Tobacco Documents Online

ICANCER RESEARCH 33, 69-77, January 19731 I Chemical Induction of Subcutaneous Tumors in BALB/c and Swiss Mice Infected with Wild Type C RNA Viruses Derived from BALB/c Tissues 1 Department of Viral-Chemical Oncology, fficrobiological .4ssociates, Inc.. Bethesda, alaryland 20014 (R. A. S.. C. E. W./, and Department of Zoology. Universitv oj .blarvland. College Park. M1farvland 20742 /G. .N. R./ Ronald A. Salerno,2 Gordon M. Ramm, and Carrie E. Whitmire SUMMARY The effect of exogenous type C RNA viruses derived from BALB/c mice on the chemical induction of s.c. tumors in mice was determined in relation to host strain, age at treatment, sex, carcinogen dose, and virus strain. Viruses that were cocarcinogenic in Swiss mice were anticarcinogenic or without effect in BALB/c mice under similar treatment conditions. Such effects of viruses on tumor induction by 100 µg 3-methylcholanthrene occurred when the chemical was given at newborn but not weanling age. In BALB/c mice, the mean effect of all viral inocula was related to sex and to the dose of 3-methylcholanthrene administered. Virus decreased the induction of tumors by 25 µg 3-methylcholanthrene in males or by 100 µg 3-methylcholanthrene in females; however, virus ~ increased tumor induction by 25 µg 3-methylcholanthrene in (~ females or by 100 µg 3-methylcholanthrene in males. Viruses derived from tumor-bearing BALB/c mice were more co- and anticarcinogenic than viruses derived from nontumorous mice. Mixing viruses isolated from neoplasms in equal proportions with viruses isolated from normal mice averaged the effects of the individual isolates. This study indicated that wild type C RNA virus may significantly influence chemical carcinogenesis; however, the type of modifying effect depended on the mouse strain, sex and age at treatment, carcinogen dose, and virus strain. INTRODUCTION Oncogenic type C RNA viruses, referred to as oncornaviruses (31), have been found to be a natural cause of various cancers in mice, hamsters, fowl, and cats (18, 42, 43, 48). The activation and isolation of leukemogenic type C RNA viruses from chemically and physically induced neoplasms have also been reported (1, 12, 21, 22). These and other results led Huebner et al. (17-19) and Whitmire et al. (55) to propose that host genotype and environmental factors (such as `This study was conducted under Contracts NIH 70-2068 and NIH-43-67-697 within the Special Virus-Cancer Program of the National Cancer Institute, NIH, USPHS, at Microbiological Associates, Inc., and The Council for Tobacco Research. =Submitted to the faculty of the University of Maryland in partial fulttliment of the requirements for the Ph.D. degree. Received June 21, 1972; accepted October 4, 1972. JANUARY 1973 chemical carcinogens) may derepress type C RNA oncogene and virogene expression(s). Recent support for this theory includes the in vitro studies by Freeman et al. (11), Price et a!. (37), and Rhim et a1. (39-4I ), who demonstrated that, mouse, rat, and hamster tissue culture cell lines were transformed by chemicals when the cells were preinfected with nontransforming type C RNA viruses, while the cells treated with either virus or chemical alone were not transformed. The results of in vivo investigations have conflicted with regard to the combined effects of a type C RNA virus and a chemical carcinogen. Studies that demonstrated cocarcinogenesis (9, 25), anticarcinogenesis (23), and no effect between type C virus and the chemical (10, 13, 25) differed in experimental design, materials, and methodology. In our laboratory, preliminary experiments indicated that the effect of added type C RNA virus on tumor induction by 3MC3 in mice depended on certain treatment conditions. To investigate this further, we undertook a highly controlled study of the role of exogenous, potentially oncogenic type C RNA virus(es) derived from BALB/c tissues in the induction by 3MC of s.c. tumors in mice. The effects of mouse strain, treatment age and sex, carcinogen dose, and virus strain were found to be significant factors on the virus-carcinogen-host interaction. MATERIALS AND METHODS Mice. BALB/c° mice were obtained from Microbiological. Associates, Inc., Bethesda, Md., and Swiss-Webster randombred [N:NIH(SW)) mice were obtained from the NIH, Bethesda, Md. When received, the mice were 1 to 3 days old, and they were randomized 8/mother. At 4 weeks of age, they were weaned and grouped 5/cage/sex. The care, feeding, and housing of the animals were described previously (52). BALB/c Type C RNA Viral Pools. Infectious type C RNA 'The abbreviations used are: 3MC, 3-methylcholanthrene; HBSS, Hanks' balanced salt solution; VIB, veal infusion broth; NB, newborn (mice, 1 to 3 days of age); WL, wean6ng (mice, 28 to 30 days of age); g; group specific; CF, complement fixation; MHV, mouse hepatitis virus; MVM, minute virus of mice; CI, carcinogenic index; S.E., S.E. equals S.D. divided by the square root of the sample size. •These mice were provided underConuact PH-43-66-914 within the Special Virus-Cancer Program of the National Cancer Institute, NIH, USPHS. This colony was established from germ-free mice from the A. R. Schmidt Co., Inc. (Madison, Wis.), and the Charles River Breeding Laboratories, Inc. (Wilmington, Mass.). 69

