); (b) an agentsuch as bleomycin or a nitrosourea would seem to
be a reasonable addition to such therapy to specifically attack
anynonproliferating oxygenated cells present in the tumor
(i.e.,plateau phase-like cells); and (c) X-irradiation and/or an
agentor mixture of agents with specificity for actively
proliferatingaerated cells. The drug(s) selected to attack these
cellularcomponents of the malignant tumor obviously must be
capableof achieving biochemical lesions which lead to cell
death.
REFERENCES
1. Bachur, N. R. Anthracycline antibiotic pharmacology and
metabolism.Cancer Treat. Rep.. 63. 817-820. 1979.
2. Bachur, N. R., Gordon. S. L.. and Gee, M. V. Anthracycline
antibioticaugmentation of microsomal electron transport and free
radical formation.Mol. Pharmacol.. 13: 901-910, 1977.
3. Bachur, N. R., Gordon, S. L., and Gee, M. V. A general
mechanism formicrosomal activation of quinone anticancer agents to
free radicals. CancerRes., 38. 1745-1750, 1978.
4. Baker, L. H.. Opipari. M. I., and Izbicki, R. M. Phase II
study of mitomycinC, vincristine, and bleomycin in advanced
squamous cell carcinoma of theuterine cervix. Cancer (Phila.). 38.
2222-2224, 1976.
5. Baker, L. H., Opipari, M. I.. Wilson. H., Bottomley, R., and
Coliman, C. A.Mitomycin C, vincristine, and bleomycin therapy for
advanced cervicalcancer. Obstet. Gynecol., 5ÃŽ.146-150, 1978.
6. Barranco. S. C., and Novak, J. K. Survival responses of
dividing andnondividing mammalian cells after treatment with
hydroxyurea. arabinosyl-cytosine. or Adriamycin. Cancer Res.. 34:
1616-1618. 1974.
7. Barranco, S. C., Novak, J. K.. and Humphrey, R. M. Response
of mammalian cells following treatment with bleomycin and
1,3-bis(2-chloroethyl>-1-nitrosourea during plateau phase.
Cancer Res., 33. 691-694, 1973.
8. Sasaga, S. H.. Dunlop, J. R., Searle, A. J. F., and Willson.
R. L. Metroni-dazole (Flagyl) and misonidazole (Ro-07-0582)
reduction by faculativeanaerobes and cytotoxic action of hypoxic
bacteria and mammalian cellsin vivo. Br. J. Cancer, 37(Suppl. 3).
132-135, 1978.
9. Bedford, J. S., and Mitchell, J. B. The effect of hypoxia on
the growth andradiation response of mammalian cells in culture Br.
J. Radiol.. 47: 687-696. 1974
10. Berlino. J. R. (ed.). Folate antagonists as Chemotherapeutic
agents. Ann.N. Y. Acad. Sci.. Õ86.5-519, 1971.
11. Berlino, J. R. Folate antagonists. In: A. C. Sartorelli and
D. G. Johns (eds.),Antineoplastic and Immunosuppressive Agents,
part 2, pp. 468-483. Berlin: Springer-Verlag, 1975.
12. Berlino. J. R., Booth. B. A.. Cashmore. A.. Bieber. A. L..
and Sartorelli. A.C. Studies on the inhibition of dihydrofolate
reducÃ-aseby folate antagonistsJ. Biol. Chem.. 239. 479-485,
1964.
13. Bhuyan. B. K.. Fräser,T. J., and Day, K. J. Cell
proliferation kinetics anddrug sensitivity of exponential and
stationary populations of cultured L1210cells. Cancer Res., 37.
1057-1063, 1977.
14. Born, R., Hug, O., and Trott. H.-R. The effect of prolonged
hypoxia ongrowth and viability of Chinese hamster cells. Int. J.
Radiât.Oncol Biol.Phys.. J. 687-697. 1976.
15. Brown, J. M. Cytotoxic effects of the hypoxic cell
radiosensitizer Ro-07-0582 10 ujmor relis in
-
S. A. Teicher et al.
infused 5-fluorouracil in the treatment of disseminated
gastrointestinalcarcinomas. Med. Pediatr. Oncol.. 4: 35-42,
1978.
