[CANCER RESEARCH 36, 631-639, February 1976]

Treatment of Spontaneous Leukemia in AKR Mice with Chemotherapy, Immunotherapy, or Interferon'

J. George B e k e s i , '~ Julia P. Roboz, E u g e n e Z i m m e r m a n , and J a m e s F. Hol land

Department of Neoplastic Diseases, Mount Sinai School of Medicine and Hospital of the City University of New York, New York, New York 10029 [J. G. B., J. P. R., J. F. H.], and Litton Bionetics, Kensington, Maryland 20795 [E. Z.]

Summary

AKR mice are genetically destined to develop Gross (RNA) v i rus- induced lymphatic leukemia. Leukemic AKR mice treated with combinat ion vincristine, cyclophospha- mide (Cytoxan), and 1 - ( 2 - c h l o r o e t h y l ) - 3 - ( t r a n s - 4 - m e t h y l c y -

clohexyl ) - l -n i t rosourea sustained a 180% increase of life- span. Combina t ion chemotherapy plus immunizat ion with neuraminidase-treated allogeneic (Gross virus-induced) G.~G leukemic cells intradermally resulted in 35% of animals surviving beyond 150 days wi thout evidence of the disease. It is s igni f icant that allogeneic E2G leukemic cells as im- munogen were as effective in prolonging the life-span of the immunized leukemic AKR mice as were syngeneic leukemic thymocytes.

Vwazole (1 -/3- D- r ibofuranosyl- 1,2,4-tr iazole-3- carbox- amide), an antiviral compound, alone showed no appar- ent an t i tumor effect. However, in experiments in which the clinically d iagnosed leukemic AKR mice received a combina- tion of cytoreduct ive therapy [vincristine plus prednisone or, more effect ively, vincristine, Cytoxan plus 1-(2-chloro- e t h y l ) - 3 - ( t r a n s - 4 - m e t h y l c y c l o h e x y l ) - l - n i t r o s o u r e a , fol lowed by Virazole], there was a noticeable reduction of the viral titer, a delay in the reappearance of viable clonogenic cells, and an increase in the survival t ime for the leukemic AKR mice as compared to those receiving cytoreductive therapy alone.

The effectiveness of purified mouse interferon in AKR mice was a lso examined. The decrease in the viral titer of animals that received interferon treatment was markedly greater than of those receiving a combinat ion of cytoreduc- tive therapy wi th Virazole or immunotherapy. The adminis- tration of mouse interferon had a direct effect on the appear- ance of the spontaneous leukemia in AKR mice. The median life-span of the control animals was 36 weeks, whereas 45% of the AKR mice treated with five doses of 5 • 104 units of interferon are still alive at 54 weeks of age. Thus, interferon not only reduces the Gross murine leukemia virus titer in the chronica l ly infected AKR mice but also signif icantly delays the appearance of the primary lymphoma.

Introduction

Increased immunogenic i ty of transplantable (8, 33, 34) and autochthonous MuLV 3 (Gross) and methylcholan-

' Presented at the symposium "Immunological Control of Virus-associated Tumors in Man: Prospects and Problems," April 7 to 9, 1975, Bethesda, Md. This work was supported by the National Cancer Institute Cancer Virus Program, Contract 1 CP 43225.

2 Presenter.

threne-induced) tumors after treatment with Vib r i o c h o l e r a e

neuraminidase has been demonstrated in syngeneic mice, result ing in immunospeci f ic rejection of untreated synge- neic tumors (2, 3, 29, 30, 40, 42). Mult iple injections of neuraminidase-treated leukemic L1210 cells reduced the lethality rate and increased the survival of mice with estab- lished leukemia L1210 tumor grafts (36, 37). Immunizat ion with neuraminidase-treated syngeneic L1210 cells fo l lowing cytoreductive therapy in DBA/2 mice with L1210 tumor re- sulted in cure of 25 to 55% of treated animals, while less than 10% of mice treated with chemotherapy alone survived free of disease (2-4). The immunoprotect ion evoked by neuraminidase-treated cells was found to be specif ic for the tumor used for immunizat ion (2-6, 29, 30, 36, 37). These experiments led to good understanding of the factors that determine the opt imal condi t ions for therapy in a rapidly growing neoplasm in highly inbred animals. Recently, con- siderable attention has been directed to the study of the response to combined modali ty therapy of spontaneous leukemia of viral or igin (26-28, 35, 41-43, 45). In many respects spontaneous leukemic AKR mice have characteris- tics similar to those of humans with leukemias. It is well documented that the vertically transmitted MuLV is the etio- logical agent for lymphoma in AKR mice (17, 19, 21-23, 25).

This work shows the effectiveness of cytoreductive drugs in combinat ion with specif ic immunotherapy with neuramin- idase-treated syngeneic and al logeneic leukemia cells in AKR mice. The effectiveness of preventive immunotherapy in MuLV-infected AKR mice as well as the therapeut ic value of Virazole in combinat ion with chemotherapy was tested. The suppressive effects of purif ied mouse interferon on the expression of Gross (RNA) leukemia virus and the appear- ance of primary lymphoma in AKR mice are currently under investigation.

Mater ia ls and M e t h o d s

Animals. Male DBA/2/Ha mice, 8 to 10 weeks old, were obtained from the West Seneca Laboratory of Roswell Park Memorial Institute, Buffalo, N. Y. Male C57BL and female AKR mice, 7 to 8 weeks old, and female retired AKR breed- ers, 5 to 7 months old, were supplied by The Jackson Lab- oratories, Bar Harbor, Maine. The mice were kept in a room

a The abbreviations used are: MuLV, murine leukemia virus; i.d., intrader- mally; palmO-ara-C, 1-/3-o-arabinofurahosylcytosine 5'-palmitate; methyl- CCNU, 1-(2-chloroethyl)-3-(trans-4-cyclohexyl)- l -nitrosourea.

FEBRUARY 1 976 631

J. G. Bekesi et al.

thermostat ical ly control led at 20-22 ~ and al lowed Purina chow (breeders) and tap water ad l ibi tum.

Experimental Tumors. Leukemia L1210 tumor cells have been maintained as highly virulent leukemia by weekly i.p. passage in DBA/2 mice. Gross virus-induced E2G leu- kemia was maintained by weekly i.v. passage of 0.5 to 1 x 108 viable leukemic cells in syngeneic C57BL mice. Sponta- neous leukemic AKR mice were selected from a colony of 3500 to 4000 female retired breeders. Clinical diagnosis of spontaneous leukemia in the AKR mice was made with 95% accuracy by splenic and lymph node palpat ion, fol lowed by leukocyte counts. Animals were considered to be leukemic when their leukocyte counts were greater than 17,000/cu mm and they had splenic and lymph node enlargement. At the t ime of diagnosis, the average splenic weight was 470 mg, the thymus weight was 650 mg, and the average leuko- cyte count was 28,000/cu mm.