_ R. A. Saletno, G. M. Ramm, and C. E. Whiimfne Table 1 The history and nomeeclature of the viml inocula Viral nomenclature BALB/c mouse code tissue source were reported positive for MHV by mouse antibody production test. Serum antibody titers of 1:20 or greater for MVM were found in 6 of 18 BALB/c mice that were tested 4 months after their inoculation (when NB) with NI or N2 virus preparations; all other preparations were negative. HBSS-VIB control mice were negative at 4 months for MVM or MHV, NI Spleen from a normal 24-month- whereas control mice that received normal spleen inoculum old female BALB/c mouse were positive for MVM and/or MHV at titers of 1:20 to 1:40. N2 Spleen of a normal 24-month- old female BALB/c mouse There was no correlation ' between the presence of MVM TI Tumor from spontaneous fibro- and/or MHV and the experimental treatment. sarcoma of a 15-month-old female Detection of Type C RNA Virus gs Antigen in Mouse BALB/c mouse Tissues. Tumor, spleen, and muscle (gastrocnemius) tissues T2 Spleen of reticular cell sarcoma were aseptically removed, weighed, ground, and prepared as of an 18-month-old female BALB/c mouse 10% extracts (w/v) in HBSS-VIB diluent and were stored at NITI 1:1 mixture of N1 and TI -70°, as previously described (42, 51). The antiserum (Pool N2T2 1:1 mixture of N2 and T2 20) used for CF tests was prepared in Fischer rats that carried Control spleen Four-week-old virus-negative transplanted syngeneic sarcomas induced by murine sarcoma BALB/c spleen virus (14, 16). The serum pool was positive for antibodies at viruses isolated from the above-described BALB/c mouse colony by Dr. R. Peters (Microbiological Associates) were received as partially purified spleen preparations. Two isolates were derived from neoplastic BALB/c tissues and 2 were derived from normal BALB/c spleen tissues (Table 1). These viral preparations were injected i.p. into BALB/c NB mice. After' 4 weeks, the 4 viruses were partially purified and . concentrated from the spleen tissue by the Moloney procedure (28), modified and described by Huebner et al. (16), and were stored at -120°. The following 2 control inocula were used: (a) HBSS-VIB consisting of 80% HBSS, 20% VIB, 300 units penicillin per ml, 300 µg streptomycin per nzl, and 60 µg polymyxin B per ml; and (b) 4-week-old normal BALB/c spleen tissue processed in the same manner as the infected spleens. Samples were used within 3 months after preparation. Viruses were given to BALB/c and Swiss NB and WL mice at a'rate of one hundred 50% infective doses per 0.05 ml for BALB/c NB mice. We calculated the 50% infective dose by the Reed-Muench method (38), using the number of spleens positive for the gs antigen detected by CF test (14, 46) after 4 weeks as the criterion of infection. All viral preparations were determined to be BALB/c-trophic in tissue culture, according to the procedures of Hartley et al. (15). None of the viral inocula produced detectable neoplasm in the mice during the 8-month observation period. The viral inocula were tested, through the Microbiological Associates, Inc., Diagnostic Testing Service,s for contamination with extraneous murine viruses by the mouse antibody production test (33, 44). All preparations were negative for pneumonia virus of mice, reovirus type 3, K virus, Sendai virus, polyoma virus, mouse adenovirus, lymphocytic choriomeningitis, and ectromelia vaccinia. Although no antibody against MHV was found in the sera of BALB/c mice 4 months after their inoculation (when NB) with the viral preparations, the TI and T2 viral concentrate preparations ° This setvioe rvas provided under Contract PH-43-67-700 within the Special VirusCancer Program bf the National Cancer Institute, NIH, USPHS. =>1:80 for murine leukemic viruses (AKR and Rauscher leukemia virus) grown in tissue culture and murine sarcoma virus rat tumor antigens, and negative at z 1:40 with normal 4-week-old NIH Swiss mouse spleen extract. Carcinogen Preparation and Inoculation. The carcinogen 3MC (Eastman Organic Chemicals, Rochester, N. Y.) was prepared at concentrations of 25 and 100 µg/0.05 nzl trioctanoin (Eastman Organic Chemicals). Solutions were made once and stored in sealed 6-ml vials in the dark at room temperature and were 3 to 4 months old at the time of use. The 3MC was administered s.c. in the intrascapular region. ' Histopathological Preparation. Tissue samples for light microscopy included tumor, spleen, liver, thymus, heart, lungs, and any obviously abnormal tissues. Tissue preparation has been described previously (55). Analysis of Data. The s.c. tumor incidence and latency periods were calculated (55) for the number of mice at risk for 8 months. The CI (20) for each tumor was calculated by dividing the days of the latent period into the tumor incidence for 240 days and multiplying the quotient by 100. The type C RNA virus gs antigen incidence was calculated (55) from positive results by CF test at the designated final tissue dilution. The latency period and CI parameters were statistically analyzed by a 3-way factorial analysis of variance. Specific comparisons between viral and control inocula were tested according to the orthogonal contrast requirements. Tumor ' incidence data were statistically tested according to a 2-way contingency table analysis. Only those mice that received 3MC were included in this analysis. RESULTS Experiments 1 and 2. In Experiment 1, the effect of BALB/c type C RNA viruses N1 and N2 on 3MC tumor induction by 100 µg 3MC in BALB/c mice was tested in relation to mouse sex and age at treatment with virus and 3MC. In Experiment 2, the same factors were tested in the Swiss mouse. Both experiments were designed in a 2' X 3 X 4 factorial arrangement of sex, virus, and age factors. 70 CANCER RESEARCH VOL. 3

