The Synergistic Anti-Neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine Or @.-F1uorouracip

CANCER RESEARCH

VOLUME 25 OCTOBER 1965 NUMBER 9

The Synergistic Anti-neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine or @.-F1uorouraciP

ALAN C. SARTORELLI AND BARBARA A. BOOTH Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut

SUMI@1ARY Mitomycin C or porfiromycin (methyl mitomycin) in combination with either 6- thioguanine or 5-fluorouracil produced synergistic inhibition of the growth of both Sar coma 180 and Lymphoma L1210 ascites cell neoplasms. i\Iitomycin C and porfiromy cm possessed the same therapeutic index in the L1210 lymphoma system, but mitomycin C was about 5 times more potent. Doses of mitomycin C that induced pro nounced inhibition of thymidine-3H incorporation into deoxyribonucleic acid (DNA) did not decrease the average DNA content of treated cells. The degree of inhibition of ribonucleic acid (RNA) synthesis by mitomycin C was less pronounced than that of DNA, and lysine-1-'4C fixation into residual protein was insensitive to the antibiotic. 6-Thioguanine induced a marked depression in the rate of incorporation of thymidine 3H and orotic acid-6-'4C into DNA and RNA, respectively. The combination of these agents produced subadditive to additive inhibition of the formation of nucleic acids and also caused inhibition of protein synthesis; incorporation of thymidine into DNA ap peared to be the metabolic path most sensitive to the combination of these agents. Measurement of thymidine kinase and thymidylate kinase activity in cells treated with these agents indicated that these enzymes were relatively resistant to the action of mi tomycin C ; however, some inhibition of thymidylate kinase activity was induced by the thioguanine treatment. It is hypothesized that these combinations inhibit cell reproduction by a complementary process involving DNA, the mitomycins acting to cross-link existing DNA molecules, thereby interfering with primer functions, and the anti-metabolites providing a more complete blockade of the synthesis of DNA by in hibition of specific enzymatic sites on the biosynthetic route.

In previous reports from this laboratory (29, 30) it was cells (29). That the metabolic attack was indeed directed suggested that synergistic drug combinations might be against RNA was supported by the finding that the 2 drugs selected rationally by combining agents that bind and in in combination caused a subadditive depletion of the total activate cellular informational molecules (i.e., DNA and cellular RNA content ; presumably some fraction of RNA RNA) or enzymes with drugs capable of altering biosyn was decreased to a level which became limiting for cell thetic pathways leading to the formation of the damaged survival. polymers. This mechanism, termed â€œcomplementary in This rationale was extended further with DNA as the hibition,â€• was illustrated by the demonstration that corn â€œtargetsiteâ€•(31). Mitomycin C or porfiromycin (methyl binations of ribonuclease and aetinomycin D produced mitomycin) was used to attack specifically the pre-existing synergistic inhibition of the growth of Sarcoma 180 ascites molecules of DNA, because these antibiotics have been 1 This investigation was supported by USPHS Research Grant indicated to function by cross-linking complementary CA-02817 from the National Cancer Institute and by Grants ACS strands of the DNA double helix and by inducing a de IN-31E-806 and ACS IN-31F-905 from the American Cancer Society. polymerization of the DNA itself (39). The mitomycins Received for publication February 10, 1965. were combined with either 6-thioguanine or 5-fluorouracil, 1393