18. Buroker, T., Kim, P. N., Groppe. C.. McCracken, J., O'Bryan,
R., Panet
tiere, F., Coliman, C., Bottomley, R., Wilson, H., Bonnet. J.,
Thigpen, T.,Vaitkevicius, V. K., Hoogstraten. B., and Heilbrun, L.
5-FU infusion withmitomycin C versus 5-FU infusion with methyl-CCNU
in the treatment ofadvanced colon cancer. Cancer (Phila.). 42:
1228-1233. 1978.
19. Buroker, T., Kim, P. N., Groppe, C.. McCracken, J.. O'Bryan,
R.. Panetière.
F., Costanzi. J.. Bottomley, R.. King. G. W.. Bonnet, J ,
Thigpen, T..Whitecar, J., Haas, C., Vaitkevicius, V. K.,
Hoogstraten, B., and Heilbrun,L. 5-FU infusion with mitomycin C
versus 5-FU infusion with methyl-CCNU
in the treatment of advanced upper gastrointestinal cancer.
Cancer (Phila.),44: 1215-1221. 1979.
20. Chahinian, A. P.. Mandel, E. M., Holland. J. F., Jatfrey. I.
S., and Teirstein,A. S. MACC (methotrexate. Adriamycin,
cyclophosphamide and CCNU) inadvanced lung cancer. Cancer (Phila.),
43: 1590-1597, 1979.
21. Cohen, S. S. On the nature of thymineless death. Ann. N. Y.
Acad. Sci..186: 292-301, 1971.
22. Cone, R.. Hasan, S. K.. Lown, J. W., and Morgan. A. R. The
mechanism ofthe degradation of DMA by streptonigrin. Can. J.
Biochem.. 54: 219-223.
1976.23. Creasey, W. A. Vinca alkaloids and colchicine. In: A.
C. Sartorelli and D. G.
Johns (ed.), Antineoplastic and Immunosuppressive Agents, Part
2, pp.670-694. Berlin: Springer-Verlag. 1975.
24. Crooke. S. T.. and Bradner, W. T. Mitomycin C: A review.
Cancer Treat.Rev., 3. 121-139. 1976.
25. DeJager, R. L., Magill, G. B.. Golbey. R. B.. and Krakoff,
l. H. Combinationchemotherapy with mitomycin C, 5-fluorouracil, and
cytosine arabinosidein gastrointestinal cancer. Cancer Treat. Rep..
60. 1373-1375. 1976.
26. Denekamp, J.. and Harris, S. R. The response of a
transplanted tumor tofractionated irradiation. I. X-Rays and the
hypoxic cell radiosensitizer Ro-07-0582. Radiât.Res., 66. 66-75,
1976.
27. Denekamp, J.. and McNally, N. J. The magnitude of hypoxic
cell cytotox-icity of misonidazole in human tumors Br. J. Radiol.,
51: 747-748, 1978.
28. Digenis, G. A., and Issidorides. C. H. Some biochemical
aspects of A/nitroso compounds. Bioorg. Chem.. 8. 97-137, 1979.
29. DiMarco, A. Adriamycin (NSC-123,127): mode and mechanism of
action.Cancer Chemother. Rep. Part III. 6. 91-106. 1975.
30. Donehower. R. C.. Myers, C. E., and Chabner, B. A. New
developments onthe mechanisms of action of antineoplastic drugs.
Life Sci.. 25. 1-14,
1979.31. Einhorn, L. H., and Williams. S. D. Combination
chemotherapy with c/s-
dichlorodiammineplatinum (II) and Adriamycin for testicular
cancer refractory to vinblastine plus bleomycin. Cancer Treat.
Rep.. 62. 1351-1353,
1978.32. Foster, J. L. Differential cytotoxic effects of
metronidazole and other nitro-
heterocyclic drugs against hypoxic tumor cells. Int. J.
Radiât.Oncol. Biol.Phys., 4: 153-156, 1978.
33. Foster, J. L., Conroy, P. J., Searle. A. J., and Wilson, R.
L. Metronidazole(Flagyl): characterization as a cytotoxic drug
specific for hypoxic tumorcells. Br. J. Cancer. 33. 485-490.