Viral inocula Ni and N2 were compared to each other and to the control spleen inoculum. The treatment sequences of the viral or control inoculum and 100 ug 3MC were as follows: (a) the viral inoculum was given to NB mice 3 hr before administration of 3MC: (b) virus was given to NB's; 3MC was ¢iven to WL's: (c) virus was given to WL's 3 hr before 3MC was ¢iven: (d) 3MC was given to NB's: virus was given to WL's. Each viral and control inoculum was administered to 240 mice. Of these, 120 received the inoculum as NB's and 120 as tiy;.'s. In each of these 2 age groups, 3MC was administered to 40 NB and 40 WL mice. The remaining 40 mice were not treated with 3MC. and these served as virus or normal spleen Combined Effects of Type C RNA Virus and 3MC controls. One month after treatment with virus, 10 (5 male and 5 female) mice were selected at random from each inoculum group to determine virus infectivity. The mice were weaned and separated according to sex at 4 weeks of age (approximately 15 mice/experimental unit). The results of Experiment I are shown in Table 2. Viruses Ni and N2 delayed tumor induction without affecting the tumor incidence in BALB/c. mice treated with 3MC as NB's. This effect was determined significant (p < 0.05) by an analysis of variance of special treatment comparisons between 3MC-treated NB and WL mice (p < 0.05), and it was dependent on the virus strain and mouse sex. Latency was Table 2 Incidence and latency periods of s.c. tumors in BALB/c mice inoculated with 100 ug 3 bfC and BALB/c type C R.VA virus or control spleen inoculum at different ages and sequences Results, as affected by both age and sequence of viral or control inoculum and 3MC treatment 3MC as NB's; virus as NB's 3MC as NB's; virus as WL's 3MC as WL's; virus as NB's 3MC as WL's; virus as WL's Tumor Tumor Tumor Tumor latency latency latency latency Viral Inci- period Inci- period Inci- period Inci- period Sex inocula dence oa (days) dence % (days) dence % (days) dence % (days) Male Control (spleen) 9/126 75 127 - 16` 11/14 79 99 ± 7 13/15 87 113 t 12 15/15 100 96 - 7 Ni 14/19 74 109 ± 8 11/12 92 111 ± 13 13/15 87 124 ± 11 13/15 87 99 2 8 N2 11/13 85 142 ± 16 12/15 80 124 ± 10 15/15 100 107 ± 10 15/15 100 101 +_ 12 I Female Control (spleen) 16/18 89 106 ± 7 13/15 92 108 ± 8 14/15 93 103 t 10 14/15 93 111 _ 15 Ni 8/11 73 144 ± 22 16/18 89 129 t 11 15/15 100 110 t 11 14/15 93 99 z 6 N2 14/17 82 119 ± 11 14/15 93 106 ± S 14/15 93 100 ± 8 13/15 87 84 - 5 ° Percentage of tumors induced 8 months after 3MC treatment. b Number of tumor-bearing mice/total number of mice that were at risk to 3MC for 8 months. ° Mean ± S.E. Table 3 Incidence and latency period of s.c. tumors in Swiss mice inoculated with 100 t,g 3MC and BALB/c type C RNA virus or control spleen inoculum at different ages and sequences I Results as affected by both age and sequence of viral or control inoculum and 3MC treatment 3MC as NB's; 3MC as NB's; 3MC as WL's; 3MC as WL's; virus as NB's virus as WL's virus as NB's virus as WL's Tumor Tumor Tumor Tumor latency latency latency latency Viral Inci- period Inci- period Inci- period Inci- period Sex inocula dence %° (days) dence % (days dence % (days) dence % (days) Male Control b 10/15 67 c 118 t 13 12/15 80 136 _ 13 0 3 1/1 73 22 + 8 1 13115 87 114 + 8 - (spleen) Ni 13/15 87 102 ± 3 11/15 73 121 ± 12 11/15 73 109 t 8 14/15 93 127 ± 14 N2 13/15 87 118 t 9 10/15 67 106 ± 5 14/15 93 109 ± 10 13/15 87 97 ± 5 Female Control 9/15 60 152 r 14 12/15 80 113 s 7 11/15 73 126 = 14 12/15 80 118 ± 12 (spleen) N1 13/15 87 118 t 8 3/15 20 118 z 16 13/15 87 128 t 13 12/15 80 110 = 10 N2 12/15 80 113 s 43 9/15 - 60 126 ± 13 13/15 87 122 ± 14 13/15 87 121 ± 11 ° Percentage of tumors induced 8 months after 3MC treatment. b Number of tumor-bearing mice/total number of mice that were at risk to 3MC for 8 months. ` Mean ± S.E. JANUARY 1973 71

R. A. Salemo, G. M. Ramm, and C. E. Whitmire increased in males by N2 virus and in females by Nl virus. The tumor latency was not altered by viral treatment when 3MC treatment was given at WL age, regardless of the time of viral inoculation. In BALB/c mice treated with virus as NB's, all tumors were positive for gs antigen at dilutions of 1:20 to 1:80, while very few tumors of mice treated as WL's were gs positive unless 3MC treatment had been given at NB age. One of 10 NB mice treated with 3MC and given control inoculum as a WL was positive for gs antigen at a 1:80 tissue dilution, whereas 7 of 20 mice inoculated with N I and N2 virus were positive. Normal muscle tissue in 45 to 56% of the mice infected with virus as NB's was also gs positive. The results of Experiment 2 are shown in Table 3. Viral inocula N1 and N2 affected the incidence and not the latency of tumors induced with 3MC in NB Swiss mice. When NB mice were treated with viral or control (spleen) inoculum and 3MC, both NI and N2 enhanced 3MC tumor induction by 20% in males and 25% in females over that produced by the control (spleen) inoculum (0.10 > p> 0.05). When viral or control inocula were given to WL mice (after having been given 3MC at NB age), the tumor incidence in females was significantly decreased (60% by N1 and 20% by N2). The tumor incidence and latency in both male and female mice treated with 3MC as WL's was not significantly affected by virus given at NB or WL age. Viral inocula, mouse age, and sex were not important factors, separately or in combination, in affecting the incidence of gs antigen in tumor tissue of Swiss mice. The gs antigen incidence in 1:80 dilution of tumor tissue was 3:40, 3:40, 4:40 for mice inoculated with NI, N2, and control (spleen), respectively. In no instance was muscle tissue positive forgs antigen at a 1:20 dilution. The combined effects of tumor incidence and tumor latency expressed as the CI are given in Table 4 for both BALB/c and Swiss mice treated as NB's with virus and 3MC. High significant levels indicated that the effect of virus on 3MC tumor induction was dependent on mouse strain and sex (Table 5). Experiment 3. A randomized 5 X 2 X 2 factorial arrangement of viral and control inocula. 