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[CANCEK RESEARCH 28, 684-888, April 1968]
Virus Particles in Renal Tumors Obtained from SpringRana pipiens of Known Geographie Origin1
Robert Gilmore McKinnell and Joseph ZambernardDepartment of Biology, Newcomb College of Tulane University, New Orleans, Louisiana 70118, and Department of Anatomy,University of Colorado Medical Center, Denver, Colorado 80220
SUMMARY
Virus particles were found to be characteristically associatedwith spontaneous renal adenocarcinoma cells obtained fromRana pipiens collected from several localities in Minnesotaduring early spring 1967 while the afflicted frogs were still inoverwintering lakes or in breeding ponds. The lake water temperature from which the tumor-bearing frogs were obtainedwas 2-7°C. Eleven renal tumors were examined with the elec
tron microscope; intranuclear, cytoplasmic, and extracellularvirus particles were observed. One renal tumor was studiedwith the light microscope only; its histology resembled thatof tumors of frogs maintained in a cold laboratory environment.
These results are in contrast to the variable cytology andfine structure reported concerning renal tumors obtained infrogs from commercial sources.
INTRODUCTION
The renal tumor of Rana pipiens, first described by Lücke(8), is becoming increasingly interesting because of its possibleviral etiology (9, 10). Light microscope observations of primary tumors reveal that some tumors appear to have nuclearinclusions characteristic of viral infection, in contrast to othertumors which are free of such inclusions (9, 21). The cytologyof frog renal tumors appears to depend upon the temperatureof the laboratory where they are maintained or the season ofthe year.
Enlarged cells, many mitotic figures, and nuclei which containa large amount of intensely staining Feulgen-positive materialare characteristic of tumors of frogs kept in the laboratory at20-25°C. These tumors have been contrasted to tumors con
sisting of cells which appear to be necrotic or degenerating,with nuclei which stained faintly with Feulgen. Some cellsof the latter tumor type contain an irregularly shaped nuclearaggregate which stained faintly with Fast Green. The secondtumor type was characteristic of frogs which had been storedin the laboratory at 5°C for at least 2 months (24). Lücke
reported that there was seasonal variation in the cytology ofthe tumor (10).
1Supported by American Cancer Society Research GrantE-369-A (R.G.M.) and University of Colorado Medical CenterResearch Grant GRS-325 Fluid Research (J.Z.).
Received August 28, 1967; accepted December 25, 1967.
684
Electron microscopic studies of the fine structure of frogrenal tumors also reveal a variable cytology. Fawcett (7)examined 4 frog renal tumors with the electron microscope andreported that some tumor cells contained nuclear and cytoplasmic structures which he believed to be virus. Other tumorcells appeared to be free of the particles. Lunger et al. (14)reported that 80% of frog tumors obtained from a Wisconsindealer and 85.7% of tumors from Vermont contained virusparticles. All tumors were found to contain nuclear and cytoplasmic virus particles in a study of 13 renal tumors of theburnsi mutant of R. pipiens (29). The virus appears to be lostif the host frog is maintained at elevated temperature for afew days in the laboratory (28).
All of the studies of the fine structure of the Lückerenaladenocarcinoma have been made on frogs purchased fromdealers. Although a "life cycle" of the tumor has been proposed
(20, 21), absolutely nothing is known concerning the fine structure of tumors obtained from natural populations. Since alldealers must store their frogs, and since it is now known thattumor cytology varies depending on how the animals are maintained, it becomes apparent that the knowledge that we haverelates to stored frogs and we still know nothing concerningthe fine structure of this variable tumor obtained from frogsin their natural habitat. It would appear that studies of field-collected frogs bearing spontaneous tumors would be essentialto provide knowledge of the biology of the tumor and to confirm the proposed life cycle of the virus. The present reportis designed to provide some of this needed information.
A high incidence of renal tumors in north-central UnitedStates frogs obtained from a dealer was reported by McKinnell(16). It was clear from the study that these frogs were exceptionally susceptible to renal tumor formation. The spontaneous tumor rate of 8.5% (92 tumors from 1,088 frogs) wasamong the highest ever reported for any untreated group ofanimals. Because of the high spontaneous rate, it seemed to usthat the population of frogs would be ideal for studying therelationship of environmental factors associated with distribution and fine structure of the tumor. Accordingly, we begana study which involved the collection and autopsy of frogs takenfrom natural populations in the state of Minnesota. Collectionsover a period of several years suggest a seasonal fluctuationin the prevalence of tumors, with the highest rate occurringduring the cold months. The collections are described elsewhere(17-19). The present study concerns collections of frogs madein Minnesota during the Spring of 1967.