1976.
34. Geard, C. R., Povals, S. F., Astor, M. B., and Hall, E. J.
Cytological effectsof 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol
(misonidazole) on hypoxic mammalian cells in vitro. Cancer Res.,
38. 644-649, 1978.
35. George, K. C.. Hirst, D. G., and McNally, N. J. Effect of
hyperthermia oncytotoxicity of the radiosensitizer Ro-07-0582 in a
solid mouse tumor. Br.J. Cancer, 35: 372-377, 1977.
36. Goldberg. I. H. Actinomycin D. In: A. C. Sartorelli and D.
G. Johns (eds.),Antineoplastic and Immunosuppressive Agents. Part
2. pp. 582-592.Berlin: Springer-Verlag, 1975.
37. Greenberg, B. R., Kardinal, C. G.. Pajak, T. F., and
Bateman, J. R.Adriamycin versus Adriamycin and bleomycin in
advanced epidermoidcarcinoma of the cervix. Cancer Treat. Rep., 61:
1383-1384, 1977.
38. Hahn. G. M.. Gordon, U. F.. and Kurkjian, D. A. Responses of
cycling andnoncycling cells to 1.3-bis(2-chloroethyl)-1-nitrosourea
and to bleomycin.Cancer Res., 34: 2373-2377. 1974.
39. Haller. D. G.. Woolley. P. V., MacDonald. J. S., Smith, L.
F., and Schein.P. S. Phase II trial of 5-fluorouracil. Adriamycin,
and mitomycin C inadvanced colorectal cancer. Cancer Treat. Rep..
62. 563-565, 1978.
40. Handa, K.. and Sato, S Generation of free radicals of
quinone group-containing anti-cancer chemicals in NADPH-microsome
system as evidenced by initiation of sulfite oxidation. Gann. 66.
43-47, 1975.
41. Harris. J. W.. and Shrieve. D. C. Effects of Adriamycin and
X-rays oneuoxic and hypoxic EMT6 cells in vitro. Int. J.
Radiât.Oncol. Biol. Phys.,5: 1245-1248, 1979.
42. Heidelberger. C. Fluorinated pyrimidines and their
nucleosides. In: A. C.Sartorelli and D. G. Johns (eds.),
Antineoplastic and ImmunosuppressiveAgents, part 2, pp. 193-231.
Berlin: Springer-Verlag, 1975.
43. Hilton, J., Maldorelli. F., and Sargent. S. Evaluation of
the role of isocyan-ates in the action of therapeutic nitrosoureas.
Biochem. Pharmacol., 27.1359-1363, 1978.
44. Howell, S. D.. Ensminger. W. D., Kristan, A., and Frei E..
III. Thymidinerescue of high-dose methotrexate in humans. Cancer
Res.. 38. 325-330.
1978.
45. Hryniuk, W. M., Fischer. G. A., and Berlino. J. R. S-phase
cells in rapidlygrowing and resting populations: differences in
response to methotrexate.Mol. Pharmacol.. 5. 557-564, 1969.
46. Iyer, V. N., and Szybalski. W A molecular mechanism of
mitomycin action:linking of complimentary DNA strands. Proc. Nati.
Acad. Sei. U. S. A., 50.355-362. 1963.
47. Iyer, V. N., and Szybalski, W. Mitomycin and porfiromycin:
chemicalmechanism of activation and cross-linking of DNA. Science
(Wash. D. C.),145: 55-58, 1964.
48. Jensen, D. E. Reaction of DNA with alkylating agents.
Differential alkylationof poly[dA-dT] by Methylnitrosourea and
ethylnitrosourea. Biochemistry,77:5108-5113, 1978.
49. Jensen, D. E.. and Reed. D. J. Reaction of DNA with
alkylating agents.Quantitation of alkylation by ethylnitrosourea of
oxygen and nitrogen siteson poly(dA-dT] including phosphotriester
formation. Biochemistry, 17:5098-5107, 1978.