3MC dosage, and BALB/c sex was used. The viral preparations used were: T 1, T2, 1:1 mixture of N 1 and T I, and 1:1 mixture of N2 and T2 (Table 1). The control inoculurn was HBSS-VIB. Each virus and control inoculum was given to 120 NB BALB/c mice; 40 were treated with 25 µg 3MC, 40 received 100 ug 3MC, and the remaining 40 were observed for tumor induction by the Table 4 The effects of BALB/c type C RNA viral inocula on s.c. tumor induction in BALB/c and Swiss mice treated as.VB's Mouse Strain Sex Inocula CI cc ACI° BALB/c Male Control (spleen) 67 ± 8° Ni 71t5 +6 N2 68t7 +2 Female Control (spleen) 93 ± 6 N1 61t9 -34 N2 76 ± 7 -18 , Swiss Male Control (spleen) 62 ± 5 N1 87±4 +40 N2 78 t 5 +26 Female Control (spleen) 44 ± 3 N1 78 4 4 , +77 N2 78 ± 7 +77 a Percentage change in Cl =(CI of viral inoculum - Cl of control inocultun)/(CI of control inoculum) X 100. b Mean ± S.E. Table 5 Analysis of variance of the CI's ofNB BALB/c and Swiss mice with s.c. tumors These mice were treated with 100 µg 3MC in a 2 X 2 x 3 factorial arrangement of mouse strain, sex, and BALB/c type C RNA viral or control inoculum. Source of variation Sum of squares - d.f. Mean square F ratio p Main effects Error 56,497.73 130 43459 Mouse straina 104.45 1 104.45 . 0.24 < 0.625 Sex 140.76 1 140.76 0.32 < 0.570 Viral or control inoculumb 886.75 2 443.37 1.12 < 0.363 Interactions Mouse x sex 3,215.40 1 3,215.40 7.39 < 0.007c Mouse X inocula 11,491.47 2 5,745.73 13.22 < 0.0010 Sex x inocula 1,531.15 2 765.57 1.76 < 0.176 Mouse x sex X inocula 2,961.39 2 1,480.69 3.40 < 0.036c Interactions of mouse strain and sex with specific inocula comparison NI and N2 vs. control inocula X 2,854.55 2,854.55 6.56 < 0.012C mouse X sex a Swiss and BALB/c. b Inocultun = N 1, N2, and control spleen. ' Significant p values. 72 CANCER RESEARCH VOL 33 t r 4k / ,

Combined Effects of Type CRNA Virus and 3MC Table 6 The effects of BALBIc type C RNA viral inocula on s.c. tumor induction b_v 25 and 100 mg 3MC in BALBIc mice treated as NB's Dose Sex Inocula Tumor incidence -° Latency (days) C[ o a Ctb 25 Male HBSS-VIB l I115° 73 149 '_ 11d 52 - 4 NITI 7/13 54 168n20 34s3 -35 TI 5/15 33 189 - 30 22 - 2 -57 T2 5/13 31 163 ~ 27 20 - 2 -61 N2T2 5/15 33 189 ~ 30 18 = 2 -65 Female HBSS-V1B 3/15 20 218 s 39 10 ~ 2 NITI 4/13 30 149- 15 20-2 +14)0 N2T2 5/15 33 125 - 10 27 _ 3 +17r) TI 4/10 40 198=28 29-3 +191) T2 9/15 63 149 •_ 15 42 - 3 +320 100 lfale HBSS-VIB 11/14 79 128 ~ 12 65 z 5 NITI 10/12 83 118•_18 70+-6 +8 T2 13/ 14 93 132 } 15 81 - 10 +25 N2T2 13/15 87 110 t 10 87 : 7 +33 TI 15/1S 100 98 f 9 113 _ 9 +74 Female HBSS-VIB 14/16 87 110 ± 10 84 ± 6 TI 13/14 93 112± 13 93z8 +1I NITI 18/18 100 131 ± 7 82 ± 5 -2 N2T2 9/14 64 111 - 16 64 - 6 -24 T2 10/ 15 67 149 ± 21 54 - 8 -40 M t I ° Percentage of tumors induced 8 months after 3MC treatment. b Percentage change in CI =(CI of viral inoculum - CI of control inoculum)/(CI of control inoculum) X 100. ` Number of tumor-bearing mice/total number of mice that were at risk to 3MC for 8 moTths. Mean ± S.E. Table 7 Analysis of variance of the CI's of BALB/c mice treated as NB's with 25 or 100 pg 3MC in a 5 X 2 X 2 factorial arrangement of viral or control inocula, 3MC dosage, and sex Source of variation Sum of squares d.f. Mean square F ratio p Main effects Error 75,798.89 168 451.18 5.61 Viral or control inoculuma 10,134.37 4 2,533.90 5.61 < 0.001C Dosageb 107,245.64 1 107,245.64 237.69 <0.001° Sex 944.16 1 944-16 2.09 < 0.150 Interactions Inocula x sex 1,748.77 4 437.19 0.96 < 0.426 Inocula X dose 13,837.20 4 3,459.30 7.66 < 0.001C Dose X sex 181.87 1 181.87 0.40 < 0.526 Inocula X dose X sex 15,702.84 4 3,925.71 8.70 < 0.001C Interactions of dose and sex with specific inocula comparisons T1 vs. T2 X dose 11,081.93 1 11,081.93 24.56 <0.001C HBSS-VIB vs. all viral inocula X dose 2,229.74 1 2,229.74 4.94 < 0.028c TIT2 vs. NITI and N2T2 x dose x sex 2,296.17 1 2,296.17 5.08 < 0.025c T1 vs. T2 X dose X sex 698.82 1 698.82 1.54 < 0.215 NITI vs. N2T2 X dose and sex 2,756.98 1 2,756.98 6.11 < 0.014c HBSS-VIB vs. all viral inocula x dose x sex 9,950.87 1 9,950.87 22.05 < 0.001° a Inocula = Tl, T2, N1T1, N2T2, or control (HBSS-VIB). b Dosage = 25 or 100 Ng 3MC. c Significant p values. JANUARY 1973 73

R..A. Salemo~ G bE Ransuk ®.d CE. A+hitmae virus and control inocula in the absence of 3MC. One month after virus inoculation, mice were weaned and tested for viral infectivity as described above. Approximately 15 animals per viral or control inoculum per sex per 3MC dose remained on test. A total of 288 mice treated with 3MC, virus, and control inocula were analyzed. Table 6 presents the co- and anticarcinogenic effects of each viral inocula (relative to sex and 3MC dose) on tumor incidence, latency, and CI index. Statistical analysis of CI data (Table 6) indicated that viral inocula, 3MC dose, and sex all were highly significant modifying factors (Table 7). The interactions of viruses, sex, and 3MC dose were highly significant in comparisons that contrasted the 2 viral inocula (T1 and T2) to the 2 mixed viral inocula (N1T1, N2T2); the 2 mixtures of viral inocula against each other; and all 4 viral inocula to the HBSS-VIB control inoculum (Table 7). This latter interaction demonstrated that tumor induction with 25 µg 3MC was inhibited by virus in males (control Cl 52 versus average virus CI 24) and enhanced in females (control CI 10 versus average virus CI 30). On the other hand, viruses enhanced tumor induction by 100 µg 3MC in males (control CI 65 versus average virus CI 88) and inhibited tumor induction in females (control CI 88 versus average virus CI 72). An analysis of tumor latency periods indicated a 3-way significant interaction (p < 0.05) of virus, sex, and 3MC dose. An analysis of contrast between