anti-met abolites that, following activation , possess the ving 50 days and the @)ercentage of regressions of tumor potential to retard DNA synthesis presumably at the nu growth were unequivocally greater than the sum of these cleotide level. This approach gains support from the effects produced by each drug alone. All mice surviving finding that cytosine arabinoside, an agent that also in over 50 days were calculated as 50-day survivors in deter hibits DNA formation (6), caused synergistic inhibition of mination of the average survival time. neopla.stic cell growth in combination with porfiromycin Drug-induced metabolic effecls.â€”Anirnals bearing 6-day (8). The data in this report describe the synergistic growths of Sarcoma 180 a.scites cells were each given a growth-inhibitory effects exhibited by combinations of the single i.p. dose of either mitornycin C, 6-thioguanine, or a mitornycins with 6-thioguanine or 5-fluorouracil on tratis combination of the 2 drugs. At selected time intervals planted rodent neopla.srns and provide evidence supporting thereafter, either 200 @gof thyniidine methyl-3H (5.4 X the hypothesis that these combinations may function to 10@c))ni/@Lg), 100 @gof orotic acid-6-'4C hydrate (2.1 X destroy these cells by a complementary process that in 10@cJ)fli/@ug), or 150 @hgof DL-lysine-1-'4C monohydrate volves the nucleic acids. (2.2 X 10@cpm/@g)were administered by i.p. injection to each mouse and were allowed 1 hr for metabolic utilization. MATERIALS AND METHODS In experiments involving the incorporation of thymidine Tumor lransplantalion.â€”Experi metits were performed 3H and orotic acid-6-'4C into nucleic acids, sodium nude on 9- to 11-week-old female Ha/ICR Swiss mice (A. R. ates were isolated by the method of Tyner ci al. (41) and Schmidt Company, Madison, Wisconsin) bearing Sarcoma hydrolyzed with 70 % perchloric acid for 1.5 hr (22). Ex @@ 180 a.scites cells, male C57BL x DBA F1 (hereafter tracts were desalted on charcoal columns, and the desired called BDF1) Ii1iC(@(Cumberland View Farms, Cumber pyrirnidine components of the nucleic acids were purified land, Tennessee) bearing L1210 lymphorna cells. Trans and analyzed as described by Danneberg et al. (7). After plantation of flâ‚¬@O))[email protected] out by withdrawing exposure of cells to lysine-1-'4C, residual protein was iso peritoneal fluid from a donor mouse bearing a 7-day tumor lated and analyzed as previously described (3). Radio growth. The suspension was centrifuged for 2 miii activity was measured with a Packard Tri-Carb liquid (1600 x g), the supernatant peritoneal fluid was decanted, scintillation spectrometer in all cases except those in which a 10-fold dilution with isotonic saline was made, and 0.1 the radioactivity of residual protein was measured; in the ml of the resulting cell suspension was injected i.p. into latter a Nuclear-Chicago gas flow counter equipped with a each aninial. Microniil window was employed. The PhosPhor solution Drug 1herapy.â€”F@orany one experiment, animals were used in the liquid scintillation counting contained 100 mg distributed into groups of 5â€”10mice of comparable weight of p-bis [2-(5-phenyloxazolyl)] benzene and 8 gm of 2,5- and maintained throughout the course of the experiment diphenyloxazole dissolved in 2 liters of toluene and 1 liter on Purina laboratory chow and water ad libiluin. Ex of absolute ethanol. periments with more thati 5 mice were performed at least The DNA and RNA content of the cells was measured 2 times, and the results obtained were averaged. by pentose analyses (33) in which deoxyadenosine and Drugs were administered by i.p. injection once daily for adenosine, respectively, were the standards. Cell volumes 6 consecutive days beginning 24 hr after tumor implanta were measured in hematocrit tubes by determining the tion, and combination treatments were administered simul volume occupied by a known number of cells. The cell taneously. 1\Iitomycin C, porfiromycin, and 5-fluorouracil number was determined with a Coulter particle counter, were administered iii solution in isotonic saline, whereas model A. 6-thioguanine was solubilized in isotonic saline with sodium Enzyme extracts were prepared from 6-day growths of hydroxide and adjusted to pH 7â€”8with hydrochloric acid. Sarcoma 180 ascites cells ; all procedures were carried out Solutions of mitornycin C and porfiromycin were freshly at 4Â°C. Cells were treated for 3 miii with 4 volumes of prepared every 3â€”fldays; during this time the antibiotics 0.2 % sodium chloride to lyse erythrocytes ; subsequently, were protected from light and were stored at 4Â°C. All an equal volume of 1.6 % sodium chloride was added and drugs were administered in volumes of 0.25â€”0.5ml. Con the cells were collected by centrifugation at 1000 X g. Af trols given injections of comparable volumes of saline were ter 1 wash with isotonic saline, the cells were suspended in included in each experiment. Mice were weighed during 4 volumes of 0.05 Mtris (hydroxymethyl) aminomethane the course of the experiment, and the percentage of change buffer, 1)11 7.5, containing 0.05 Mmole of thymidine/mi. in body weight from onset to termination of therapy was The cell suspension was disrupted with a Branson sonifier used as an indication of drug toxicity. at 8 amp. by one 30-sec burst; tubes containing the cell Evaluation with a.scitic iieoplasnis of combinations of suspensions were immersed in a methanol-ice bath during drugs as synergists could not be based solely on the pro sonication. The sonicates were centrifuged at 105,000 X longation of the survival time afforded by drug treatments, @1for 1 hr, and the supernatant fraction was collected. The since therapy which Produces a significant number of re protein content of the extracts was determined by the bi gressions of tumor growth (complete eradication of neo uret method (18) with twice recrystallized bovine albumin plastic cells) results in difficulty in expressing meaningful as the standard. Thymidine kina.se, thymidylate kinase, average survival times. Therefore, 2 additional criteria and thymidylate nucleotidase activities were measured by the number of animals surviving 50 days and the number incubating the following mixture at 37Â°C: either thymi of regressions of tumor growthâ€”also were used to assess dine-3H, 0.1 @mole(2.9 X 10@cpm) or thymidylate-2-'4C, the effectiveness of drug treatments. Drugs were con 0.1 @mole (8.0 X 1O@cpm) ; adenosine triphosphate, 5.5 sidered synergists when the percentage of animals survi @moles; magnesium chloride, 5.5 @moles; tris(hydroxy

