Virus-like Particles Associated with the Rous Sarcoma as ... · GAYL0RD—Virus-like Particles...
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Virus-like Particles Associated with the Rous Sarcoma as Seen in Sections of the Tumor* %MLLIAM H. GAYLORD, JR. (Departments of Microbiology and Pathology, Yale University School of Medicine, New Haven, Conn.) In 1947, Claude, Porter, and Pickels (4) demon strated that a virus-like particle is associated with the Rous sarcoma. They observed round, dense particles in the cytoplasmic portions of cells cul tured from two different tumors. The particles had diameters of 67—80mp and were uniform in ap pearance with a tendency to occur in pairs or clus ters. These observations were not confirmed, how ever, in spite of repeated efforts by a group in France (13). After 5 years of obtaining only ques tionable results, Bernhard and Oberling (8) re ported failure to demonstrate the particles in cul tured cells, and they suggested that the virus might be present in a masked form. Shortly after this paper appeared, Bernhard et al. (@) reported more favorable results stemming from an experi ment designed to test the effects of x-rays, ultra violet radiation, and isotopes on cells cultured from the tumor. Particles analogous to those de scribed by Claude, Porter, and Pickels were seen both in irradiated cells and in the controls. It was their conclusion that the particles are always pres ent but only in a small percentage of cells. The work reported here was undertaken to de termine whether particles were present in the mass of a tumor as it develops in its natural host and, if so, to study the relationship of the particles to tumor cells. The technic of sectioning tissue for electron microscopy offers many advantages over that of direct viewing of cultured cells. Observa tions are not limited by thick nuclei to the periph eral portions of the cytoplasm, and the observer is able to see more detail in all parts of the cell. METHODS Ti3siue specimens.'—Three different Rous sar coma tumors were studied. The first tumor, about 1 centimeter in diameter, was from a @0-day-old chicken inoculated 6 days previously. Another was * Aided by a grant #11% from the Jane Coffin Childs Memo rial Fund for Medical Research and by a research grant USPHS C1886 M & G from the National Cancer Institute of the National Institutes of Health, Public Health Service. Received for publication July 22, 1954. about @ centimeters in diameter, and it came from a fl-day-old chicken inoculated 15 days pre viously. The third tumor was 16 days old and came from a 4—5-week-old chicken. All inocula tions were cell suspensions. For comparison, a piece of 18-day chick chorioallantoic membrane and a methyicholanthrene-induced mouse sar coma were studied. Fixation and imbedding.—Small pieces of tissue were fixed in buffered osmium tetroxide (14) for @ hours and imbedded in a 1 : 10, methyl to butyl, methacrylate mixture (11). Cutting.—Sections were cut with glass knives (10) on a commercial model of the microtome de scrib@d by Porter and Blu.m (17). Sections were routinely flattened by flotation on water at 50°C. for several hours before mounting on collodion covered, 80-mesh, Cu-Ni grids. RESULTS Many particles resembling those reported pre viously (@, 4, 1@) were seen in the 6-day tumor (Figs. 1, @, 8, 5, 6). A few apparently identical par tides were observed in the 15-day tumor (Fig. 4) from the young chick, and none was seen in any of the other preparations. The particles in the 6-day tumor were uniform, round, and dense to electrons. They consisted of thin outer â€oemembranes― surrounding a denser cen tral mass (Fig. 1). The particle diameters varied from 450 to 600 A, and the central mass usually occupied about a third of the particle volume. This very dense central portion was usually round, but at times it appeared to be star-shaped (Figs. 1, 6). Particles occurred individually (Fig. 4) or in small clusters (Figs. 1, @, 4—6) on the outer surface of cell membranes. Pairing of particles was com mon when they occurred in clusters (Figs. 1, 3, 6), but isolated pairs were seldom observed (Fig. 4). Occasionally, clusters were observed which seemed to be within cells. In many instances it 11 am indebted to Dr. Francisco Duran-Reynals for the Rous sarcoma and to Dr. Henry Bunting for the mouse sarco ma specimens. 80 Research. on November 15, 2020. © 1955 American Association for Cancer cancerres.aacrjournals.org Downloaded from
Transcript of Virus-like Particles Associated with the Rous Sarcoma as ... · GAYL0RD—Virus-like Particles...
Virus-like Particles Associated with the Rous Sarcomaas Seen in Sections of the Tumor*
%MLLIAM H. GAYLORD, JR.
