Effects of Elevated Temperatures and Drugs on the ... · complete Fischer or Spinner media at...

[CANCER RESEARCH 30, 1623â€”1631, June 1970]

survival of the animals tested (33, 40). Furthermore, theresponse of Ll2lO leukemia to tumor-inhibitory drugs isconsidered to be similar to that of many human tumors(18). We have treated the cells in vitro because it wasnecessary to use temperatures higher than mice can tolerate.The viability of the cells after treatment was assayed byreinoculation into mice, rather than by the dye exclusiontest (36) used by von Ardenne and others (2â€”6) in similarexperiments. We have found that the viability determined bythe dye exclusion method does not agree with the bioassay,which is a more precise and direct method.

Experimental studies have shown that some metabolicprocesses are preferentially affected by heat : respiration (7,8, 29, 30, 43), DNA synthesis (16, 17, 28, 29), RNAsynthesis (28, 29), and protein synthesis (28, 29). Stehlin(41) has demonstrated that the chemotherapeutic effect ofphenylalanine mustard, administered by closed circuitperfusion to human melanomas of the limbs, is considerablyincreased by raising the temperature of the perfusing fluidabove 42Â°.

These findings have prompted us to investigate moreextensively the possibility of using chemicals in combinationwith heat. We have tested metabolic inhibitors of DNAsynthesis [FUDR4 (20, 21)] , RNA synthesis [actinomycin D(23)] , protein synthesis [DHBA (19)1 , glycolysis [DLglyceraldehyde (26, 27), 2-deoxy-D-glucose (10, 46)],sodium oxamate (35), mitotic poisons [vinbiastine (34)1,and an alkylating agent [PAM (1 1, 32)1.

MATERIALS AND METHODS

Cells and Media. L1210 leukemia, obtained from theWi sc o n 5in Alu m n i Foundation , Madison , Wis., wasmaintained in the ascites form by weekly transplants infemale BDF1 mice purchased from A. R. Schmidt Co.,Madison, Wis. For maintenance, 0.1 ml of 6-day-old ascitesdiluted 1: 10 with 0.9% NaC1 solution was injected i.p.

Cells, 5 to 10 days after passage, were suspended in Fischermedium (14) plus 10% horse serum and antibiotics, or inMEM Spinner medium plus 10% calf serum and antibiotics.The cells were then counted in a hemocytometer and the

4The abbreviations used are: FUDR, 5-fluoro-2@-deoxyuridine;DHBA, L-erythro-aj3-dihythoxybutyraldehyde; PAM, L-phenylalanine mustard; MEM, minimal essential medium.

JUNE 1970 1623

Effects of Elevated Temperatures and Drugs on the Viability ofLi 210 Leukemia Cells'

Beppino C. Giovanella,2 Wendy A. Lohman, and Charles Heidelberger3

McArdle Laboratory for Cancer Research, The Medical School, University of Wisconsin, Madison, Wisconsin 53706

SUMMARY

The lethal action of elevated temperatures on neoplastic cellshas been determined quantitatively by means of an in vitro-invivo system of L1210 leukemia cells. Temperatures from 37Â°to 40Â°have little effect on these cells. Prolonged exposure at41Â° impairs their viability. Between 41Â° and 42Â° the lethaleffect increases markedly; in Fischer medium, a 4-log kill isachieved at 42Â°in 3 hr. The exposure of Ll210 leukemia cellsto a temperature of 42Â°for a short time sensitizes them to theaction of lower temperatures. A 3-hr exposure at 40Â°has ahighly lethal effect when applied after an initial treatment at42Â°. If the sequence of heating is reversed, no such effect isobserved. Many drugs have been tested for combined therapy.L.eiythro-aj3-Dthydroxybutyraldehyde has been found to actsynergistically with heat. DL-Glyceraldehyde, L-phenylalaninemustard, actinomycin D, and sodium oxamate are also activein combination with heat. However, if toxicity to humans isconsidered, only DL-erythro-ajl-dthydroxybutyraldehyde,DL-glyceraldehyde, and phenylalanine mustard can beconsidered as suitable for clinical trial in combination withheat.