TABLE 1 EFFECT OF COMBINATIONS OF MITOMYcIN C AND INHIBITORS [email protected] 1.8 OF NUCLEIC ACID BIOSYNTHESIS ON THE SURVIVAL TIME OF MICE BEARING SARCOMA 180 ASCITES wz @o 1.6 CELLS

[email protected](mg/kg)Â° @ Av. Av. SURVIVAL No. OF No. or 1.4 CHANGE IN (days)Â± SE. 50-DAY >> WT.b (%) SURVIVORSC @ENS@c Antimetabolitemycin Mito C (0(1) >@ 1.2 None 0 +25.4 13.1 Â±0.4 0/50 0/50 0.1 +33.5 18.4 Â± 1.0 0/10 0/10 0.2 +6.5 23.2 Â± 2.4 1/15 1/15 0.4 +2.4 24.6 Â± 1.3 1/30 1/30 POR F I R 0 MYC I N 4 8 12 16 0.6 +0.4 30.1 Â± 1.9 4/29 2/29 MITOMYCIN C 0.8 1.6 2.4 3.2 0.8 +0.9 34.0 Â± 3.8 3/10 2/10 mg/kg 1.0 +0.1 40.9 Â± 2.5 5/15 3/15 1.5 â€”5.0 17.5 Â± 1.4 0/10 0/10 CHART 1.â€”Comparative effects of porfiromycin arid initomycin 2.0 â€”9.6 11.6 Â±0.9 0/5 0/5 C on the survival time of mice bearing L1210 lymphoma. Drugs 6-Thioguanine were administered daily at the indicated dosage for 6 consecutive days, beginning 24 hr after tumor implantation. Each figure 0.5 0 +32.2 15.9 Â± 1.1 0/10 0/10 represents the mean value of results obtained from 5â€”20mice. 1.0 21.7 Â± 1.3 0 +21.4 0/10 0/10 Porfiromycin, 0â€”0; mitomycin C, Sâ€”S. 0.5 0.2 +9.2 31.9 Â± 3.9 2/10 2/10 0.5 0.4 +1.4 36.9 Â±2.3 7/15 4/15 1.0 0.2 +4.7 37.6 Â±4.2 5/10 4/10 lose (exchange capacity approximately 0.5 niEq/gm). 1.0 0.4 +1.2 41.7 Â± 2.7 6/15 5/15 Thymidine was eluted with water (70 ml when thymi 1.0 0.6 â€”2.3 45.7 Â± 2.6 7/10 4/10 dine-3H [email protected] and 50 ml when thymidylate-'4C 1.0 0.8 â€”4.1 40.2 Â± 3.5 4/10 2/10 was the substrate) ; thymidine nionophosphate with 0.01 5-Fluorouracil N HC1 (50 ml) when thymidine-3H was the substrate @ 5 0 +11.2 17.3 0.5 0/15 0/15 and with 0.02 N HC1 (70 ird) when thymidylate-'4C was 10 0 +3.9 21.6 Â± 1.4 0/15 0/15 the substrate ; thymidine diphosphate with 50 ml of 20 0 â€”5.0 24.2 Â± 1.0 0/15 0/15 0.05 N HC1; and thymidine triphosphate with 50 ml of 30 0 â€”7.3 18.8 Â±4.0 0/5 0/5 0.5 N HC1. The concentration of thyrnidine or thymi 5 0.2 +2.3 28.0 Â±5.7 1/5 0/5 dine nucleotide in each fraction was determined by meas 5 0.4 0.0 37.4 Â±3.0 2/10 0/10 5 0.6 +3.5 46.6 Â±2.0 7/10 3/10 urement of the radioactivity therein. 10 0.4 +0.4 42.9 Â± 2.6 8/15 4/15 RESULTS 10 0.6 â€”2.0 38.2 Â±4.0 5/10 2/10 Uracil mustard The effects of niitomycin C, alone and in combination 0.25 0 +0.8 25.5 Â± 1.9 1/15 1/15 with some inhibitors of nucleic acid biosynthesis, on the 0.25 0.1 â€”1.2 32.5 Â±3.7 2/10 1/10 survival time of mice bearing Sarcoma 180 a.scites cells is 0.25 0.2 â€”6.6 32.0 Â±2.9 1/10 1/10 shown in Table 1. Mitomycin C caused pronounced in 0.25 0.4 â€”6.7 40.7 Â± 2.9 4/10 3/10 hibition of this neoplasm; with the maximum effective 0.25 0.6 â€”4.4 42.1 Â±2.8 4/10 3/10 dose, 1.0 mg/kg daily, 33 % of the tumor-bearing animals survived 50 days. By previously described criteria (32), a Administered once daily for 6 consecutive days, beginning 20 % of these mice were found to be tumor free. The op 24 hr after tumor implantation; combined treatments were given simultaneously. timum effective doses of 6-thioguanine in this neoplastic b From onset to termination of drug treatment. cell line are 1.0â€”2.0mg/kg of body weight (4). However,