Purification of V. cholerae Neuraminidase. The enzyme is purif ied as described by Ada et al. (1) using V. cholerae f i l trate (obtained from Sigma Chemical Co., St. Louis, Mo.) as a source of crude enzyme preparation. The enzyme showed maximum activity at pH 5.6 with dif ferent substrates and was free of other proteolyt ic activity including neurami- noaldolase. One unit of neuraminidase activity is defined as the amount of enzyme that hydrolyzes 1/~g of N-acetylneura- minic acid from N-acetylneuraminosyl lactose or orosomu- coid for 15 min at 37 ~ .

Preparation of Tumor Cells for Neuraminidase Treat- ment. Leukemia L1210 cells were harvested from DBA/2 mice 6 to 7 days after the animals were inoculated with 10 '~ or 108 leukemic cells i.p. The tumor cells were freed from contaminat ing RBC by a 5-sec osmot ic shock treatment. Tumor cell preparat ions were consistently free of erythro- cytes, and 98% of the leukemic cell populat ion excluded trypan blue. E2G leukemic cells were obtained from the spleens of C57BL mice 7 days after transplantat ion of 1,000,- 000 leukemic cells i.v. The spleens were removed and placed immediately in a plastic Petri dish containing Ros- well Park Memorial Institute Medium 1640 supplemented with 20% heat-deactivated fetal bovine sera. The edges of the spleens were split at regular spacings and gently squeezed between blunt forceps to force the contents into the surrounding media. Cell suspensions were drawn through a 20-gauge needle in the plastic syringe to produce a single-cell suspension. This was fi ltered through a double layer of gauze to remove any aggregates of cells. Cells were washed twice with Roswell Park Memorial Institute Medium 1640. The separation of viable from nonviable tumor cells was achieved by f lotat ion on bovine serum albumin as de- scribed by Wepsic (46). Viable single-cell suspensions from normal spleens or thymus and AKR lymphoma were achieved by the method described for the preparation of E2G leukemic cells. The final viabil ity of leukemic cells was 92 to 98% as determined by the trypan blue exclusion method.

Incubation of Normal and Leukemic Cells with Neura- minidase. Normal and leukemic cells were incubated at 37 ~ in sodium acetate buffer (0.05 M sodium acetate, 0.154 M sodium chloride, and 0.005 M calcium chloride) at pH 5.6 to 6 in the presence of 25 to 35 units of neuraminidase per 2.5 x 107 cells per ml each. In certain experiments, tumor cells

were incubated with neuraminidase in the presence of mito- mycin C (25/~g/108 cells). At the terminat ion of incubation the sil iconized reaction flask was removed, cooled rapidly, and spun at 800 to 1000 x g for 55 sec in a cl inical centri- fuge. Cells thus incubated were washed twice with 50 vol- umes of 0.9% NaCI solut ion containing 0.005 M EDTA and used for immunizat ion experiments. Supernatant and wash- ings were combined and used for the quant i tat ion of neuraminidase-susceptible N-acetylneuraminic acid.

Prophylactic Therapy of MuLV-infected AKR Mice with Neuraminidase-treated Syngeneic and AIIogeneic Leuke- mia Cells. Eight-week-old female AKR mice were random- ized into 6 groups of 40 mice each. Group 1 received 0.9% NaCI solut ion injections every 15 days for a total of 9 times. Group 2 received 2 x 107 neuraminidase-treated leu- kemia L1210 cells per injection i.d. Group 3 received 2 x 107 neuraminidase-treated allogeneic E2G cells per injection i.d. Group 4 received 2 x 107 neuraminidase-treated normal AKR thymocytes per injection. Group 5 received 2 • 107 neuraminidase-treated leukemic AKR thymocytes i.p. while animals in Group 6 were immunized i.d. wi th the same number of neuraminidase-treated normal thymocytes per injection. The mice were checked daily for mortal i ty until the termination of the experiment (450 days after the date of their birth). The thymus and spleens were removed from mice that died, and the weights were recorded . . . . .

Production of Mouse Interferon. Interferon was pro- duced at 20,000 units/ml in C-243 mouse tissue culture line infected with Newcastle disease virus. Subsequently, the interferon was concentrated to approximately 1 x 108 units/ml. The concentrated mouse interferon preparation then was subjected to column chromatographic purif ication on a Sephadex G-200 column. The fractions representing the interferon were pooled and rechromatographed on a Sephadex G-100 column. With this simple gel fi ltration method, a relatively pure interferon preparat ion ('[ x 107 interferon units/mg of protein) was obtained.

Randomization of AKR Mice into Interferon Experiment. Three-month-old female AKR mice were randomized into 5 equal groups of 20 animals each. Group 1 served as the control and received at the time of randomizat ion and 2, 4, 6, and 8 days later 1 ml of 0.9% NaCI solution i.p. Group 2 received at the t ime of randomization and 2, 4, 6, and 8 days later 1 ml of 10% fetal bovine sera i.p. Group 3 received 5 x 10 '~' units of interferon per injection i.p. at the t ime of ran- domizat ion and 2, 4, 6, and 8 clays later Group 4 received 5 x 104 units of interferon per injection i.p. at randomizat ion and 2, 4, 6, and 8 days later and Group 5 received 5 x 10 ,~ units of interferon per rill per injection i.p. at randomizat ion and 2, 4, 6, and 8 days later.

The MuLV titer in the experimental animals was deter- mined by obtaining a 2% extract of a short sect ion (4 to 6 mm) of mouse tails from 10 individual animals in each group. The samples were obtained prior to the treatment, at the time of randomizat ion, dur ing the treatment (on Days 4, 6, and 8), and at posttreatment periods (10, 30, and 85 days). Each tail specimen was individually assayed by the XC focus-forming assay as described by Rowe et al. (32). The MuLV titer is expressed per ml of 2% extract of a 4- to 6- mm section of AKR mouse tail.

Mice were checked for mortality daily. All animals were

632 CANCER RESEARCH VOL. 36

Various Treatments o f Spontaneous Leukemia in AKR Mice

Table 1 Change of MuLV titer in AKR mice after chemotherapy or chemotherapy +

immunotherapy

Virus titer"

During treatment (drug or drug + immu- nization)

Pre- Post- treatment Day Day Day Day Day treatment

Experimental groups (Day 0) 4 8 13 17 21 (Day 45)

Untreated leu kem ic 1260 1317 1 0 6 0 1 5 2 0 1290 1620 AKR mice

Chemotherapy + 0.9% 1310 1020 890 756 1100 960 NaCI solution

Chemotherapy + immu- 950 680 420 notherapy

1390

385

MuLV titer is expressed per 0.2 ml of 2% extract of a short section (4 to 6 ram) of mouse tails obtained from animals at days indicated, using XC focus assay.