INTRODUCTION

A selective lethal effect of elevated temperatures on cancercells has been demonstrated both experimentally (6, 9 , 15 , 37,38) and clinically(7, 8, 12, 24, 25, 41, 42). For a moreextensive bibliography see Ref. 7 . We have tried to determinethis lethal effect quantitatively and to explore the possibilityof enhancing it by exposing neoplastic cells to heat andchemicals simultaneously.

As an experimental model, we have chosen an in vitro-invivo system. Ll2lO leukemia cells, suspended in a completeculture medium, are treated in vitro and then tested forviability by reinoculation into groups of BDF1 female mice.The L1210 leukemia has been selected because of the preciseinverse correlation between the number of cells injected and

iThis work was supported in part by Grant CA7175, from theNational Cancer Institute, NIH.

2Present address: Stehlin Foundation, 834 Hermann ProfessionalBuilding, Houston, Texas.

3American Cancer Society Professor of Oncology.Received November 12, 1969; accepted January 26, 1970.

on April 3, 2021. © 1970 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

B. C. Giovanella, W. A. Lohman, and C. Heidelberger

suspension adjusted by dilution to the required concentration. The combination of these media plus serum andantibiotics will henceforth be referred to as complete media.It has been reported that Ll2lO leukemia cells can be grownin culture in shaker flasks with various media (13 , 31) and insoft agar with Fischer medium (22). We have grown themsuccessfully in shaker culture with Fischer medium + 10%horse serum for 5, 5, 10, 12, and 20 passages. In the Spinnermedium, L1210 leukemia cells do not reproduce, but can bemaintained at 37Â° without loss of viability for 24 hr ormore. For avoidance of agglutination, in some cases, 0.04%Pluronic F 68 was added after it had been shown that suchan addition did not change the sensitivity to heat of theleukemic cells. All the media and sera were purchased fromthe Grand Island Biological Co., Grand Island, N.Y.

Drugs. The drugs used in this work were: FUDR (Hoffmann La Roche, Inc., NuiJey, N. J.), actinomycin D (K andK Laboratories, Inc., Plainview, N. Y.), DHBA (donated byFarmitalia, Milano, Italy), DL-glyceraldehyde and 2-deoxyD-glucose (Aldrich Chemical Co., Milwaukee, Wis.), sodiumoxamate (jrepared from oxamic acid; Sigma Chemical Co.,St. Louis, Mo.), vinblastine sulfate (Eli Lilly and Co.,Indianapolis, md.), and PAM (donated by BurroughsWeilcome and Co., Tuckahoe, N. Y.).

Heat Treatment. L12 10 leukemia cells were suspended incomplete Fischer or Spinner media at concentrations of 10to iO@cells/rnl. From 20 to 50 ml of these suspensions wereagitated in 50- or 100-mi Erlenmeyer flasks, closed withground-glass stoppers, in a thermostated gyrotory water bathshaker (Model G76, New Brunswick Scientific Co., NewBrunswick, N. J.) for various lengths of time at the desiredtemperature. The flasks were agitated at 90 rpm. At the endof the treatment, 0.1 ml of the cell suspension was injectedi_p. into groups of 10 to 20 female BDF1 mice. The injectedanimals were inspected daily and the deaths were recorded.

For the dye exclusion test (36), the following techniquehas been used. To a drop on a microscope slide of asuspension containing 1 X 106 cells/mi, a small amount ofdry Eosin Y was added and the suspension was agitated untilthe dye dissolved. A coverslip was superimposed, and thepercentage of stained cells was determined by observation at400X of 400 cells.