C Mice surviving more than 50 days and tumor-free animals such dose levels of thioguanine cause only a prolongation were calculated as 50-day survivors in determination of the of survival time, and no 50-day survivors are produced. average survival time. Similarly, 5-fluorouracil did not prolong life for 50 days; maximum anti-neopla.stic activity, expressed as an ap methyl)aminomethane, 37.5 @moles (pH 7.5) ; and enzyme proximate doubling of the survival time, occurred at a extract (0.25 ml containing approximately 2 mg of pro daily level of 20 mg/kg of body weight. Several combina tein) ; total volume, 1.3 ml. Reaction vessels containing tions of mitomycin C with either thioguanine or fluorou thymidine-3H as substrate were incubated for 30 mm, and racil possessed tumor-inhibitory properties greater than those containing thymidylate-2-'4C were incubated for 20 those equivalent to the sum of the maximally effective mm ; during these periods reaction rates were linear. Re levels of the individual drugs. The most effective therapy actions were terminated by the addition of 0.13 ml of 40 % with the combination of mitomycin C and thioguanine re perchloric acid, precipitated protein was removed by cen quired 0.6 mg/kg and 1.0 mg/kg of each agent, respec trifugation, and the collected supernatant solutions were tively ; 70 % of the tumor-bearing animals receiving this neutralized to 1)H 7 with potassium hydroxide. After re regimen survived 50 days, and 40 % of the treated animals moval by centrifugation of precipitated potassium per were found to be tumor free. A dose of 0.6 mg of mito chlorate, supernatant solutions were subjected to chroma mycin C/kg and 5 mg of fluorouracil/kg was ol)timal for tography oil 1.4 X 9 cm columns of ECTEOLA-cellu this combination ; in this instance, 70 % of the treated sar

theseEFFECT TABLE 2coma-bearing animals survived 50 days, and 30 % of OF COMBINATIONS OF PORFIROMYCIN ANDmice were tumor free. Toxicity, as measured by a de 6-THIOGUANINEresultsBEARING ON THE SURVIVAL TIME OF MICEcrease in body weight, was minimal. Thus, the L1210 LYMPHOMA CELLS that therapeutic synergism (10) was produced by combinations of mitomycin C with either thioguanine or DAILY DOSAGE (mg/kg/i A@. CHANGE Av. SURVIVAL (days) No. 50-DAY fluorouracil. Therapy with a combination of mitomycin IN WT. b (â€˜Jr) Â±SE. SURVIVORSCindicate @ 6-Thjo [5-bis(2-chloroethyl)aminouraciljcausedand uracil mustard Porfiromycin guanine tumor0/45 40 % 50-day survivors, of which 30 % were ; optimum therapeutic levels of uracil mustard alone 0 0 +13.1 7.2 Â±0.1 are capable of producing 20 % 50-day survivors and 20% 1.0 0 + 10.3 8.4 Â±0.4 0/5 tumor-free animals (4). 2.0 0 +14.6 8.3 Â± 0.2 0/15 4.0 0 +7.0 9.5 Â± 0.4 0/20 Since Wagner and Gitterman (42) reported that with 6.0 0 +5.2 10.7 Â±0.8 0/10 human adenocarcinoma cells (HAd 1) grown on the cho 8.0 0 +0.9 11.3 Â± 0.5 0/20freeporfiromycin0/10appearedrioallantoic membrane of embryonated eggs 10 0 â€”9.6 10.5 Â± 0.6 to have twice the therapeutic potency of mito 12 0 â€”8.6 9.8 Â± 0.6 therapeutic0/100/10mycin C, it was of interest to compare the 14 0 â€”7.8 8.6 Â± 0.5 of these 2 agents in the present system. The re 0 0.5 + 14.9 10.3 Â± 0.5 0/10 sults in Chart 1 show that porfiromycin and mitomycin C 0 1.0 +3.0 19.8 Â±5.8 0/20 possess the same therapeutic index when employed in mice +2.6 13.2 Â±0.2 0 2.0 bearing the L1210 lymphoma; in this system mitomycin 0 4.0 â€”7.6 12.9 Â± 0.2 @gC is approximately 5 times more potent than porfiromycin. 1.0 0.5 +14.2 15.5 Â± 3.4 0/10 2.0 0.5 +11.6 16.4 Â± 2.9 0/9 That porfiromycin is able to synergize effectively with 4.0 0.5 +5.4 25.4 Â± 4.9 1/10 thioguanine is shown in Table 2. Greater than additive 1.0 1.0 +3.4 23.8 Â±3.6 optimum5/25effective1/10efficacyeffects were produced by such combinations; the 2.0 1.0 +6.6 28.4 Â±2.7 porfiromycin/0/15kg therapeutic regimen was 2.0 mg of 4.0 1.0 0.0 18.7 Â±2.4 tumor-bearing0/10and 1.0 mg of thioguanine/kg daily. Of 6.0 1.0 â€”2.9 16.5 Â± 0.8 treated with these doses of the 2 drugs, 20 % sur vived for at least 50 days, while in contrast neither drug a Administered once daily for 6 consecutive days, beginning 24 possessed the ability to prolong life for 50 days when used hr after tumor implantation; combined treatments were given Simultaneously. alone. Several investigators have reported that the mitomycins b From onset to termination of drug treatment.

