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[CAN@KRRESEARCH @,15g6-1@4,Aug@M19681

SUMMARY

Different isolates of bovine papilloma virus (BPV) originating from different cases induced well-differentiated subcutanoons fibrosarcomas in 67 to 100% of inoculated hamsters afteran induction period ranging from 25 to 56 weeks. Age at timeof inoculation had little if any effect upon the incidence orsubsequent development of the BPV-induced tumors. Disseminated tumor nodules occurred in about 10% of the animals. Several tumors were transplanted to other hamsters. Areview of reports on other viruses oncogenic for the hamsterrevealed that the incidence of tumors decreased when olderanimals were inoculated.

INTRODUCTION

Bovine papilloma virus (BPV) can induce tumors in bothepithelial and connective tissues of the bovine as well as connective tissue tumors in other species of animals. The lesioninduced by BPV in the bovine skin may properly be called afibropapilloma since it is a fibroma underlying a benign neoplastic epithelium. This feature was first described by Creechin 1929 (10) and has been observed by others (4, 9). Bovinepapulioma virus can induce fibroblastic tumors independent ofepithelial hyperplasia in the urinary bladder (28) and genitalmucosa of cattle (26), as well as in the dermis of the horse(27) and C3H/eB mice (6). The hamster is susceptible to theoncogenic action of BPV and develops connective tissue tumorsin the dermis (6, 8, 25, 36) and brain (8, 25). Various factorsinfluencing tumor induction by BPV in the hamster and othercharacteristics of the tumors were examined in this study.These factors included the use of different BPV isolates, dose,different sites of subcutaneous inoculation, and variations ofthe age of the hamster at time of inoculation. In addition,several of the BPV-induced tumors were transplanted to otherhamsters.

MATERIALS AND METHODS

Cutaneous bovine fibropapillomas from several differentanimals were used. Isolate 247 had been carried in 3 successivebovine animal passages. Tissue from Isolate 247 was storedfor several years at 4C in 50% glycerin and saline prior to its

1 Published with the approval of the Director of the Wisconsin

Agricultural Experiment Station. Supported in part with the aidof RG-C4627 and USPHS Training Grant 817, NIH.

Received December 4, 1967; accepted April 15, 1968.

use in this experiment. Tissue from field Isolates 305, 306, and310 had been stored at 70C.The fibropapilloma tissue wasground in a mortar and pestle with sterile sand and saline tomake a 10% tissue suspension. This suspension was sonifiedat a setting of 3, with 3 amp for 2 minutes in an ice water bath(Model LS-75, Branson Inst. Co., Danbury, Conn.). The suepension was centrifuged at 3500 x g for 10 minutes and thesupernatant fluid used as the inoculum. A suspension of normalbovine skin was prepared in the same manner and injectedinto control hamsters.

Hamsters were obtained from Con Olson Co. Inc., Madison,Wisconsin. One-tenth ml of supernatant fluid of the appropriateinoculum was injected subcutaneously over the dorsal midlinein the thoracic and/or lumbar area (Table 1) . Isolates 305,306, and 310 were inoculated into the pinna of the ear as well.The hamsters were kept in individual cages to avoid canniballam and observed at 2-week intervals for the duration of theirexperimental life.

The induction period (interval from inoculation until the firstappearance of tumors) was determined by palpation of therestrained animal. Tumors were measured in two diametersand size was expressed as the average diameter. Several of thetumors were removed surgically, and small pieces of tumorwere transplanted subcutaneously into young (less than 14day old) as well as one-year-old normal hamsters.

Representative tissues were selected at necropsy for histologic examination. These were fixed in formalin, processed byconventional paraffin embedding, and sections were stained bythe hematoxylin and eosin, van Gieson, Giemsa, and Kingsleytechnics.

RESULTS

Death from non-neoplastic disease occurred in less than 10%of the hamsters before tumors developed, and these animalswere not considered in the results of the respective experiments.

Tumors developed in 86% (37/43) of the hamsters inoculatedat ages ranging from 2 to 52 weeks. The induction period averaged 39 weeks and ranged from 25 to 50 weeks (Table 1, Experiment1).

All 13 animals inoculated with dilutions of 10@ to 108from Isolate 247 developed tumors. Only 1 of 4 hamsters developed a tumor from the 104dilution, while the 10@ dilution failed to induce tumors in 5 hamsters. The induction periodaveraged 43 weeks and ranged from 36 to 56 weeks (Table 1,Experiment 2) . There was no appreciable difference in the

induction period with respect to the dilution of the viralsuspension.