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Virus Particles in Renal Tumors
MATERIALS AND METHODS
Frogs. R. pipiens were collected during April, 1967. Themonth was chosen because the ice cover of most Minnesotalakes melts and frogs emerge from hibernation during this time.Leopard frogs can be captured with relative ease as they congregate near the margin of the lakes in preparation for leaving.Pairs of frogs in amplexus can be caught in small transientbreeding ponds adjacent or near the overwintering lakes.
Frogs were autopsied between 12 and 24 hours after capture.Autopsy was performed under a large magnifying glass, aroundwhich was mounted a circular fluorescent lamp which provided high intensity shadowless lighting.
Electron Microscopy. Fragments of tumor tissue measuring1-2 cu mm were cut with a razor blade from infected kidneysfound in autopsy. The tissue was placed in cold 4% S-collidine-buffered gluteraldehyde for 1 hour. The fixed tumor fragmentwas then rinsed and stored in S-collidine buffer until it wasshipped to Denver for examination.
The tissue was rapidly dehydrated through a graded seriesof ethyl alcohol and embedded in Epon 812 (11). Thin sectionswere cut with a Porter-Blum MT-2 microtome and placed onuncoated 200-mesh copper grids. The sections were stained for10 minutes at 60°C in 2% aqueous uranyl acetate, rinsed, andstained for 2-5 minutes in lead citrate (23). The sections wereexamined in a Philips EM-200 electron microscope.
RESULTS
Collections
Renal tumors of R. pipiens were discovered in three separatecounties. Frogs were collected in two other counties, but notumors were discovered. A description of each collection ispresented separately.
Otter Tail County. Date of collection: 12 April 1967. Placeof collection: Block Lake located 7 miles west of ParkersPrairie in southeastern part of county (the lake is designated"Black Lake" on the 1953 Brainerd, Minnesota 1:250,000 U. S.
Geological Survey Quadrangle). Most of the frogs were caughtfrom 10 to 15 feet from the shore in water about 2 feet deepwith a temperature of 2°C.A total of 100 frogs consisting of
52 males and 48 females were taken; of this number, 9 werefound to have renal adenocarcinomas. The tumors were designated SRT-1-67 through SRT-9-67 (SRT, spring renal tumor).The mean head-body length of all frogs in the Otter TailCounty collection was 67 mm. The mean head-body length ofthe tumor-bearing frogs in the sample was 68 mm. Seven ofthe tumor-bearing frogs were female, 2 were male. One of thetumor-bearing frogs was a juvenile female with a head-bodylength of 57 mm and an ovary without pigmonted ova.
Todd County. Date of collection: 16 April 1967. Place ofcollection: Cedar Lake located adjacent to U. S. Highway 71about 1 mile south of Little Sauk in the southern part of thecounty. Most of the frogs were caught within a foot of theshore in shallow water with a temperature of 7°C.Eighty-five
frogs comprised the collection; 37 were male and 48 were female. The mean head-body length was 56 mm. One tumor(SRT-10-67) was found in the sample. It was discovered in afemale frog with a head-body length of 71 mm.
Kandiyohi County. Date of collection: 18 April 1967. Placeof collection : In a breeding pond adjacent to east bank of Diamond Lake near County Road 137 in the eastern part ofCounty. No frogs could be found in the lake on that date.Frogs were calling and many pairs were seen in amplexus.Twenty-six males and 7 females comprised the sample whosemean head-body length was 69 mm. Two tumors (SRT-11-67and SRT-12-67) were found in frogs of the collection; bothfrogs were male and each measured 67 mm head-body length.
Itasca State Park. Date of collection: 20 April 1967. Placeof collection: On roads in Hubbard and Clearwater Countieswithin Itasca State Park. Frogs were collected from twilightuntil 10 P.M. as they migrated across the roads. Air temperaturewas 9°C. Forty-six frogs (30 males and 16 females) whose
mean head-body length was 66 mm were collected. No tumorswere found.
Hubbard County. Date of collection: 26 April 1967. Fifteenfrogs (mean head-body length 68 mm) were taken from breeding ponds. No tumors were found.
Fine Structure
Eleven tumors were examined with the electron microscope.All were found to contain virus particles. Intranuclear, cyto-plasmic, and extracellular virus particles similar to those whichhave been described elsewhere (7, 12-14, 27-29) were observed(Figs. 1-3). Thus, it would seem that virus particles which areresolvable with the electron microscope are characteristicallyassociated with spontaneous frog renal tumors derived from anearly spring cold water environment in Minnesota. One tumorwas examined with the light microscope only. It was similarin histology to the cold temperature frogs of Roberts (24).