C Mice surviving more than 50 days were calculated as 50-day induce a depolymerization and a partial loss of the DNA survivors in determination of the average survival time.animals of exposed cells (9, 13, 15â€”17,24, 27, 28, 34, 36â€”38); how

TABLE 3

THE EFFECT OF MITOMYCIN C AND 6-THIOGUANINE ON THE CELLULAR CONTENT OF DNA AND RNA Mice bearing 6-day implants of Sarcoma 180 ascites cells received a single i.p. dose of either mito mycin C, 6-thioguanine, or a combination of the 2 drugs administered simultaneously. At selected time intervals thereafter, the average DNA and RNA content and the average volume of the cells were determined. Each figure represents the mean value (Â±S.E.) of results obtained from the sepa rate analyses of cells from 3 to 35 mice.

after deoxyribose ribose (mpmoles/ volume 10')None30.8TreatmentDose (mg/kg)Time drug (hr)DNA (mpmoles/i0' cells)RNA 10' cells)Cell (cu es/cell X

0.1Mitomycin Â±0.9202.2 Â±9.62.93 Â±

C0.5 Â±3.0 Â± 16.9 Â± 1.2 1.0 12 36.2 Â± 3.7 213.1 Â± 21.4 6.68 Â± 1.8 2.0 6 31.7 Â± 1.5 2.0 12 32.1 Â± 3.9 224.4Â± 12.5 5.19 Â± 1.0 6.0 6 35.8 Â± 1.8 213.3 Â± 19.8 3.03 Â± 0.1 6.0 12 37.9 Â± 1.9 196.4 Â± 9.6 4.74 E 0.3 6.0 24 37.6 Â±1.3 183.9 Â± 10.3 4.63 Â± 0.3 20 6 27.9 Â± 0.6 140.9 Â± 7.9 3.29 Â±0.2 0.36-Thioguanine1012342012 1231.0 27.5 Â± 2.6164.0 155.4 Â± 13.34.11 5.00 Â±