1596 CANCER RESEARCH VOL.28

Oncogenic Action of Bovine Papilloma Virus in Hamsters'

Martin 0. RobI and Carl Olson

Department of Veterinary Science, University of Wisconsin, Madison, WiscOnsin 58706

Research. on February 14, 2019. 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Age at Number PercentNumber rnoc@i- devel- tumor WO@k8to

Inoculum inocu- lation oped devel- fI@ gToWthand dilution lated (wk.) tumors opment AverageRangeExperiment

1. Influence of age on tumorsusceptibility247@10'8 2 7 88 3425-36247@101

8 4 7 88 3425-37247@10'8 8 6 75 4436-50247@101

4 16 3 75 363340247@10'6 24 6 100 3526-46247@10'3 36 3 100 342638247@1016 52 5 83 3933-43Total

43 37 86 3925SOExperiment

2. Titration of a bovine papilloma virussuspension247@1O15 3 5 100 443652247@1024 3 4 100 523854247@1034 3 4 100 5240-56247@1O@5 3 1 20 4242247@105

5@ 3 00Total23 14 61 4336-56Experiment

3. Comparison of BPV isolates inoculated into subcutis of ear andback305@1O16(ear) 3 4 67 302434305

@ 101 6 (back) 3 6 100 3628-40306@1016(ear) 3 2 33 322836306

@ 101 6 (back) 3 4 67 4028-50310@1O'7(ear) 3 4 57 322440310@1O'7(back) 3 6 86 3826-52Total

19(ear) 10 53 3124-4019(back) 16 84 3826-52Experiment

4. ControltrialsBovinesldn@10'3 3 00Glycerin@2%3 3 00None

20 3 00Formalinized247 7 3 00Formalin@OS%

3 3 00Total36 0 0 a

of various ages with different

Bovine Papilloma Virus in Hamsters

Table 1

Incidence and induction of tumors produced in hamstersisolates of bovine fibropapilloma suspensions.

a Animals were kept under observation until death, which

20 months.occurred at an average age of

Three different field isolates (305, 306, and 310) induced tumors in 83% (16/19) of the hamsters inoculated subcutaneouslyon their backs, while 53% (10/19) developed a tumor at theinoculation site in the pinna of the ear (Table 1, Experiment3) . Animals that developed tumors in the ear also developedtumors on the back.

Suspensions of normal bovine skin (10%) and BPV (10%)inactivated by formalin (0.5%), as well as glycerin (2%) andformalin (0.5%) solutions, failed to induce tumors in hamsters.Twenty uninoculated animals did not develop tumors (Table 1,Experiment 4) . The control animals were kept in the same environment and under similar conditions as the test animals.They were examined periodically until death, which occurredat an average age of 20 months. As an additional controlmeasure, serums from several of the noninoculated and tumorbearing hamsters were collected at different times during thecourse of the study. The serums were tested for antibodiesagainst viral and tumor-specific antigens of SV4O, polyoma,

adenovirus type 12, and Rous sarcoma virus, as well as lymphocytic choriomeningitis virus. Complement-fixing antibodiesfor these agents were not detected in any of the hamster serums.Hamster tumors induced by BPV were examined for both viraland tumor-specific antigens by using the appropriate complement-fixing antiserums against the above oncogenic viruses andtumors induced by them. No such antigens were found. Thesecomplement fixation tests were done by Drs. Paul Black andRobert J. Huebner of the National Institutes of Health.

Tumors in the dermis first appeared as small (2-5 mm indiameter) , firm, circumscribed nodules after a long, variableincubation period. Initially, tumor growth was slow for the next4 to 14 weeks when most tumors grew to 10 to 20 mm indiameter. The rate of growth usually increased after the tumorsreached this size. Twenty to 24 weeks after first appearance,most of the tumors measured 40 to 50 mm in diameter andconsisted of either a single large mass or a cluster of varioussized nodules (Fig. 1) . Variation in growth rate of the tumors

AUGUST 1968 1597

Research. on February 14, 2019. 1968 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Martin G. Robi and Carl Olson

is represented in Chart 1. All of the tumors grew progressivelyand none regressed.

The epithelium covering the tumors frequently became erodedwith superficial necrosis when they were 20-50 mm in diameter.The firm tumors were usually separated from the adjacent normal subcutis by a loose connective tissue. .Occasionally the tumor growth extended into the abdominal cavity (Fig. 2) . Thetumor mass was usually irregular in shape and had a nodularappearance. Large tumors that weighed from 50 to 90 gm werenot uncommon at time of death, and in several hamsters thetumor weighed more than the host (Fig. 1) . One tumor weighed150 gm and the rest of the hamster weighed only 100 gm. Thecut surface of the tumors was usually pale yellow, but an cccasional tumor had circumscribed black nodular areas. Disseminated tumor nodules were found in approximately 10% of th

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