(Departments of Microbiology and Pathology, Yale University School of Medicine, New Haven, Conn.)
In 1947, Claude, Porter, and Pickels (4) demonstrated that a virus-like particle is associated withthe Rous sarcoma. They observed round, denseparticles in the cytoplasmic portions of cells cul
tured from two different tumors. The particles haddiameters of 67—80mp and were uniform in appearance with a tendency to occur in pairs or clusters. These observations were not confirmed, however, in spite of repeated efforts by a group inFrance (13). After 5 years of obtaining only questionable results, Bernhard and Oberling (8) reported failure to demonstrate the particles in cultured cells, and they suggested that the virusmight be present in a masked form. Shortly afterthis paper appeared, Bernhard et al. (@) reportedmore favorable results stemming from an experiment designed to test the effects of x-rays, ultraviolet radiation, and isotopes on cells culturedfrom the tumor. Particles analogous to those described by Claude, Porter, and Pickels were seenboth in irradiated cells and in the controls. It wastheir conclusion that the particles are always present but only in a small percentage of cells.
The work reported here was undertaken to determine whether particles were present in the massof a tumor as it develops in its natural host and, ifso, to study the relationship of the particles totumor cells. The technic of sectioning tissue forelectron microscopy offers many advantages overthat of direct viewing of cultured cells. Observations are not limited by thick nuclei to the peripheral portions of the cytoplasm, and the observer isable to see more detail in all parts of the cell.
METHODSTi3siue specimens.'—Three different Rous sar
coma tumors were studied. The first tumor, about1 centimeter in diameter, was from a @0-day-oldchicken inoculated 6 days previously. Another was
* Aided by a grant #11% from the Jane Coffin Childs Memo
rial Fund for Medical Research and by a research grantUSPHS C1886 M & G from the National Cancer Institute ofthe National Institutes of Health, Public Health Service.
Received for publication July 22, 1954.
about@ centimeters in diameter, and it came froma fl-day-old chicken inoculated 15 days previously. The third tumor was 16 days old andcame from a 4—5-week-old chicken. All inoculations were cell suspensions. For comparison, apiece of 18-day chick chorioallantoic membraneand a methyicholanthrene-induced mouse sarcoma were studied.
Fixation and imbedding.—Small pieces of tissuewere fixed in buffered osmium tetroxide (14) for@hours and imbedded in a 1 : 10, methyl to butyl,methacrylate mixture (11).
Cutting.—Sections were cut with glass knives(10) on a commercial model of the microtome describ@d by Porter and Blu.m (17). Sections wereroutinely flattened by flotation on water at 50°C.for several hours before mounting on collodioncovered, 80-mesh, Cu-Ni grids.
RESULTSMany particles resembling those reported pre
viously (@, 4, 1@) were seen in the 6-day tumor(Figs. 1, @,8, 5, 6). A few apparently identical partides were observed in the 15-day tumor (Fig. 4)from the young chick, and none was seen in any ofthe other preparations.
The particles in the 6-day tumor were uniform,round, and dense to electrons. They consisted ofthin outer “membranes―surrounding a denser central mass (Fig. 1). The particle diameters variedfrom 450 to 600 A, and the central mass usuallyoccupied about a third of the particle volume.This very dense central portion was usually round,but at times it appeared to be star-shaped (Figs. 1,6). Particles occurred individually (Fig. 4) or insmall clusters (Figs. 1, @,4—6)on the outer surfaceof cell membranes. Pairing of particles was common when they occurred in clusters (Figs. 1, 3, 6),but isolated pairs were seldom observed (Fig. 4).
Occasionally, clusters were observed whichseemed to be within cells. In many instances it
11 am indebted to Dr. Francisco Duran-Reynals for theRous sarcoma and to Dr. Henry Bunting for the mouse sarcoma specimens.
80
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GAYL0RD—Virus-like Particles and the Rous Sarcoma 81
could be shown that these clusters were actuallyextracellular (Fig. 5), but infrequently groups ofparticles were seen in locations considered to beintracellular (Figs. 5, 6).
Structures in the cytoplasm having dimensionssimilar to those of the virus-like particles butshowing less density were seen in tumor cells andin the two tissues studied for comparison (Fig. @).