the 4 viral inocula and the HBSS-VIB control indicated that tumor latency was increased by viruses in males and was decreased in females treated with 25 µg 3MC. Conversely, at 100 µg 3MC, a shorter tumor latency period occurred in males and a longer latency occurred in females than in control mice (p < 0.005). The same relationships between virus, sex, and 3MC dose were demonstrated by the tumor incidence data (Table 6). Contingency table analyses demonstrated differences in frequency of tumors among inocula for males treated with 25 µg 3MC and females treated with 25 or 100 µg 3MC (p <0.1). No differences were significant among the inocula in male mice treated with 100 µg 3MC. Overall, 95% (78:82) of the tumors from mice treated with virus were positive for gs antigen at a final dilution of 1:80; the remaining 4 tumors were positive at 1:40. In comparison, tumors from control mice treated with 3MC were 19% (6:33), 24% (8:33), and 24% (8:33) at tumor extract dilutions of 1:20, 1:40, and 1:80, respectively. Muscle tissues from mice treated with HBSS-VIB and 3MC were negative at a 1:20 dilution for gs antigen. However, 60% (46:78) of virus-treated mice had detectable levels of gs antigen in normal muscle at a 1:20 dilution. In all 3 experiments, histological studies of the tumors demonstrated no difference in the type of tumors related to mouse strain, sex, age at treatment, virus strain, or 3MC dose. The majority of tumors were classified as fibrosarcomas. DISCUSSION The chemical induction of s.c. tumors in mice was either augmented, inhibited, or not affected by wild type C RNA viral inocula derived from BALB/c tissues. The significant responses were dependent on mouse strain, age at treatment, sex, carcinogen dose, and viral strain. Effect of Mouse Strain and Age. Co- and anticarcinogenic effects of BALB/c type C RNA viruses on chemical carcinogenesis in BALB/c mice occurred when the carcinogen 3MC (100 gg) was given at NB but not at WL age. This effect of age was the same as that observed when the same viral inocula were given to Swiss mice; however, the observed host response to virus and 3MC differed. In BALB/c mice, NI and N2 viral inocula decreased the CI in female mice by 18 and 34%, respectively; the CI in males was slightly augmented by either viral inocula. In Swiss mice, the mean CI was increased by 77% in females by both N1 and N2, as compared with control (spleen) inocula, and was increased 26% by N 1 and 40% by N2 inocula in males. When Swiss female mice were inoculated with virus 28 days after being treated with 3MC as NB's, tumor incidence was significantly decreased. This decrease was not observed in similarly treated BALB/c mice. Hence, both mouse strain and age at treatment were significant factors in determining the, effects of these type C RNA viral inocula on s.c. tumor induction by 3MC. Previously reported viral-chemical carcinogen-host systems indicated that maximum interactions occurred when the carcinogen and virus infection occurred at the same time (6, 9. 49). For instance, when type C RNA viruses were given simultaneously with carcinogens at NB, WL, or young adult age, a cocarcinogenic effect occurred (9, 25). However, when these viruses were given at NB age and the carcinogen was given at WL or young adult age, no interaction between virus and carcinogen was observed (10, 13, 25). Our results do not explain why only NB's given 3MC were susceptible to the effects of virus, but one may, suggest that the immaturity of metabolic and defense systems are the determining factors (35, 54). The influence of mouse strain on viral-chemical carcinogenesis has also been observed (1, 7, 8, 10). For example, Fiore-Donati et al. (10) reported that a combined treatment with a leukemia virus derived from the C57BL/6 mouse and urethan enhanced the incidence of leukemia in the natural C57BL/6 host of the virus but not in the unnatural C3H/Fg host, as compared with the results with either agent alone. Effect of Sex and 3MC Dose. When 25 kg 3MC were used for tumor induction in BALB/c mice, viruses were generally cocarcinogenic in females and anticarcinogenic in males. On the other. hand, when 100 µg 3MC were inoculated into BALB/c mice, viruses were cocarcinogenic in males and anticarcinogenic in females. Sex reportedly influences both viral and chemical carcinogenesis when used separately but not when combined (24, 32, 34, 42). In our system, BALB/c males without virus were more susceptible to s.c. tumor induction than were females treated with 25 Kg 3MC but were not more susceptible than females treated with 100 µg 3MC. Price et aL (37) reported that the dose of 3MC quantitatively affected transformation of rat embryo cells in vitro in combination with Rauscher leukemia virus. Low doses of 3MC were more effective than high doses in accelerating transformation of infected cells. 74 CANCER RBSBAAQi- VOI.. 33