Table 2 Change of MuLV in AKR mice after chemotherapy or chemotherapy + Virazole

Virus titer ~

During treatment Pre- Post-

treatment Day Day Day Day Day treatment Experimental groups (Day 0) 4 8 13 17 21 (Day 45)

Untreated leukemic AKR mice

Chemotherapy + 0:9% NaCI solution

Chemotherapy + Vira- zole

1260

1120 1020 890 975 1100 960 1310

780 640 550 410

a MuLV titer is expressed per 0.2 ml of 2% extract of a short section (4 to 6 mm) of mouse tails at days indicated using XC focus assay.

autopsied. Spleens, lymph nodes, and thymus of dead ani- mals were removed, and the weights were recorded.

Combined Antiviral Chemoimmunotherapy in AKR Mice with Spontaneous Leukemia, The studies of the effective- ness of antiviral therapy alone or in combinat ion with chem- otherapy with or wi thout immunotherapy using neuramini- dase-treated al logeneic E2G leukemic cells in AKR mice with spontaneous lymphoma were carried out after positive con- f irmation of the disease (palpation and WBC count).

In the 1st experiment, diagnosed mice were treated with vincristine, 0.75 mg/kg on Day 1; palmO-ara-C, 125 mg/kg on Day 3; methyI-CCNU, 25 mg/kg on Day 7. On Day 9 they were randomized into the fo l lowing groups: chemotherapy alone; chemotherapy fol lowed by immunizat ion at mult iple sites with 2 x 107 neuraminidase-treated allogeneic E2G leukemic cells i.d.; and chemotherapy plus immunizat ion with 2 • 10 ~ X-irradiated (5000 R) al logeneic E2G leukemic cells i.d. at the times indicated.

In the 2nd experiment, AKR mice were treated with vincris- tine, 0.75 mg/kg on Day 1; or combinat ion therapy with vincristine, 0.75 mg/kg on Day 1, and prednisone on Days I , 2, 3, and 4; or with vincristine, 0.75 mg/kg on Day 1, Cy- toxan, 125 mg/kg on Day 3, and methyI-CCNU, 25 mg/kg on Day 7. Two days after the termination of chemotherapy, the animals were randomized into the fo l lowing experimental

groups: chemotherapy alone, chemotherapy fo l lowed by treatment with Virazole, either 150 or 200 mg/kg i.p. at t imes indicated. In each experimental group the MuLV titer was determined at the t ime of clinical diagnosis, dur ing the treatment and posttreatment periods as indicated in Tables 1 and 2 using the XC focus-forming assay (32).

Results

Prophylactic Therapy of MuLV-infected AKR Mice with Neuraminldase-treated Syngeneic and AIIogeneic Leu- kemic Cells. The total covatently bound N-acety lneuraminic acid per 108 cells is: leukemia L1210, 0.11 /~mole; E2G leuke- mia, 0.135 /~mole; normal or leukemic AKR thymocytes, 0.020 to 0.025 /~mole. Upon incubat ion of normal or leu- kemic cells under opt imal condit ions, about 65 to 70% of N- acetylneuraminic acid was removed by neuraminidase.

Optimal neuraminidase concentrat ion for E2G, L1210, and AKR leukemic thymocytes was establ ished at 25 to 35 units/2.5 x 107 tumor cells/ml of media. This enzyme con- centration represents about 15- to 30-fold excess of the number of neuraminidase-susceptible N-acety lneuraminic acid molecules available on the L1210 or E2G or leukemic thymocytes. After incubat ion with neuraminidase 75 to 85%

FEBRUARY 1976 633

J. G. B e k e s i e t al .

of the t reated cel ls exc luded t rypan blue but cou ld not in i t ia te t u m o r g rowth in a syngene ic host.

The ef f icacy of us ing a l logene ic leukemia L1210 and M u L V - i n d u c e d E~G leukemia cel ls t reated wi th neuramin i - dase as i m m u n o g e n as p rophy lac t i c t rea tmen t of AKR mice was compared to the data ob ta ined wi th neuramin idase- t reated syngene ic normal or leukemic t hymocy tes f rom AKR mice. It is s ign i f i can t tha t bo th neuramin idase- t rea ted synge- neic leukemic AKR thymocy tes (Chart 1) and the a l logene ic E=G leukemic cel ls (Chart 2) were equal ly ef fect ive in delay- ing the appearance of pr imary l ymphoma in AKR mice (p < 0.05). Immun iza t i on carr ied out wi th neuramin idase- t rea ted L1 210 leukemic cel ls or neuramin idase- t rea ted normal AKR thymocy tes i.d. was inef fec tua l , and the t reated mice d ied of leukemia at abou t the same rate as did the con t ro l g roup . Th is c lear ly es tab l ishes the i m m u n o p r o p h y l a c t i c and thera- peut ic value of neuramin idase- t rea ted M u L V - i n d u c e d spon- t aneous and a l logene ic leukemic cells.

Immunotherapy in Spontaneous Leukemic AKR Mice after Cytoreductive Therapy. Wi thou t cy to reduc t i ve ther- apy 50% of AKR mice d ied by 14 days af ter d iagnos is of s p o n t a n e o u s leukemia , 90% died by 33 days, and 96% died by 56 days. I m m u n o t h e r a p y a lone us ing neuramin idase- t reated s p o n t a n e o u s leukemic thymocy tes or neuramin i - dase- t reated a l logene ic E~G leukemic cel ls i.p. w i t hou t pr ior t r ea tmen t wi th cy to reduc t i ve the rapy did not change the survival pat tern of leukemic AKR mice (Chart 3).

Good remiss ion i nduc t i on was achieved wi th the fo l low- ing d rug c o m b i n a t i o n : v incr is t ine on Day 1, p lus palmO-ara- C or Cytoxan on Day 3, p lus methyI -CCNU on Day 7. Signi f i - can t reduc t ion in the t hymus and spleen we ights was ob- served after chemo the rapy . Exp lan ta t i on of t hymus and spleen of an imals in the c h e m o t h e r a p y g roup to young n o n l e u k e m i c AKR mice fai led to show ev idence of viable neop las t i c cel ls before Day 11 for spleen and before Day 21 for thymus.

Leukemic AKR mice that received comb ina t i on therapy us ing v incr is t ine, pa lmO-ara-C, and methyI -CCNU susta ined an increased l i fe-span of abou t 180%, but less than 7% of the mice surv ived beyond 100 days (Chart 4). These an imals then died of l y m p h o m a as a resul t of r e induc t i on of a sec- onda ry l y m p h o m a where the spleen rather than thymus was

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l llll l __ I l l I I

O 70 IOO 200 300 400 LIFE[SPAN IN DAYS

Chart 1. Effect of immunotherapy with neuraminidase (N'ase)-treated nor- mal and leukemic thymocytes on the appearance of primary leukemia in AKR mice, Mice were immunized at days indicated with 2 x 10 r cells with normal thymocytes (El), leukemic thymocytes injected i.p. (O), leukemic thymocytes injected i.d. (O), Control-Saline, Control-0.9% NaCl.