In some areas of the 6-day-old tumor almostevery cell was associated with particles, whereasother areas of the same section lacked particles. Ithas not been possible to correlate the presence ofparticles with any cell type or stage of division.Many elongated fibroblast-like cells contained twonuclei (Fig. 6), and particles were sometimes in association with such cells. When mitotic figureswere observed, particles were sometimes present,sometimes not. The particles were also commonlyassociated with rounded cells having large monocyte-like nuclei. No unusual intracellular changescould be correlated with the presence of particles.Unusual but not necessarily pathological conditions observed in tumor cells included pores in thenuclear membranes (Fig. 6), an intricate endoplasmic reticulum, reduced numbers of mitochondria, and many circular structures in the cytoplasm which may have been vacuoles or sectionedinvaginations of the cell wall. Lipid-like depositsof osmiophilic material were encountered occasionally in the cytoplasm of some cells, possibly macrophages.
In the other tumor in which particles were seen(Fig. 4), the frequency of particle occurrence wasvery low. Prolonged searching revealed one or twoparticles on a very few cells with no observablepattern to their occurrence.
DISCUSSIONIt is clear that the finding of particles in sections
of the Rous sarcoma tumor is a matter of chance.In a 6-day-old tumor, particles were abundant inparts of every section studied. In another tumor,15 days old, the paucity of similar particles mightbe cause for rejecting the observations if the parallel study were not so convincing. The only reasonthat they were not rejected as anomalous findingswas the fact that occasional virus-like particlesclinging to the outer surface of a cell membraneresembled, in every way, those seen in great numbers in the 6-day-old tumor (Fig. 4). The thirdtumor studied showed no particles. In electronmicroscopy the sampling technic is such that onlyminute portions of the whole are studied; as a consequence, such studies are difficult to treat statistically, and negative results may be misleading. It isof interest, however, to note that particles were
found in large numbers in the youngest tumor froma young host. Fewer particles were found in anolder tumor from a young host, and no particleswere found in an equivalent aged tumor from anolder host. Duran-Reynals and Freire (6) haveshown that virus activity is dependent on both theage of the tumor and the age of the host, but itwould be presumptuous to say that this finding hasbeen verified by the small number of samples inthe present study.
In actuality, the second and third tumors werestudied merely to confirm the results obtainedfrom the first, and no difficulty was expected. Thefact that so few particles were found would lendstrong support to the early work of Oberling et al.(@,13).Theydid,indeed,finda fewparticlesbutfelt that they could not credit them because oftheir infrequent occurrence. Why they should suddenly see particles in large numbers or whyClaude, Porter, and Pickels (4) should have originally seen large numbers is, of course, not known.Selby has reported difficulty in repeating observations of particles in ascites tumor cells (18). It wasevident in the 6-day Rous tumor that the particlesoccurred in localized areas or foci, and, perhaps,the over-all lack of particles is merely a reflectionof a reduced number of such foci. The problem is atroublesome one in view of the fact that positiveidentification of the particles in more than onetumor is the only evidence available for ruling outthe possibility of a contaminant. The statementthat a lackof results is meaningless must, of course,be extended to any experimental controls, and,furthermore, a control for tumor tissue is not easilydefined.2
If the particles described here are to be acceptedas the virus of the Rous sarcoma, then their predominant extracellular location presents a problem. It is generally assumed that viruses are intracellular parasites, and if the present finding is to beunderstood then the following points must be considered for further investigation:
a) The virus might multiply extracellularly.Both previous reports of the particles (3, 4, 1@)mentioned that, in the case of cultured cells, it wasnot possible to know whether particles were in thecytoplasm or on the surface of a cell. Furthermore,Claude, Porter, and Pickels (4) drew attention tothe fact that the particles observed by them often
Bang reported at the 1954 meeting of the Electron Microscope Society of America (J. Applied Physics [in pressl) thatparticles similar to those associated with Rous sarcoma couldbe found in about 10 per cent of his tissue cultures which hadbeen grown from “normal―chick embryo. Because they werenot found in cells cultured from other sources, he feels thatthey could, in fact, be the Rous sarcoma virus being transmitted by eggs.