Combined Effects of Type C RNA Virus and 3MC t , I b Effect of Virus Strain. In BALB/c mice, the viral inocula derived from tumor-bearing mice (compared with that from normal mice) were more active in affecting the mean CI's resulting from 100 µg 3MC. In males, Ti had the greatest cocarcinogenic effect, followed by T2: NI and N2 were without effect. In females, T2 virus was most anticarcinogenic, followed by NI and N2: TI did not alter the Cl results (Chart 1). Also, the cocarcinogenic effect in males treated with 100 µg 3MC was obtained with viruses derived from solid tumors (TI and T2), whereas the anticarcinogenic effect occurred with viruses derived from the reticuloendothelial system (T2, v I. and N2) (Table 1). Mixing viruses of tumorous and nontumorous origin averaged the effectiveness of individual viral inoculum on the mean Cl with 25 and 100 µg 3MC (Table 6). When the viral mixtures were prepared at a 1:1 ratio, the concentration of •9o .w~ •m -a~ -ao 100 0q rMc Tt 1 T2 N1 N2 VNAL MqCUTA Chart 1. Effect of type C RNA viral inocula on 3MC tumor induction in BALB/c mice treated as NB's. Percentage change in Cl (Cl =of viral inoculum - Cl of control inoculum)/(CI of control inoculum) x 100. JANUARY 1973 each virus was diluted by 5035, thus reducing the activity of the more active components. This dose reduction and the consequent decrease in viral-3MC interaction may be analogous to that reported by Tanaka and Southam (52). who found that the combined effect of 3MC and herpes simplex was related to viral dosage. The strain variation of the BALB/c type C RNA viruses demonstrated in these studies suggests that the diversity in the reported oncogenic virus-chemical, cocarcinogenic-host systems (6, 26, 45, 49) may be due, in part, to differences in viral properties and characteristics. Hypothesis. The observed augmentation, inhibition, or lack of effect on chemical carcinogenesis by the viral inocula possibly may be explained in terms of the oncogene theory (18. 55) and host defense reactions (2, 4. 47, 51). According to the oncogene theory, the control of the host over the oncogenic expressions of the type C RNA viral genome is derepressed after the administration of 3MC. Consequently, the addition of viral oncogenes should have accelerated the transformation of normal cells into cancerous ones. Such a reaction has been demonstrated in vitro, whereby the transformation of various mouse, rat, and hamster fibroblast cells with low doses of carcinogens was enhanced after such cells were infected with nontransforming type C RNA viruses (11, 37, 39-41). The fact that certain viruses inhibited as well as enhanced chemical tumor induction in mice used in this study suggests that the growth of the induced (transformed) cancer cells was permitted or inhibited by the host, depending on its ability to respond to the induced tumor and viral antigens of the specific viral inocula. This ability may invofve mechanisms of dose-dependent carcinogen immunosuppression (3, 36, 50), sex-dependent immune responsiveness (29, 53), immunological enhancement (2, 47), viral immunological protection, therapy, and interference phenomena (4, 5, 27, 51), as well as other undefined cellular control mechanisms. The host immune reaction may be the major underlying reason for the effects of mouse sex, 3MC dose, and viral strain in determining the actions of exogenous type C virus on 3MC s.c. tumorigenesis. In our system, the immune response of BALB/c mice given 25 µg 3MC may have been greater than that of mice given 100 µg 3MC, because the spleens were significantly larger (p < 0.006) at the lower dose. The spleen weight data (not reported in "Results") indicated that the spleens of mice that were given 100 µg 3MC weighed 160 mg, as compared to the 216-mg spleens of mice given 25 µg 3MC. Recently, Pollack (34) demonstrated that BALB/c females had a greater immune response to sarcoma induction by Moloney sarcoma virus than did males. Theoretically, one may argue that the increase in tumors induced by 100 µg 3MC was observed in BALB/c males inoculated with type C RNA viral inoculum, as compared with a control inoculum, because the male immune system was unable to check the induced viral and tumor antigens; however, the female system (possibly more immunocompetent) prevented the enhanced s.c. tumor growth. On the other hand, with 25 µg 3MC, males may have been immunocompetent and responded as did the females to 100 µg 3MC, so that they were able to overcome the added viral and tumor antigens. The defense system of the females, which probably was not affected by 25 µg 3MC, overresponded to the viruses and led to immunological 75

R. A. Salerno, G. M. Ramm, and C. E. Whilmire enhancement and consequent s.c. tumor growth. Recent unpublished data in our laboratory indicated that ovariectomy blocks this viral enhancement of tumors induced by 25 µg 3MC. Extrapolation of these same hypotheses of the BALB/c mouse system to Swiss mice may not be made for the following reasons. First, only N1 and N2 viral inocula and 100 µg 3MC were studied; 2nd, these viruses were not infectious in Swiss mice; and 3rd, the effect of these viruses was significantly different in the 2 mouse strains. The viral inocula may have acted in an additive or sypergistic manner with 3MC to derepress oncogenic expression of endogenous Swiss type C RNA virus. Although the gs antigen incidence data do not support the idea of enhanced virogene expression, oncogenic expression may have been significantly affected, since both oncogene and virogene expressions are independent (19). However, in WL Swiss mice that were treated with 3MC as NB's, the viruses may have acted as nonspecific agents. The inoculation of the viral inocula may have stimulated the reticuloendothelial system to attack the developing tumor cells, resulting in the observed increased latency and decreased tumor incidence. The stimulus could have been viral envelope antigens which are capable of eliciting an immune response (19). This enhancement of the defense system against the induced tumors is similar to that [reported by Nilsson et al. (30)] of increased latency and decreased incidence of osteosarcomas in 9° Sr-exposed hosts when Mycobacterfum bovis [strain Bacillus Calmette-Guerin (BCG)] was given at the time of suspected tumor appearance. From the above discussion, one may speculate that the host-defense systems to tumor induction in both Swiss and BALB/c mice that were inoculated with type C RNA virus may very well be the basis for the significance of the mouse strain, sex, age, viral