DISCUSSION
Interest in the relationship of viruses to cancer has beenstimulated by studies of a malignant tumor in children ofAfrica known as Burkitt's lymphoma (2). Virus particles have
been resolved by electron microscopy in cells cultured frompatients afflicted with Burkitt's lymphoma (5, 25). Of par
ticular interest to the present study is the fact that the morphology of the virus associated with cells derived from patients with Burkitt's tumor bear a striking similarity to virus
particles which have been observed in the renal tumor of theleopard frog. Indeed, Epstein, Toshima, and their associatescall attention to the impressive resemblances of virus particlesassociated with the human lymphoma and the frog renal adeno-carcinoma (6, 26). In support of this view, we would call attention to the similar morphologies, as revealed in Fig. 3 ofEpstein et al. (6) and Fig. 15 of Zambernard et al. (29).
Tumor natural history studies (i.e., epidemiology) may contribute to an understanding of the relationship of proposedetiologic agents, their mode of transmission, and periods of hostsusceptibility. Accordingly, Burkitt has made tumor "safaris"(3, 4). The "safaris" are in effect geographic biopsies. They
are trips in an effort to survey and delimit portions of Africainto tumor-bearing areas and tumor-free areas.
The present study of frog tumors resulted from "frog tumor safaris" conducted over a period of several years. We are
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Robert Gilmore McKinnell and Joseph Zambernard
not the first to make safaris in search of frog tumor epidemiologie data. Mateyko (15) was unable to find tumors amongMontana R. pipiens. Auclair (1) reported finding tumor-bearing leopard frogs near Lake Champlain in Vermont, but noreport was made of the fine structure of the tumors. K. A.Rafferty, Jr. and one of the authors of this paper (R. G. M.)collected R. pipiens in the northern United States and partsof Canada during the summer of 1963. Renal tumors developedin frogs stored in the laboratory for 8 months which had beencollected in several areas including Minnesota and SouthDakota (22).
A variable fine structure of the frog renal tumor has beenreported, i.e., virus particles can be resolved in some but notother renal tumors (7, 14). The question to which we addressedthe present study was: Do spontaneous renal tumors obtainedfrom frogs of similar cold water environments also show thevariable fine structure? Our answer is that, in this study ofeleven renal tumors examined with the electron microscope,we do not find any tumors free of virus particles.
This report comprises the first study of the fine structureof the renal tumor of R. pipiens which has been made on material which has not been stored by a dealer or held for anyappreciable time in a laboratory. All the tumors were foundto have virus particles associated with the tumor cells. Webelieve that the presence of virus particles in tumor cells istypical of the early spring Minnesota renal tumors. Additionally, we feel that the presence of virus particles in allLücketumors is probably also typical of the winter condition.We believe this because, although the frogs were collected inthe spring, many of the lakes still had ice cover and the temperature periodically plummeted precipitously. Examples of"winter-like" conditions in Minnesota taken from our notes in
clude: Frogs were collected in Itasca State Park on 20 April1967 at an air temperature of 9°C.Two days later, the temperature dropped to —9°Cand there were 2 inches of snow
on the ground. Ice covered most of Leech Lake in Cass Countyas late as 15 April 1967. Cedar Lake in Todd County wascovered with ice on 9 April 1967. We collected 85 frogs fromCedar Lake six days later. Ice covered most of Sand Lake,Prairie Lake, and Lake Lida north of Pelican Rapids in OtterTail County on 8 April 1967. One hundred frogs were takenin the county 4 days later.
The fine structure of renal tumors of frogs taken directlyfrom warm weather (i.e., summer and early fall) populationsis not known. We predict, on the basis of laboratory studies(24) and field studies (19), that warm weather tumors will befree of virus particles which can be resolved with the electronmicroscope. We are presently studying the fine structure ofrenal tumors obtained from frogs captured and autopsied inthe field during late summer and early fall (Zambernard andMcKinnell, manuscript in preparation).
ACKNOWLEDGMENTS
The authors thank Dr. William H. Marshall, Director, FieldBiology Program, University of Minnesota, for his courtesy inmaking laboratory and housing facilities available for this studyat the University of Minnesota, Lake Itasca Forestry and BiologyStation, Lake Itasca, Minnesota.
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Fig. 1. A section of a tumor nucleus containing immature (IV) and mature (MV) virus particles. Note that the mature particlesare enclosed with a thin membranous sac. Uranyl acetate and lead citrate stain, X 13,000.
Fig. 2. Virus particles in the cytoplasm show distinct double-membraned capsids and viral nucleoid. Note that they are enclosedwithin a membranous envelope. Uranyl acetate and lead citrate stain, X 17,500.
Fig. 3. The fine structure of extracellular virus particles found within the lumens of the tumor tubules are almost identical to thecytoplasmic particles, with the exception that they contain a dense amorphous material in the space between the viral envelope andcapsid (arrows). Uranyl acetate and lead citrate stain, X 48,000.
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688 CANCER RESEARCH VOL. 28
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1968;28:684-688. Cancer Res Robert Gilmore McKinnell and Joseph Zambernard
of Known Geographic OriginpipiensRanaVirus Particles in Renal Tumors Obtained from Spring
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