50. Kennedy, K. A., Rockwell, S., and Sartorelli. A. C.
Preferential activationof mitomycin C to cytotoxic metabolites by
hypoxic tumor cells. CancerRes., 40: 2356-2360. 1980.
51. Kennedy. K. A., and Sartorelli, A. C. Metabolic activation
of mitomycin Cby isolated liver nuclei and microsomes. Fed. Proc.,
38. 443, 1979.
52. Kennedy, K. A., Teicher, B. A.. Rockwell, S., and
Sartorelli, A. C. Thehypoxic tumor cell: a target for selective
cancer chemotherapy. Biochem.Pharmacol.. 29. 1-8. 1980.
53. Kinami, Y.. and Miyazaki, I. The superselective and the
selective one shotmethods for treating inoperable cancer of the
liver. Cancer (Phila.) 41:1720-1727, 1978.
54. Koch. C. J., Kruuv, J., Frey. H. E.. and Snyder. R. A.
Plateau-phase ingrowth induced by hypoxia. Int. J. Radiât.Biol.
Relat. Stud. Phys. Chem.Med. 23. 67-74, 1973.
55. Krauss, S.. Sonoda, T., and Solomon, A. Treatment of
advanced gastrointestinal cancer with 5-fluorouracil and mitomycin
C. Cancer (Phila.), 43:1598-1603. 1979.
56. Langenbach, R. J., Dannenberg, P. V., and Heidelberger, C.
Thymidylatesynthetase: mechanism of inhibition by
5-fluoro-2'-deoxyuridylate. Biochem. Biophys Res. Commun., 48:
1565-1571. 1972.
57. Lin, A. J., Cosby, L. A., and Sartorelli. A. C. Potential
bioreductive alkylatingagents. In: A. C. Sartorelli (ed.). Cancer
Chemotherapy, pp. 71-86. Washington: American Chemical Society,
1976.
58. Un, A. J., Pardini, R. S., Cosby, L. A., Lillis. B. J.,
Shansky, C. W., andSartorelli. A. C. Potential bioreductive
alkylating agents. 2. Antitumor effectand biochemical studies of
naphthoquinone derivatives. J. Med. Chem..16: 1268-1271, 1973.
59. Lokich. J. J.. Skarrini, A. T., Mayer, R. J.. Henderson. I.
C.. Blum, R. H.,and Frei E., III. Adriamycin plus alkylating agents
in the treatment ofmetastatic breast cancer. Cancer (Phila.). 40:
2801-2805. 1977.
60. Lown, J. W.. Begleiter, A., Johnson, D.. and Morgan, R.
Studies related toantitumor antibiotics. Part V. Reaction of
mitomycin C with DNA examinedby ethidium fluorescence assay. Can.
J. Biochem., 54. 110-119, 1976.
61. Lown, J. W.. and Sim. S.-K. The mechanism of the
bleomycin-inducedcleavage of DNA. Biochem. Biophys. Res. Commun.,
77. 1150-1157.1977.
62. Lown. J. W.. Sim. S.-K.. Majumdar. K. C.. and Chang, R.-Y.
Strand scissionof DNA by bound Adriamycin and daunorubicin in the
presence of reducingagents. Biochem. Biophys. Res. Commun., 76.
705-710, 1977.
63. MacDonald. J. S., Woolley, P. V.. Smythe. T., Veno, W.,
Hoth. D., andSchein. P. S. 5-Fluorouracil. Adriamycin. and
mitomycin C (FAM) combination chemotherapy in the treatment of
advanced gastric cancer. Cancer(Phila.). 44: 42-47. 1979.
64. Mandel, H. G., Klubes, P., and Fernandes. D. J. New
challenges with anold drug, 5-fluorouracil. In: S. K. Carter. A.
Goldin. K. Kuretani, G. Mathé.Y. Sakurai. S. Tsukagoshi. and H.
Umezawa (eds.). Advances in CancerChemotherapy, pp. 255-266.
Baltimore: University Park Press. 1978.
65. Misra, N. C., Jaiswal. M. S. D.. Singh, R. V., and Das. B.
Intrahepaticarterial infusion of a combination of mitomycin C and
5-fluorouracil intreatment of primary and metastatic liver
carcinoma. Cancer (Phila.). 39.1425-1429, 1977.