strain, and 3MC dosage factors in determining the effect of exogenous type C RNA virus on 3MC s.c. tumor induction. Significantly, however, this study in viral-chemical carcinogenesis defines the conditions under which chemical carcinogenesis may be accelerated or inhibited by means of the addition of potentially oncogenic wild type C RNA virus in a specific biological system. ACKNOWLEDGMENTS The authors thank Dr. R. J. Huebner, Dr. R. E. Kouri, Dr. B. Talbot, Dr. R. Peters, Dr. R. Nims; and Dr. M. L. Vernon for their criticism of the manuscript. The technical assistance of Mr. H. C. Turner, Mrs. E. Von Kaenel, Mr. W. Boykins, Mrs. G. Feeser, Mr. H. Ratrie, Mr. S. Zelnio, Miss H. Sipe, and Mr. L. Lauer was also appreciated. Assistance in computer analysis of the data was obtained from Dr. D. Waggoner, Dr. T. L. Connell, Dr. M. C. Dayton, and Mrs. W. Varratto. Computer time and usage was funded by the University of Maryland. The authors express their appreciation to Mr. T. Black and his staff for preparation of the histopathological specimens and to Dr. L. S. Rabstein for histopathologicai diagnosis. REFERENCES 1. Ageenko, A. I. Filterable Virus-like Agent Isolated from Tumors Induced by Carcinogenic Substanees. Folia BioL Prague, 8: 7-11, 1962. 2. Amos, D. B., Cohen, I., and Kiein, W. j., Jr. Mechanisms of Immunologic Enhancement. Transplant. proc., 2: 68-75, 1970. 3. Ball, J. K. Immunosuppresaion and Carcinogenesis Contrasting Effects with 7,12-Dimethylbenz(a)anthracene, Benz(a)pyrene and 3-Methylcholanthrene. J. Nail. Cancer Inst., 44: 1-10. 1970. 4. Barbieri, D., Belehradek, J., Jr., and Barski, G. Decrease in Tumor-producing Capacity of Mouse Cell Lines following Infection with Mouse Leukemia Viruses. Intern. J. Cancer, 7: 364-371. 1971. 5. Barski, G.. and Youn, J. K. Protection of Mice against Gross Leukemia by Interfering Action of Non-leukemogenic C-type Murine Viruses Inoculated into Newborns. J. NatL Cancer Inst., 47: 575-583, 1971. 6. Duran-Reynals, M. L. Combined Effects of Chemical Carcinogen Agents and Viruses. Progr. Exptl. Tumor Res.. 3: 148-185, 1963. 7. Duran-Reynals, M. L. Combined Neoplastic Effects of Vaccinia Virus and 3-Methylcholanthrene. 1. Studies with Mice of Different Inbred Strains. J. Nail. Cancer Inst., 48: 95-I04, 1972. 8. Duran-Reynals, M. L., and Lilly, F. The Role of Genetic Factors in the Combined Neoplastic Effects of Vaccinia Virus and Methylcholanthrene. Transplant. Proc., 33: 1243-1246, 1971. 9. Engle, C. G., and Groupe, V. Effect of Chemical Carcinogens on Virus-induced Rous Sarcoma. Cancer Res., 29: 1345-1349, 1969. 10. Fiore-Donati, L., Chieco-Bianchi, L., and de Benedictis, G. Chemical and Viral Interactions in Leukaemogenesis: Effect of Urethane on the Induction of Leukemia by Graffi Virus in Adult Mice. In: P. Emmelot and 0. Muhibach, (eds.), Cellular Control Mechanisms in Cancer, pp. 268-271. New York: American Elsevier Publishing Co., Inc., 1964. 11. Freeman, A. E., Price, P. J., Igel, H. J., Young, J. C., Maryak, J. M., and Huebner, R. J. Morphological Transformation of Rat Embryo Cells Induced by Diethylnitrosamine and Murine Leukemia Viruses. J. Natl. Cancer Inst., 44: 65-78, 1970. 12. Gross, L. Attempt to Recover Filterable Agent from X-ray Induced Leukemia. Acta HaematoL,19: 353-361, 1958. 13. Gross, L., Roswit, B., Mada, E. R., Dreyfuss, Y., and Moore, L. A. Studies on Radiation-induced Leukemias in Mice. Cancer Res., 19: 316-320, 1959. 14. Hartley, J. W., Rowe, W. P., Capps, W. I., and Huebner, R. J. Complement Fixation and Tissue Culture Assays for Mouse Leukemia Viruses. Proc. Natl. Acad. Sci. U. S., 53: 931-938, 1965. 15. Huebner, R. J. In Vitro Methods for Detection and Assay of Leukemia Virnses. In: Carcinogenesis: A Broad Critique, M. D. Anderson Hospital and Tumor Institute at Houston, pp. 23-47. Baltimore: The Williams & Wilkins Co., 1967. 16. Huebner, R. J., Hartley, J. W., Rowe, W. P., Lane, W. T., and Capps, W. 1. Rescue of the Defective Genomes of Moloney Sarcoma Virus from a Non-infectious Hamster Tumor and the Production of Pseudo-type Sarcoma Viruses with Various Murine Leukemia Viruses. Proc. Nail. Acad. Sci. U.S., 57: 1164-1169, 1966. 17. Huebner, R. J., Kelloff, G. J., Sarma, P. S., Lane, W. T., and Turner, H. C. Group-specific (gs) Antigen Expression during Embryogenesis of the C-type RNA Tumor Virus: Implications for Ontogenesis and Oncogenesis. Proc. NatL Acad. Sci. U. S., 67: 366-376,1970. 18. Heubner, R. J., and Todaro, G. J. Oncogenes of RNA Tumor Viruses as Determinants of Cancer. Proc. Nail. Acad. Sci U. S., 64: 1087-1094, 1969. 19. Huebner, R. J., Toduo, G. J., Sarma, P. S., Hartley, J. W., Freeman, A. E., Peoers, R. L., Whitmire, C. E., Meier, H., and Gilden, R. V. "SwitchedofP" Vertically Transmitted C-type RNA Tumor Viruses U Determiaants of Spontaneous and Induced Cancer. A New Hypothesis of Viral Carcinogenesis. In: 76 CANCER RESEARCH VOL. 33 I