We have used the following technique for culturing theleukemic cells : a suspension of Li 2 10 leukemia in completeFischer medium plus 0.04% Pluronic F 68 was saturatedwith a mixture of 95% 02 and 5% CO2 and was maintainedat 37Â° in Gyrotory Model G25 shaker incubator (NewBrunswick Scientific Co., New Brunswick, N. J.) set at 140rpm. From 1 to 5 X i0@ cells/mi were added to start theculture. After a lag period of 2 to 3 days, the culture wasestablished and was maintained by a 1:5 dilution with freshmedium when it had reached a level of 5 X i0@ cells/mi.

RESULTS

In several reports (2â€”5), the viability of cells after treatment with heat and chemicals has been assayed by dyeexclusion methods. We have also applied this technique,which has the advantages of simplicity and rapidity . As

shown in Chart 1, a good correlation between exposure toelevated temperatures and number of stained cells wasobserved. Nevertheless, we decided to test the validity ofthese results by reinoculation of the heated cells into mice.This will henceforth be referred to as the bioassay. TheL1210 leukemia in ascites form is particularly suitable forquantitative experiments of this type because there is adirect inverse correlation between the number of cellsinj ected into mice and their survival, as elegantlydemonstrated by Skipper et aL (39, 40) and Dixon et aL(13). Wilkoff et aL (44) have used a similar bioassay todetermine the response of cultured L1210 cells to antileukemic drugs.

We have checked this relationship in our strain of L1210leukemia and found, in a series of experiments involving1350 mice (Chart 2), that there is such a correlation, but thefluctuation among different experiments was quite large. It

I00

Chart 1. Percentage of L1210 leukemic cells unstained by eosin Yafter incubation at the temperatures indicated as a function of time.The cells were suspended in MEM Spinner complete medium. Eachpoint has been determined by counting 400 cells.

37.

50

40

U)-J-jIiiC-)

0Iii

z

C,)z

I0

5

A

43@

42

I 2 3 4 5HR

1624 CANCER RESEARCH VOL.30



Survivors atmoreNo.

of cellsinjectedNo.

of animalsinjectedthan

40daysNo.%1x104

1x1031x102ixiol320

4503402200

635620

110

28S206301382771

Effects ofHeat and Drugs on Leukemia Cells

Table1

The number offemale BDF1 mice survivingmore than 40 days afterinjection ofLl2lO leukemia cells

(320)

(450)

(340)

(220)

p -@@@oOT

..-.-â€”â€”..3OMINA.@ INN

aâ€”-â€”.. 21*Q.â€”..â€”o41*.â€”â€”. 81*

S

I00

IX IO@

IX IO@

IX i02

IX 10

50( I)(20)

05 0

DAYS2 4 6 8 IC 2 14 6 18 20 22

DAYS Chart 3. The percentage of surviving mice as a function of timeafter i.p. injection of 1 X i0@ L1210 cells that had been incubated at37 in complete MEM Spinner medium for the lengths of timeindicated. Ten female BDF1 mice were used for each line.

survival curve identical with that obtained when only a singlecell was injected. This is equivalent to a 4-log kill.

There was a major discrepancy between the results of thebioassay and those obtained by dye exclusion. By the lattermethod, the critical temperature at which the major killingeffect appears is between 40Â°and 41Â°(Chart 1); the 41and 42Â° curves were almost equivalent. By the bioassay, thetemperature at which a massive lethal effect appears isbetween 41Â° and 42Â° (Charts 4 and 5). The quantitativedifference is also quite striking. Using the dye exclusionmethod, after 2 hr at 43Â°, 15% of the cells were stillunstained, which is equivalent to less than a 2-log kill (Chart1), whereas with the bioassay we found that, under the sameconditions and the injection of 1 X i0@ treated cells intomice, there were no deaths (Table 2). This is equal to morethan a 4-log kill. After fmding this 100-fold discrepancybetween these 2 methods, we decided to adopt the bioassaybecause it is more direct and precise, although considerablymore tedious. Having observed the lethal effect of heat in a