0.3Mitomycin .4 Â±0 .9194 .2 Â± 11.33 .64 Â±

1.26-thioguanineC +6 +101242.5 Â± 4 .2221 .3 Â± 5.97 05 Â±

ever, Iyer and Szybalski (13) and Schwartz et al. (34) have to the antibiotic or to thioguanine (Table 4). Thymidyl provided evidence that in microbial cells and in rodent ate nucleotida.se activity was also estimated under these tissues, respectively, the depolymerization of DNA is a conditions. The activity of thyrnidine kina.se was not ef secondary phenomenon. The results shown in Table 3 fected by the addition of 5 X 10@ M mitomycin C to the would corroborate this conclusion, for only a slight de enzyme ; however, exposure of tumor-bearing mice to 2 mg crease (approximately 10 %) in the average DNA content of mitomycin C/kg 5â€”12hr prior to preparation of the en of Sarcoma 180 a.scitescellsoccurred when a massive dose zyme resulted 111an increase in the total enzyme activity of of 20 mg of mitomycin C/kg was employed. A corre the cells. Pretreatment of Sarcoma 180 with thioguanine sponding decrease in the cellular content of RNA was also 12 hr prior to preparation of the enzyme did not alter its obtained under these conditions. When smaller doses of activity. Addition of mitomycin C (5 X 10@ i@i)to thy mitomycin C were administered (1â€”flmg/kg), slight in midylate kinase caused a 15 % inhibition of this enzyme creases 111the DNA content of the cells were observed. In activity ; however, this degree of inhibition could not ac addition, the exposure of mitomycin C-treated cells to count for the pronounced decrease in the rate of incorpora thioguanine caused a further accumulation of DNA. Such tion of thymidine-3H into the DNA of intact cells. A accretions of nucleic acids were accompanied by an increase 12-hr pretreatment of Sarcoma 180 cells with mitoniycin C in the average volume of the treated cells. analogous to that described for the thymidine kinase also At the dose level of 1.0 mg of mitomycin C/kg, pro caused an elevation in thymidylate kinase activity. This nounced inhibition of thymidine-3H incorporation into occurred without alteration of the thymidylate nucleotid DNA thymine occurred (Chart 2). 1\Iarked inhibition of ase activity of the preparation. Conversely, thioguanine DNA synthesis, which appeared to be relieved almost fully pretreatment of the neoplastic cells caused a decrease in 15 hr after the drug, also was produced by 10 mg of thio the rate of phosphorylation of thymidylate and a pro guanine/kg. The simultaneous exposure of the cells to nounced increase in the activity of thymidylate nucleo mitomycin C and thioguanine caused a greater decrease in tidase. the specific activity of DNA thymine than was produced The effects of these drugs on the formation of RNA was by either drug alone. To determine whether the inhibi estimated by determining alterations iii the rate of incor tion of DNA synthesis induced by mitomycin C is mani poration of orotic acid-6-'4C into the uracil of RNA (Chart fest at the level of the enzymes that phosphorylate thy 3). I\Iitomycin C produced considerably less inhibition of midine, thyniidine kinase and thymidylate kinase activities RNA formation than of the synthesis of DNA ; however, were assayed in extracts prepared from cells exposed either thioguanine caused a similar degree of inhibition of both RNA and DNA formation. Combination of the 2 drugs produced subadditive to additive inhibition of RNA for mation, depending upon the time after the drugs at which RNA synthesis was measured. Table 5 showsthe relative insensitivity of I)rotein synthesis to these agents, as meas

oJ ured by lysine-1-â€•C incorporation into residual protein. 0 â€˜C TABLE 4 4, TIIYMIDINE KINASE, THYMIDYLATE KINASE, AND THYMIDYLATE 0 E NUCLEOTIDASE ACTIVITY OF SARCOMA 180 CELLS TREATED WITH EITHER MITOMYCIN C OR E 6-THIOGUANINE 0. U Six-day growths of Sarcoma 180 ascites cells from untreated mice or from those treated i.p. with either 2 mg of mitomycin C/kg or 10 mg of 6-thioguanine/kg were used as the enzyme source; the figure given in parentheses indicates the time after the drug at which the enzyme was prepared. Each value represents HOURS the mean value of 2â€”4determinations.

CHART 2.â€”Incorporation of thymidine-'H into the deoxyribo KINASE ACTIVITY NUCLEO nucleic acid (DNA) of Sarcoma 180ascites cells exposed to a corn LINASE DRUG TREATMENT PRIOR TO TIDASE bination of mitomycin C and 6-thioguanine. Mice bearing 6-day ACTIVITY ENZYME PREPARATIONTHYICIDINE (rn5i moles! ACTIVITY implants of Sarcoma 180 ascites cells received a single intra msmoles/ m@anoles/ (m5moles/ hr/mg hr/10' hr/mg)ThY'C@DYLATE hr/mg)Untreated peritoneal dose of either 1.0 mg of mitomycin C/kg, 10 mg of protein cellsTRYMIDYLATE 6-thioguanine/kg, or a combination of the 2 drugs administered @ simultaneously. At selected time intervals thereafter, 200 of thymidine-'H (5.4 X 10' cpm/jsg) were administered intra peritoneally to each mouse and 1 hr was allowed for metabolic Untreated + 5 X 10@ M 11.9 23.4 60.2 8.2 utilization. The DNA thymine was isolated and its specific mitomycin C (added to radioactivity was determined. Each figure represents the mean flask) value of results obtained from the separate analyses of cells from Mitomycin C (2.5 hr) 11.5 19.8 3â€”38mice. The results obtained with untreated cells are shown Mitomycin C (5 hr) 14.8 30.3 by the solid line (control specific activity, 97 cprn/pmole of DNA Mitomycin C (12 hr) 17.7 39.2 80.5 9.3 thymine x 10â€”'). Mitomycin C, â€¢ â€¢; thioguanine, 6-Thioguanine (12 hr)10.5 10.019.6 19.569.2 50.19.7 15.6 A@â€”-â€”A;mitomycinC + thioguanine,0â€”â€”â€”0.