I00

bJ > --I

W u 50

�9 o o o

~~ ~ o o ~

o o ~176 ~

o oo ~ o o ~ - E2G leukem,c cells (, d )

~ oo C .. . . . . . solme(id } ~IZ10% O O 000 0

o X o o o ~ Leukern,o ~

~ o ~ LI210 (id) ~176 o

X o o ~

I I I l l I [ l i ~ 1 7 6 o { ; ; ; ; ; ; ; ; -, ,~

llll I I l __ I I

0 0 I00 200 300 400 LIFESPAN IN DAYS

Chart 2. Effect of preimmunization with neuraminidase-treated alloge- neic E=G or L1210 leukemic cells on the appearance of spontaneous leuke- mia in AKR mice. Mice were immunized at days indicated with 2 x 107 cells with E2G leukemic cells i.d. (0), or leukemia L1210 cells i.d. (D). Control- Saline, Control-0.9% NaCI.

IR,_ IR, ]~ Je,. J~ i~

i 0 0 ~ ' ~ _>

t~ ' ~ 5 �9

; -%

i- t

�9 20 40 60 80 POSTD,AGNOS,S ,N DAYS

Chart 3. Life-span of AKR mice with spontaneous leukemia after immu- notherapy with neuraminidase-treated spontaneous leukemic thymocytes or allogeneic E2G leukemic cells. Mice were immunized at multiple sites each time with 2 x 107 neuraminidase-treated leukemic cells i.d. at days indicated by the arrows. Immunization with E2G leukemia (O) or spontaneous leukemic thymocytes (�9 saline (0.9% NaCI) control, - -

I00 -

> / " '~176 o

ILl ~eo~ o O 5 0 ~ . "" �9 o o o Chemolher0py+ .

\ ,, . . . . o ~ ~9 ee o o o o E2G leukem,c 0fief N ose

-~ �9 �9 o o o b_ O i o o

I \ " , ' . ooo o . . . . o o o o . . . . o "-~__ �9 �9 �9 o �9

~. �9 Chem01her0py "~ - ~ .. ._., e_ ~ E 2 G leukemtc cell

~ Chem0fher0py ~ " - " �9 �9

l?m:n,tol,on (,, d) i ~ . ~ _ l ~ " _ _ . e e�9 e l

o i ~ ~,I ~, ~ , ~ " ' f - ' ~ , , , t 7"'# , , t t t 50 ,00 ,50

~,~z% POST-DIAGNOSIS IN DAYS Chart 4. Immunization of spontaneous leukemic AKR mice with neuramin-

idase (N'ase)-treated allogeneic E=G leukemic cells after vincristine, palmO- ara-C and methyI-CCNU treatment. AKR mice with spontaneous leukemia received, on Day 0, vincristine, 0.75 mg/kg; on Day 3, palmO-ara-C, 130 mg/kg ; and on Day 7, methyI-CCNU, 25 mg/kg. Animals were randomized into 3 equal groups: Group 1, drug control ( - - - ) ; Group 2, immunized i.d. at multiple sites with 2 x 107 X-irradiated E=G leukemia (O); Group 3, immu- nized i.d. at multiple sites with 2 x 107 neuraminidase-treated E=G leukemia (O) at days indicated by arrows on the abscissa. Saline (0.9% NaCI) control,

634 CANCER RESEARCH VOL. 36

the pr imary organ involved in the repopu la t ion of c lono- genic lymphocytes after chemotherapy.

Combinat ion chemotherapy fo l lowed by immun iza t ion with 2 x 107 neuraminidase- t reated a l logeneic E,G leuke- mic cells injected at mul t ip le sites at the days indicated in Chart 4 resulted in the survival of 30% of treated mice free of disease beyond 120 days. Immuniza t ion alone carr ied out wi th 2 x 10 ~ untreated a l logeneic E2G leukemic cells used as an immunogen was ineffectual , and the treated mice died at about the same rate as did the drug contro l g roup (Chart 4).

Using the above exper imenta l pro toco ls we establ ished the relat ionship between the AKR virus titer and the type and cl inical eff icacy of the t reatment. The results presented in Table 1 indicate that cytoreduct ive therapy alone did not s igni f icant ly alter the viral t i ter in the treated AKR mice. Chemotherapy and immunotherapy wi th neuramin idase- treated leukemic thymocytes i.d. p roduced a not iceable decrease in the viral t i ter after the 13th day of the t reatment, and this remained at a lower level at 45 days after the ini t iat ion of the t reatment .

Effect of Virazole in Combination with Cytoreductive Therapy in Leukemic AKR Mice. When Virazole was tested for antiviral activity against MuLV in vitro by the XC focus- forming assay, 80% inhib i t ion of the plaque format ion was achieved wi th as low as 20 /~g Virazole per ml of cul ture medium. For this reason we began to study the effect of Virazole wi th or w i thou t cytoreduct ive therapy in leukemic AKR mice. Virazole in doses as high as 800 mg/kg showed no quant i tat ive or qual i tat ive effect on the lymphocy te func- t ion of the treated animals (WBC, phy tohemagg lu t in in , pokeweed blastogenesis, or thymus-der ived lymphocytes) . Virazole alone showed no apparent an t i t umor effect in cl ini- cally d iagnosed AKR mice, and only a sl ight increase in the survival t ime was observed when used in comb ina t ion wi th vincrist ine. When c l in ical ly d iagnosed AKR mice received combinat ion cytoreduct ive therapy [v incr ist ine (0.75 mg/kg on Day 1), prednisone (30 mg /kg on Days 1, 2, 3, and 4), fo l lowed by Virazole (150 mg /kg on Days 9, 10, 11, and 12)], there was a delay in the reappearance of viable c lonogen ic l ymphoma cells as measured by the exp lantat ion-sp leno- megaly assay and a moderate increase in the survival t ime of these animals as compared to the ones receiving cytore- ductive therapy alone (Chart 5). The most effective com- binat ion was vincr ist ine plus Cytoxan plus methyI-CCNU fo l lowed by 3 doses of Virazole (200 mg/kg) , on Days 9, 10, and 11. The increase of l i fe-span wi th chemotherapy alone was 183%, whi le wi th chemotherapy plus Virazole it was 280% (Chart 6). There was a short delay in the reappearance of the viable c lonogen ic l ymphoma cells as measured by the exp lanta t ion-sp lenomegaly assay as compared to animals that received cytoreduct ive therapy alone (Chart 7).