Chart 2. Fifty % survival of mice as a function of the number ofL1210 leukemic cells injected i.p. The numbers of animals used are inparentheses. Central points, arithmetical averages; solid bars,deviations found in various groups tested in each experiment. Foreach point of every experiment, groups of 10 to 20 mice were used.The total number of mice was 1350.

was also clear that the injection of a single cell gave a 20 to40% mortality (Table 1). Accordingly, we can state that a4-log kill has taken place when 20 to 40% of the mice diedafter inoculation of 1 X i04 treated cells/mouse . Charts 3,4, and 5 show the survival of mice inoculated with 1 X l0@L1210 cells that had been exposed to heat for variouslengths of time in MEM Spinner. At 37Â°(Chart 3), there wasno effect on the viability of the leukemic cells; no differencewas found in the survival of the control group and the onegiven injections of cells incubated for 13 hr. At 41Â° (Chart4), some of the effects of the heat become apparent. In the13-hr group, there was a significant increase in survival time,which according to Chart 2, corresponds approximately to a2-log kill. The lethal effect becomes dramatically evident at42Â° (Chart 5), at which exposure of the cells for 4 hr gave a

JUNE 1970 1625



TemperatureTime (hi)Survivors@Z%Complete

MEMSpinner42Â°

424242424343430.5

12470.5120

02080

1000

60100Complete

Fischermedium42

4242424243430.5

12370.510

55295

100100100

B. C. Giovanella, W. A. Lohman, and C. Heidelberger

Table 2oâ€”o OTâ€¢.-â€”â€”â€”..3OMIN

fr A IHR The conditions required to achieve a 3. to 4-log killof L1210 leukemic

frâ€”.â€”_-'2 HR cells by hyperthermia alone

o..â€”-a 4HR Ten thousand cells were injected i.p. into groups of 10 to 20 female.â€”â€”-. 81* BDFi mice after treatment in vitro. The survival was determined 40

Ã¸â€”_..â€”-@I3 HR days after inoculation. For this table, 540 mice were used.

S

@00

50

0

Chart 4. The percentage of surviving mice as a function of timeafter i.p. injection of 1 X i0@ L1210 cells that had been incubated at41 in complete MEM Spinner medium for the lengths of timeindicated. Ten mice were used for each line.

medium (MEM Spinner) in which the Ll2iO cells survive butdo not reproduce, we chose to investigate their heatsensitivity in Fischer medium, in which these cells can becultured in shaker flasks.

Li210 leukemia cells exposed to elevated temperatures incomplete Fischer medium have a greater sensitivity to heatthen those in complete MEM Spinner medium. Charts 5 and6 show 2 identical experiments, except that in 1 thesuspending medium was Fischer (Chart 6) and in the other itwas MEM Spinner (Chart 5). A striking difference betweenthe 2 complete media was found. As shown in Chart 5,inoculation of mice with cells that had been subjected to 2hr of exposure to 42Â°led to a survival of 20%, but in Chart6 (Fischer) the survival was 80% at 42 . Whereas Charts 5and 6 illustrate the results of individual experiments, Table 2lists the average percentages of surviving mice, according tothe length of exposure to 42Â° and 43Â° of the cells withwhich they were inoculated. It is evident from this table thatat both temperatures there was a significant difference in thesensitivity to heat, depending on the medium in which the

DAYS

@100%survivors = less than 1 viable cell present more than 4-logkill; 60 to 80% survivors 1 viable cell present 4-log kill; 20 to 40%survivors= 10 viablecellspresent 3-logkill.

Chart 5. The percentage of surviving mice as a function of timeaft,@ri.p. injection of 1 X i0@L1210 cells that had been incubated at42 in complete MEM Spinner medium for the lengths of timeindicated. Ten mice were used for each line.