TABLE 6

TOxIcITY OF A SINGLE DOSE OF MITOMYCIN C AND 2 60 6-THIOGUANINE TO SARCOMA 180 ASCITES CELLS Mice bearing 5-day implants (0 hr) of Sarcoma 180 ascites cells

SC received a single i.p. injection of either mitomycin C, 6-thio 4) 40 guanine, or a combination of the 2 drugs. Cells were removed 0 E quantitatively from the peritoneal cavities at selected intervals after exposure to the drugs, and the number was determined with E 20 a Coulter model A particle counter. Each figure represents the 0. U mean value (Â±S.E.) of results obtained from the separate analyses of cells from 5â€”iSmice.

C- 2.5 5 10 10'MitoDOSE (mg/kg)TOTAL CELLS/MOUSE X HOURS hr0 hr.12 hr15 hr24 CHART 3.â€”Incorporation of orotic acid-6-'@C into the ribo mycin C6â€”Thioguanineo nucleic acid (RNA) of Sarcoma 180 ascites cells exposed to a corn bination of mitomycin C and 6-thioguanine. Experiments were conducted @ti;described for Chart 2, except that 100 jsg of orotic 0 10 353 Â± 2 387 Â± 60 327 Â± 26 acid-6-1@Chydrate (2.1 X 104 cpm/@sg)were administered to each 2 0 332 Â± 19 276 Â± 13 297 Â± 23 mouse, and the specific radioactivity of RNA uracil was deter 6 0 266Â±47 180Â±52 mined. Eachfigure represents the mean value of results obtained 2 10 308Â±20 261Â±22 225Â±33 from the separate analyses of cells from 3â€”24mice. The results 10290Â±17330Â±9 obtained with untreated cells are shown by the solid line (control 60 128Â±37340Â±1583Â±24397Â±28 specific activity, 58 cpm/@tmole of RNA uracil X l02). Mito mycin C, â€¢â€”S; thioguanine, Aâ€” - â€”A; mitomycin C + thioguanine, 0â€”â€”â€”0. in the peritoneal cavity, mitomycin C was carcinolytic; following an injection of 6 mg of mitomycin C/kg the cell TABLE 5 number decreased with time. Addition of thioguanine to PROTEIN SYNTHESIS IN SARCOMA 180 ASCITES CELLS this level of mitomycin caused a faster rate of cell death TREATED WITH MITOMYCIN C AND 6-THIOGUANINE than did mitomycin C alone, indicating that drug syner Mice bearing 6-day implants of Sarcoma 180 ascites cells gism was also produced under these experimental condi received a single i.p. dose of either 1 mg of mitomycin C/kg, 10 tions. mg of 6-thioguanine/kg, or a combination of the 2 drugs ad DISCUSSION ministered simultaneously. Twelve hours later 150 @sgof DL lysine-1-'4C monohydrate (2.2 X 10@cpm/@sg)were administered The biochemical effects produced by the mitomycins are i.p. to each mouse, and incorporation was permitted for 1 hr. directed primarily towards DNA metabolism ; inhibition of Each figure represents the mean value (Â±S.E.) of results obtained synthesis appears to be the primary biochemical event (13, from the separatemice.Treatmentcpm/mg analyses of cells from 4 34, 39). Such metabolic insult in some systems is aceom panied or followed by a depolymerization of the DNA itself 102None protein X (9, 13, 15â€”17,24, 27, 28, 34, 36â€”38). The inhibition of DNA biosynthesis by these antibiotics may be due to in Â± 3.2 Mitomycin C 38.5 Â± 5.2 terference with the primer function of DNA in the DNA 6-Thioguanine 37.9 Â± 6.3 polymerase reaction, since the mitomycins have been Mitomycin C + 6-thioguanine37.8 28.9 Â± 3.6 shown to cross-link the complementary strands of the DNA double helix (13, 14, 20, 21, 23, 39, 43). In support of this concept, isolation of DNA from mitomycin-treated Twelve hr after the administration of either mitomycin C cells and subsequent testing of the DNA for primer activ or thioguanine, flO inhibitory effects were produced. In ity in polymerase reactions have shown inhibition of combination, however, at doses which cause profound in primer activity in both the DNA and the RNA polym hibition of nucleic acid formation, a decrease of approxi erase systems (25, 26). However, in contrast, it has mately 24 % in the specific activity of proteins was induced also been reported that DNA isolated from niitomycin by the agents. This differential sensitivity of nucleic acid treated cells showed no decreased ability to prime the and protein synthesis to these agents implies that the pro DNA polymerase reaction (40). No explanation is nounced inhibition of the formation of DNA was not the available for these conflicting results ; whether these result merely of cell death. differences are due to the degree of binding of the drug, To ascertain whether cell death was produced by a single the physical state of the DNA, or other factors cannot exposure of the neoplastic population to these agents, be decided from data now available. That inhibition of mice bearing 5-day implants of Sarcoma 180 ascites cells DNA synthesis by the mitomycins is not due to a direct were treated 1.1).with a single dose of the various agents blockade of the enzymes that phosphorylate thymidine under conditions analogous to the biochemical studies, and and thymidylate or of the DNA polymerase enzyme has the iiurnber of surviving cells was measured at intervals been shown in this report and by other workers (1, 12, thereafter by quantitatively collecting and determining 24, 35, 40). Thus, although inhibition may conceivably the cell number. The data in Table 6 show that, whereas be the result of an additional enzymatic block that limits thioguanine did not markedly decrease the number of cells the availability of an essential metabolite for DNA forma