In order to test whether weekly in ject ions of Virazole could fur ther increase the survival of leukemic mice, 30 AKR mice were first treated with a combina t ion of v incr ist ine plus Cytoxan plus methyI-CCNU fo l lowed by 3 doses of Virazole on Days 9, 10, and 11 fo l lowed by t reatment wi th an addi t ional 200-mg/kg dose of Virazole once a week. Results presented in Chart 6 clearly indicate that after the 7th injec- t ion of Virazole the morta l i ty rate changed and most of the

Various Treatments of Spontaneous Leukemia in AKR Mice

Rondom- IOO izotion

hi >_ -J 50 o - \

X ~ ~ + Virozole o~ " ' - , ~ 1 7 6 f '

Soline control I:~ -/olone ~" ..m~ m o S~ c~176 X R'~ /~ ,. _'~[~,~

K:oo 0 i l I ~ I i I I I I

~ 20 4 0 60 80 IOO

V POSTDIAGNOSIS IN DAYS

Chart 5. Antileukemic effect of vincristine + prednisone and vincristine + preclnisone + Virazole on spontaneous leukemic AKR mice. Rx, vincristine, 0.75 mglkg, on Day 0, and prednisone, 30 mglkg, on Days 0, I, 2, and 3. V, Virazole, 150 mglkg, on Days 9, 10, 11, and 12. Control-Saline, control-0.9% NaCI

Rondorn- I00 'zli'~176

~ J ~ ~

~ ) \ \ k \ < k- %, "~ ~-\',,-- R, ......... ~8 ..... ,

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O | 1~l L I --I o ~ _ , u - ] u __-- H t t2of t hot 6o 8o IOO ~,a,2 R, 3 v v v v v

POSTDIAGNOSIS IN DAYS Chart 6. The effect of vincristine + Cytoxan + methyl-CCNU and vincris-

tine + Cytoxan + methyl-CCNU + Virazole on the life-span of spontaneous leukemic AKR mice. Rx,, vincristine, 0.75 mg/kg; Rx2, Cytoxan, 125 mg/kg; Rx3, methyI-CCNU, 25 mg/kg; V, Virazole, 200 mg/kg; Control-Saline, control- 0.9% NaCI, on Days 9, 10, and 11, while in the 2nd experimental group Virazole was injected on Days 9, 10, 11,18, 25, 32, 39, and 46 after diagnosis.

remain ing treated AKR mice died, p resumab ly because of V i razo le- induced drug tox ic i ty .

The results in Table 2 show that chemo the rapy plus Vira- zole produced a decrease in the viral t i ter ev ident af ter the 13th day of t reatment .

Effectiveness of Purified Mouse Interferon on MuLV Ti- ter and Subsequent Change on the Appearance of Pri- mary Lymphoma in AKR Mice. In the l ight of our part ial success in spon taneous AKR leukemia using c h e m o i m m u - notherapy or ant iviral agents in comb ina t ion w i th cy toreduc- rive therapy, we invest igated the ef fect iveness of pur i f ied exogenous mouse inter feron on the express ion of Gross leukemia virus and the appearance of p r imary l y m p h o m a in AKR mice. The viral t i ter was de te rmined pr io r to and dur ing the t rea tment as wel l as 10, 30, and 85 days after in i t iat ion of t rea tment wi th inter feron. Table 3 summar i zes the results obta ined f rom each exper imenta l g roup . The viral t i ter in exper imenta l g roups that received 0.9% NaCI

FEBRUARY1976 635

J. G. Bekesi et al.

SPONTANEOUS AKR LEUKEMIA 8OO

6OO

=

u') 4 0 0

2 0 0

o z~ V C M V2 ~ o

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t f t t 2 t z'0 3o V C M V2 T I M E , D A Y S

Chart 7. Change of organ weights in leukemic AKR mice after cytoreduc- tire therapy + Virazole. A, mice treated with vincristine + Cytoxan + methyl- CCNU (VCM). O, spleen; A, thymus. B, mice treated with vincristine + Cytoxan + methyI-CCNU + Virazole. (VCM V~). 0, spleen; A, thymus.

I O 0 - -

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_ J

I , I

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20 30 40 50

LIFE SPAN IN WEEKS Chart 8. The effect of exogenous mouse interferon on the appearance of

spontaneous leukemia in AKR mice. Three-month-old female AKR mice were randomized into 5 equal groups of 20 animals each. Control group, 0.9% NaCI solution injection on day of randomization, then 2, 4, 6, and 8 days later (x); Group 1, 5 x I(P units of interferon at randomization, 2, 4, 6, and 8days later (1:3); Group 2, 5 x 10' units of interferon at randomization, 2, 4, 6, and 8 days later (O); Group 3, 5 x 103 units of interferon at randomization, 2, 4, 6, and 8 days later. Open symbols, animals that died of tumor; closed symbols, animals that died of organ atrophy.

solut ion or fetal bovine sera did not change during the course of exper imentat ion. On the other hand, exogenous mouse interferon showed a profound effect on the viral titer. This was part icularly apparent in the group of animals that received 5 x 10 .~ or 5 x 104 units interferon and, to a lesser degree, in animals receiving 5 • 10 '~ units of inter- feron. It is signif icant that the reduction of viral titer from 2000 to less than 100 was not transient, since animals tested 85 days after the init iat ion of t reatment showed no signifi- cant increase as compared to the viral t iter obtained 4 days after starting the interferon therapy.

Similarly, administrat ion of exogenous mouse interferon had a direct effect on the appearance of primary sponta- neous leukemia in AKR mice. The median life-span of AKR

mice receiving 0.9~ NaCI solution or fetal bovine sera after randomization was 34 and 38 weeks, respectively (Chart 8). All the animals in both control groups died of primary lym- phoma which was conf irmed by autopsy. While in Group 3, which received the highest concentrat ion of interferon, the viral t iter (Table 3) was drastically reduced or eliminated, the animals died at the same rate as did the animals receiv- ing 0.9% NaCI solut ion or fetal calf sera injection, but only 6 of 20 animals died of leukemia. The remaining animals showed marked signs of atrophy of the spleen (5 to 10 mg) or thymus. On the other hand, 45% of animals in Group 4, which received 5 injections of 7 x 104 units of interferon, were still alive at 52 weeks of age. In this experimental group, only 2 animals showed any sign of organ atrophy. A, lower concentrat ion of interferon, that is, 7 x 103 units of interferon, also showed beneficial effects, and 25% of ani- mals were still alive at 52 weeks of age. Thus, exogenous mouse interferon used in optimal doses not only reduces the viral titer in the chronical ly infected AKR mice but also signif icantly delays the appearance of primary lymphoma.

D i s c u s s i o n

Immunotherapy alone using neuraminidase-treated spon- taneous leukemic thymocytes or al logeneic E2G leukemic cells i.d. wi thout prior treatment with cytoreductive therapy d id not change the survival pattern of the leukemic AKR mice.