DAYS

Chart 6. The percentage of surviving mice as a function of timeafter i.p. injection of 1 X i0@ Li 210 cells that had been incubated at42Â°in complete Fischer medium for the lengths of time indicated.Ten mice were used for each line.

leukemic cells were suspended. In MEM Spinner 2 hr at 43Â°was as lethal as 0.5 hr in Fischer medium, and 4 hr at 42Â° inMEM Spinner give fewer surviving mice than did 3 hr inFischer. In Table 2, the killing effect of heat was estimatedfrom the number of mice surviving for more than 40 days.From the data in Table i , it is evident that the injection of1 live cell into groups of mice gave approximately 70%survivors, and the injection of 10 cells gave about 30%.Taking into account some variability from experiment toexperiment, we can safely assume that if, in a group of micegiven injections of L1210 leukemia cells, 90 to 100% of theanimals survive, less than 1 live cell per animal was presentin the inoculum. If an average of 60 to 80% survive, 1 livecell has been injected. If 20 to 40% survive, the live cellsinjected were 10 per mouse. If the inoculum contains 1 X




Stage I (1ff)

Â°42Stage

II (Kr)

Â°40. Survivors(%)1

110.510

12440

6080

010040Â°42Â°4

440

120

00


10@cells , these percentages correspond , respectively , to morethan a 4-log kill (90 to 100% survivors), to a 4-log kill (60to 80% survivors), and to a 3-log kill (20 to 40% survivors).

Chart 7 summarizes the results obtained when L1210 cellssuspended in Fischer medium were heated at varioustemperatures. The amount of cell killing was calculated fromChart 2 at the 50% survival time of the mice inoculated withthe treated cells. These results are in good agreement withthose in Table@ 2 and 5 , where the leukemic cell kill wascalculated from the number of survivors at 40 days. In ouropinion, the latter is more accurate, but also requires manymore animals. The only significant discrepancy betweenthese 2 methods is found in the 43Â° curve, at which

Chart 7. The survival of L1210 cells calculated according to theplot of Chart 2. Each point corresponds to a group of 10 mice giveninjections of 1 X i0'@ cells which had been incubated in completeFischer medium at the temperatures and for the lengths of timeindicated. The total number of mice used was 240.

temperature an exposure of 70 mm produced a 4-log kill(Chart 7). In Table 2, 30 mm at 43Â° was sufficient toachieve the same effect. This discrepancy may be explainedby the fact that, when the period of incubation becomes tooshort, exact quantitative analysis becomes more difficult. Asshown in Chart 7 , temperatures of 41 Â°or less are largelyineffective. Nevertheless, we have observed that the lethaleffect of such temperatures became considerable if theleukemic cells were treated sequentially, first with a shortperiod of incubation of 42Â°and then followed by incubationat 40Â° (Table 3). This result was not observed when thesequence of treatments was reversed.

Table 4 summarizes the results of the combination ofhyperthermia and some compounds. At the concentrationsand under the experimental conditions used, DHBA,DL-glyceraldehyde, actinomycin D, PAM, and sodiumoxamate exhibited some lethality in combination with heat.Vinblastine, FUDR, and 2-deoxy-D-glucose did not kill thesecells, whether applied alone or in association with elevatedtemperatures. With the exception of PAM and vinblastine,which were tested only at the 2 levels listed in Table 4, all

Table 3

The difference in lethal effect causedby reversingthe sequence ofexposure ofLl2lO leukemia cells in cgmplete Fischermedium to 40

and 42The cell suspension was incubated first as in Sta@eI and immediately

afterwards as in Stage II. After treatment, 1 X 10 cells were injectedi_p. into groups of 20 mice. The number of survivors was determined at40 days after inoculation. For this table, 300 mice were used.

the other chemicals were tested at various concentrations fordifferent lengths of time. This was done in order todetermine the minimum concentration capable of giving100% survival after incubation at 37Â°. Once this concentration had been established, it was tested for shorter periodsof incubation at 42Â°. We also tested lower concentrations ofthe same drug for various times at 42Â°.