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occurred in pairs, suggestive of binary fission. Inthe present study pairing was noted when the particles were in clusters or under conditions whichwould promote cohesion by mechanical pressingtogether. (b) The virus could have been releasedfrom other cells and absorbed to the cells understudy. This possibility seems unlikely because theparticles were never seen scattered through the intercellular spaces. In fact, they were usuallytightly bound to cell membranes. In a study ofvaccinia virus (7) it was possible to follow a concentration gradient of extracellular particles, thenumbers being greatest near the center of a lesion.There were always areas where both extracellularand intracellular virus could be seen. (c) A thirdpossibility would be that the particles were formedwithin a cell and released as rapidly as they wereproduced. A system of influenza virus “extrusion―has been postulated where the virus is pinched offfrom the ends of long cytoplasmic protrusions carrying with it a portion of the cell membrane (9).Furthermore, no clear demonstration of intracellular influenza virus has yet been presented (8).
Bang (1) has suggested that an avirulent strain ofNewcastle disease virus might multiply at the surface of a host cell, in the ectoplasm. If a similarmechanism were operating here, then the intracellular particles of the Rous sarcoma might be thesame as those seen extracellularly but few in number, or they might be entirely different. In thelatter circumstance, differences could be in size,density, or both. Certain structures of the samesize range as the extracellular particles, but lessdense, can be seen in tumor cells (Fig. 2), but theycannot be differentiated from similar structures innormal cells (16). In addition, small, dense granules are seen concentrated in the nucleolus andscattered through the nucleus and cytoplasm (Fig.2). They resemble the basophilic component described by Palade (15). If clusters of these or similar particles were surrounded by a bit of cell membrane and then precipitated by the fixative, thepeculiar dense centers of the particles might be theresult (Fig. 1). Long strands radiating from thecenter of many of the particles seem to indicateeither internal organization or, more probably, a
FIG. 1.—Large cluster of virus-like particles observed insections of a 6-day-old Rous sarcoma tumor. The cluster issituated on the outer surface of a cell membrane in an invagination. The particles are uniform, round, and dense toelectrons. They consist of thin outer “membranes,―about500 A in diameter, and very dense centers. The central massis round or stellate. Several paired particles are present. Pairing is common when particles are in dusters and may resultmerely from cohesion. X71,000.
Fso. 2.—Medium-sized cluster clinging to the exterior of acell membrane. Intracellular structures marked by arrowsresemble virus-like particles in size but not density. Similarvesicles appear in normal cells, where they may be the resultof pinocytosis. Granules clustered in the cytoplasm (A) havedensities similar to the virus-like particles, but they are smaller. These granules are present in large numbers in some cellsof the tumor and may have RNA associated with them.X47,000.
FIG. 3.—Unusual intracellular cluster surrounded by deli
cate circles but no membrane. Pair of particles marked byarrows shows definite transverse septum indicating presenceof two individual particles. X39.000.
Fzo.4.—Fourparticles on surfaceofa cellfroma 15-day-oldtumor. This was the greatest concentration of particles observed in sections of the tumor. The picture is also representative of the major part of the 6-day-old tumor, all the otherother figures being especially selected for interest. X26,000.
Fia. 5 (lower flgure).—Portion of tumor cell showing partides at the surface and apparently intracellularly as well. Clusters of A, B, and C are actually situated on the outside in membrane folds. The space at D is extracellular, and, by inference,the cluster of particles atEcould beextracellular. Single partidemarked by arrow appears to be actually in the cytoplasm.Other single particles at left and right are on the outside.Round structures in the cytoplasm near the nudeus are prosumably cross-sectioned endoplasmic reticulum. X28,000.
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FIG. 6.—Elongated fibroblast-like cell with two nuclei(inset). Upper nucleus (n) has been cut almost at a tangentleaving only a cap in which prominent porelike structures arevisible. Clusters of virus-like particles at A, B, and (‘seem tobe intracellular. All other particles are extracellular. Intracellular particles are rarely encountered. The clusters at Band C include some circles of low density. Large extraceHularcluster at left contains particles with round central masses,star-shaped centers, and some with no dense centers. Someseem to have small vacuoles instead. Pair of particles at extreme tip of cell (D) is unusual, because it is not in a cluster.This pair has one particle with a dense center attached to avacuolated particle which is, in turn, attached to the cell bya stalk. X39,000; inset X@,3OO.
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GAm0RD—Virus-like Particles and the Rous Sarcoma 83
5. DAVIDSON, J. N. The Biochemistry of the Nucleic Acids,pp. 186—88.London: Methuen & Co., Ltd., 1950.
6. Duaasr-Ravr@a, F., and Fasinm@.,P. M. The Age of theTumor-bearing Hosts as a Factor Conditioning the Transmissibility of the Rous Sarcoma by Filtrates and Cells.Cancer Research, 13:879-8%, 1958.