T h e efficacy of using allogeneic MuLV-induced E2G leu- kemic cells as immunogen with or wi thout treatment with neuraminidase was compared to the data obtained using AKR leukemic thymocytes in leukemic AKR mice that were brought to remission with vincristine plus Cytoxan plus methyI-CCNU. As a result of immunizat ion of AKR mice i.d. with neuraminidase-treated E2G leukemic cells, approxi~. mately 30% of the treated leukemic mice survived beyond 160 days wi thout evidence of the disease.

Comparable observations were made on immunoprotec- t ion in young AKR mice. The use of both neuraminidase- treated spontaneous leukemic thymocytes and allogeneic E=G leukemic cells resulted in a signif icant delay of the appearance of primary lymphoma in AKR mice. Immuniza- tion performed with untreated E=G leukemic cells or neura- minidase-treated L1210 tumor cells or normal (AKR) thymo- cytes was ineffectual, and the treated mice died at about the same rate as did the 0.9% NaCI solution control.

It is particularly signif icant that the al logeneic (MuLV- induced) E=G leukemic cells used as immunogen in animals brought into remission with combinat ion chemotherapy was as effective in prolonging the life-span of the leukemic AKR mice as were the syngeneic leukemic thymocytes (2, 3). This system may therefore represent the best model for human leukemia for working out optimal condit ions f o r chemoimmunotherapy using allogeneic leukemic blast cells as immunogen.

Combinat ion drug therapy plus immunotherapy resulted in considerable prolongat ion of the life-span of AKR mice diagnosed with spontaneous lymphoma. Longer observa- tion of the treated mice, however, showed that despite an

636 CANCER RESEARCH VOL. 36

Various Treatments of Spontaneous Leukemia in AKR Mice

Table 3 Change of MuLV titer in AKR mice after treatment with exogenous mouse interferon

Virus titer ~

During treatment Posttreatment ere-

treatment Day Day Day Day Day Day Experimental groups a (Day 0) 4 6 8 10 30 85

Control (0.9% NaCI solu- 2020 2330 1725 2455 2230 1995 2735 tion)

Co ntrol (fetal bovine se- 2245 2030 2000 3850 2645 1655 2100 rum)

Interferon (7 x 105 2010 50 30 135 40 0 5 units/injection)

Interferon (7 x 104 3010 35 190 180 305 85 75 units/injection)

Interferon (7 x 103 2655 85 135 290 800 495 220 units/injection)

a Treatment of AKR mice was performed after randomization by injection of 0.9% NaCI solution or 10% fetal bovine sera, or exogenous mouse interferon i.p. on Days 0, 2, 4, 6, and 8.

b MuLV titer is expressed per ml of 2% extract of a short section (4 to 6 mm) of AKR mouse tails obtained from animals at days indicated, using the XC focus assay.

apparent "ce l l cure" relapse nonetheless fol lows, probably due to viral reinduct ion. For this reason, we studied the effectiveness of Virazole, a broad-spectrum antiviral agent (18, 20, 38, 39, 44, 47). The effectiveness of purif ied mouse interferon was also tested in vivo and in vitro against the MuLV.

Virazole showed antiviral activity against MuLV in vitro as determined by the XC focus-forming assay. A concentra- tion of <10/~g Virazole resulted in 80% inhibi t ion of MuLV replication w i thou t any apparent cytotoxici ty or cell death of the treated mouse embryo cells. Experiments performed in clinically d iagnosed spontaneous leukemic AKR mice with Virazole alone showed no apparent ant i tumor effect. Leu- kemic AKR mice pretreated with combinat ion cytoreductive therapy, e.g. , vincristine plus prednisone fol lowed by 4 courses of Virazole, resulted in a delay of the reappearance of viable c lonogenic lymphoma cells and a moderate in- crease in the life-span for the animals as compared to the ones receiving cytoreductive therapy alone. Better therapeu- tic effectiveness was achieved when a combinat ion of vin- cristine plus Cytoxan plus methyI-CCNU was fol lowed by 3 doses of Virazole. There was a considerable increase in life- span and a delay in the reappearance of the viable clono- genic lymphoma cells as compared to those AKR mice that received cytoreduct ive therapy alone. Virazole inhibited MuLV repl icat ion in vitro as well as in combinat ion with chemotherapy in vivo and produced a decrease of viral titer in the treated AKR mice. Animals that received chemother- apy alone showed no change in their viral titer.

We tested the effects of purified mouse interferon on the expression of MuLV, the etiological agent for spontaneous leukemia in AKR mice. The viral t iter was determined prior, during and 10, 30, and 85 days after the treatment with various dosages of purified mouse interferon. The MuLV titer, as measured by the XC focus-forming assay uti l izing mouse tail extracts, showed no change in experimental groups where the animals received 0.9% NaCI solution or

10% fetal bovine sera. There was a marked decreased in the viral titer of animals that received interferon treatment. This was particularly true in mice that received mult iple injec- t ions of 5 • 10 '~ or 5 • 104 units interferon. It is s igni f icant that the reduction of viral t iter (2000 to <100) was not a transient one, since the animals tested on day 85 showed no signif icant dif ference from their MuLV titer observed only 4 days after the init iat ion of the interferon therapy. The magni- tude of MuLV titer reduct ion in AKR mice achieved by exogenous interferon is far greater than that by the combi- nation of cytoreductive therapy and Virazole with or w i t hou t immunotherapy using syngeneic or al logeneic leukemic cells. Administrat ion of purif ied mouse interferon in chroni- cally infected AKR mice had a direct effect on the appear- ance of the primary spontaneous leukemia. The median life- span of the control groups (0.9% NaCI solut ion or fetal bovine sera) was 36 weeks. All animals in these groups died of primary lymphoma as conf i rmed by autopsy. AKR mice treated with the highest dose of interferon (5 courses of 7 • 10 ~ units of interferon) died at a faster rate than did animals given injections of 0.9% NaCI solut ion or fetal calf sera. However, only 7 of 20 mice died of leukemia; and the remain- ing ones showed marked signs of organ atrophy, became athymic, and had abnormal ly small spleens (5 to 10 mg) at the t ime of death. There were defini te signs of wast ing in these animals. On the other hand, 45% of the AKR mice treated with 5 doses of 5 x 104 units of interferon are stil l alive at 54 weeks of age. In this experimental group only 4 animals of 11 that died showed any sign of organ atrophy. Thus, interferon used under opt imal condi t ions not only reduces the MuLV titer in the chronical ly infected AKR mice but also signif icant ly delays the appearance of primary lym- phoma. Our studies substant iate and further extend obser- vations reported by several laboratories that interferon treat- ment appears to offer a certain degree of protect ion in animals infected with oncogenic viruses, and it appears to inhibit the growth of some type of tumors (9-16, 24, 31).