Having discovered that DHBA has considerable lethalactivity when combined with heat, we wished to determinewhether there is a synergistic relationship between theseagents. We then titrated the lethal effects of differentdosages of DHBA and heat applied separately and concomitantly (Table 5). This was done essentially by the samemethod used in Table 2 , with the difference that in theexperiments summarized in Table 5 variable inocula wereused instead of a fixed one (1 X i0@ cells), as in Table 2. Inthis way it was possible to detect and determine quantitatively the lethal effects of less than 3 logs on L1210leukemia cells, because as shown in Table 2 , it was possibleto measure a minimum kill of 3 logs when 1 X i0@ cellswere injected. Similarly, a 2-log kill could be measured byinjection of 1 X 10@ cells, and so on. We found (Table 5)that 30 mm at 42 in combmation with 0.1 M DHBAresulted in more than a 4-log kill, whereas the sum of thelethal effects of both treatments administered separately wasless than 2 logs. From these results, we have established thata synergism of action exists between DHBA and heat, whichincreases the killing effect on L12 10 leukemia cells by afactor of about 100.

DISCUSSION

The results reported here clearly demonstrate that thelethal effects of heat on L1210 leukemia in ascites form canbe effectively determined quantitatively by measuring thenumber of cells viable after heat treatment by inoculation

HOURS

JUNE 1970 1627



DrugConcentration (M)TemperatureTime of exposure (hi)Survivors(%)Actinomycin

D2 X 10@8 [email protected] X @37Â°

3737421

211100

1000

100PAM3.3X10@

[email protected] 10@637

37421

11100

0100DL-Glyceral

dehyde2.2 X [email protected] X i0@337

37428

11100

0100DHBA6

X i0@31x10_31x10_31x10_31 X @37

373741421

5120.5100

1000

100100

B. C. Giovanella, W. A. Lohman, and C. Heidelbe,ger

Table 4

The results obtained by combined treatment ofLl2lO leukemia cells suspendedin complete Fischer medium with hyperthermia and drugs

Groups of 10 to 20 female BDFi mice were used for each determination. Ten thousand cells wereinjected i.p. after treatment. The number of survivorswas determined at 40 days after inoculation.

(weight/mi incubation medium to weight/kg body weight)only the first 3 drugs could be considered effective incombination with heat (Table 6). Stehlin (41) hasdemonstrated enhanced effectiveness of PAM againstmalignant melanomas of the limbs when the drug is usedclinically in heated regional perfusions, at doses andtemperatures of the same order of magnitude as in theseexperiments. Sodium oxamate is also quite effective incombination with heat at doses that inhibit 90% of theglycolytic activity of neoplastic cells. This is not surprising inview of the fact that heat strongly inhibits respiration intumor cells (7, 29, 30). Thus, the simultaneous applicationof heat and sodium oxamate deprives the leukemic cells ofboth their sources of energy. Unfortunately, the concentration of sodium oxamate required to produce this effect isso high that this observation has no practical value. However,these results indicate that it might be worthwhile to searchfor more efficient glycolytic inhibitors to use in combinationwith hyperthermia.

Of the chemicals that we have tested, only DHBA producesa synergistic lethality in combination with heat. This drug isalso the least toxic for man, as is shown in Table 6.DL-Glyceraldehyde would be as effective as DHBA if thedosages reported by von Ardenne (1) have really been usedin humans. Unfortunately, it is impossible to tell from hispaper whether those doses are purely theoretical or havebeen tested successfully in man.

