Classification of Antineoplastic Agents by their Selective … · Classification of Antineoplastic Agents by their Selective Toxicities toward Oxygenated and Hypoxie Tumor Cells1

[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 Â¡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 oxygÃ©nation 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 oxygÃ©nationand 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 37Â°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 37Â°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 (â€¢)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 oxygÃ©nation

Preferential toxic-ity to aerobic cells

(type 1)Preferential toxicity to hypoxic

cells (type 2)Minimal or no selectivity based on cel

lular oxygÃ©nation (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 OxygÃ©nation

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, (Â£>)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 Â¡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 /Â¿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 {â€¢)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 (â€¢)and hypoxic (O) EMT6 cells treated for 1 hrwith various concentrations of procarbazine in cell culture.

Chart 4. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hrwith various concentrations of actinomycin D in cell culture.

JANUARY 1981 75



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 Â±0.89%for aerobic cells and 6.59 Â± 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, Â¿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/Â¿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 (â€¢)and hypoxic (O) EMT6 cells treated for 1 hrwith various concentrations of vincristine in cell culture.

Chart 6. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hrwith various concentrations of mitomycin C in cell culture.





5-FLUOROURACIL

0.0001 0.001 0.01 O.I 1.0

DRUG CONCENTRATION, Â¿iM

Chart 7. Survival of aerobic (â€¢)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 (â€¢)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 oxygÃ©nation.

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

JANUARY 1981 77



ÃŸ.A. Teicher et al.

METHOTREXATEi.oo -

001 0.1 1.0 IO 100

DRUG CONCENTRATION, MM

1000

Chart 9. Survival of aerobic (â€¢)and hypoxic (O) EMT6 cells treated for 1 hrwith various concentrations of methotrexate in cell culture.

cis-DIAMMINEDICHLOROPLATINUM (H)

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oooooi ooooi IOO0.001 0.01 O.I 10

DRUG CONCENTRATION,/iM

Chart 10. Survival of aerobic (â€¢)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 oxygÃ©nation. A similar result was obtainedwith CCNU.

100

BCNU

O.I IO IO

DRUG CONCENTRATION, MM

IOO

Chart 11. Survival of aerobic (â€¢)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





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.. Ã•86.5-519, 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.. FrÃ¤ser,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. RadiÃ¢t.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.. PanetiÃ¨re.

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. RadiÃ¢t.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. RadiÃ¢t.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. RadiÃ¢t.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. RadiÃ¢t.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. MathÃ©.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.RadiÃ¢t.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.





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. RadiÃ¢t.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. RadiÃ¢t.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., KÃ¼hn,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. RadiÃ¢t.Res.. 75: 541-555. 1978.

JANUARY 1981 81



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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