FEBRUARY1976 637

J. G. B e k e s i e t a l .

I n t e r f e r o n h a s b e e n s h o w n to i n h i b i t t h e m u l t i p l i c a t i o n a n d

t r a n s f o r m a t i o n in v i t r o a n d i n v i vo of m a n y R N A a n d D N A

v i r u s e s (7). O u r o b s e r v a t i o n s b a s e d o n t h e A K R s y s t e m are

n o w b e i n g t r a n s l a t e d in o u r l a b o r a t o r y i n t o v a r i o u s t h e r a -

o e u t i c m o d e l s in v a r i o u s a n i m a l s y s t e m s .

T h e c o m b i n e d m o d a l i t y o f t h e r a p y c l e a r l y o f f e r s a m a j o r

m p r o v e m e n t in t h e c o n t r o l o f s p o n t a n e o u s M u L V - i n d u c e d

e u k e m i a . T h e c o m b i n e d u s e s o f c h e m o t h e r a p y , a n t i v i r a l

a g e n t s a n d i n t e r f e r o n , a n d i m m u n o t h e r a p y d i r e c t e d a g a i n s t

t u m o r c e l l s a n d v i r a l e t i o l o g i c a l a g e n t s h a v e d i r e c t i m p l i c a -

t i o n s f o r h u m a n n e o p l a s t i c d i s e a s e s .

References

1. Ada, G. L., French, E. L., and Lind, P. E. Purification and Properties of Neuraminidase from Vibrio cholerae. J. Gen. Microbiol., 24: 409-421, 1961.

2. Bekesi, J. G., and Holland, J. F. Combined Chemotherapy and Immuno- therapy of Transplantable and Spontaneous Murine Leukemia in DBA/2 and AKR Mice in Investigation and Stimulation of Immunity in Cancer Patients. Recent Results Cancer Res., 47: 357-369, 1974.

3. Bekesi, J. G., Roboz, J. P., and Holland, J. F. Immunotherapy with Neuraminidase-treated Murine Leukemia Cells after Cytoreductive Ther- apy in Leukemic Mice. In: M. A. Chirigos (ed.), Modulation of Host Im- mune Resistance in the Prevention or Treatment of Induced Neoplasias. Natl. Cancer Inst. Monograph, 1975.

4. Bekesi, J. G., Roboz, J. P., Walter, L., and Holland, J. F. Stimulation of Specific Immunity against Cancer by Neuraminidase-treated Tumor Cells. Symposium on Neuraminidase. Behringwerk-Mitt., 55: 309-321, 1974.

5. Bekesi, J. G., St.-Arneault, G., and Holland, J. F. Increase of Leukemia L1210 Immunogenecity by Vibrio cholerae Neuraminidase Treatment. Cancer Res., 31: 2130-2132, 1971.

6. Bekesi, J. G., St.-Arneault, G., Walter, L., and Holland, J. F. Immunogen- icity of Leukemia L1210 Cells after Neuraminidase Treatment. J. Natl. Cancer Inst., 49: 107-118, 1972.

7. Carter, W. A., and DeClercq, E. Viral Infection and Host Defense. Sci- ence, 186: 1172-1178, 1974.

8. Currie, G. A., and Bagshawe, K. D. The Role of Sialic Acid in Antigenic Expression: Further Studies of the Landschutz Ascites Tumor. Brit. J. Cancer, 22: 843-853, 1968.

9. Gresser, I. Antitumor Effects of Interferon. Advan. Cancer Res., 16: 97- 140, 1972.

10. Gresser, I., and Bourali, C. Antitumor Effect of Interferon Preparations in Mice. J. Natl. Cancer Inst., 45: 365-376, 1970.

11. Gresser, I., Bourali, C., Levy, J. P., Fontaine-Brouty-Boye, D., and Thomas, M. P. Increased Survival in Mice Inoculated with Tumor Cells and Treated with Interferon Preparations. Proc. Natl. Acad. Sci. U. S., 63: 51-57, 1969.

12. Gresser, I., Coppey, J., Falcoff, E., and Fontaine, D. Interferon and Murine Leukemia I. Inhibitory effect of Interferon Preparations on the Development of Friend Leukemia in Mice. Proc. Soc. Exptl. Biol. Med., 124: 84-91, 1967.

13. Gresser, I., Coppey, J., Fontaine-Brouty-Boye, D., Falcoff, E., Falcoff, R., Zagdela, F., Bourali, C., and Thomas, M. P. The Effect of Interferon Preparations on Friend Leukemia in Mice. In: G. A. Wolstenholme and J. O'Connor (eds.), Ciba Symposium on Interferon, pp. 240-248. London: J. & A. Churchil l, Ltd., 1967.

14. Gresser, I., Falcoff, R., Fontaine-Brouty-Boye, D., Zagdela, F., Coppey, K., and Falcoff, E. Interferon and Murine Leukemia. IV. Further Studies on the Efficacy of Interferon Preparations Administered after Inoculation of Friend Virus. Proc. Soc. Exptl. Biol. Med., 126: 791-797, 1967.

15. Gresser, I., Fontaine-Brouty-Boye, D., Bourali, C., and Thomas, M. P. A Comparison on the Efficacy of Endogenous, Exogenous and Combined Endogenous-Exogenous Interferon in the Treatment of Mice Infected with Encephalomyocarditis Virus. Proc. Soc. Exptl. Biol. Med., 130: 236-242, 1969.

16. Gresser, I., Maury, C., and Brouty-Boye, D. Mechanism of the Antitumor Effect of Interferon in Mice. Nature, 239: 167-168, 1972.

17. Gross, L. Mouse Leukemia. In: Oncogenic Viruses, Ed. 24, pp. 286-291. New York: Pergamon Press, 1970.

18. Huffman, J. H., Sidwell, R. W.., Khare, G. P., Witkowski, J. T., Allen, L. B., and Robins, R. K. In Vitro effect of 1-/8-D-Ribofuranosyl-1,2,4-triazole- 3-carboxamide (Virazole, ICN 1229) on Deoxyribonucleic Acid and Ribo- nucleic Acid Viruses. Antimicrobial Agents Chemotherapy, 3: 235-241, 1973.

19. Ihle, J. N., Yurconic, M., Jr., and Hanna, M. G., Jr. Autogenous Immunity

22.

23. . .

24.

to Endogenous RNA Tumor Virus: Radioimmune Precipitation Assay of Mouse Serum Antibody Levels. J. Exptl. Med., 38: 194-208, 1973.

20. Khare, G. P., Sidwell, R. W., Witkowski, J. T., Simon, L. N., and Robins, R. K. Suppression by 1-/~-o-Ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole, ICN 1229) of Influenza Virus-induced Infections in Mice. Antimi- crobial Agents Chemotherapy, 3: 517-522, 1973.