r Defectivenps, Rescue and Stimulation of Oncogenic Viruses, pp. 33-47. Paris: Editions du Centre National de la Recherche Scientifique, 1970. 20. Iball, J. The Relative Potency of Carcinogenic Compounds. Am. J. Cancer, 35: 188-190, 1939. 21. Igel. H. J., Huebner, R. J., Turner, H. C., Kotin, P., and Falk, H. L. Mouse Leukemia Virus Activation by Chemical Carcinogens. Science, 166: 1624-1636, 1969. 22. Irino, S., Ota, A., Sezaki, T., and Suzaki, K. Cell-free Transmission 23. 24. of 20-,Methylcholanthrene Induced RF Mouse Leukemia and Electron Microscopic Demonstration of Virus Particles in Its Leukemic Tissue. Gann, 54: 225-237, 1963. Law. L. W., and Precerutti. A. Inhibition by Urethane (Ethyl Carbonate) of Virus Induction of Leukemia in C3H Mice. Nature, 200: 692-693. 1963. Leiter, J.. and Shear, M. J. Quantitative Experiments on the Production of Subcutaneous Tumors in Strain A Mice with Marginal Doses of 3,4-Benzpyrene. J. Natl. Cancer Inst., 3: • 455-477, 1943. 25. Lieberman, M., Haran-Ghera, N., and Kaplan, H. S. Potentiation of I Virus Leukemogenesis in C57BL Mice by X-irradiation or Urethane. Nature, 203: 420-422, 1964. 26. Martin, C. 14. Interactions of Hydrocarbon Carcinogens with Viruses and Nucleic Acids in Vivo and in Vitro. Progr. Exptl. Tumor Res., 5: 134-156, 1964. 27. Mayyasi, S. A., Bulfone, L., Foster, H., Shibley, G., and Aldenderfer, P. H. The Tumorigenic and Leukemogenic Activity of a!viouse Tissue Culture Cell Line Chronically Infected with Murine Leukemic Viruses. Proc. Am. Assoc. Cancer Res., 10: 56, 1956. 28. Moloney, J. B. Biological Studies on the Rous Sarcoma Virus. V. Preparation of Improved Standard Lots of the Virus for Use in Quantitative Investigations. J. Natt. Cancer Inst., 16: 877-888, 1956. r , 29. Nicol, T., Bilbey, D. L. J., Charles, L. M., Cordingly, J. L., and . Vernon-Roberts, B. Oestrogen: The Natural Stimulant of Body Defense. Endocrinology, 30: 277-291, 1964. 30. Nilsson, A., Revesz, L., and Stjernswird, J. Suppression of Strontium-90-induced Development of Bone Tumors by Infection with Bacillus Calmette-Guerin, BCG. Radiation Res., 26: 378-382, 1965. 31. Nowinski, R. C., Old, L. V., Sarkar, H. H., and Moore, D: H. Common Properties of the Oncogenic RNA Viruses. Virology, 42: 1152-1157,1970. 32. Old, L. J., Boyse, E. A., Clarke, D. A., and Carswell, E. A. Antigenic Properties of Chemically Induced Tumors. Ann. N. Y. Acad. ScL, 101: 80-106, 1968. 33. Parker, J. C., and Reynolds, R. K. Natural History of Sendai Virus Infection in Mice. Am. J. EpidemioL, 88: 112-125, 1968. 34. Pollack, S. B. Effect of Host Sex and Splenectomy on Moloney Virus-induced Sarcomas. Intern. J. Cancer, 8: 264-271, 1971. 35. Porta, D. G., and Terracino, B. Chemical Carcinogenesis in Infant. Animals. Progr. Expti. Tumor Res., 11: 334-363, 1969. 36. Prehn, R. T. Function of Depressed Immunologic Reactivity during Carcinogenesis. J. NatL Cancer Inst., 31: 791-805, 1963. 37. Price, P. J., Freeman, A. E., Lane, W. T., and Huebner, R. J. Morphological Transformation of Rat Embryo Cells by the Combined Action of 3-Methylcholanthrene and Rauscher Leukemia Virus. Nature New Biol., 230: 144-146, 1971. 38. Reed, L. Z., and Muench, H. A Simple Method of Estimating Fifty Percent Endpoints. Am. J. Hyg., 27: 493-497, 1938. 39. Rhim, J. S., Creasy, B., and Huebner, R. J. Production of Altered JANUARY 1973 Combined Effects of Type C RNA Virus and 3MC Cell Foci by 3-Methylcholanthrene in Mouse Cells Infected with AKR Leukemia Virus. Proc. NatL Acad. Sci. U. S., 68: 2212-2216, 1971. 40. Rhim, J. S., Huebner, R. J., Lane, W. T., and Vernon, M. L. Neoplastic Transformation of Rat Embryo Cells Induced in Vitro by Rauscher Leukemia Virus. Proc. Soc. ExptL Biol. Med., 133: 914-920, 1970. 41. Rhim, 1. S., Vass, W., Cho, H. Y., and Huebner, R. J. Malignant Transformation Induced by 7,12-Dimethylben2(a)anthracene in Rat Embryo Cells Infected with Rauscher Leukemia Virus. Intern. J. Cancer, 6: 65-74, 1971. 42. Rich, M. A. Virus Induced Murine Leukemia. In: Experimental Leukemia, p. 142. New York: Appleton-Century-Crofts, Inc., 1968. 43: Richard, G., Post, J. E., Noronha, F., and Barr. L. r1. A Transmissible Virus-induced Lymphocytic Leukemia of the Cat. J. Natl. Cancer Inst., 42: 987-1014, 1969. 44. Rowe, W. P.. Hartley, J. W., and Huebner, R. J. Polyoma and Other Indigenous Mouse Viruses. In: R. C. J. Harris (ed.), Problems of Laboratory Animal Diseases, pp. 131-142. New York: Academic Press, Inc., 1962. 45. Salaman, M. H., Rowson, K. E. K., Roe, F. J. C., Ball, J. K., Harvey, J. J., and De Benedictis, G. The Combined Action of Viruses and Other Carcinogens. In: Viruses, Nucleic Acids and Cancer, M. D. Anderson Hospital and Tumor Institute at Houston, pp. 544-558. Baltimore: The Williams & Wilkins Co., 1963. 46. Sever, J. L. Application of a Microtechnique to Viral Serological Investigations. J. Immunol., 88: 320-329, 1962. 47. Smith, R. T. Tumor-specific Immune Mechanism (continued). New Engl. J. Med., 278: 1268-1331, 1968. 48. Snyder, S. P., and Theilan, G. H. Transmissible Feline Fibrosarcomas. Nature, 221: 1074-1075, 1969. 49. Southam, C. M., Tanaka, S., Arata, T., Simkovic, 0., Miura. M., and Petropulos, S. F. Enhancement of Responses to Chemical Carcinogens by Nononcogenic Viruses and Anti-metabolites. Progr. Expti. Tumor Res.,11: 194-212, 1967. 50. Stjernsward, J. Effect of Bacillus Calmerte-Guerin and/or Methylcholanthrene on the Antibody Forming Cells Measured at the Cellular Level by a Hemolytic Plaque Test. Cancer Res., 26: 1591-1594, 1966. 51. Svet-Moldavsky, G. J., and Hamburg, V. P. An Approach to the Immunological Treatment of Tumors by Artificial Heterogenization. In: Specific Tumor Antigens, VICC Monograph Series, VoL 2, pp. 323-327. Copenhagen, Denmark: Munksgaard, 1967. 52. Tanaka, S., and Southam, C. M. Joint Action of Herpes simplex Virus and 3-Methylcholanthrene in Production of Papillomas in Mice. J. Natl. Cancer Inst., 34: 441-451, 1965. 53. Terres, F., Morrison, S. L., and Habicht, G. S. A Quantitative Difference in the Immune Responses between Male and Female Mice. Proc. Soc. ExptL Biol. Med.,127: 664-667, 1968. 54. Toth, B. A Critical Review of Experiments in Chemical Carcinogenesis Using Newborn Animals. Cancer Res., 28: 727-738, 1968. 55. Whitmire, C. E., Salerno, R. A., Rabstein, L. S., Huebner, R. J., and Turner, H. C. RNA Tumor Virus Antigen Expression in Chemically Induced Tumors. Virus Genome Specified Common Antigens Detected by Complement-fixation in Mouse Tumors Induced by 3-Methylcholanthrene. J. Nat1 Cancer Inst., 47: 1255-1265,1971. 77