The treatment of Ll210 leukemia cells with simultaneoushigh temperature and DHBA is 100-fold more effective thaneach applied separately. We are presently investigating themechanism of this synergism, which is potentially veryimportant in future clinical applications. The use of drugswith low toxicity that exhibit synergism in combination withsequential heating (first at high temperatures for a short

into mice and observing their survival. This can be done byutilizing the 50% survival curve (Chart 2), or, more exactly,by calculating the percentages of long-term survivors afterinoculation of different numbers of treated cells (Table 5).We have seen that the relationship between temperatureincrease and lethality is not a linear one. With the periods ofheatmg used, the killing effect becomes evident between 41and 42Â°. At lower temperatures, this effect is minimal ornonexistent. The medium in which the cells are suspendedhas a significant effect on their heat sensitivity. Hyperthermia has a greater lethal effect on cells suspended incomplete Fischer medium than those in MEM Spinner. Inthe Fischer medium, Ll2 10 leukemic cells can be cultured,whereas in MEM Spinner, they are unable to multiply. Thisdifference suggests that some of the synthetic processesrelated to cell division are particularly sensitive to heatinjury. Such a conclusion is in good agreement with ourbiochemical studies (16, 17) and with those of Mondovi etaL (28, 29), which have shown that DNA synthesis isselectively inhibited by heat.

We have found that the effectiveness of hyperthermia of alesser degree is increased when applied after a short burst ofhigher temperature, but the reverse is not true. We do nothave an explanation for this phenomenon, but its implications relative to clinical uses of heat for cancer therapydeserve further consideration.

It has been shown that several chemicals have an additivelethal effect with heat on L1210 leukemia cells. It isnecessary, however, to consider their toxicity in evaluatingthese results in view of potential clinical applications.Accordingly, the drugs tested can be placed in the followingorder: DHBA, DL-glyceraldehyde, PAM, actinomycin D, andsodium oxamate . If the concentrations used in our experiments are extrapolated to clinically effective doses




AConcentration

(M)%

Survivors1

X 10â€•cells 1 X i0@ cells1 X 102 cells1 X [email protected]

0.20.40.60.81.00

00 00 0

100 100100 100100 1000

3020

1001001000

5590

100100

100BTime

ofheating(Kr)%

Survivors1

X@ cells 1 X i0@cells1 X 102 cells1 x 10'cells0.5

130

105 45

1006010090100CTime

ofheating (hi)Concentration (M)No. of cells%survivors0.5

10.1 0.11X i0@

1X104100 100

DrugExperimental doseÂ°ClinicaldosebReferenceDHBA8

g20â€”30 g(E. Ciaranfi, personalcommunication)DL-Glyceraldehyde16

g48â€”72 g?(1)PAM80mg25mg(45)Actinomycin

D32 mg8mg(33)Sodiumoxamate880 gNot determined


Table 5

The synergisticeffect ofDHBA and heat on L1210 leukemia cells incubatedin complete Fischer medium

The percentages of survivors are shown as a function of the number of cells inoculated. The number ofsurvivors was determined at 40 days after inoculation. For this table, 800 mice were used.

In Section A L1210 leukemia cells were treated with 6 different concentrations of DHBA andincubated at 37b for I hr. In Section B, L1210 leukemia cellswere incubated at 42Â°for different lengthsof time without DHBA. In Section C, L1210 leukemia cells were heated at 42Â°in combination with 0.1 MDHBA.

Table 6

A comparison between maximum clinical doses and the experimental doserequired to kill 1 X 104 L1210 leukemia cells in combination with

I hr ofexposure to heatingat 42Â°

aExp@iment@ dose quantity by weight of the drug required to kill more than99.99% of L1210 leukemia cells present at a concentration of 1 X 105/mi in 80 litersFischer medium in combination with 1 Krof heating at 42Â°.

bÃ§jfr.j@j dose maximum daily dose tolerated by an 80-kg man (i.v. injection orinfusion). Data are taken from the literature.

JUNE 1970 1629



B. C. Giovanella, W. A. Lohman, and C. HeideTherger

period of time, followed by a much longer exposure to alower degree of hyperthermia) is, in our opinion, the mostpromising way of increasing the effectiveness of whole-bodyheat therapy for disseminated human cancers.