21. Law, L. W. Increase in Incidence of Leukemia in Hybrid Mice Bearing Thymic Transplants from a High Leukemic Strain. J. Natl. Cancer Inst., 12: 789-796, 1952. Law, L. W. Present Status of Nonviral Factors in the Etiology of Reticular Neoplasms of the Mouse. Ann. N. Y. Acad. Sci., 68: 616-825, 1957. Law, L. W., and Miller, J. H. Observations on the Effect of Thymectomy on Spontaneous Leukemias in Mice of the High Leukemic Strains, R1L and C58. J. Natl. Cancer Inst., 11: 253-262, 1950. Lindahl, P., Leary, P., ~nd Gresser, I. Enhancement by Interferon of the Specific Cytotoxicity of Sensitized Lymphocytes. Proc. Natl. Acad. Sci. U. S., 69: 721-725, 1972.

25. Margis, B. J., and Malkiel, S. Immunocapacity of the AKR Mouse. Cancer Res., 32:291-297, 1972.

26. Omine, M., and Perry, S. Perturbation of Leukemic Cell Population in AKR Mice due to Chemotherapy. Cancer Res., 33: 2596-2602, 1973.

27. Omine, M., Sarha, G. P., and Perry S. Composition of Leukemic Cell Populations in AKR Leukemia and Effects of Chemotherapy. European J. Cancer, 9: 557-565, 1973.

28. Pollard, M., and Sharon, N. Prevention and Treatment of Spontaneous Leukemia in Germ Free AKR Mice. Proc. Soc. Exptl. Biol. Med., 137: 1494-1497, 1971.

29. Rios, A., and Simmons, R. L. Immunospecific Regression of Various Syngeneic Mouse Tumors in Response to Neuraminidase-treated Tumor Cells. J. Natl. Cancer Inst., 51: 637-644, 1973.

30. Rios, A., and Simmons, R. L. Active Specific Immunotherapy of Minimal Residual Tumor: Excision Plus Neuraminidase-treated Tumor Cells. In- tern. J. Cancer, 13: 71-81, 1974.

31. Rossi, G. B., Marchegiani, M., Matarese, G. P., and Gresser, I. Inhibitory Effect of Interferon on Multiplication of Friend Leukemia Cells In Vivo. J. Natl. Cancer Inst., 54: 993-996, 1975.

32. Rowe, W. P., Pugh, W. E., and Hartley, J. W. Plaque Assay Techniques for Murine Leukemia Viruses. Virology, 42: 1136-1139, 1970.

33. Sanford, B. H. An Alteration in Tumor Histocompatibility Induced by Neuraminidase. Transplantation, 5: 1273-1279, 1967.

34. Sanford, B. H., and Codington, J. F. Further Studies on the Effect of Neuraminidase on Tumor Cell Transplantability. Tissue Antigens, 1:153- 161, 1971.

35. Schabel, F. M., Jr., Skipper, H. E., Trader, M. W., Lester, W. R., Jr., and Simpson-Herren, L. Spontaneous AKR Leukemia (Lymphoma) as a Model System. Cancer Chemotherapy Rept., 53: 329-344, 1969.

36. Sethi, K. K., and Brandis, H. Neuraminidase Induced Loss in the Trans- plantability of Murine Leukemia L1210 Induction of !mmunoprotection and the Transfer of Induced Immunity to Normal DBA/2 Mice by Serum and Peritoneal Cells. Brit. J. Cancer, 27: 106-113, 1973. "; "~" ~

37. Sethi, K. K., and Teschner, M. Neuraminidase Induced Immunospecific Destruction of Transplantable Experimental Tumors. Postepy Hig. Med. Doswialdczalne, 28: 103-120, 1974.

38. Sidwell, R. W., Allen, L. B., Khare, G. P., Huffman, J. H., Witkowski, J.T., Simon, L. N., and Robins, R. K. Effect of 1-/~-D-Ribofuranosyl-l,2,4- triazole-3-carboxamide (Virazole, ICN 1229) on Herpes and Vaccinia Keratitis and Encephalitis in Laboratory Animals. Antimicrobial Agents Chemotherapy, 3: 242-246, 1973.

39. Sidwell, R. W., Huffman, J. G., Khare, G. P., Allen, L. B., Witkowski, J. T., and Robins, R. K. Broad-spectrum Anti-viral Activity of Virazole: 1-/~- D-Ribofuranosyl-l,2,4-triazole-3-carboxamide. Science, 177: 705-706, 1972.

40. Simmons, R. L., and Rios, A. Immunospecific Regression of Methylchol- anthrene Fibrosarcoma Using Neuraminidase. II1. Synergistic Effect of BCG and Neuraminidase-treated Tumor Cells. Ann. Surg., 176: 188-194, 1972.

41. Skipper, H. E., Schabel, F. M., Jr., and Trader, M. W. Basic and Thera- peutic Trial Results Obtained in the Spontaneous AKR Leukemia (Lym- phoma) Model End of 1971. Cancer Chemotherapy Rept., 56: 273-314, 1972.

42. Skipper, H. E., Schabel, F. M., Jr., Trader, M. W., and Lester, W. R., Jr. Response to Therapy of Spontaneous, First Passage, and Long Passage Lines of AKR Leukemia. Cancer Chemotherapy Rept., 53: 345-366, 1969.

43. Straus, M. J., Choi, S. C., and Goldin, A. Increased Lifespan in AKR Leukemia Mice Treated with Prophylactin Chemotherapy. Cancer Res., 33:1724-1728, 1973.

44. Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell, R. W., Bauer, R. J., Robins, R. K., and Simon, L. N. Mechanism of Action of 1-/~-D- Ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole), a New Broad- Spectrum Anti-viral Agent. Proc. Natl. Acad. Sci. U. S., 70: 1174-1178, 1973.

45. Ungaro, P. C., Drake, W. P., and Mardiney, M. R., Jr. Repetitive Adminis-

638 CANCER RESEARCH VOL. 36

tration of BCG in Prevention and Treatment of Spontaneous Leukemia in AKR Mice. J. Natl. Cancer Inst., 50: 125-128, 1973.

46. Wepsic, H. T. Separation of Viable Tumor Cells from Non-viable Tumor Cells by Flotation on Bovine Serum Albumin. J. Natl. Cancer Inst., 45: 1031-1033, 1970.

Various Treatments of Spontaneous Leukemia in AKR Mice

47. Witkowski, J. T., Robins, R. K., Sidwell, R. W., and Simon, L. The Design, Synthesis, and Broad-Spectrum Anti-viral Activity of 1-/~-D- Ribofuranosyl-l,2,4-triazole-3-carboxamide and Related Nucleosides. J. Med. Chem., 15: 1150-1154, 1972.

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