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. SARTORELLI AND BooTHâ€”Synergistic Activity of Mitomycins and Antimetabolites 1399 tion it appears likely that the mitomycins act in sensitive sis under the proper conditions. The apparent inhibition cells by a direct binding of the DNA and that this process of synthesis, as measured by thymidine-3H incorporation yields primer molecules with some decrease in function. into the thymine of DNA at 12 hr after treatment of cells The slight accumulation of DNA in Sarcoma 180 cells with the mitomycins, must, however, to some extent rep treated with relatively low doses of mitomycin C is accom resent dilution at the polymer level; but the magnitude of panied by some cell death and therefore suggests that de the inhibition of thymidine-3H utilization for DNA for polymerization of DNA is not the primary toxic event in mation caused by mitomycin C cannot be completely ex these cells. It is possible, however, that depolymerization plained by metabolic dilution at the polymer level. has occurred and that low molecular weight fragments of Therefore, we have tentatively assumed that the subaddi DNA have been retained. Although the rate of synthesis tive depression of isotopic thymidine incorporation into of DNA, as measured by thymidine-3H uptake, is mark DNA that is induced by combinations of mitomycin C and edly depressed by mitomycin C, the slightly greater cellu thioguanine truly represents a profound depression in the lar content of DNA suggests that inhibition of cell division rate of synthesis of DNA. Since lesser inhibition of pro occurs in cells with some synthetic activity. In agreement tein and RNA biosynthesis occurs, a metabolic imbalance with this concept, 1\Iagee and Miller (21) reported that in that results in the death of the neoplastic cells may thus HeLa cells in culture, cell division was more sensitive to be created. low concentrations of mitomycin C or porfiromycin than ACKNOWLEDGMENTS was the synthesis of DNA. The role of the increased ac The authors are indebted to Miss Sheila J. Feld and Miss Judith tivity of both thymidine and thymidylate kinases, as well Albaum, who carried out certain aspects of the work; to Dr. as the increase in DNA polymerase activity reported for Harry B. Wood, Jr., and Dr. Howard W. Bond, Cancer Chemo other mammalian cells treated with the mitomycins (1), therapy National Service Center, and to the Kyowa Hakko Kogyo in the slight accretion of DNA remains to be elucidated. Company, Ltd., Tokyo, Japan, for mitomycin C; to Dr. Robert The synergistic anti-neoplastic activity of combinations M. Smith, the Upjohn Company, for porfiromycin; and to Dr. Dorris J. Hutchison, Sloan-Kettering Institute for Cancer Re of the mitomycins with either 6-thioguanine, 5-fluorouradil, search, for a transplant of the L1210 lymphoma. or cytosine arabinoside (8) may be the result of different biochemical modes of action, or the anti-metabolites, by REFERENCES virtue of distinct enzymatic blocks in the same metabolic 1. Bach, M. K., and Magee, W. E. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. The Synergistic Anti-neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine or 5-Fluorouracil

Alan C. Sartorelli and Barbara A. Booth

Cancer Res 1965;25:1393-1400.

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