ACKNOWLEDGMENTS

We gratefully acknowledge the skilled technical assistanceof Mrs.Lois Griesbach and Miss Joyce Liegel.

REFERENCES

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2. Ardenne, M. von. Syncarcinokolyse in Gestalt der MehrschrittChemotherapie. In: Aktuelle Probleme aus dem Gebiet derCancerologie, pp. 128â€”143.Berlin: Springer-Verlag, 1966.

3. Ardenne, M. von. Theoretische und Experimentelle Grundlagender Krebs-MeKrscKritte-Therapie,pp. 1â€”365.Berlin: Veb VerlagVolk and Gesundheit, 1967.

4. Ardenne, M. von, and KrUger, W. Messungen zur IrreversiblenScEÃ¡digung der Atmung von Krebszellen durch Extremhyperthermie. Z. Naturforsch., 21b: 836â€”840, 1966.

5 _ Ardenne, M. von, and Reitnauer, P. G. In Vitro Thermosensibilisierungvon EKrlichM@Ã¡use-Ascites-Krebszellendurch VitaminK3 and K5. Z. Naturforsch., 22b: 422â€”435,1967.

6. Bender, E., and Schramm, T. Untersuchungen zur ThermosensibiliiÃ¤tvon Tumor- und Normalzellen in Vitro. Acta Biol.Med. Ger., 17: 527â€”543,1966.

7. Cavaliere, R., Ciocatto, E., Giovanella, B. C., Heidelberger, C.,Johnson, R., Margottini, M., Mondovi, B., Moricca, G., and RossiFanelli, A. Selective Heat Sensitivity of Cancer Cells. Cancer, 20:1351â€”1381,1967.

8. Cavallere, R., Giovanella, B. C., Margottini, M., Mondovi, B.,Moricca, G., and Rossi-Fanelli, A. Sensibilita Selettiva ddeCellule Neoplastiche a! Calore. Ricerche Biochimiche edApplicazioni Terapeutiche. Rend. Accad. Naz. Lincei, 42:164â€”176,1967.

9. Chen, T. T., and Heideiberger, C. Quantitative Studies on theMalignant Transformation of Mouse Prostate Cells by Carcinogemc Hydrocarbons in Vitro. Intern. J. Cancer, 4: 166â€”178,1969.

10. Cramer, F. B., and Woodward, G. E. 2-Desoxy-D-glucose as anAntagonist of Glucose in Yeast Fermentation. J. Franklin Inst.,253: 354â€”360,1952.

11. Crathorn, A. R., and Hunter, G. D. The Effect of NitrogenMustards on the Incorporation of Amino Acids into Protein byStaphylococcus aureus. Biochem. J., 67: 37â€”41,1957.

12. Crile, G., Jr. Selective Destruction of Cancers after Exposure toHeat. Ann. Surg., 156: 404â€”407, 1962.

13. Dixon, G. J., Dulmadge, E. A., and Schabel, F. M., Jr. GrowthRequirements and Biological Characteristics of Leukemia L1210Cells in Culture. Cancer Chemotherapy Rept., 50: 247â€”254,1966.

14. Fischer, G. A., and Sartorelli, A. C. Development, Maintenanceand Assay of Drug Resistance. Methods Med. Res., 10: 247â€”262,1964.

15. Friedgood, H. B. On the Thermal Death Point of Sarcoma andNormal Mononuclear Cells (Walker Rat Tumor). Arch. Exptl.Zellforsch., 7: 243â€”248, 1928.

16. Giovanella, B. C, and Heidelberger, C. Biochemical and BiologicalEffects of Heat on Normal and Neoplastic Cells. Proc. Am.Assoc. Cancer Res., 9: 24, 1968.

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JUNE 1970 1631



1970;30:1623-1631. Cancer Res Beppino C. Giovanella, Wendy A. Lohman and Charles Heidelberger L1210 Leukemia CellsEffects of Elevated Temperatures and Drugs on the Viability of

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