7. GAYLORD,W. H., Ja., and Mmricx, J. L. IntracellularForms of Pox Viruses as Shown by the Electron Microscope (Vaccinia, Ectromelia, MoUuscum contagiosum). J.Exper. Med., 98:157—72, 1953.
8. Har@x, W. Multiplication of Influenza Virus in the Entodermal Cells of the Chick Embryo. In: Saarni, K. M., andLAiIFFEB, M. (eds.), Advances in Virus Research, 1:141-
227. New York: Academic Press, 1958.9. HomE, L. The Multiplication of Influenza Viruses in the
Fertile Egg. J. Hyg., 48:277—97, 1950.10. LATrA,H., and H&av@ie, J. F. Use of a Glass Edge in
Thin Sectioning for Electron Microscopy. Proc. Soc.Exper. Blot. & Med., 74:436—39, 1950.
11. Nawawi, S. B.; Boaysxo, B.; and SWERDLOW,M. NewSectioning Techniques for Light and Electron Microscopy.Science, 110:66—68, 1949.
12. OBERLING,C.; B@un, W.; Doirreunra, A.; andVionsa, P. Observation et étude quantitative de corpuscules d'aspect virusal dana des cultures de sarcome deRous. Experientia, 10:1-8, 1954.
13. OBERLING,C.; BzimxuAlw,W.; Gu@IuN,M.; and HAREL,J. Images de cellules cancéreusesau microscope électronique. Bull. du Cancer, 37:96—109, 1950.
14. PALADE,G. E. A Study of Fixation for Electron Microscopy. J. Exper. Med., 95:285—98,1952.
15. . A Small Particulate Component of the Cytoplasm.J. Applied Physics, 24:1419, 1953.
16. . Fine Structure of Blood Capillaries. Ibid., p.1424.
17. Powrmi, K. R., and BLtmr,J. A Study in Microtomy forElectron Microscopy. Anat. Rec., 117:685-710, 1953.
18. Ssaaiy, C. C. The Electron Microscopy of Normal andNeoplastic Cells. Texas Rep. Blot. & Med., 11:728-44,1954.
19. Sm@z@, W. M. Biochemistry and Biophysics of Viruses.In: Doama, R., and HAi.I@&vEn,C. (eds.), Handbuch derVirusforschung, pp. 468-67. Vienna: Julius Springer, 1038.
shrinkage of the internal mass of material. In anyevent, the small intracellular granules are the onlystructures with a density comparable to that of theparticle centers except for osmiophilic lipids. Ribonucleic acid is reportedly the only type of nucleicacid found in Rous sarcoma virus preparations (5),and the presence of lipid is questionable (19).
SUMMARY
Virus-like particles have been observed in sections of two Rous sarcoma tumors and not in athird. The particles were numerous in the youngesttumor, occurring singly, in pairs, or in small clusters usually on the outer surface of the cell membranes. The particles were round, dense, and about500 A in diameter. They consisted of thin limiting“membranes―and a very dense central masswhich was found or stellate. Occasionally, particles appeared vacuolated without a central mass.
The implications of the predominant extracellular location of particles has been discussed.
REFERENCES
1. B@ia, F. B. The Development of Newcastle DiseaseVirus in Cells of the Chorlo-allantoic Membrane asStudied by Thin Sections. Bull. Johns Hopkins Hoap., 92:809—28,1953.
2. Banxaaiw, W.; DoxTcasrr, A.; OBEBLING, C.; andVzoiua, P. Corpuacules d'aspect virusal dana lea cellulesdu sarcomede Rous. Bull. du Cancer, 40:311—21,1953.
8. BEaz@us1w,W., and OBEELING,C. Echec de Ia mise enevidence de corpuscules-virus dana lea celles du sarcome deRous. Bull. du Cancer, 40:178-85, 1953.
4. Cz.Aulm,A.; Poam, K R.; and Picxsa.s, E. ElectronMicroscope Study of Chicken Tumor Cells. Cancer Besearch, 7:421—80, 1947.
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1955;15:80-83. Cancer Res William H. Gaylord, Jr. in Sections of the TumorVirus-like Particles Associated with the Rous Sarcoma as Seen
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