66. Miyamoto, T., Takabe, Y., Watanabe, M.. and Terasima. T.
Effectivenessof a sequential combination of bleomycin and mitomycin
C on an advancedcervical cancer. Cancer (Phila.), 41: 403-414,
1978.
67. Mohindra. J. K., and Rauth. A. M. Increased cell killing by
metronidazoleand nitrofurazone of hypoxic compared to aerobic
mammalian cells. CancerRes.. 36. 930-936, 1976.
68. Momparler, R. L. in vitro systems for evaluation of
combination chemotherapy. Pharmacol. Ther. Part A Chemother.
Toxicol. Metab. Inhibitors. 8.21-35, 1980.
69. Montgomery, J. A., James, R., McCaleb, G. S., Kirk, M. C.,
and Johnston,T. P. Decomposition of
N-(2-Chloroethyl)-N-nitrosoureas in aqueous media.J. Med. Chem..
18: 568-571, 1975.
70. Moore, B. A.. Palcic. B.. and Skarsgard. L. D.
Radiosensitizing and toxiceffects of the 2-nitroimidazole
Ro-07-0582 in hypoxic mammalian cells.Radiât.Res.. 67. 459-473.
1976.
71. Moore, H. W. Bioactivation as a model for drug design
bioreductivealkylation. Science (Wash. D. C.), 797. 527-532.
1977.
80 CANCER RESEARCH VOL. 41
on June 18, 2021. © 1981 American Association for Cancer
Research. cancerres.aacrjournals.org Downloaded from
http://cancerres.aacrjournals.org/
-
Drug Cytotoxicity and Cellular Oxygénation
72. Newman, H. K., and Quan. S. H. Q. Multi-modality therapy for
epidermoidcarcinoma of the anus. Cancer (Phila.), 37: 12-19,
1976.
73. Oberley, L. W.. and Buettner, G. R. The production of
hydroxyl radical bybleomycin and iron (II). FEBS Lett., 97: 47-49.
1979.
74. Olive, P. L., and Durand, R. E. Activation of
radiosensitizers by hypoxiccells. Br. J. Cancer. 37(Suppl. 3).
124-128. 1978.
75. Overgaard, J. Effect of hyperthermia on malignant cells in
vivo. Cancer(Phila.), 39. 2637-2646. 1977.
76. Presant, C. A., Amburg, A. V., and Klahr. C. Adriamycin.
1,3-bis(2-chlo-roethylM-nitrosourea (BCNU, NSC-409,962) and
cyclophosphamide therapy of drug-resistant metastatic breast
cancer. Cancer (Phila.), 40: 987-993, 1977.
77. Presant, C. A.. Ratkin, G., and Klahr, C. Phase II study of
mitomycin C.cyclophosphamide and methotrexate in drug-resistant
colorectal carcinoma. Cancer Treat. Rep., 62: 549-550, 1978.
78. Reed, D. J., May. H. E., Boose. R. B., Gregory. K. M., and
Beilstein, M. A.2-Chloroethanol formation as evidence for a
2-chloroethyl alkylating intermediate during chemical degradation
of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and
1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1 -nitrosourea.
Cancer Res.. 35 568-576. 1975.
79. Roberts, J. J., and Pascoe, J. M. Cross-linking of
complementary strandsof DNA in mammalian cells by antitumor
platinum compounds. Nature(Lond.). 235. 282-284, 1972.
80. Roberts, J. J., and Thompson, A. J. The mechanism of action
of antitumorplatinum compounds. Prog. Nucleic Acid Res. Mol. Biol.,
22: 71-133,1979.
81. Roberts, P. B. Radiosensitization of E. coli B/r by the
cytotoxic agentprocarbazine: a hypoxic cell sensitizer
preferentially toxic to aerobic cellsand easily oxidized. Br. J.
Cancer. 39. 755-760, 1979.
82. Rockwell, S. In vivo-in vitro tumor systems: new models for
studying theresponse of tumors in therapy. Lab. Anim. Sci., 27:
831-851, 1977.
83. Rockwell, S.. Kallman, R. F., and Fajardo. L. F.
Characteristics of a seriallytransplanted mouse mammary tumor and
its tissue culture-adapted derivative. J. Nati Cancer Inst., 49:
735-749, 1972.
84. Rockwell, S., and Kennedy. K. A. Combination therapy with
radiation andmitomycin C. Preliminary results with EMT6 tumor cells
in vitro and in vivo.Int. J. Radiât.Oncol. Biol. Phys.. 5:
1673-1676, 1979.
85. Roizin-Towle, L., and Hall. E. J. Studies with bleomycin and
misonidazoleon aerated and hypoxic cells. Br. J. Cancer, 37:
254-260, 1978.
86. Samson, M. K., Comis, R. L., Baker, L. H., Ginsberg. S.,
Fraile. R. J., andCrooke, S. T. Mitomycin C in advanced
adenocarcinoma and large cellcarcinoma of the lung. Cancer Treat.
Rep., 62: 163-165, 1978.
87. Santi, D. V.. McHenry, C. S., and Sommer, H. Mechanism of
interaction ofthymidylate synthetase with 5-fluorodeoxyuridylate.
Biochemistry, )3.471-481. 1974.
88. Sato, S.. Iwaizumi. M., Manda, K., and Tamura. Y. Electron
spin resonancestudy on the mode of generation of free radicals of
daunomycin, Adriamycinand carboquone in NAD(P)H-microsome system.
Gann, 68: 603-608,1977.
89. Sausville. E. A., Peisach, J.. and Horwitz, S. B. Effect of
chelating agentsand metal ions on the degradation of DNA by
bleomycin. Biochemistry. T7:2740-2746, 1978.
90. Sausville, E. A., Stein, R. W., Peisach, J., and Horwitz, S.
B. Propertiesand products of the degradation of DNA by bleomycin
and iron(ll). Biochemistry. 17: 2746-2754, 1978.
91 Schwartz, H. S. Pharmacology of mitomycin C: III. In vitro
metabolism byrat liver. J. Pharmacol Exp. Then, 736: 250-258.
1962.
92. Schwartz, H. S.. Sodergren. J. E.. and Philips, F. S.
Mitomycin C: chemicaland biological studies on alkylation. Science
(Wash. D. C.), 142: 1181-1183, 1963.
93. Singer, B.. Bodell, W. J.. Cleaver. J. E., Thomas, G. H.,
Rajewsky, M. F.,and Thon, W. Oxygens in DNA are main targets for
ethylnitrosourea innormal and xeroderma pigmentosum fibroblasts and
fetal rat brain cells.Nature (Lond.). 276 85-88, 1978.
94. Smith, E.. Stratford, I. J., and Adams, G. E. The resistance
of hypoxicmammalian cells to chemotherapeutic agents Br. J. Cancer,
40: 316,
1979.95. Song, C. W., Clement. J. J., and Levitt, S. H.
Preferential cytotoxicity of 5-
thio-D-glucose against hypoxic tumor cells. J. Nati. Cancer
Inst.. 57. 603-
605. 1976.96. Song, C. W.. Clement, J. J., and Levitt, S. H.
Elimination of hypoxic
protection by 5-thio-D-glucose in multiceli spheroids. Cancer
Res., 38:4499-4503, 1978.
97. Song, C. W., Sung, J. H., Clement, J. J.. and Levitt, S. H.
Cytotoxic effectof 5-thio-p-glucose on chemically hypoxic cells in
multiceli spheroids. BrJ. Cancer, 37(Suppl. 3): 136-140, 1978.
98. Sridhar, R., Kocj, C. J., Stroude, E. C.. and Inch. W. R.
Cell survival in V79multiceli spheroids treated with
dehydroascorbate. 5-thio-D-glucose, and2-deoxy-n-glucose. Br. J.
Cancer, 37(Suppl. 3). 141-144, 1978.
99. Sugiura, Y. Production of free radicals from phenol and
tocopherol bybleomycin-iron(ll) complex. Biochem. Biophys. Res.
Commun., 87. 649-
653. 1979.100. Sutherland, R. M. Selective chemotherapy on
noncycling cells in an in vitro
tumor model. Cancer Res.. 34: 3501-3503, 1974.101. Sutherland,
R. M., Eddy, H. A., Bareham, B., Reich, K., and Vanantwerp.
D. Resistance to adriamycin in multicellular spheroids. Int. J.
Radiât.Oncol.Biol. Phys., 5. 1225-1230. 1979.
102. Tannock, I. F. Chemotherapy for hypoxic tumor cells:
metronidazole andmisonidazole in combination with conventional
anti-cancer drugs. Proc.
Am. Assoc. Cancer Res.. 20 8, 1979.103. Taylor, Y. C., and
Rauth, A. M. Differences in the toxicity and metabolism
of the 2-nitroimidazole misonidazole (Ro-07-0582) in HeLa and
Chinesehamster ovary cells. Cancer Res., 38: 2745-2752, 1978.
104. Tranum. B., Hoogstraten. B.. Kennedy. A.. Vaughn. C. B.,
Samal. B..Thigpen, T., Rivkin, S.. Smith, F., Palmer, R. L.,
Constanzi. J.. Tucker, W.G., Wilson, H.. and Maloney, T. R.
Adriamycin in combination for thetreatment of breast cancer. Cancer
(Phila.), 41: 2078-2083. 1978.
105. Trowbridge, R. C.. Kennedy, B. J., and Vosika, G. J
CCNU-Adriamycintherapy in bronchogenic carcinoma. Cancer (Phila.),
41: 1704-1709,1978.
106. Twentyman. P. R. Comparative chemosensitivity of
exponential versusplateau-phase cells in both in vitro and in vivo
model systems. CancerTreat. Rep.. 60. 1719-1722, 1976.
107. Varghese, A. J., Gulyas. S.. and Mohindra, J. K.
Hypoxia-dependentreduction of
1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol by Chinesehamster
ovary cells and KHT tumor cells in vitro and in vivo. Cancer
Res.,36:3761-3765, 1976.
108. Vaupel, P. Hypoxia in neoplastic tissue. Microvasc. Res.,
13: 399-408.
1977.109. Vaupel, P., and Thews, G. P0, distribution in tumor
tissue of DS-carcino-
sarcoma. Oncology (Basel), 30: 475-484, 1974.110. Waksman, S. A.
(ed.). Conference on actinomycins: their potential for
cancer chemotherapy. Cancer Chemotherap. Rep., 58: 1-122,
1974.111. Waterfield. W. C., Tasima. C. K.. Hortobagyl. G. N..
Blumenschein, G. R ,
Buzdar, A. U.. and Burgess, M. A Adriamycin and
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the
treatment of metastatic breastcancer. Cancer (Phila.), 41:
1235-1239, 1978.
112. Weinkam. R. J.. and Shiba. D. A. Metabolic activation of
procarbazine. LifeSci., 22. 937-946. 1978.
113. Wilson, L., Anderson. K. A., and Chin. D. Nonstoichiometric
poisoning ofmicrotubule polymerization: a model for the mechanism
of action of thevinca alkaloids, podophyllotoxin and colchicine.
Cold Spring Harbor Conf.Cell Proliferation. 3: 1051-1064, 1976.
114. Wise, G. R., Kühn,I. N.. and Godfrey. T. E. Mitomycin C in
large infrequentdoses in breast cancer. Med. Pediatr. Oncol., 2.
55-60. 1976.
115. Wodinsky, I., Johnson, R. K., and Clement. J. J. Enhanced
activity againstmurine tumors of cyclophosphamide in combination
with the hypoxic cellradiosensitizer misonidazole. Proc. Am. Assoc.
Cancer Res., 20: 230,1979.
116. Wong, T. W., Whitmore, G. F.. and Gulyas, S. Studies on the
toxicity andradiosensitizing ability of misonidazole under
conditions of prolonged incubation. Radiât.Res.. 75: 541-555.
1978.
JANUARY 1981 81
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1981;41:73-81. Cancer Res Beverly A. Teicher, John S. Lazo and
Alan C. Sartorelli Toxicities toward Oxygenated and Hypoxic Tumor
CellsClassification of Antineoplastic Agents by their Selective
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